**Understanding and Manipulating Endogeneous Healing of Tissues**

10 Tissue Regeneration – From Basic Biology to Clinical Application

Vandenburgh, H.H. et al., 1996. Mechanical stimulation of organogenic cardiomyocyte

**1** 

*Italy* 

**The Role of Physical Factors** 

**Tissue Repair and Regeneration** 

*Dept. of Clinical Physiopathology, University of Florence* 

Physical factors may induce significant biological effects, therefore they can be applied in biomedical and biotechnological fields in order to drive and modulate biological processes. It is well known that both humoral and physical factors (in particular, but not limited to the mechanical ones) are necessary for maintaining tissue homeostasis. Both biochemical and physical factors can induce the cells to reprogram their functions to adapt dynamically to

It is evident, therefore, that the only way to approach functional tissue regeneration and repair is to supply combined humoral and physical stimuli in a dose- and time-dependent manner. For example, in vitro studies have shown that a biomimetic environment simulating pulsatile flow is an indispensable condition for the tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Static controls show

Studies on the role of physical factors in tissue repair and regeneration cover a very broad field that extends from investigations aimed at deepening our understanding of the physiological mechanisms of tissue repair and regeneration to biotech advances in tissue engineering, such as development of biocompatible scaffolds, 3D cell culture systems and bioreactors, which in the future must integrate the delivery of biochemical factors with the provision of physical stimuli that are equally necessary. In this chapter, far from providing a comprehensive overview of this field of studies, we introduce some issues concerning the application of physical factors in biomedicine and biotechnology and report the results of our research on the application of various physical stimuli (gravitational and mechanical stresses, laser radiation, electromagnetic fields (EMF)) for modulating cell commitment and differentiation, cell adhesion/migration, production and assembly of extracellular matrix (ECM) components, with the final aim of understanding when and how physical stimuli can be useful for promoting tissue repair and formation of functional tissue constructs. We also briefly mention how, in past centuries, the role of physical factors in biological processes has been understood and

morphological alterations and weaker mechanical properties (Hoerstrup et al., 2002).

physical stimuli have been applied for therapeutic purposes.

**1. Introduction** 

the environmental conditions.

**in Cell Differentiation,** 

Monica Monici and Francesca Cialdai *ASAcampus Joint Laboratory, ASA Res. Div.* 

## **The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration**

Monica Monici and Francesca Cialdai *ASAcampus Joint Laboratory, ASA Res. Div. Dept. of Clinical Physiopathology, University of Florence Italy* 

#### **1. Introduction**

Physical factors may induce significant biological effects, therefore they can be applied in biomedical and biotechnological fields in order to drive and modulate biological processes. It is well known that both humoral and physical factors (in particular, but not limited to the mechanical ones) are necessary for maintaining tissue homeostasis. Both biochemical and physical factors can induce the cells to reprogram their functions to adapt dynamically to the environmental conditions.

It is evident, therefore, that the only way to approach functional tissue regeneration and repair is to supply combined humoral and physical stimuli in a dose- and time-dependent manner. For example, in vitro studies have shown that a biomimetic environment simulating pulsatile flow is an indispensable condition for the tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Static controls show morphological alterations and weaker mechanical properties (Hoerstrup et al., 2002).

Studies on the role of physical factors in tissue repair and regeneration cover a very broad field that extends from investigations aimed at deepening our understanding of the physiological mechanisms of tissue repair and regeneration to biotech advances in tissue engineering, such as development of biocompatible scaffolds, 3D cell culture systems and bioreactors, which in the future must integrate the delivery of biochemical factors with the provision of physical stimuli that are equally necessary. In this chapter, far from providing a comprehensive overview of this field of studies, we introduce some issues concerning the application of physical factors in biomedicine and biotechnology and report the results of our research on the application of various physical stimuli (gravitational and mechanical stresses, laser radiation, electromagnetic fields (EMF)) for modulating cell commitment and differentiation, cell adhesion/migration, production and assembly of extracellular matrix (ECM) components, with the final aim of understanding when and how physical stimuli can be useful for promoting tissue repair and formation of functional tissue constructs. We also briefly mention how, in past centuries, the role of physical factors in biological processes has been understood and physical stimuli have been applied for therapeutic purposes.

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 15

shown to be affected by cell shape (Roskelley, 1994; McBeath, 2044; Watt 1988; Spiegelman, 1983). Internal and external forces regulate cell shape and studies have shown that cell shape can control apoptosis, gene expression, and protein synthesis, in addition to stem cell fate

The cells belonging to tissues that resist the effects of gravity are particularly sensitive to mechanical and gravitational stimuli, which play a key role in the development and homeostasis of these tissues. Lack of gravitational and mechanical stresses leads to the formation of impaired tissues with lower mechanical properties and reduced function.

It is well established that bone adapts its mass and architecture in accordance with the external mechanical loads applied and osteocytes, terminally differentiated cells of the osteoblastic lineage, may be considered "mechanosensory cells" (Vatsa et al., 2010). They are sensitive to both stretching and fluid flow. Mechanical stimulation of osteocytes induces intercellular signaling which results in the modulation of osteoblast and osteoclast activity (Chow et al., 1998; Turner et al., 1997). Interestingly, it has been shown that the stimulation

Osteoblastic differentiation can be induced by applying mechanical stress, for example by stretching the surface on which the cells are attached (Cavalcanti-Adam et al., 2002). Many studies revealed that the micromotions at the interface between bone and artificial scaffolds play a key role in scaffold integration: they can promote tissue differentiation or induce bone resorption (Prendergast et al., 1997; Carter et al., 1998; Buchler et al., 2003; Stadelmann et al., 2008; Jasty et al.,1997). In a recent paper on biomechanics of scaffolds for bone tissue engineering applications, it has been stated that in the development of a scaffold it is important to take in account not only the structural integrity but also the load transmitted to

A recent review of studies which investigated the importance of loading in maintaining the balance of matrix turnover in the intervertebral disk, reported about the possible role of overloading in the initiation and progression of disc degeneration and proposed a physiological/beneficial loading range as a basis on which to design loading regimes for testing tissue constructs or favouring differentiation of stem cells towards "discogenic" cells

An overview of studies on the role of mechanical stimuli in chondrogenesis showed that uniaxial loading induces the upregulation of genes associated with a chondrogenic phenotype while multiaxial loading results in a broader pattern of chondrogenic gene upregulation, revealing that not only intensity, but also direction and other parameters which characterize the stimulation are relevant for the achievement of the final effect. The physiological multiaxial pattern of loading within articulating joints is so complex that currently, even with the most sophisticated bioreactors, it would be impossible to simulate the in vivo situation. Therefore, it has been suggested to use the body as an "in vivo

Conditions of gravitational unloading, both real and modeled by a Random Positioning Machine (RPM), negatively affect cellular organization and ECM production in cartilage

constructs, even if at different extent. (Stamenkovic et al., 2010).

of a single osteocyte activates many surrounding cells (Vatsa et al., 2007).

the cells via the scaffold deformation (Pioletti, 2011).

for tissue engineering (Chan et al., 2011)

bioreactor"(Grad et al., 2011).

(Chen, 1997; Thomas, 2002).

#### **2. Mechanical stresses**

The importance of gravitational and mechanical factors in modulating biological processes has been known for a long time: from Galileo Galilei onwards, studies on functional adaptation of the skeleton demonstrated that bone loss or gain is related to the magnitude, direction and frequency of the stress acting upon the skeleton during application of loads (Galilei, 1632; Wolff, 1985; Rubin, 1985; Frost, 1988; Rubin, 1984; Ingber, 1998).

Within the body, cells are subject to mechanical stimulation, caused by blood circulation, ambulation, respiration, etc., which give rise to a variety of biochemical responses. It has been demonstrated that changes in inertial conditions, shear stress, stretching, etc. can strongly affect cell machinery. Cells may sense mechanical stresses through changes in the balance of forces that are transmitted across transmembrane adhesion receptors that link the cytoskeleton to the ECM and to other cells. These changes, in turn, alter the ECM mechanics, cell shape and cytoskeletal organization (Ingber, 1998, 1999). A great deal of information has revealed that the ECM is a highly dynamic and elastic structure which undergoes continuous remodelling, in particular during development, angiogenesis, wound healing and other tissue repair processes. The ECM interacts with cells to provide relevant microenvironmental information, biochemically through the release of stored soluble and insoluble factors, and physically through imposition of structural and mechanical constraints (Carson, 2004). On the other hand, mechanical stimuli modulate ECM homeostasis: mechanical forces strictly regulate the production of ECM proteins indirectly, by stimulating the release of paracrine growth factors, or directly, by triggering intracellular signalling pathways leading to the activation of genes involved in ECM turnover (Chiquet, 2003).

Mechanical stimuli affect cells through poorly understood mechanotransductive pathways that lead to changes in morphology and orientation, modulation of gene expression, reorganization of cell structures and intercellular communication through both secretion of soluble factors and direct intercellular contact (Maul et al., 2011; Kang et al., 2011; Park et al., 2006; Papachroni et al., 2009; Wall & Banes, 2005; Bacabac et al., 2010; Hughes-Fulford & Boonstra, 2010). Over the past decade, in vitro studies have indicated that the transduction of physical stimuli involves the ECM-integrin-cytoskeleton network and also calcium channels, guanosine triphosphatases (GTPases), adenylate cyclase, phospholipase C (PLC) and mitogen-activated protein kinases (MAPKs), all of which play important roles in early signaling (Rubin et al., 2006; Hoberg et al., 2005; Adachi et al., 2033; Mobasheri et al., 2005; Chiquet et al., 2009; Bacabac et al., 2010; Hughes-Fulford & Boonstra, 2010). It has been demonstrated that, in endothelial cells, different genetic programs leading to growth, differentiation and apoptosis can be mechanically switched. Cells grow when they spread, die when fully retracted, and differentiate into capillary tubes if maintained at a moderate degree of extension (Chen, 1997).

The in vitro application of mechanical stretch, simulating the mechanical load to whom heart cells are exposed in vivo, initiated in adherent cultures of neonatal cardiomyocytes morphological alterations similar to those occurring during in vivo heart growth (Vandenburg, 1996). Stem cell commitment, the process by which a cell chooses its fate, and differentiation, the resulting development of lineage-specific characteristics, have been

The importance of gravitational and mechanical factors in modulating biological processes has been known for a long time: from Galileo Galilei onwards, studies on functional adaptation of the skeleton demonstrated that bone loss or gain is related to the magnitude, direction and frequency of the stress acting upon the skeleton during application of loads

Within the body, cells are subject to mechanical stimulation, caused by blood circulation, ambulation, respiration, etc., which give rise to a variety of biochemical responses. It has been demonstrated that changes in inertial conditions, shear stress, stretching, etc. can strongly affect cell machinery. Cells may sense mechanical stresses through changes in the balance of forces that are transmitted across transmembrane adhesion receptors that link the cytoskeleton to the ECM and to other cells. These changes, in turn, alter the ECM mechanics, cell shape and cytoskeletal organization (Ingber, 1998, 1999). A great deal of information has revealed that the ECM is a highly dynamic and elastic structure which undergoes continuous remodelling, in particular during development, angiogenesis, wound healing and other tissue repair processes. The ECM interacts with cells to provide relevant microenvironmental information, biochemically through the release of stored soluble and insoluble factors, and physically through imposition of structural and mechanical constraints (Carson, 2004). On the other hand, mechanical stimuli modulate ECM homeostasis: mechanical forces strictly regulate the production of ECM proteins indirectly, by stimulating the release of paracrine growth factors, or directly, by triggering intracellular signalling pathways leading to the activation of genes involved in ECM turnover (Chiquet,

Mechanical stimuli affect cells through poorly understood mechanotransductive pathways that lead to changes in morphology and orientation, modulation of gene expression, reorganization of cell structures and intercellular communication through both secretion of soluble factors and direct intercellular contact (Maul et al., 2011; Kang et al., 2011; Park et al., 2006; Papachroni et al., 2009; Wall & Banes, 2005; Bacabac et al., 2010; Hughes-Fulford & Boonstra, 2010). Over the past decade, in vitro studies have indicated that the transduction of physical stimuli involves the ECM-integrin-cytoskeleton network and also calcium channels, guanosine triphosphatases (GTPases), adenylate cyclase, phospholipase C (PLC) and mitogen-activated protein kinases (MAPKs), all of which play important roles in early signaling (Rubin et al., 2006; Hoberg et al., 2005; Adachi et al., 2033; Mobasheri et al., 2005; Chiquet et al., 2009; Bacabac et al., 2010; Hughes-Fulford & Boonstra, 2010). It has been demonstrated that, in endothelial cells, different genetic programs leading to growth, differentiation and apoptosis can be mechanically switched. Cells grow when they spread, die when fully retracted, and differentiate into capillary tubes if maintained at a moderate

The in vitro application of mechanical stretch, simulating the mechanical load to whom heart cells are exposed in vivo, initiated in adherent cultures of neonatal cardiomyocytes morphological alterations similar to those occurring during in vivo heart growth (Vandenburg, 1996). Stem cell commitment, the process by which a cell chooses its fate, and differentiation, the resulting development of lineage-specific characteristics, have been

(Galilei, 1632; Wolff, 1985; Rubin, 1985; Frost, 1988; Rubin, 1984; Ingber, 1998).

**2. Mechanical stresses** 

2003).

degree of extension (Chen, 1997).

shown to be affected by cell shape (Roskelley, 1994; McBeath, 2044; Watt 1988; Spiegelman, 1983). Internal and external forces regulate cell shape and studies have shown that cell shape can control apoptosis, gene expression, and protein synthesis, in addition to stem cell fate (Chen, 1997; Thomas, 2002).

The cells belonging to tissues that resist the effects of gravity are particularly sensitive to mechanical and gravitational stimuli, which play a key role in the development and homeostasis of these tissues. Lack of gravitational and mechanical stresses leads to the formation of impaired tissues with lower mechanical properties and reduced function.

It is well established that bone adapts its mass and architecture in accordance with the external mechanical loads applied and osteocytes, terminally differentiated cells of the osteoblastic lineage, may be considered "mechanosensory cells" (Vatsa et al., 2010). They are sensitive to both stretching and fluid flow. Mechanical stimulation of osteocytes induces intercellular signaling which results in the modulation of osteoblast and osteoclast activity (Chow et al., 1998; Turner et al., 1997). Interestingly, it has been shown that the stimulation of a single osteocyte activates many surrounding cells (Vatsa et al., 2007).

Osteoblastic differentiation can be induced by applying mechanical stress, for example by stretching the surface on which the cells are attached (Cavalcanti-Adam et al., 2002). Many studies revealed that the micromotions at the interface between bone and artificial scaffolds play a key role in scaffold integration: they can promote tissue differentiation or induce bone resorption (Prendergast et al., 1997; Carter et al., 1998; Buchler et al., 2003; Stadelmann et al., 2008; Jasty et al.,1997). In a recent paper on biomechanics of scaffolds for bone tissue engineering applications, it has been stated that in the development of a scaffold it is important to take in account not only the structural integrity but also the load transmitted to the cells via the scaffold deformation (Pioletti, 2011).

A recent review of studies which investigated the importance of loading in maintaining the balance of matrix turnover in the intervertebral disk, reported about the possible role of overloading in the initiation and progression of disc degeneration and proposed a physiological/beneficial loading range as a basis on which to design loading regimes for testing tissue constructs or favouring differentiation of stem cells towards "discogenic" cells for tissue engineering (Chan et al., 2011)

An overview of studies on the role of mechanical stimuli in chondrogenesis showed that uniaxial loading induces the upregulation of genes associated with a chondrogenic phenotype while multiaxial loading results in a broader pattern of chondrogenic gene upregulation, revealing that not only intensity, but also direction and other parameters which characterize the stimulation are relevant for the achievement of the final effect. The physiological multiaxial pattern of loading within articulating joints is so complex that currently, even with the most sophisticated bioreactors, it would be impossible to simulate the in vivo situation. Therefore, it has been suggested to use the body as an "in vivo bioreactor"(Grad et al., 2011).

Conditions of gravitational unloading, both real and modeled by a Random Positioning Machine (RPM), negatively affect cellular organization and ECM production in cartilage constructs, even if at different extent. (Stamenkovic et al., 2010).

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 17

These results, in agreement with those of other authors (Kaneuji et al., 2011; Wang et al., 2010; Searby et al., 2005) reveal that mechanical/gravitational stresses induce osteoblastic differentiation while gravitational unloading and loss of mechanical stress favour

In cultures of fibroblasts exposed for 3 hours to hypergravity (10x*g*), we observed enhanced expression of collagen I and fibronectin (20% and 30% more than control, respectively), while chondrocytes exposed to the same treatment showed a marked increase in collagen II, aggrecan and Sox 9, a transcription factor which plays a key role in chondrogenesis. Therefore, after definition of optimal range of intensity and force direction, loading can be used to stimulate ECM production by cells of the connective tissues and to favour

A series of experiments we carried out with the aim of studying the effect of gravitational unloading on processes involved in tissue remodelling demonstrated that the loss of mechanical stress causes a disregulation in laminin and fibronectin (FN) production by fibroblasts and endothelial cells (Fig. 2B) (Monici et al., 2011). In particular, FN forms a disordered and intricate network, reproducing the typical condition of fibrous scars. We hypothesized that the altered FN fibrillogenesis could be a cause of impaired ECM rebuilding and altered cell adhesion/migration and could contribute to the impairment of

wound healing observed in microgravity (Midura & Androjna, 2006; Delp, 2008).

Fig. 2. FN expression in CVECs (analysed by immunofluorescence microscopy): A) control, B) exposed for 72 h to modelled microgravity and C) treated with pulsed Nd:YAG laser (1064 nm). In figure B a tight network of FN fibrils appears while in figure C the FN fibrils

Studying the behaviour of aortic endothelial cells cultured in micro- and hypergravity we found that the exposure to simulated microgravity conditions for 72 hours (angular velocity of rotation 60°/s) caused a reduction in coronary venular endothelial cell (CVEC) number. Genomic analysis revealed that proapoptotic signals increased, while antiapoptotic and proliferation/survival genes were downregulated by the absence of gravity. Activation of apoptosis was accompanied by morphological changes, with mitochondrial disassembly and organelles/cytoplasmic NAD(P)H redistribution, as evidenced by autofluorescence analysis. Moreover, cells were not able to respond to angiogenic stimuli in terms of

adipogenesis, osteoclastogenesis and bone resorption.

chondrogenesis.

are parallel and ordered (see arrows).

migration and proliferation (Morbidelli et al., 2005)

Our group is conducting for several years research on the role of gravitational and mechanical stimuli in cell differentiation, tissue repair and regeneration, with particular attention to the remodelling phase.

Our studies demonstrated that gravitational unloading favours the differentiation of osteoclastic precursors (FLG 29.1 cells). After 72 hours exposure to conditions of microgravity, modelled by a RPM (angular velocity of rotation 60°/s), the cells showed a dramatic increase in apoptosis, but the viable ones showed osteoclastic-like morphology, cytoskeletal reorganization, significant changes in gene expression profile. The expression of the major osteoclastic markers Receptor Activator of Nuclear Factor Kappa-B (RANK) and Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) strongly increased and cells showed the ability to resorb bone (Fig. 1) (Monici et al., 2006).

Fig. 1. Scanning electron microscopy of a bone slice exposed to FLG 29.1 cells cultured in modelled microgravity. Adherent cells on the bone surface can be observed. Arrows indicate the sealing zone.

Analysing the gene expression profile of human mesenchymal stem cells (HMSC) in loading conditions (3 hours exposure to 10x*g* in hyperfuge), we found overexpression of genes involved in osteoblastogenesis (*GLI1, NF1, MEN1*) and downregulation of genes involved in adipogenesis (*PPAR, FABP4*) (Tab. 1) (Monici et al., 2008a).


Table 1. Gene expression profile in HMSCs.

Our group is conducting for several years research on the role of gravitational and mechanical stimuli in cell differentiation, tissue repair and regeneration, with particular

Our studies demonstrated that gravitational unloading favours the differentiation of osteoclastic precursors (FLG 29.1 cells). After 72 hours exposure to conditions of microgravity, modelled by a RPM (angular velocity of rotation 60°/s), the cells showed a dramatic increase in apoptosis, but the viable ones showed osteoclastic-like morphology, cytoskeletal reorganization, significant changes in gene expression profile. The expression of the major osteoclastic markers Receptor Activator of Nuclear Factor Kappa-B (RANK) and Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) strongly increased and cells

Fig. 1. Scanning electron microscopy of a bone slice exposed to FLG 29.1 cells cultured in modelled microgravity. Adherent cells on the bone surface can be observed. Arrows

Analysing the gene expression profile of human mesenchymal stem cells (HMSC) in loading conditions (3 hours exposure to 10x*g* in hyperfuge), we found overexpression of genes involved in osteoblastogenesis (*GLI1, NF1, MEN1*) and downregulation of genes involved in

**Gene** Control RPM 10 x g Nd:YAG

*, FABP4*) (Tab. 1) (Monici et al., 2008a).

**FABP4 27 304 3 9 PPARG 12 587 11 14 GLI1 47 45 789 547 NF1 25 9 241 258 MEN1 48 25 158 258** 

attention to the remodelling phase.

indicate the sealing zone.

adipogenesis (*PPAR*

Table 1. Gene expression profile in HMSCs.

showed the ability to resorb bone (Fig. 1) (Monici et al., 2006).

These results, in agreement with those of other authors (Kaneuji et al., 2011; Wang et al., 2010; Searby et al., 2005) reveal that mechanical/gravitational stresses induce osteoblastic differentiation while gravitational unloading and loss of mechanical stress favour adipogenesis, osteoclastogenesis and bone resorption.

In cultures of fibroblasts exposed for 3 hours to hypergravity (10x*g*), we observed enhanced expression of collagen I and fibronectin (20% and 30% more than control, respectively), while chondrocytes exposed to the same treatment showed a marked increase in collagen II, aggrecan and Sox 9, a transcription factor which plays a key role in chondrogenesis. Therefore, after definition of optimal range of intensity and force direction, loading can be used to stimulate ECM production by cells of the connective tissues and to favour chondrogenesis.

A series of experiments we carried out with the aim of studying the effect of gravitational unloading on processes involved in tissue remodelling demonstrated that the loss of mechanical stress causes a disregulation in laminin and fibronectin (FN) production by fibroblasts and endothelial cells (Fig. 2B) (Monici et al., 2011). In particular, FN forms a disordered and intricate network, reproducing the typical condition of fibrous scars. We hypothesized that the altered FN fibrillogenesis could be a cause of impaired ECM rebuilding and altered cell adhesion/migration and could contribute to the impairment of wound healing observed in microgravity (Midura & Androjna, 2006; Delp, 2008).

Fig. 2. FN expression in CVECs (analysed by immunofluorescence microscopy): A) control, B) exposed for 72 h to modelled microgravity and C) treated with pulsed Nd:YAG laser (1064 nm). In figure B a tight network of FN fibrils appears while in figure C the FN fibrils are parallel and ordered (see arrows).

Studying the behaviour of aortic endothelial cells cultured in micro- and hypergravity we found that the exposure to simulated microgravity conditions for 72 hours (angular velocity of rotation 60°/s) caused a reduction in coronary venular endothelial cell (CVEC) number. Genomic analysis revealed that proapoptotic signals increased, while antiapoptotic and proliferation/survival genes were downregulated by the absence of gravity. Activation of apoptosis was accompanied by morphological changes, with mitochondrial disassembly and organelles/cytoplasmic NAD(P)H redistribution, as evidenced by autofluorescence analysis. Moreover, cells were not able to respond to angiogenic stimuli in terms of migration and proliferation (Morbidelli et al., 2005)

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 19

EMFs are widely used to treat musculoskeletal diseases and many studies indicated that the most effective devices use pulsed EMFs with frequencies from 1 to 100 Hz, which induce EF of the order of V/cm (Pilla, 2002). Therefore, physiological effects may be induced by EMFs characterized by low frequencies ( optimal range 8-60 Hz) and amplitudes 1 G

It has been demonstrated that pulsed EMFs can increase osteoblastic differentiation and activity and, on the other hand, inhibit osteoclastogenesis, thus shifting the balance towards

Studies aimed at evaluating the possibility to apply EMFs to favour ligament healing and repair demonstrated that, after exposure to pulsed EMF, fibroblasts from calf anterior cruciate ligament increased migration speed and showed enhanced collagen I expression. On the contrary, static EMF had an inhibitory effect on wound healing, which was reversed

EMFs can modulate cell proliferation. The literature indicates that both intensity and frequency of the EMF are important in determining the final effect. Kwee and Raskmark (Kwee & Raskmark, 1995) have found an increase in the proliferation of human fibroblasts exposed to 0.08 mT, while Kula and Drozdz (Kula & Drozd, 1996) have shown inhibition of cell growth in murine fibroblasts exposed to 20 mT. Even trials carried out by exposing cultures of human lymphocytes have given different effects (increase, decrease or no effect in the proliferation) depending on the intensity of the applied EMF (Paile et al., 1995; Scarfì

As regards frequency, many authors reported increases in proliferation of different cell

Numerous studies have addressed the interaction between EMFs and calcium fluxes, because calcium is a principal regulator of several cellular processes. It is an activator of cyclic AMP, key molecule in triggering intracellular metabolic processes. It has been observed that the exposure to EMFs can modulate calcium concentration in a way which

The effects of EMFs on cell differentiation have been studied too. A progressive inhibition of enzyme activity and differentiation in MC-3T3 osteoblast-like cells, after exposure to 30 Hz EMF, was described by McLeod and Collazo (McLeod & Collazo, 2000). In HMSCs exposed to EMFs during chondrogenic differentiation, increase in collagen II and glycosaminoglycan (GAG)/DNA content was observed (Mayer-Wagner et al., 2010). Therefore EMFs might be a way to stimulate and maintain chondrogenesis of HMSCs and provide a new step in

In recent experiments aimed at studying the effects of EMFs on neuroblasts and understanding whether these effects can be useful in promoting tissue regeneration, we found that in neuroblasts (SHSY5Y human cell line derived from neuroblastoma) exposed to low frequency EMF (50 Hz; 2 mT, 3 hours) synaptophysin and TAU (microtubule-associated proteins) were overexpressed while Microtubule-Associated Protein 2 (MAP2) was downregulated. Synaptophysin participates in the formation of the channel for neurotransmitter release. TAU is associated with the protofilaments in neurites and MAP2 is a microtubule-associated protein found predominantly in the cell body. MAP2 function is

osteogenesis (Otter et al., 1998; Hartig et al., 2000; Chang et al., 2004).

types at 50 Hz frequency (Scarfì et al., 1991; Cossarizza et al., 1993).

regenerative medicine regarding tissue engineering of cartilage.

depends on cell type and field intensity. (Farndale, 1987; Walleczek, 1990).

(Funk et al., 2009).

et al., 1999).

by pulsed EMF (Chao et al., 2007).

In contrast, after exposure to hypergravity (10x*g*), no significant changes were observed in cell morphology and energy metabolism. Cells remained adherent to the substrate, but integrin distribution was modified. Accordingly, the cytoskeletal network reorganized, documenting cell activation. There was a reduction in expression of genes controlling vasoconstriction and inflammation. Proapoptotic signals were downregulated. Overall, the results documented that hypergravity exposure maintained endothelial cell survival and function by activation of adaptive mechanisms. The behavior of cells derived from microcirculation was somewhat different, because the above described effects were associated with increased anaerobic metabolism and cell detachment from the substrate (Morbidelli et al., 2009). These findings demonstrate that gravitational/mechanical stress can strongly affect endothelial function and neoangiogenesis and the biological response could also depend on the different vascular districts.

#### **3. Electromagnetic fields**

It is said that in the first century AD an "electric fish" was used to cure headache. Paracelsus (1493-1542) studied the medical use of lodestone and, in the sixteenth century, Sir Kenelm Digby described the magnetic cure of wounds. At the end of the seventeenth century, Galvani, with his famous experiments on bioelectricity, opened the way for modern studies on physiological EMFs and the effects of external EMFs on the body.

Over the past forty years, important advances have been made in research on bioelectricity: differences in electrical potentials of plants, animals and humans have been measured (Burr, 1972), changes in voltage gradients have been correlated with morphogenetic events in plants and animals (McCaig et al., 2005), physiological currents have been found to be signals for key processes in development (Levin, 2007).

An extensive discussion on electromagnetic effects from cell biology to medicine is presented in a recent review written by Funk et al. (Funk et al., 2009), where the coupling between physical mechanisms and cell biology is discussed in depth. In a nutshell, EMFs can cause polarization of bound charges, orientation of permanent dipoles (which results in topographycal changes in molecules), drift and diffusion of conduction charges, ion bound or release from proteins, ion-channel or receptor redistribution, conformational changes of voltage-sensitive enzymes, modulation of binding kinetics, reorientation of membrane phospholipids and changes in activation kinetics of ion channels (Funk et al., 2009).

Endogenous EMFs in living tissues are generated by physiological activities, for example movements of the musculoskeletal system structures. Vibrations of human muscles induce mechanical strains and currents have been measured both during postural muscle activity (5–30 Hz) and walking (<10 Hz) (Antonsson & Mann, 1985). Muscle contractions induce in the underlying bone tissue EMFs which are important for maintaining bone mass. Bone cells are selectively sensitive to low frequencies, in particular those ranging from 15 to 30 Hz. In this narrow range of frequencies, fields as low as 0.01mV/cm affect the remodelling activity (McLeod & Rubin, 1993). It has been found that EM current densities produced by mechanical loading (e.g. 1Hz during walking) in bone lie in the range 0.1–1.0 mA/cm2 (Lisi et al., 2006). Generally, physiological EMFs are characterized by extremely low frequencies (ELF), from 0 to 300 Hz, and have low intensity.

In contrast, after exposure to hypergravity (10x*g*), no significant changes were observed in cell morphology and energy metabolism. Cells remained adherent to the substrate, but integrin distribution was modified. Accordingly, the cytoskeletal network reorganized, documenting cell activation. There was a reduction in expression of genes controlling vasoconstriction and inflammation. Proapoptotic signals were downregulated. Overall, the results documented that hypergravity exposure maintained endothelial cell survival and function by activation of adaptive mechanisms. The behavior of cells derived from microcirculation was somewhat different, because the above described effects were associated with increased anaerobic metabolism and cell detachment from the substrate (Morbidelli et al., 2009). These findings demonstrate that gravitational/mechanical stress can strongly affect endothelial function and neoangiogenesis and the biological response

It is said that in the first century AD an "electric fish" was used to cure headache. Paracelsus (1493-1542) studied the medical use of lodestone and, in the sixteenth century, Sir Kenelm Digby described the magnetic cure of wounds. At the end of the seventeenth century, Galvani, with his famous experiments on bioelectricity, opened the way for modern studies

Over the past forty years, important advances have been made in research on bioelectricity: differences in electrical potentials of plants, animals and humans have been measured (Burr, 1972), changes in voltage gradients have been correlated with morphogenetic events in plants and animals (McCaig et al., 2005), physiological currents have been found to be

An extensive discussion on electromagnetic effects from cell biology to medicine is presented in a recent review written by Funk et al. (Funk et al., 2009), where the coupling between physical mechanisms and cell biology is discussed in depth. In a nutshell, EMFs can cause polarization of bound charges, orientation of permanent dipoles (which results in topographycal changes in molecules), drift and diffusion of conduction charges, ion bound or release from proteins, ion-channel or receptor redistribution, conformational changes of voltage-sensitive enzymes, modulation of binding kinetics, reorientation of membrane phospholipids and changes in activation kinetics of ion channels (Funk et al.,

Endogenous EMFs in living tissues are generated by physiological activities, for example movements of the musculoskeletal system structures. Vibrations of human muscles induce mechanical strains and currents have been measured both during postural muscle activity (5–30 Hz) and walking (<10 Hz) (Antonsson & Mann, 1985). Muscle contractions induce in the underlying bone tissue EMFs which are important for maintaining bone mass. Bone cells are selectively sensitive to low frequencies, in particular those ranging from 15 to 30 Hz. In this narrow range of frequencies, fields as low as 0.01mV/cm affect the remodelling activity (McLeod & Rubin, 1993). It has been found that EM current densities produced by mechanical loading (e.g. 1Hz during walking) in bone lie in the range 0.1–1.0 mA/cm2 (Lisi et al., 2006). Generally, physiological EMFs are characterized by extremely low frequencies

could also depend on the different vascular districts.

signals for key processes in development (Levin, 2007).

(ELF), from 0 to 300 Hz, and have low intensity.

on physiological EMFs and the effects of external EMFs on the body.

**3. Electromagnetic fields** 

2009).

EMFs are widely used to treat musculoskeletal diseases and many studies indicated that the most effective devices use pulsed EMFs with frequencies from 1 to 100 Hz, which induce EF of the order of V/cm (Pilla, 2002). Therefore, physiological effects may be induced by EMFs characterized by low frequencies ( optimal range 8-60 Hz) and amplitudes 1 G (Funk et al., 2009).

It has been demonstrated that pulsed EMFs can increase osteoblastic differentiation and activity and, on the other hand, inhibit osteoclastogenesis, thus shifting the balance towards osteogenesis (Otter et al., 1998; Hartig et al., 2000; Chang et al., 2004).

Studies aimed at evaluating the possibility to apply EMFs to favour ligament healing and repair demonstrated that, after exposure to pulsed EMF, fibroblasts from calf anterior cruciate ligament increased migration speed and showed enhanced collagen I expression. On the contrary, static EMF had an inhibitory effect on wound healing, which was reversed by pulsed EMF (Chao et al., 2007).

EMFs can modulate cell proliferation. The literature indicates that both intensity and frequency of the EMF are important in determining the final effect. Kwee and Raskmark (Kwee & Raskmark, 1995) have found an increase in the proliferation of human fibroblasts exposed to 0.08 mT, while Kula and Drozdz (Kula & Drozd, 1996) have shown inhibition of cell growth in murine fibroblasts exposed to 20 mT. Even trials carried out by exposing cultures of human lymphocytes have given different effects (increase, decrease or no effect in the proliferation) depending on the intensity of the applied EMF (Paile et al., 1995; Scarfì et al., 1999).

As regards frequency, many authors reported increases in proliferation of different cell types at 50 Hz frequency (Scarfì et al., 1991; Cossarizza et al., 1993).

Numerous studies have addressed the interaction between EMFs and calcium fluxes, because calcium is a principal regulator of several cellular processes. It is an activator of cyclic AMP, key molecule in triggering intracellular metabolic processes. It has been observed that the exposure to EMFs can modulate calcium concentration in a way which depends on cell type and field intensity. (Farndale, 1987; Walleczek, 1990).

The effects of EMFs on cell differentiation have been studied too. A progressive inhibition of enzyme activity and differentiation in MC-3T3 osteoblast-like cells, after exposure to 30 Hz EMF, was described by McLeod and Collazo (McLeod & Collazo, 2000). In HMSCs exposed to EMFs during chondrogenic differentiation, increase in collagen II and glycosaminoglycan (GAG)/DNA content was observed (Mayer-Wagner et al., 2010). Therefore EMFs might be a way to stimulate and maintain chondrogenesis of HMSCs and provide a new step in regenerative medicine regarding tissue engineering of cartilage.

In recent experiments aimed at studying the effects of EMFs on neuroblasts and understanding whether these effects can be useful in promoting tissue regeneration, we found that in neuroblasts (SHSY5Y human cell line derived from neuroblastoma) exposed to low frequency EMF (50 Hz; 2 mT, 3 hours) synaptophysin and TAU (microtubule-associated proteins) were overexpressed while Microtubule-Associated Protein 2 (MAP2) was downregulated. Synaptophysin participates in the formation of the channel for neurotransmitter release. TAU is associated with the protofilaments in neurites and MAP2 is a microtubule-associated protein found predominantly in the cell body. MAP2 function is

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 21

red light in preventing suppuration and scarring in patients with smallpox: the basis for the modern phototherapy were laid (Barnard & Morgan, 1903; Finsen, 1901; McDonagh, 2001). The extensive application of UV, visible, infrared (IR) radiation in biological and medical fields led to the development of suitable light sources. Actually, lasers are the latest and

The advantages of lasers, compared to other sources, are the high intensity of radiation emitted, the directionality (which allows efficient coupling to optical fibers and focus), the monochromaticity (if needed) and, with pulsed lasers, the possibility to transfer large

Following the widespread clinical use, many studies have been conducted to investigate at the cellular and molecular level the mechanisms underlying the systemic effects produced by exposure to laser radiation. Propagation and absorption of radiation in a biological sample or in a tissue may produce photochemical, photothermal and photomechanical

The actual laser systems, because of their versatility, are particularly suitable for application in biomedicine and biotechnology and the use of lasers to modulate biological and

Hsu (Hsu et al., 2010) proved that endothelial cells pre-exposed to red-emitting laser (632 nm) and then seeded on a biomaterial surface (biomedical grade poly (carbonate) urethane) increase matrix secretion and are more resistant to flushing (greater cell retention on the

An overview on the state of the art in the photoengineering of bone repair showed that infrared (IR) radiation increases osteoblastic proliferation, collagen deposition and bone

A recent review in which the photoengineering of tissue repair in skeletal and cardiac muscles is discussed, reported that exposure to lasers with red/near infrared (NIR) emission is effective in favouring muscle repair: the application of He-Ne laser irradiation significantly enhanced muscle regeneration in rats, while Ga-Al-As laser radiation reduced muscle degeneration in the ischemia/reperfusion injury in skeletal leg muscle. Photoexposure also favoured proliferation of myogenic satellite cells. In mouse, rat, dog and pig ischemic heart models, phototherapy significantly reduced (50% - 70%) the formation of scar tissue after induction of myocardial infarction. Ventricular dilation was also reduced

In order to evaluate the effectiveness of light emitted by lasers and other sources in enhancing cell proliferation, Alghamdi and colleagues (Alghamdi et al., 2011) reviewed the literature in this specific field from 1923 to 2010. They concluded that light with wavelength ranging from 600 to 700 nm is helpful in enhancing the proliferation rate of various cell types, stem cells included. The increase in proliferation was generally associated with increased synthesis of ATP, RNA and DNA. The reviewed data indicated that the optimum value of energy density was between 0.5 and 0.4 J/cm2. The possibility to develop phototreatments aimed at favouring cell proliferation could be very useful in the production of vaccines and hybrid cell lines as well as in tissue engineering and regenerative medicine.

most advanced type of light source.

amounts of energy controlling the thermal effects.

biotechnological processes has been proposed.

neoformation (Pinheiro & Gerbi, 2006)

graft) in comparison with non laser-exposed controls.

and ATP in the infarcted area increased (Oron, 2006).

effects, which are able to induce a biological response (Jacques, 1992).

not required when the cell disassembles microtubules in the cell body to give rise to the formation of neurites, while TAU is required to add new subunits to microtubules which are forming in the neurites. Moreover, in the treated neuroblasts, we observed rearrangement of microtubules and actin microfilaments, with formation of cones and cytoplasmic extensions (Fig.3), and increase of neurofilaments, a marker of neurogenic differentiation (not yet published data). Therefore we hypothesize that EMFs can favour differentiation. The expression of synaptophysin, TAU and MAP2 returned to control values 24 h after exposure. Instead, the formation of neurites continued to progress even after 24 h, with the appearance of branched extensions. This means that the changes in protein expression are part of a complex biological response that, once triggered by exposure to the EMF, proceeds even after the cessation of the stimulus (Cerrato et al., in press)

Fig. 3. Actin expression in SHSY5Y cells (analysed by immunofluorescence microscopy): control (A) and cells exposed to EMF (50 Hz, 2 mT, 3h) analysed immediately (B) and 24h (C) after the treatment. The formation of neurites can be observed in figures B and C.

Preliminar experiments on the effect of pulsed EMF on fibroblast behaviour in wound healing models showed, in agreement with other authors (Sunkari et al., 2011), that EMF can accelerate or slow the migration of fibroblasts, depending on the properties of the applied field (data not yet published). The possibility of modulating fibroblast migration during wound healing could be very interesting: in fact it might be useful to enhance the migration of fibroblasts to promote wound healing in chronic ulcers and, in general, in cases where healing is slow, while to inhibit the migration would be beneficial to prevent the formation of fibrous scars.

#### **4. Light**

Ancient civilizations had learned that light can have effects on the tissues of the body: both Romans and Greeks widely used the exposure to sun for therapeutic purposes.

In ancient China, a ritual to attain immortality in use during the Tang dynasty (fifth century AD) prescribed exposure to the sun holding in the right hand a piece of green paper with the character representing the sun in red. Subsequently, the paper previously exposed to the sun should be soaked in water and eaten for "trapping" in the body the essence of the sun. At the turn of the nineteenth century and beginning of the twentieth, it was discovered the lethal effect of ultraviolet (UV) component of sunlight on microorganisms and the efficacy of

not required when the cell disassembles microtubules in the cell body to give rise to the formation of neurites, while TAU is required to add new subunits to microtubules which are forming in the neurites. Moreover, in the treated neuroblasts, we observed rearrangement of microtubules and actin microfilaments, with formation of cones and cytoplasmic extensions (Fig.3), and increase of neurofilaments, a marker of neurogenic differentiation (not yet published data). Therefore we hypothesize that EMFs can favour differentiation. The expression of synaptophysin, TAU and MAP2 returned to control values 24 h after exposure. Instead, the formation of neurites continued to progress even after 24 h, with the appearance of branched extensions. This means that the changes in protein expression are part of a complex biological response that, once triggered by exposure to the EMF, proceeds

Fig. 3. Actin expression in SHSY5Y cells (analysed by immunofluorescence microscopy): control (A) and cells exposed to EMF (50 Hz, 2 mT, 3h) analysed immediately (B) and 24h (C) after the treatment. The formation of neurites can be observed in figures B and C.

Preliminar experiments on the effect of pulsed EMF on fibroblast behaviour in wound healing models showed, in agreement with other authors (Sunkari et al., 2011), that EMF can accelerate or slow the migration of fibroblasts, depending on the properties of the applied field (data not yet published). The possibility of modulating fibroblast migration during wound healing could be very interesting: in fact it might be useful to enhance the migration of fibroblasts to promote wound healing in chronic ulcers and, in general, in cases where healing is slow, while to inhibit the migration would be beneficial to prevent the formation

Ancient civilizations had learned that light can have effects on the tissues of the body: both

In ancient China, a ritual to attain immortality in use during the Tang dynasty (fifth century AD) prescribed exposure to the sun holding in the right hand a piece of green paper with the character representing the sun in red. Subsequently, the paper previously exposed to the sun should be soaked in water and eaten for "trapping" in the body the essence of the sun. At the turn of the nineteenth century and beginning of the twentieth, it was discovered the lethal effect of ultraviolet (UV) component of sunlight on microorganisms and the efficacy of

Romans and Greeks widely used the exposure to sun for therapeutic purposes.

even after the cessation of the stimulus (Cerrato et al., in press)

of fibrous scars.

**4. Light** 

red light in preventing suppuration and scarring in patients with smallpox: the basis for the modern phototherapy were laid (Barnard & Morgan, 1903; Finsen, 1901; McDonagh, 2001). The extensive application of UV, visible, infrared (IR) radiation in biological and medical fields led to the development of suitable light sources. Actually, lasers are the latest and most advanced type of light source.

The advantages of lasers, compared to other sources, are the high intensity of radiation emitted, the directionality (which allows efficient coupling to optical fibers and focus), the monochromaticity (if needed) and, with pulsed lasers, the possibility to transfer large amounts of energy controlling the thermal effects.

Following the widespread clinical use, many studies have been conducted to investigate at the cellular and molecular level the mechanisms underlying the systemic effects produced by exposure to laser radiation. Propagation and absorption of radiation in a biological sample or in a tissue may produce photochemical, photothermal and photomechanical effects, which are able to induce a biological response (Jacques, 1992).

The actual laser systems, because of their versatility, are particularly suitable for application in biomedicine and biotechnology and the use of lasers to modulate biological and biotechnological processes has been proposed.

Hsu (Hsu et al., 2010) proved that endothelial cells pre-exposed to red-emitting laser (632 nm) and then seeded on a biomaterial surface (biomedical grade poly (carbonate) urethane) increase matrix secretion and are more resistant to flushing (greater cell retention on the graft) in comparison with non laser-exposed controls.

An overview on the state of the art in the photoengineering of bone repair showed that infrared (IR) radiation increases osteoblastic proliferation, collagen deposition and bone neoformation (Pinheiro & Gerbi, 2006)

A recent review in which the photoengineering of tissue repair in skeletal and cardiac muscles is discussed, reported that exposure to lasers with red/near infrared (NIR) emission is effective in favouring muscle repair: the application of He-Ne laser irradiation significantly enhanced muscle regeneration in rats, while Ga-Al-As laser radiation reduced muscle degeneration in the ischemia/reperfusion injury in skeletal leg muscle. Photoexposure also favoured proliferation of myogenic satellite cells. In mouse, rat, dog and pig ischemic heart models, phototherapy significantly reduced (50% - 70%) the formation of scar tissue after induction of myocardial infarction. Ventricular dilation was also reduced and ATP in the infarcted area increased (Oron, 2006).

In order to evaluate the effectiveness of light emitted by lasers and other sources in enhancing cell proliferation, Alghamdi and colleagues (Alghamdi et al., 2011) reviewed the literature in this specific field from 1923 to 2010. They concluded that light with wavelength ranging from 600 to 700 nm is helpful in enhancing the proliferation rate of various cell types, stem cells included. The increase in proliferation was generally associated with increased synthesis of ATP, RNA and DNA. The reviewed data indicated that the optimum value of energy density was between 0.5 and 0.4 J/cm2. The possibility to develop phototreatments aimed at favouring cell proliferation could be very useful in the production of vaccines and hybrid cell lines as well as in tissue engineering and regenerative medicine.

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 23

**MECHANICAL STRESS** 

Neonatal rat cardiomyocytes

Human and bovine capillary endothelial cells

150N, 1 Hz Female rats Increased bone

MC3T3-E1 osteoblastlike cells

> MC3T3-E1 osteoblastic cell

> > Fibroblasts

Human osteoblastlike osteosarcoma cell line MG-63 and primary human osteoblasts

Coronary venular endothelial cells

Connective tissue cells

**model Effects Author** 

Cardiomyocytes organized into parallel arrays, increased binucleation and hypertrophy.

Modulation of growth, differentiation and apoptosis

Reorganization of the cytoskeleton and increased expression of alpha(v)- beta3 integrin receptor

Changes in intracellular Ca2+ concentration and actin microfilament organization

Production of ECM components (in particular tenascin-C)

Increased proliferation rates and activation of PLC beta 2 expression

Decreased cell number, increased proapoptotic signals and down regulation of antiapoptotic and proliferation/survival genes

Activation of mechanotransduction pathways, Ca2+ signalling, stretchactivated channels, voltage-activated channels

Vandenburg, 1996

Chen, 1997

Cavalcanti-Adam et al., 2002

Adachi et al., 2003

Chiquet et al., 2003

Hoberg et al., 2005

Morbidelli et al., 2005

Wall and Banes., 2005 **Review** on Early responses to mechanical loading in connective tissue cells

formation Chow et al., 1998

**Physical** 

Mechanical stress lengthening

Mechanical stress topographycal stimulation

Mechanical stress loading

Mechanical stress stretching

Mechanical stress

Mechanical stress stretching

Mechanical stress stretching

Gravitational stress microgravity

Mechanical stress

**stimulus Parameters Experimental** 

Lengthening of the substratum on which cells adhered (5 mm/day resulting in a 94-110% increase in 4 days

Micropatterned substrates with ECM-coated adhesive islands

2% biaxial strain applied cyclically, 0.25 Hz.

Mechanical perturbation using a glass microneedle

Cyclic stretching, 5% to 15% elongation, 0.3 to 1 Hz

30 cycles of uniaxial stretching, 1 Hz, 4000 με

Microgravity modelled by RPM, angular velocity of rotation 60°/s, 72 h

Different mechanical stresses fluid flow, strain, shear and combinations of them

In a review concerning the literature from 1960 to 2008 on the use of laser treatments for the improvement of tissue repair, the authors stated that, despite the difficulty in comparing results obtained with different laser sources, treatment protocols and experimental models, the majority of the reviewed reports clearly indicated that laser irradiation (the most frequently used is red/NIR radiation) speeds up tissue repair (da Silva et al., 2010).

In order to evaluate the possibility to use laser treatments as a tool to stimulate cell differentiation processes and cell functions involved in tissue repair, we studied the effect of NIR-emitting lasers on HMSCs, endothelial cells and cells of connective tissues. We found that in HMSCs treated with NIR pulses emitted by a high power Nd: YAG laser (1064 nm wavelength, 200 μs pulse duration, 10 Hz repetition rate, 458.65 mJ/cm2 energy fluence, 73 sec exposure) genes involved in osteoblastogenesis (*GLI1, NF1, MEN1*) appeared upregulated while *PPAR*, which is a major marker of adipogenesis, and *FABP4* were downregulated, suggesting that the treatment can favour osteoblastogenesis and inhibit adipogenesis (Tab. 1). Interestingly the results obtained with laser treatment are very close to those obtained by exposure of HMSCs to hypergravity (10x*g*) (Monici et al., 2008b).

Moreover, the Nd:YAG laser pulses resulted effective in enhancing the production of ECM molecules, such as collagen I, collagen II, aggrecan and FN in cultures of connective tissue cells. A similar increase in ECM molecules was found when the cells were exposed to hypergravity (10x*g*) (Monici et al., 2008b, Basile et al., 2009).

The results of experiments in which we compared the effects of gravitational loading (10x*g*) and laser pulses on cells belonging to tissues with antigravitational function are consistent with the hypothesis that, using suitable laser pulses, it is possible to induce transient ECM rearrangements in the cell microenvironment (cell niche) which, in turn, act as indirect "photomechanical" stimuli on the cells (Rossi et al., 2010).

Experiments carried out on fibroblasts and endothelial cells, which are responsible for ECM production and neoangiogenesis in the remodelling phase of wound healing, demonstrated that NIR pulses not only increase (30%), FN production, but also favour the ordered assembly of FN fibrils in fibrillogenesis (Fig. 2C) (Monici et al., 2011). This effect is interesting because it could improve the quality of the neoformed ECM: in fact, FN fibrils act as a template for the formation of collagen fibers and strongly affect ECM properties (Shi et al., 2010). Moreover, we observed that NIR laser pulses can also favour the formation of ordered monolayer of endothelial cells (Monici et al., 2011). This effect could be of consequence in neoangiogenesis. Finally, data obtained with preliminary experiments carried out in our laboratory show that pulsed NIR radiation enhances the production of inflammation cytochines (not yet published data). The treatment could thus have the effect of accelerating the transition from inflammatory to the remodelling phase in tissue repair.

Advanced laser systems allow to apply more complex treatment protocols, to try to potentiate or to exploit synergistically the effects produced by emissions with different characteristics.

After exposure of myoblasts to a Multiwave locked System (MLS) laser, emitting at 808 and 905 nm (continuous/interrupted and pulsed mode, respectively) we observed increased activity of enzymes involved in cellular energy metabolism and enhanced expression of MyoD (Vignali et al., 2011), an early marker of muscle differentiation. These results provide an interesting premise for the future application of Multiwave systems to favour muscle tissue repair.

In a review concerning the literature from 1960 to 2008 on the use of laser treatments for the improvement of tissue repair, the authors stated that, despite the difficulty in comparing results obtained with different laser sources, treatment protocols and experimental models, the majority of the reviewed reports clearly indicated that laser irradiation (the most

In order to evaluate the possibility to use laser treatments as a tool to stimulate cell differentiation processes and cell functions involved in tissue repair, we studied the effect of NIR-emitting lasers on HMSCs, endothelial cells and cells of connective tissues. We found that in HMSCs treated with NIR pulses emitted by a high power Nd: YAG laser (1064 nm wavelength, 200 μs pulse duration, 10 Hz repetition rate, 458.65 mJ/cm2 energy fluence, 73 sec exposure) genes involved in osteoblastogenesis (*GLI1, NF1, MEN1*) appeared

downregulated, suggesting that the treatment can favour osteoblastogenesis and inhibit adipogenesis (Tab. 1). Interestingly the results obtained with laser treatment are very close to those obtained by exposure of HMSCs to hypergravity (10x*g*) (Monici et al., 2008b).

Moreover, the Nd:YAG laser pulses resulted effective in enhancing the production of ECM molecules, such as collagen I, collagen II, aggrecan and FN in cultures of connective tissue cells. A similar increase in ECM molecules was found when the cells were exposed to

The results of experiments in which we compared the effects of gravitational loading (10x*g*) and laser pulses on cells belonging to tissues with antigravitational function are consistent with the hypothesis that, using suitable laser pulses, it is possible to induce transient ECM rearrangements in the cell microenvironment (cell niche) which, in turn, act as indirect

Experiments carried out on fibroblasts and endothelial cells, which are responsible for ECM production and neoangiogenesis in the remodelling phase of wound healing, demonstrated that NIR pulses not only increase (30%), FN production, but also favour the ordered assembly of FN fibrils in fibrillogenesis (Fig. 2C) (Monici et al., 2011). This effect is interesting because it could improve the quality of the neoformed ECM: in fact, FN fibrils act as a template for the formation of collagen fibers and strongly affect ECM properties (Shi et al., 2010). Moreover, we observed that NIR laser pulses can also favour the formation of ordered monolayer of endothelial cells (Monici et al., 2011). This effect could be of consequence in neoangiogenesis. Finally, data obtained with preliminary experiments carried out in our laboratory show that pulsed NIR radiation enhances the production of inflammation cytochines (not yet published data). The treatment could thus have the effect of accelerating the transition from inflammatory to the remodelling phase in tissue repair. Advanced laser systems allow to apply more complex treatment protocols, to try to potentiate or to exploit synergistically the effects produced by emissions with different

After exposure of myoblasts to a Multiwave locked System (MLS) laser, emitting at 808 and 905 nm (continuous/interrupted and pulsed mode, respectively) we observed increased activity of enzymes involved in cellular energy metabolism and enhanced expression of MyoD (Vignali et al., 2011), an early marker of muscle differentiation. These results provide an interesting

premise for the future application of Multiwave systems to favour muscle tissue repair.

, which is a major marker of adipogenesis, and *FABP4* were

frequently used is red/NIR radiation) speeds up tissue repair (da Silva et al., 2010).

upregulated while *PPAR*

characteristics.

hypergravity (10x*g*) (Monici et al., 2008b, Basile et al., 2009).

"photomechanical" stimuli on the cells (Rossi et al., 2010).


The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 25

Chondrogenic cells

MC3T3-E1 preosteoblasts

Mesenchymal stem cells

Fibroblast and endothelial cells

Many different experimental models

0.01 mV/cm Bone cells Affect remodelling

Osteoblast-like primary cells

Osteoblast-like primary cells

**ELECTROMAGNETIC FIELDS** 

**model Effects Author** 

Upregulation of genes normally associated with a chondrogenic phenotype with uniaxial loading, upregulation of a broader pattern of chondrogenic genes with multiaxial loading

Acceleration of matrix

Increased smooth muscle cells expression with cyclic stretch and endothelial cells expression with cyclic pressure, and laminar shear stress

> Disregulation in laminin and fibronectin production

> Many effects are reported

> > activity

Increased proliferation, enhancement of alkaline phosphatase activity, enhanced synthesis and secretion of ECM proteins

Increased proliferation, OPG upregulation and RANKL downregulation

maturation Kang et al., 2011

Grad et al., 2011 **Review** on Chondrogenic cell response to mechanical stimulation in vitro

Maul et al., 2011

Monici et al., 2011

Funk., 2009 **Review** on Electromagnetic effects from cell biology to medicine

McLeod & Rubin, 1993

Hartig et al., 2000

Chang et al., 2004

**Physical** 

Mechanical stress loading

Mechanical stress

Mechanical stress

Gravitational stress microgravity

EMF

Pulsed EF

**stimulus Parameters Experimental** 

Uniaxial and multiaxial, hydrostatic pressure 7 to 10 Mpa, average tension, 3.8% radial and 2.1% circumferential tensile strains, compression 0.5 to 7.7 MPa

Cyclic strain, 1Hz, 10 % strain of 3D culture + ultrasound 1.0 MHz and 30 mW/cm2

Cyclic stretch 5%, 1Hz; cyclic pressure 120/80mmHg, 1Hz; shear stress 10 dynes*/*cm2.

Microgravity modelled by RPM, angular velocity of rotation 60°/s, 72 h

Many different parameters and treatment protocols

100V external voltage, 16 Hz, EF across cell membrane 6 kV/m (estimated by computer simulation)

EF 2 mV/cm

ELFEMF 15 to 30 Hz,

Pulsed EMF 15 Hz, 0.1 mT,


Osteoclastic precursors

Ligament fibroblasts

Osteoblasts, osteoclasts, osteocytes and cells of the vasculature

Osteocytes

Human mesenchymal stem cells

Human fetal fibroblast and human chondrocytes

Coronary venular endothelial cells

Intervertebral disc (IVD) and stem cells

**model Effects Author** 

Increased apoptosis, osteoclast-like morphology, cytoskeleton reorganization, overexpression of osteoclastic markers

Enhanced cell proliferation and collagen production

Many effects are reported

Upregulation of NO production in the stimulated cell and in the sourrading osteocytes

Overexpression of genes involved in osteoblastogenesis

resorption

Increased production

Activation of adaptive mechanisms, increased anaerobic metabolism, detachment from the substrate

Possible role of loading to favour differentiation of stem cells toward "discogenic" phenotype

of ECM molecules Basile et al., 2009

Bone implant Activation of bone

Monici et al., 2006

Park et al., 2006;

Rubin et al., 2006 **Review** on Molecular pathways mediating mechanical signalling in bone

Vatsa et al., 2007

Monici et al., 2008a

Stadelmann et al., 2008

Morbidelli et al.,2009

Chan et al., 2011 **Review** on The effects of loading on IVD and stem cells

**Physical** 

Gravitational stress microgravity

Mechanical stress stretching

Mechanical stress

Mechanical stress

Gravitational stress hypergravity

Mechanical stress compression

Gravitational stress hypergravity

Gravitational stress hypergravity

Mechanical stress compression

**stimulus Parameters Experimental** 

Microgravity modelled by RPM, angular velocity of rotation 60°/s, 72 h exposure

Cyclic stretching 0.5 Hz , magnitude 8% (8 % deformation of cell-seeded silicone substrate)

Mechanical load, strain, pressure, different parameters

> Mechanical stimulation by microneedles

5 periods of 10 min at 10 x g spaced with 10 min recovery periods at 1 x g

> Compression 0.5 Mpa, sinusoidal micromotion 100 μm, 1 Hz

5 periods of 10 min at 10 x g spaced with 10 min recovery periods at 1 x g

5 periods of 10 min at 10 x g spaced with 10 min recovery periods at 1 x g

Compressive strain of 5% to 20% elongation, 0.15 to1 Hz, 1 to 12 h/day hydrostatic pressure 0.1 to 10 Mpa, 0.25 to 1 Hz


The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 27

Human mesenchymal stem cells

Human fetal fibroblasts and human chondrocytes

Different experimental models

Endothelial cells preexposed to laser and then seeded on a biomaterial surface

Various cell types

Fibroblasts and endothelial cells

Table 1. Based on the studies cited in this chapter, the table lists physical factors, treatment

**model Effects Author** 

Increment of osteoblastic proliferation, collagen deposition and bone neoformation

Upregulation of genes involved in osteoblastogenesis, downregulation of genes involved in adipogenesis

Increased production

Promotion of tissue repair

Increase in ECM secretion and increased resistance to flushing

Increase in proliferation associated with enhanced synthesis of ATP, RNA and DNA

Increased production of fibronectin and ordered assembly of FN fibrils in fibrillogenesis

Enhanced expression of MyoD and increased activity of enzymes involved in cellular energy metabolism

of ECM molecules Monici et al., 2008b

Pinheiro & Gerbi, 2006 **review** state of the art on photoengineering of bone repair using laser therapy

Monici et al., 2008a

Da Silva et al., 2010 **Review** on Lasertherapy in tissue repair processes

Hsu et al, 2010

Alghamdi et al., 2011 **Review** on The use LLLT for enhancing cell proliferation

Monici et al., 2011

Vignali et al., 2011

**Physical** 

Light IR radiation

Light High power Nd: YAG laser

Light High power Nd: YAG laser

Light

Light He-Ne laser

> Light Red radiation

Light High power Nd: YAG laser

Light MLS laser

**stimulus Parameters Experimental** 

and parameters Bone cells and tissues

Various protocols

λ 1064 nm, 200 μs pulse duration, 10 Hz repetition rate, 458.65 mJ/cm2 energy fluence, 73 sec exposure

λ 1064 nm, 200 μs pulse duration, 10 Hz repetition rate, 458.65 mJ/cm2 energy fluence, 73 sec exposure

Different laser sources and treatment protocols

λ 632.8 nm, average energy on sample 1.18 J/cm2

λ from 600 to 700 nm energy density 0.4 - 0.5 J/cm2

λ 1064 nm, 200 μs pulse duration, 10 Hz repetition rate, 458.65 mJ/cm2 energy fluence, 73 sec exposure

λ 808 and 905 nm,

1500 Hz Myoblasts

parameters applied, experimental models used and observed effects.


Calf anterior cruciate ligament (ACL) fibroblasts

Human lymphocytes

Rat thymic lymphocytes

like cells

Human mesenchymal stem cells (hMSCs)

SHSY5Y neuroblast model

Injured muscles in rat Ischemic leg muscles Ischemic heart model of mouse, rat, dog and pig Myogenic satellite cells (SC)

**LIGHT** 

Human fibroblasts Activation of

Human lymphocytes No effect on

EMF 50 Hz, 25 to 180 μT Human fibroblasts Increased

ELFMF 50 Hz, 0.020 T Murine fibroblasts Inhibition of cell

**model Effects Author** 

Increased migration speed and enhanced collagen I expression with pulsing direct current (DC) EF, inhibition in wound healing with static direct current (DC) EF

proliferation

Slight decrease of cell proliferation at the intensities tested

Modulation of

Progressive inhibition of enzyme activity and differentiation

Increase of collagen II and glycosaminoglycan (GAG)/DNA content during chondrogenic differentiation

> Promotion of neurogenic differentiation

Enhanced muscle regeneration; reduced muscle degeneration; reduction in scar tissue formation after induction of myocardial infarction (MI) and in ventricular dilatation. Increment of ATP in the infarcted area; MAPK/ERK activation

Chao et al., 2007

Kwee & Raskmark, 1995

Scarfì et al., 1999

McLeod & Collazo, 2000

> Mayer-Wagner et al., 2010

Cerrato et al., 2011

Oron, 2006 **Review** on Photoengineering of tissue repair in skeletal and cardiac muscles

growth Kula & Drozd, 1996

proliferation Paile et al., 1995

calcium concentration Walleczek, 1990

fibroblast migration Sunkari et al., 2011

**Physical** 

EF

EMF

EMF

EMF

Light He-Ne and Ga-Al-As laser

**stimulus Parameters Experimental** 

Static and pulsing direct current (DC) EFs

50 Hz sinusoidal MFs intensities: 30 T, 300 T, and 1 mT

50 Hz sinusoidal MF intensities: 1.0, 0.75, 0.5, 0.25, 0.05 mT exposure 72 h

60 Hz, 44 μT

3 hours exposure

1 GHz, power density of exposure area 5 nW/cm2

Various treatment protocols and instrumental parameters

EMF 30 Hz, 1.8-mT MC-3T3 osteoblast-

MF Sinusoidal

ELFEMF 15Hz, 5mT

EMF 50 Hz; 2 mT,


Table 1. Based on the studies cited in this chapter, the table lists physical factors, treatment parameters applied, experimental models used and observed effects.

The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration 29

Chan, SC.; Ferguson, SJ., & Gantenbein-Ritter, B. (2011). The effects of dynamic loading on

Chang, WH.; Chen, LT.; Sun, JS., & Lin, FH. (2004). Effect of pulse-burst electromagnetic

Chao, PH.; Lu, HH.; Hung, CT.; Nicoll, SB., & Bulinski, JC. (2007). Effects of applied DC

Chen, CS.; Mrksich, M.; Huang, S.; Whitesides, G., & Ingber, DE. (1997). Geometric control

Chiquet, M.; Sarasa-Renedo, A.; Huber, F., & Flück M. (2003). How do fibroblast translate

Chiquet, M.; Gelman, L.; Lutz, R., & Maier, S. (2009). From mechanotransduction to

Chow, JW.; Fox, SW.; Lean, JM., & Chambers, TJ. (1998). Role of nitric oxide and

Cossarizza, A.; Capri, M.; Salvioli, S.; Monti, D.; Franceschi, C.; Bersani, F.; Cadossi, R.;

da Silva, JP.; da Silva, MA.; Almeida, AP.; Lombardi Junior, I., & Matos, AP. (2010). Laser

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#### **5. Conclusion**

Over the past twenty years, studies on molecular and cellular mechanisms that underlie biological responses evoked by physical stimuli have made great progress. However, in this fascinating field of study, many problems still remain and their solution will require further advances in our knowledge.

The results of our studies are a further, albeit modest, contribution to a large body of literature that shows how physical stimuli can be effective in modulating cellular functions and the production of ECM. It is obvious that the development and standardization of technologies for delivering appropriate physical stimuli, strictly controlled with regard to the intensity, frequency and timing of exposure, is a prerequisite for making progress in tissue engineering.

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**2** 

*China* 

**Effect of Low-Intensity Pulsed** 

**Ultrasound on Nerve Repair** 

*1Beijing Tiantan Hospital, Capital Medical University 2The 2nd Affiliated Hospital, Harbin Medical University* 

Low-intensity pulsed ultrasound (LIPUS) is a medical technology, generally utilizing 1-1.5 MHz frequency pulses, with a pulse width of 200 μs, repeated at 1-1.5 kHz, at an intensity of 10- 30 mW/cm2, 20 minutes/day. There are two main types of ultrasound effects: thermal and nonthermal. Both types are thought to first "injure" the cells, resulting in their growth retardation, and then to initiate a cellular recovery response characterized by an increase in protein production(Johns 2002). Compared to high-intensity continuous ultrasound, LIPUS is much lower in intensity and has unique characteristics such as pulsed waves, which are

Applications of LIPUS include: promoting bone fracture healing; treating orthodontically induced root resorption; regrow missing teeth; enhancing mandibular growth in children with hemifacial microsomia; promoting healing in various soft tissues such as cartilage, intervertebral disc, etc.; improving muscle healing after laceration injury. Researchers at the University of Alberta have used LIPUS to gently massage teeth roots and jawbones to cause growth or regrowth, and have grown new teeth in rabbits after lower jaw surgical lengthening (Distraction osteogenesis). As of June 2006, a device has been licensed by the Food and Drug Administration (FDA) and Health Canada for use by orthopedic surgeons. It has not yet been approved by either Canadian or American regulatory bodies and a market-ready model is currently being prepared. LIPUS is expected to be commercially available before the end of 2012. According to Dr. Chen from the University of Alberta, LIPUS may also have medical/cosmetic benefits in allowing people to grow taller by stimulating bone growth.

In recent years, data on the therapeutic effects of LIPUS have been accumulating. So far, it has been reported that LIPUS enhances cell proliferation and alters protein production in various kinds of cells such as endothelial cells, osteoblasts, chondrocytes, and fibroblasts (Ikeda, Takayama et al. 2006; Hiyama, Mochida et al. 2007; Takeuchi, Ryo et al. 2008), but there is little information on the response of Schwann cell and neurons to LIPUS irradiation. Some studies have indicated that LIPUS has positive effects on axonal regeneration during in vivo peripheral nerve injury trials (Crisci and Ferreira 2002; Chang, Hsu et al. 2005) and that its stimuli on the injured sciatic nerve can increase the number of nerve fibers compared to that of untreated injured nerves in rats (Raso, Barbieri et al. 2005). Thus, treatment with

regarded as nonthermogenic and non-destructive (Mukai, Ito et al. 2005).

**1. Introduction** 

Jiamou Li1, Hua Zhang1 and Cong Ren2


### **Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair**

Jiamou Li1, Hua Zhang1 and Cong Ren2 *1Beijing Tiantan Hospital, Capital Medical University 2The 2nd Affiliated Hospital, Harbin Medical University China* 

#### **1. Introduction**

34 Tissue Regeneration – From Basic Biology to Clinical Application

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Involvement of BMPs/Smad signaling pathway in mechanical response in

Low-intensity pulsed ultrasound (LIPUS) is a medical technology, generally utilizing 1-1.5 MHz frequency pulses, with a pulse width of 200 μs, repeated at 1-1.5 kHz, at an intensity of 10- 30 mW/cm2, 20 minutes/day. There are two main types of ultrasound effects: thermal and nonthermal. Both types are thought to first "injure" the cells, resulting in their growth retardation, and then to initiate a cellular recovery response characterized by an increase in protein production(Johns 2002). Compared to high-intensity continuous ultrasound, LIPUS is much lower in intensity and has unique characteristics such as pulsed waves, which are regarded as nonthermogenic and non-destructive (Mukai, Ito et al. 2005).

Applications of LIPUS include: promoting bone fracture healing; treating orthodontically induced root resorption; regrow missing teeth; enhancing mandibular growth in children with hemifacial microsomia; promoting healing in various soft tissues such as cartilage, intervertebral disc, etc.; improving muscle healing after laceration injury. Researchers at the University of Alberta have used LIPUS to gently massage teeth roots and jawbones to cause growth or regrowth, and have grown new teeth in rabbits after lower jaw surgical lengthening (Distraction osteogenesis). As of June 2006, a device has been licensed by the Food and Drug Administration (FDA) and Health Canada for use by orthopedic surgeons. It has not yet been approved by either Canadian or American regulatory bodies and a market-ready model is currently being prepared. LIPUS is expected to be commercially available before the end of 2012. According to Dr. Chen from the University of Alberta, LIPUS may also have medical/cosmetic benefits in allowing people to grow taller by stimulating bone growth.

In recent years, data on the therapeutic effects of LIPUS have been accumulating. So far, it has been reported that LIPUS enhances cell proliferation and alters protein production in various kinds of cells such as endothelial cells, osteoblasts, chondrocytes, and fibroblasts (Ikeda, Takayama et al. 2006; Hiyama, Mochida et al. 2007; Takeuchi, Ryo et al. 2008), but there is little information on the response of Schwann cell and neurons to LIPUS irradiation. Some studies have indicated that LIPUS has positive effects on axonal regeneration during in vivo peripheral nerve injury trials (Crisci and Ferreira 2002; Chang, Hsu et al. 2005) and that its stimuli on the injured sciatic nerve can increase the number of nerve fibers compared to that of untreated injured nerves in rats (Raso, Barbieri et al. 2005). Thus, treatment with

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 37

demonstrated that the magnitude and duration of LIPUS has a direct effect on whether the stimulus has a positive or negative effect on nerve regeneration (Chang, Hsu et al. 2005; Raso, Barbieri et al. 2005). Yet, there are currently no data about the actual types and levels of the ultrasound for the cells within the peripheral nerve. Although using similar values from studies on chondrocytes, endothelial cells, and osteoblasts would allow direct comparison between different cell lines, these values may not have any significance to neural tissue based upon the different physiologic demands of each tissue type. Thus, we chose the magnitude and duration of stimulation for these experiments based on previous work that demonstrated that ultrasound induced a biological response in Schwann cells

The purpose of this study was to evaluate how sustained LIPUS directly affects Schwann cell function. By evaluating for the expression of the pan-specific Schwann cell marker S-100 with immunohistochemistry, we determined whether Schwann cells de-differentiated after LIPUS stimulation. Schwann cell proliferation was explored using BrdU uptake assays to

Schwann cells were prepared using a method previously described with some modifications (Cai, Campana et al. 1999). Briefly, sciatic nerves were dissected from Wistar rats (n=30) at postnatal day 1–3. The epineural sheath was removed. Thereafter, the sciatic nerves were chopped into 200-μm pieces and enzymatically digested (collagenase/trypsin, 1 mg/ml, 1 hour, 37°C). The resulting cell suspensions were plated onto a six-well plate and cultured in Schwann cell medium (DMEM/10% heat-inactivated FCS/2 mM glutamine/pen/strep). Two different cell densities were prepared for subsequent experiments, a cell density of 5,000 cells/1.77 cm2 for proliferation assay and immunohistochemistry assays, and a cell density of 100,000 cells/1.77 cm2 for semiquantitative RT-PCR. The fibroblasts were eliminated by 10 μM cytosine arabinoside and complement-mediated cytolysis with the fibroblast- specific antibody Thy1.1 in conjunction with baby rabbit complement (Cedarlane, Burlington, NC). The medium was changed every other day by adding 2 μM forskolin and glial growth factor (100μg/ml) for expansion of Schwann cells for up to 14 days of cells culture. The purity of cultures was monitored by immunostaining using the Schwann cell

Schwann cells were cultured and subjected to LIPUS with modifications as previously described (Takayama, Suzuki et al. 2007). This device (Nexson-The-P41, Nexus Biomedical Devices, Hangzhou, China) generated LIPUS with a pulse width of 200 microseconds, repetition rate of 1.5 KHz, operation frequency of 1 MHz, spatial average temporal average of 100 mW/cm2, 5 minutes/day. The LIPUS treatment was started 24 hours after initiation of cells culture and repeated for 14 consecutive days. In the experimental group, LIPUS was transmitted from 35- mm diameter LIPUS transducers to the bottom of the cell culture plate via a coupling gel (Smith & Nephew, Oklahoma, CA) and was administered in an incubator (see Fig. 1). In the control group, plates were placed on the same transducers for the same

ascertain if direct LIPUS stimulation is mitogenic for Schwann cells in culture plates.

**Schwann cells culture and low-intensity pulsed ultrasound treatment** 

(Chang, Hsu et al. 2005)

**2.1 Material and methods** 

marker S-100 and the fibroblast marker Thy-1.1.

duration, but the LIPUS was not administered.

LIPUS is likely to assist the regeneration of neuronal axons. However, the mechanism of such events is unknown.

#### **2. Schwann cells that were subjected to LIPUS consistently demonstrated an increase in cell proliferation**

Ultrasound is commonly used for diagnostic imaging and physiotherapy and can exert biological effects through either thermal or mechanical mechanisms in living tissue (Choi, Pernot et al. 2007; Nahirnyak, Mast et al. 2007). In contrast to high-intensity continuous ultrasound, LIPUS (<100 mW/cm2) has much lower intensities, which are regarded as nonthermogenic and nondestructive (Ikeda, Takayama et al. 2006). Mechanical strains received by cells may result in biochemical events and increase membrane permeability (Danialou, Comtois et al. 2002). Despite the wide use of LIPUS for improving peripheral nerve tissue regeneration in animal models (Crisci and Ferreira 2002; Chang, Hsu et al. 2005; Raso, Barbieri et al. 2005), very little is known about its effects on the glial cells of peripheral nerves. It has been reported that Schwann cells respond somehow to LIPUS stimulation (Chang, Hsu et al. 2005; Raso, Barbieri et al. 2005). However, the results of previous investigations were somewhat inconclusive, particularly regarding the precise mechanism.

Fig. 1. Experimental apparatus for applying low- intensity pulsed ultrasound (LIPUS) which generated LIPUS with a SATA intensity of 10 mW/cm2, pulse width of 200 microseconds, repetition rate of 1.5 KHz, and an operation frequency of 1MHz. LIPUS irradiated neurons with two probes 24 h after in culture. A six-well plate was placed on the probes. LIPUS was transmitted to culture plate via an interposed ultrasound gel. Two transducers for the control group (sham-LIPUS; LIPUS not turned on) and two probes for the LIPUS group.

Previous studies have shown that LIPUS in the cultured cells induces significant cellular responses in nucleus pulposus cells, endothelial cells, osteoblasts, chondrocytes, and fibroblasts, (Parvizi, Wu et al. 1999; Zhou, Schmelz et al. 2004; Hill, Fenwick et al. 2005; Sena, Leven et al. 2005; Hiyama, Mochida et al. 2007) but little is known about Schwann cell response to direct LIPUS stimulation. The previous work with peripheral nerve injury have demonstrated that the magnitude and duration of LIPUS has a direct effect on whether the stimulus has a positive or negative effect on nerve regeneration (Chang, Hsu et al. 2005; Raso, Barbieri et al. 2005). Yet, there are currently no data about the actual types and levels of the ultrasound for the cells within the peripheral nerve. Although using similar values from studies on chondrocytes, endothelial cells, and osteoblasts would allow direct comparison between different cell lines, these values may not have any significance to neural tissue based upon the different physiologic demands of each tissue type. Thus, we chose the magnitude and duration of stimulation for these experiments based on previous work that demonstrated that ultrasound induced a biological response in Schwann cells (Chang, Hsu et al. 2005)

The purpose of this study was to evaluate how sustained LIPUS directly affects Schwann cell function. By evaluating for the expression of the pan-specific Schwann cell marker S-100 with immunohistochemistry, we determined whether Schwann cells de-differentiated after LIPUS stimulation. Schwann cell proliferation was explored using BrdU uptake assays to ascertain if direct LIPUS stimulation is mitogenic for Schwann cells in culture plates.

#### **2.1 Material and methods**

36 Tissue Regeneration – From Basic Biology to Clinical Application

LIPUS is likely to assist the regeneration of neuronal axons. However, the mechanism of

**2. Schwann cells that were subjected to LIPUS consistently demonstrated an** 

Ultrasound is commonly used for diagnostic imaging and physiotherapy and can exert biological effects through either thermal or mechanical mechanisms in living tissue (Choi, Pernot et al. 2007; Nahirnyak, Mast et al. 2007). In contrast to high-intensity continuous ultrasound, LIPUS (<100 mW/cm2) has much lower intensities, which are regarded as nonthermogenic and nondestructive (Ikeda, Takayama et al. 2006). Mechanical strains received by cells may result in biochemical events and increase membrane permeability (Danialou, Comtois et al. 2002). Despite the wide use of LIPUS for improving peripheral nerve tissue regeneration in animal models (Crisci and Ferreira 2002; Chang, Hsu et al. 2005; Raso, Barbieri et al. 2005), very little is known about its effects on the glial cells of peripheral nerves. It has been reported that Schwann cells respond somehow to LIPUS stimulation (Chang, Hsu et al. 2005; Raso, Barbieri et al. 2005). However, the results of previous investigations were somewhat inconclusive, particularly regarding the precise mechanism.

Fig. 1. Experimental apparatus for applying low- intensity pulsed ultrasound (LIPUS) which generated LIPUS with a SATA intensity of 10 mW/cm2, pulse width of 200 microseconds, repetition rate of 1.5 KHz, and an operation frequency of 1MHz. LIPUS irradiated neurons with two probes 24 h after in culture. A six-well plate was placed on the probes. LIPUS was transmitted to culture plate via an interposed ultrasound gel. Two transducers for the control group (sham-LIPUS; LIPUS not turned on) and two probes for the LIPUS group.

Previous studies have shown that LIPUS in the cultured cells induces significant cellular responses in nucleus pulposus cells, endothelial cells, osteoblasts, chondrocytes, and fibroblasts, (Parvizi, Wu et al. 1999; Zhou, Schmelz et al. 2004; Hill, Fenwick et al. 2005; Sena, Leven et al. 2005; Hiyama, Mochida et al. 2007) but little is known about Schwann cell response to direct LIPUS stimulation. The previous work with peripheral nerve injury have

such events is unknown.

**increase in cell proliferation** 

#### **Schwann cells culture and low-intensity pulsed ultrasound treatment**

Schwann cells were prepared using a method previously described with some modifications (Cai, Campana et al. 1999). Briefly, sciatic nerves were dissected from Wistar rats (n=30) at postnatal day 1–3. The epineural sheath was removed. Thereafter, the sciatic nerves were chopped into 200-μm pieces and enzymatically digested (collagenase/trypsin, 1 mg/ml, 1 hour, 37°C). The resulting cell suspensions were plated onto a six-well plate and cultured in Schwann cell medium (DMEM/10% heat-inactivated FCS/2 mM glutamine/pen/strep). Two different cell densities were prepared for subsequent experiments, a cell density of 5,000 cells/1.77 cm2 for proliferation assay and immunohistochemistry assays, and a cell density of 100,000 cells/1.77 cm2 for semiquantitative RT-PCR. The fibroblasts were eliminated by 10 μM cytosine arabinoside and complement-mediated cytolysis with the fibroblast- specific antibody Thy1.1 in conjunction with baby rabbit complement (Cedarlane, Burlington, NC). The medium was changed every other day by adding 2 μM forskolin and glial growth factor (100μg/ml) for expansion of Schwann cells for up to 14 days of cells culture. The purity of cultures was monitored by immunostaining using the Schwann cell marker S-100 and the fibroblast marker Thy-1.1.

Schwann cells were cultured and subjected to LIPUS with modifications as previously described (Takayama, Suzuki et al. 2007). This device (Nexson-The-P41, Nexus Biomedical Devices, Hangzhou, China) generated LIPUS with a pulse width of 200 microseconds, repetition rate of 1.5 KHz, operation frequency of 1 MHz, spatial average temporal average of 100 mW/cm2, 5 minutes/day. The LIPUS treatment was started 24 hours after initiation of cells culture and repeated for 14 consecutive days. In the experimental group, LIPUS was transmitted from 35- mm diameter LIPUS transducers to the bottom of the cell culture plate via a coupling gel (Smith & Nephew, Oklahoma, CA) and was administered in an incubator (see Fig. 1). In the control group, plates were placed on the same transducers for the same duration, but the LIPUS was not administered.

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 39

In this study, we observed that LIPUS increased Schwann cell proliferation indicating that LIPUS is mitogenic for Schwann cells in vitro (See Fig.3). The LIPUS treatment could effectively improve Schwann cell proliferation at an early stage (day 4, day 7 and day 10), while at later stages (day 14) self-renewal ability of these cells reached to a much higher level but there was no obvious difference between experimental and control groups. This increase in proliferation confirmed results with previous in vitro data, which proposes that cultured cells may be mitogenic in response to LIPUS stimulation (Mukai, Ito et al. 2005; Iwashina, Mochida et al. 2006) Furthermore, these data lend credence to the possibility that the LIPUS stimulus may directly trigger Schwann cell proliferation in the early phase.

Day 4 Day 7 Day 10 Day 14 F P

13.3± 1.67 30.53±4.98 51.93±11.56 76.70±9.67 147 0.00 experiment

16.33±2.68 40.73±7.45 71.07±9.03 80.20±11.68

LSD-t 2.35 2.788 3.20 0.06

P 0.04 0.02 0.01 0.58 The mitotic cells were identified by metabolic BrdU labeling. The results showed that experimental groups display a higher proliferation rate. LSD-t, Least Significant Difference t test; F, F value of One-

Fig. 2. LIPUS induces increased mitogenesis of in vitro cultured Schwann cells. Fluorescent images depicting the increase in the ratio of the number of BrdU-stained nuclei (green) to DAPI-stained nuclei (blue) in experimental or control cells. Note the increased number

of BrdU-positive cells in experimental cells versus the control cells.

(experimental A, B; control C, D) at day 4.

Table 1. the effect of LIPUS on the cell proliferation rate of cultured Schwann cells.

Way ANOVA; p, the p-value is the probability that the null hypothesis is true.

group

Comparison at same time point

Control group

group

Schwann cells plated at a cell density of 5,000 cells/1.77cm2 were used for the immunocytochemistry assays. At day 14 after LIPUS treatment, a total of 18 plates (nine experimental plates and nine control plates) were analyzed for S-100, NT-3, and BDNF immunostaining, respectively. The cells were fixed in plates for 10 minutes with 4% paraformaldehyde solution and then blocked in 4% goat serum with 0.25% triton in PBS. Then, the cells were incubated with either mouse anti-S100 protein monoclonal antibody (Sigma, Saint Louis, MO), mouse anti-neurotrophin-3 monoclonal antibody (Santa Cruz, Santa Cruz, CA), or mouse antibrain-derived neurotrophic factor primary antibodies (Santa Cruz, Sant Cruz, CA), then subsequently with goat anti-mouse IgG FITC (Sigma, Saint Louis, MO) or IgG TRITC (Sigma, Saint Louis, MO) for 1 hour and counterstained with DAPI (Sigma, Saint Louis, MO). The percentage of fluorescently labeled cells/DAPI-stained nuclei was counted using a fluorescent microscope-computer interface (Zeiss, Jena, Germany).

#### **Proliferation assay with 5-Bromo-2-deoxy-uridine**

The percent of proliferation was determined by the ratio of total BrdU-positive nuclei to total number of cells (DAPI-stained nuclei) as described previously (Funk, Fricke et al. 2007). Schwann cells plated at a cell density of 5,000 cells/1.77 cm2 were used for counting of each individual Schwann cell. A total of 12 plates per time point (six experimental plates and six control plates, respectively) were analyzed. At days 4, 7, 10, and 14 after LIPUS treatment, the cells in plates were treated by Brdu (Sigma, Saint Louis, MO) for 2 hours. The cells were then fixed in methanol for 10 minutes at 48ºC and treated with 1.25% proteinase K in PBS (pH 7.5) for 5 minutes. Thereafter, the cells were treated with mouse anti-BrdU monoclonal antibody (Sigma, Saint Louis, MO) for 1 hour and then with goat anti-mouse IgG FITC (Sigma, Saint Louis, MO) for 1 hour. DAPI (Sigma, Saint Louis, MO) and cover slips were added. The average proliferation percentage of the plate was counted. The average proliferation percentage was counted by examining four random images within per plate using a fluorescent microscope-computer interface (Zeiss, Jena, Germany).

#### **2.2 LIPUS stimulus may directly trigger Schwann cell proliferation in the early phase**

The Schwann cells that were subjected to LIPUS consistently demonstrated an increase in cell proliferation. Fig. 2 shows a fluorescent microscope picture demonstrating the difference in the percentage of BrdU-positive cells in both experimental and control cells at day 7. Table 1 shows the percentage of BrdU-positive cells were significantly higher in experimental groups than in control groups on day 4 (P <0.01), day 7 (P <0.01) and day 10 (P <0.01), respectively. However, this difference between experimental and control disappeared on day 14. The percentage increase in proliferation varied depending upon the control proliferation levels of BrdU uptake. For example, when the control level of proliferation was 23.2% at day 7, there was a 100% increase in the proliferation of experimental cells. While there was a 48.6% control proliferation level at day 10, the experimental cells exhibited a 43% increase in proliferation. The variation of proliferation between both groups is not secondary to changes in media or culture media additive. Moreover, the data suggest that Schwann cells are more responsive to LIPUS at different times of their cell cycle.

Schwann cells plated at a cell density of 5,000 cells/1.77cm2 were used for the immunocytochemistry assays. At day 14 after LIPUS treatment, a total of 18 plates (nine experimental plates and nine control plates) were analyzed for S-100, NT-3, and BDNF immunostaining, respectively. The cells were fixed in plates for 10 minutes with 4% paraformaldehyde solution and then blocked in 4% goat serum with 0.25% triton in PBS. Then, the cells were incubated with either mouse anti-S100 protein monoclonal antibody (Sigma, Saint Louis, MO), mouse anti-neurotrophin-3 monoclonal antibody (Santa Cruz, Santa Cruz, CA), or mouse antibrain-derived neurotrophic factor primary antibodies (Santa Cruz, Sant Cruz, CA), then subsequently with goat anti-mouse IgG FITC (Sigma, Saint Louis, MO) or IgG TRITC (Sigma, Saint Louis, MO) for 1 hour and counterstained with DAPI (Sigma, Saint Louis, MO). The percentage of fluorescently labeled cells/DAPI-stained nuclei was counted using a fluorescent microscope-computer interface (Zeiss, Jena,

The percent of proliferation was determined by the ratio of total BrdU-positive nuclei to total number of cells (DAPI-stained nuclei) as described previously (Funk, Fricke et al. 2007). Schwann cells plated at a cell density of 5,000 cells/1.77 cm2 were used for counting of each individual Schwann cell. A total of 12 plates per time point (six experimental plates and six control plates, respectively) were analyzed. At days 4, 7, 10, and 14 after LIPUS treatment, the cells in plates were treated by Brdu (Sigma, Saint Louis, MO) for 2 hours. The cells were then fixed in methanol for 10 minutes at 48ºC and treated with 1.25% proteinase K in PBS (pH 7.5) for 5 minutes. Thereafter, the cells were treated with mouse anti-BrdU monoclonal antibody (Sigma, Saint Louis, MO) for 1 hour and then with goat anti-mouse IgG FITC (Sigma, Saint Louis, MO) for 1 hour. DAPI (Sigma, Saint Louis, MO) and cover slips were added. The average proliferation percentage of the plate was counted. The average proliferation percentage was counted by examining four random images within per plate using a fluorescent microscope-computer interface (Zeiss, Jena,

**2.2 LIPUS stimulus may directly trigger Schwann cell proliferation in the early phase**  The Schwann cells that were subjected to LIPUS consistently demonstrated an increase in cell proliferation. Fig. 2 shows a fluorescent microscope picture demonstrating the difference in the percentage of BrdU-positive cells in both experimental and control cells at day 7. Table 1 shows the percentage of BrdU-positive cells were significantly higher in experimental groups than in control groups on day 4 (P <0.01), day 7 (P <0.01) and day 10 (P <0.01), respectively. However, this difference between experimental and control disappeared on day 14. The percentage increase in proliferation varied depending upon the control proliferation levels of BrdU uptake. For example, when the control level of proliferation was 23.2% at day 7, there was a 100% increase in the proliferation of experimental cells. While there was a 48.6% control proliferation level at day 10, the experimental cells exhibited a 43% increase in proliferation. The variation of proliferation between both groups is not secondary to changes in media or culture media additive. Moreover, the data suggest that Schwann cells are more responsive to LIPUS at different

Germany).

Germany).

times of their cell cycle.

**Proliferation assay with 5-Bromo-2-deoxy-uridine** 

In this study, we observed that LIPUS increased Schwann cell proliferation indicating that LIPUS is mitogenic for Schwann cells in vitro (See Fig.3). The LIPUS treatment could effectively improve Schwann cell proliferation at an early stage (day 4, day 7 and day 10), while at later stages (day 14) self-renewal ability of these cells reached to a much higher level but there was no obvious difference between experimental and control groups. This increase in proliferation confirmed results with previous in vitro data, which proposes that cultured cells may be mitogenic in response to LIPUS stimulation (Mukai, Ito et al. 2005; Iwashina, Mochida et al. 2006) Furthermore, these data lend credence to the possibility that the LIPUS stimulus may directly trigger Schwann cell proliferation in the early phase.


The mitotic cells were identified by metabolic BrdU labeling. The results showed that experimental groups display a higher proliferation rate. LSD-t, Least Significant Difference t test; F, F value of One-Way ANOVA; p, the p-value is the probability that the null hypothesis is true.

Table 1. the effect of LIPUS on the cell proliferation rate of cultured Schwann cells.

Fig. 2. LIPUS induces increased mitogenesis of in vitro cultured Schwann cells. Fluorescent images depicting the increase in the ratio of the number of BrdU-stained nuclei (green) to DAPI-stained nuclei (blue) in experimental or control cells. Note the increased number of BrdU-positive cells in experimental cells versus the control cells. (experimental A, B; control C, D) at day 4.

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 41

survival and differentiation in the absence of axons (McAllister, Katz et al. 1999), Schwann cells also contribute to the sources of BDNF during nerve regeneration and the deprivation of endogenous BDNF results in impairment in regeneration and myelination of regenerating axons (Zhang, Luo et al. 2000), BDNF also plays a role in activity-dependent neuronal plasticity (Schmidhammer, Hausner et al. 2007). The exogenous administration of these factors has protective properties for injured neurons and stimulates axonal regeneration (Lykissas, Batistatou et al. 2007). Based on these properties, these molecules may be used as therapeutic agents for treating degenerative diseases and traumatic injuries of both the

We therefore measured how LIPUS affects Schwann cells neurotrophic function by evaluated the mRNA expression of NT-3 and BDNF, two members of the neurotrophic

Schwann cells plated at a cell density of 5,000 cells/ 1.77 cm2 were used for the RT-PCR assays. At day 14 after LIPUS treatment, a total of 12 plates (six experimental plates and six control plates) were analyzed for NT-3 and BDNF mRNA expression, respectively. The cells were then incubated for 12 hours at 37°C to allow for gene transcription. Cells were then trypsinized, collected as pooled samples, and RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the protocol. The cDNA was prepared for experimental and control samples using 3μg of RNA with SuperScript II RNase reverse transcriptase (Invitrogen, Oklahoma, CA) and specific primers for NT-3 (forward: 5'- CTTATCTCCG TGGCATCCAAGG-3', reverse: 5'- TCTGAAGTCAGTGCTCGGACGT-3'), BDNF (forward: 5'-ATGGGACTCTGGAGAGCGTGAA-3', reverse: 5'-CGCCAGCCAATTCTCTTTTTGC-3'), and b-actin (forward: 5'-CCCAGAGCAAGAGAGGCATC-3', reverse: 5'-CTCAGGAGGAG

The PCR reaction conditions were consisted of one cycle of 94°C for 5 minutes, followed by 30 cycles of thermal cycling 30 seconds at 94°C, 30 seconds at T0°C, and 1 minute at 72°C. The T0 was 60°C for BDNF, 64°C for NT-3, and 58°C for b-actin. The final cycle was followed by a 5-minute extension at 72°C. Ten microliters of PCR product was then differentiated on a 1.5% agarose gel and the gel image was taken with a digital camera. ImagQuant analysis software (Stratagene Company, La Jolla, CA) was used to determine the densities of the NT-3 and BDNF bands when compared with the b-actin control for both experimental and

**3.2 Effect of LIPUS on the expression of NT-3 and BDNF mRNA in Schwann cell** 

transcriptase enzyme confirmed that there was no genomic DNA contamination.

Schwann cells that were subjected to sustained LIPUS exhibited an increase in NT-3 mRNA expression, and a decrease in BDNF mRNA expression (Fig. 4). The NT-3/-actin ratio of RT-PCR products in the experimental group was 0.56±0.13 and 0.41±0.09 in the control group. However, the BDNF/-actin ratio of RT-PCR products in the experimental group was 0.51±0.05 and 0.60±0.08 in the control group. The differences in NT-3 and BDNF products for experimental and control groups were found to be statistically significant (p<0.01 and p<0.05, respectively). Reverse transcriptase controls with no reverse

**3.1 Semiquantitative RT-PCR for detecting the BDNF and NT-3 mRNA expression** 

central and peripheral nervous system.

CAATGATCT-3') (Hatami, Oryan et al. 2007).

control samples.

factor family of the Schwann cells.

Fig. 3. Increase in proliferation of cultured Schwann cells in response to LIPUS at day 4, day 7 and day 10. No significant difference at day 14.

#### **2.3 LIPUS treatment does not change the phenotype of the Schwann cell**

In addition to the increased cell proliferation, LIPUS stimulation of cell cultures has previously been demonstrated to induce an alteration of cellular phenotype,(Ikeda, Takayama et al. 2006; Schumann, Kujat et al. 2006), but little is known about the effects of LIPUS stimulation on a Schwann cell phenotype. Thus, the initial experiments were used to determine whether LIPUS would induce a phenotype alteration to the Schwann cell or not. S-100 immunostaining results showed that Schwann cells do not de-differentiate into another cell type following LIPUS stimulation.

The immunohistochemistry study showed that more than 98% of Schwann cells were positive for the pan-specific Schwann cell marker S-100 at day 14, with or without LIPUS treatment. Moreover, immunostaining for NT-3 and BDNF shows that Schwann cells were positive in more than 98% of the evaluated cells in both the experimental and control cells at day 14. Additionally, the distribution of the positively stained cells was uniform for both the inner and outer areas of the circular plated region. These results further demonstrated that LIPUS treatment does not change the phenotype of the Schwann cell.

#### **3. Effect of LIPUS on the expression of neurotrophin-3 and brain derived neurotrophic factor in cultured Schwann cells**

Both neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) are two of key neurotrophins constituents in peripheral nervous system, NT-3 is an important regulator of neural survival, development, function, and neuronal differentiation (McAllister, Lo et al. 1995; McAllister, Katz et al. 1999). Hess (Hess, Scott et al. 2007) et al observed that NT-3 expression may modulate the number of Schwann cells at neuromuscular synapses. Otherwise, neurotrophin-3 is an important autocrine factor supporting Schwann cell

Fig. 3. Increase in proliferation of cultured Schwann cells in response to LIPUS at day 4,

In addition to the increased cell proliferation, LIPUS stimulation of cell cultures has previously been demonstrated to induce an alteration of cellular phenotype,(Ikeda, Takayama et al. 2006; Schumann, Kujat et al. 2006), but little is known about the effects of LIPUS stimulation on a Schwann cell phenotype. Thus, the initial experiments were used to determine whether LIPUS would induce a phenotype alteration to the Schwann cell or not. S-100 immunostaining results showed that Schwann cells do not de-differentiate into

The immunohistochemistry study showed that more than 98% of Schwann cells were positive for the pan-specific Schwann cell marker S-100 at day 14, with or without LIPUS treatment. Moreover, immunostaining for NT-3 and BDNF shows that Schwann cells were positive in more than 98% of the evaluated cells in both the experimental and control cells at day 14. Additionally, the distribution of the positively stained cells was uniform for both the inner and outer areas of the circular plated region. These results further demonstrated that

**3. Effect of LIPUS on the expression of neurotrophin-3 and brain derived** 

Both neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) are two of key neurotrophins constituents in peripheral nervous system, NT-3 is an important regulator of neural survival, development, function, and neuronal differentiation (McAllister, Lo et al. 1995; McAllister, Katz et al. 1999). Hess (Hess, Scott et al. 2007) et al observed that NT-3 expression may modulate the number of Schwann cells at neuromuscular synapses. Otherwise, neurotrophin-3 is an important autocrine factor supporting Schwann cell

**2.3 LIPUS treatment does not change the phenotype of the Schwann cell** 

LIPUS treatment does not change the phenotype of the Schwann cell.

**neurotrophic factor in cultured Schwann cells** 

day 7 and day 10. No significant difference at day 14.

another cell type following LIPUS stimulation.

survival and differentiation in the absence of axons (McAllister, Katz et al. 1999), Schwann cells also contribute to the sources of BDNF during nerve regeneration and the deprivation of endogenous BDNF results in impairment in regeneration and myelination of regenerating axons (Zhang, Luo et al. 2000), BDNF also plays a role in activity-dependent neuronal plasticity (Schmidhammer, Hausner et al. 2007). The exogenous administration of these factors has protective properties for injured neurons and stimulates axonal regeneration (Lykissas, Batistatou et al. 2007). Based on these properties, these molecules may be used as therapeutic agents for treating degenerative diseases and traumatic injuries of both the central and peripheral nervous system.

We therefore measured how LIPUS affects Schwann cells neurotrophic function by evaluated the mRNA expression of NT-3 and BDNF, two members of the neurotrophic factor family of the Schwann cells.

#### **3.1 Semiquantitative RT-PCR for detecting the BDNF and NT-3 mRNA expression**

Schwann cells plated at a cell density of 5,000 cells/ 1.77 cm2 were used for the RT-PCR assays. At day 14 after LIPUS treatment, a total of 12 plates (six experimental plates and six control plates) were analyzed for NT-3 and BDNF mRNA expression, respectively. The cells were then incubated for 12 hours at 37°C to allow for gene transcription. Cells were then trypsinized, collected as pooled samples, and RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the protocol. The cDNA was prepared for experimental and control samples using 3μg of RNA with SuperScript II RNase reverse transcriptase (Invitrogen, Oklahoma, CA) and specific primers for NT-3 (forward: 5'- CTTATCTCCG TGGCATCCAAGG-3', reverse: 5'- TCTGAAGTCAGTGCTCGGACGT-3'), BDNF (forward: 5'-ATGGGACTCTGGAGAGCGTGAA-3', reverse: 5'-CGCCAGCCAATTCTCTTTTTGC-3'), and b-actin (forward: 5'-CCCAGAGCAAGAGAGGCATC-3', reverse: 5'-CTCAGGAGGAG CAATGATCT-3') (Hatami, Oryan et al. 2007).

The PCR reaction conditions were consisted of one cycle of 94°C for 5 minutes, followed by 30 cycles of thermal cycling 30 seconds at 94°C, 30 seconds at T0°C, and 1 minute at 72°C. The T0 was 60°C for BDNF, 64°C for NT-3, and 58°C for b-actin. The final cycle was followed by a 5-minute extension at 72°C. Ten microliters of PCR product was then differentiated on a 1.5% agarose gel and the gel image was taken with a digital camera. ImagQuant analysis software (Stratagene Company, La Jolla, CA) was used to determine the densities of the NT-3 and BDNF bands when compared with the b-actin control for both experimental and control samples.

#### **3.2 Effect of LIPUS on the expression of NT-3 and BDNF mRNA in Schwann cell**

Schwann cells that were subjected to sustained LIPUS exhibited an increase in NT-3 mRNA expression, and a decrease in BDNF mRNA expression (Fig. 4). The NT-3/-actin ratio of RT-PCR products in the experimental group was 0.56±0.13 and 0.41±0.09 in the control group. However, the BDNF/-actin ratio of RT-PCR products in the experimental group was 0.51±0.05 and 0.60±0.08 in the control group. The differences in NT-3 and BDNF products for experimental and control groups were found to be statistically significant (p<0.01 and p<0.05, respectively). Reverse transcriptase controls with no reverse transcriptase enzyme confirmed that there was no genomic DNA contamination.

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 43

mRNA expression of GSK-3 to determine the intracellular mechanism of neurite outgrowth following irradiation by LIPUS. It is concluded that LIPUS can enhance elongation of neurites and it is possible through the decreased expression of GSK-3 (Ren, Li et al. 2010).

Cortical neurons isolated from the brain of Wistar rats were bought from ScienCell Research Laboratories (San Diego, USA). These cortical neurons were subcultured with a density of 20 000 cells/1.6 cm2 in poly-L-lysine coated 6-well plates (Costa, USA) for immunoblot and semi-quantitative RT-PCR analysis, and a density of 100 cells/0.32 cm2 in poly-L-lysine coated 96-well plates (Costa, USA) for the measurement of neurite length. The cells were cultured in neuronal medium (3 mL medium per well in the 6-well plates and 0.1mL per well in the 96-well plate; ScienCell Research Laboratories, San Diego, CA, USA) in a humidified atmosphere of 5% CO2 in air at 37 ℃. The medium was refreshed every 3 d.

A LIPUS-therapeutic apparatus, Nexson-The- P41 was constructed according to instructions from Nexus Biomedical Devices (Hangzhou, China). There were two LIPUS probes in the apparatus, both of which generated LIPUS with a SATA intensity of 10 mW/cm2, pulse width of 200 microseconds, repetition rate of 1.5 kHz, and an operation frequency of 1 MHz. The LIPUS was applied to the cultured cortical neurons after 24 h in culture through the bottom of the 6-well plates via a coupling gel (Smith & Nephew, Oklahoma, CA, USA) and was administered for 5 min every day during the span of this experiment (Fig. 1). Ultrasound signals from this generator were detected by a hydrophone system (Model OS-111; Hewlett-Packard, Japan), and the wave amplitudes of the signals passing through the tube wall were more than 90%, which resulted in more than 85% energy irradiated. Control samples were prepared in the same manner with the exception of no LIPUS treatment.

Cultured cortical neurons in 96-well plates were randomly divided into two groups: the LIPUS-treated group and the control group. After being subcultured for 24 h, the LIPUS treatment began and was administered for 5 min every day. On the third day, both the LIPUS-treated and control groups were photographed 2 h after the treatment. A Nikon Diaphot inverted microscope with a Nikon Plan 20× objective (Nikon, Tokyo, Japan) coupled to a video camera was used to obtain cell images (Carl Zeiss, Germany). Images of at least 200 neurons for each group were obtained. For each neuron, we measured its longest

There are no significant difference in morphology between LIPUS-treated group and control group except the length of neurites. In both LIPUS-treated group (Fig. 5a) and control group (Fig.5b), there were many neurons with 2-7 processes; some were thick fibers, or some were thin fibers with varicosities. We measured the length of 200 neurites in each group and most neurite measured have a length between 50 μm to 80 μm. Data showed that compared with control group, neurites in LIPUS-treated group were significant longer [(73.14 ±8.32) μm vs.

neurite with the software Image-Pro Plus 6.0 (Media Cybernetics, USA).

**4.1.2 Neurites in LIPUS-treated group were significant longer** 

**4.1 Effect of LIPUS treatment on neurite outgrowth** 

**Neurite length measurement protocol** 

**4.1.1 Materials and methods: Cell culture and ultrasound treatment** 

Fig. 4. Results from RT-PCR analysis of NT-3 and BDNF are expressed relative to -actin mRNA expression 14 days after the LIPUS stimulation. There was significantly upregulated in experimental groups compared with the control in NT-3 mRNA expression (t=2.324, P<0.05), and significantly downregulated in BDNF mRNA expression (t=2.337, p<0.05).

#### **4. LIPUS enhances elongation of neurites in rat cortical neurons through inhibition of GSK-3**

Intracellular mechanisms that enhance neurite outgrowth evidently require the reorganization of the neurite cytoskeletons including the microtubules and actin filaments (Dent and Gertler 2003). Recently, a cytoskeletal-related signaling pathway: PI 3-kinase/Atk/glycogen synthase kinase (GSK-3)/collapsin response mediator protein (CRMP-2) was reported to be important for the outgrowth of neurite, with GSK-3 being a central regulator (Jiang, Guo et al. 2005; Yoshimura, Kawano et al. 2005). GSK-3 is a multifunctional serine/threonine kinase found ubiquitously in eukaryotes (Jiang, Guo et al. 2005) and it plays key roles for various biological processes, such as the canonical Wnt signaling pathway, microtubule dynamics, and astrocyte migration (Doble and Woodgett 2003; Etienne-Manneville and Hall 2003). GSK-3 phosphorylates at least have four types of microtubule-associated proteins (MAPs), CRMP-2(Yoshimura, Kawano et al. 2005), tau (Jiang, Guo et al. 2005), adenomatous polyposis coil gene product (APC)(Frame and Cohen 2001; Grimes and Jope 2001) and MAP1B (Lucas, Goold et al. 1998; Goold, Owen et al. 1999). It modulates axial orientation during the development, differentiation, and neurite outgrowth in neurons through phosphorylation of these MAPs (Jiang, Guo et al. 2005; Yoshimura, Kawano et al. 2005; Chen, Yu et al. 2007; Conde and Caceres 2009). Some research have proved that the local inhibition of GSK-3 effectively enhances neurite/axon elongation (Kim, Zhou et al. 2006), whereas overexpression of GSK-3 could impair neurite/axon elongation (Munoz-Montano, Lim et al. 1999). During peripheral nerve regeneration, some factors such as BDNF, NT3, and laminin, locally activate the PI3 kinase/Akt/GSK-3 pathway and inhibit GSK-3, which favors neurite elongation (Kim, Zhou et al. 2006).

We measured the length of neurites to examine whether LIPUS is effective for the elongation of the neuronal processes. Then we examined the change in the activity and the

Fig. 4. Results from RT-PCR analysis of NT-3 and BDNF are expressed relative to -actin mRNA expression 14 days after the LIPUS stimulation. There was significantly upregulated in experimental groups compared with the control in NT-3 mRNA expression (t=2.324, P<0.05), and significantly downregulated in BDNF mRNA expression (t=2.337, p<0.05).

**4. LIPUS enhances elongation of neurites in rat cortical neurons through** 

Intracellular mechanisms that enhance neurite outgrowth evidently require the reorganization of the neurite cytoskeletons including the microtubules and actin filaments (Dent and Gertler 2003). Recently, a cytoskeletal-related signaling pathway: PI 3-kinase/Atk/glycogen synthase kinase (GSK-3)/collapsin response mediator protein (CRMP-2) was reported to be important for the outgrowth of neurite, with GSK-3 being a central regulator (Jiang, Guo et al. 2005; Yoshimura, Kawano et al. 2005). GSK-3 is a multifunctional serine/threonine kinase found ubiquitously in eukaryotes (Jiang, Guo et al. 2005) and it plays key roles for various biological processes, such as the canonical Wnt signaling pathway, microtubule dynamics, and astrocyte migration (Doble and Woodgett 2003; Etienne-Manneville and Hall 2003). GSK-3 phosphorylates at least have four types of microtubule-associated proteins (MAPs), CRMP-2(Yoshimura, Kawano et al. 2005), tau (Jiang, Guo et al. 2005), adenomatous polyposis coil gene product (APC)(Frame and Cohen 2001; Grimes and Jope 2001) and MAP1B (Lucas, Goold et al. 1998; Goold, Owen et al. 1999). It modulates axial orientation during the development, differentiation, and neurite outgrowth in neurons through phosphorylation of these MAPs (Jiang, Guo et al. 2005; Yoshimura, Kawano et al. 2005; Chen, Yu et al. 2007; Conde and Caceres 2009). Some research have proved that the local inhibition of GSK-3 effectively enhances neurite/axon elongation (Kim, Zhou et al. 2006), whereas overexpression of GSK-3 could impair neurite/axon elongation (Munoz-Montano, Lim et al. 1999). During peripheral nerve regeneration, some factors such as BDNF, NT3, and laminin, locally activate the PI3 kinase/Akt/GSK-3 pathway and inhibit GSK-3, which favors neurite elongation (Kim, Zhou

We measured the length of neurites to examine whether LIPUS is effective for the elongation of the neuronal processes. Then we examined the change in the activity and the

**inhibition of GSK-3**

et al. 2006).

mRNA expression of GSK-3 to determine the intracellular mechanism of neurite outgrowth following irradiation by LIPUS. It is concluded that LIPUS can enhance elongation of neurites and it is possible through the decreased expression of GSK-3 (Ren, Li et al. 2010).

#### **4.1 Effect of LIPUS treatment on neurite outgrowth**

#### **4.1.1 Materials and methods: Cell culture and ultrasound treatment**

Cortical neurons isolated from the brain of Wistar rats were bought from ScienCell Research Laboratories (San Diego, USA). These cortical neurons were subcultured with a density of 20 000 cells/1.6 cm2 in poly-L-lysine coated 6-well plates (Costa, USA) for immunoblot and semi-quantitative RT-PCR analysis, and a density of 100 cells/0.32 cm2 in poly-L-lysine coated 96-well plates (Costa, USA) for the measurement of neurite length. The cells were cultured in neuronal medium (3 mL medium per well in the 6-well plates and 0.1mL per well in the 96-well plate; ScienCell Research Laboratories, San Diego, CA, USA) in a humidified atmosphere of 5% CO2 in air at 37 ℃. The medium was refreshed every 3 d.

A LIPUS-therapeutic apparatus, Nexson-The- P41 was constructed according to instructions from Nexus Biomedical Devices (Hangzhou, China). There were two LIPUS probes in the apparatus, both of which generated LIPUS with a SATA intensity of 10 mW/cm2, pulse width of 200 microseconds, repetition rate of 1.5 kHz, and an operation frequency of 1 MHz. The LIPUS was applied to the cultured cortical neurons after 24 h in culture through the bottom of the 6-well plates via a coupling gel (Smith & Nephew, Oklahoma, CA, USA) and was administered for 5 min every day during the span of this experiment (Fig. 1). Ultrasound signals from this generator were detected by a hydrophone system (Model OS-111; Hewlett-Packard, Japan), and the wave amplitudes of the signals passing through the tube wall were more than 90%, which resulted in more than 85% energy irradiated. Control samples were prepared in the same manner with the exception of no LIPUS treatment.

#### **Neurite length measurement protocol**

Cultured cortical neurons in 96-well plates were randomly divided into two groups: the LIPUS-treated group and the control group. After being subcultured for 24 h, the LIPUS treatment began and was administered for 5 min every day. On the third day, both the LIPUS-treated and control groups were photographed 2 h after the treatment. A Nikon Diaphot inverted microscope with a Nikon Plan 20× objective (Nikon, Tokyo, Japan) coupled to a video camera was used to obtain cell images (Carl Zeiss, Germany). Images of at least 200 neurons for each group were obtained. For each neuron, we measured its longest neurite with the software Image-Pro Plus 6.0 (Media Cybernetics, USA).

#### **4.1.2 Neurites in LIPUS-treated group were significant longer**

There are no significant difference in morphology between LIPUS-treated group and control group except the length of neurites. In both LIPUS-treated group (Fig. 5a) and control group (Fig.5b), there were many neurons with 2-7 processes; some were thick fibers, or some were thin fibers with varicosities. We measured the length of 200 neurites in each group and most neurite measured have a length between 50 μm to 80 μm. Data showed that compared with control group, neurites in LIPUS-treated group were significant longer [(73.14 ±8.32) μm vs.

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 45

of GSK-3beta to determine the intracellular mechanism of neurite outgrowth following

Fig. 6. Total proteins were extracted on the third, seventh, and tenth days following daily LIPUS treatment and their activity were examined using Western blot analysis. (a) On the third day, there was no significant difference in protein levels between the control and LIPUS groups. (b) On the seventh day, the levels of p-Akt, GSK-3beta, p-GSK-3beta, and p-CRMP-2 were decreased in the LIPUS group compared to the controls. (c) On the tenth day, a remarkable decrease of p-Akt, p-GSK-3beta, and p-CRMP-2 were observed while it appeared that GSK-3beta was slightly decreased. The beta-actin in each lane served as an

For Western blot analysis, the treated and untreated cultured cells were harvested at third, seventh, and tenth days. LIPUS group cells were harvested 2 h after the last LIPUS treatment. Whole cell extracts were prepared by boiling the cells in lysis buffer (2% SDS; 10% glycerol; 10 mmol/L Tris, pH 6.8; 100 mmol/L DTT) for 10 min. Proteins were separated by electrophoresis on 4%-12% Bis-Tris gels (Novex; Invitrogen, Carlsbad, CA, USA). Separated proteins were then transferred onto PVDF membranes. The membranes were blocked with 5% nonfat dry milk in PBS, pH 7.4, and 0.1% Tween 20 (PBS-Tween) for 1 h at room temperature. The membranes were incubated with primary antibodies diluted in 5% BSA overnight at 4 ℃. The blots were washed in PBS-Tween and then incubated with diluted secondary antibodies (HRP, 1:10 000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Reactive proteins were visualized with SuperSignal West Pico chemiluminescence's reagent (Pierce Biotechnology, Rockford, IL, USA) followed by

The primary antibodies used for the Western blot analysis were as follows: rabbit anti-GSK-3beta antibody (21001-1; Signalway Antibody, Pearland, TX, USA), rabbit anti-

irradiation by LIPUS (Ren, Li et al. 2010).

internal control.

exposure to x-ray film.

**4.2.1 Materials and methods: Western blot analysis** 

(68.18 ±8.96) μm, P<0.01](Fig. 5c). We attempted to investigate how processes of the cultured neurons were extended under the influence of LIPUS. Morphological changes revealed that LIPUS could effectively enhance elongation of neurites after three days of treatment compared to the control group. However, we failed to measure the length of neurites on the seventh or tenth day because after the fifth day, most neurites reached another neurite and, consequently, the growth of those neurites stopped. Although the mechanism by which LIPUS affects the neuronal processes is likely to be complex, the regulation of the cytoskeleton is crucial for the proper growth cone motility (Dent and Kalil 2001). To clarify the intracellular mechanism of this effect, we examined the proteins related to the cytoskeletal-related signaling pathway to determine whether the proteins in the cultured neurons were changed following the LIPUS treatment.

Fig. 5. On the third day, both the LIPUS-treated and control groups were photographed with a Nikon Plan 20× objective coupled to a video camera. In both LIPUS-treated group (Fig. 5a) and control group (Fig. 5b), there were many neurons with 2-7 processes; some were thick fibers, and some were thin fibers with varicosities. Bar=50 μm. Images of at least 200 neurons for each group were obtained. For each neuron, we measured its longest neurite with the software Image-Pro plus 6.0. Most neurite measured have a length between 50 μm to 80 μm. Data showed that neurites in LIPUS-treated group were significant longer than that in control group [(73.14 ±8.32) μm vs. (68.18 ±8.96) μm, P<0.01] (Fig. 5c).

#### **4.2 Changes in protein activity related to the Cytoskeletal-signaling pathway caused by LIPUS treatment**

To investigate changes in protein activity related to cytoskeletal-signaling pathway caused by LIPUS, total proteins were extracted on the third, seventh, and tenth days following daily LIPUS treatment and their acticity were examined using Western blot analysis (Fig. 6).

To measure the length of neurites to examine whether LIPUS is effective for the elongation of the neuronal processes, we examined the change in the activity and the mRNA expression

(68.18 ±8.96) μm, P<0.01](Fig. 5c). We attempted to investigate how processes of the cultured neurons were extended under the influence of LIPUS. Morphological changes revealed that LIPUS could effectively enhance elongation of neurites after three days of treatment compared to the control group. However, we failed to measure the length of neurites on the seventh or tenth day because after the fifth day, most neurites reached another neurite and, consequently, the growth of those neurites stopped. Although the mechanism by which LIPUS affects the neuronal processes is likely to be complex, the regulation of the cytoskeleton is crucial for the proper growth cone motility (Dent and Kalil 2001). To clarify the intracellular mechanism of this effect, we examined the proteins related to the cytoskeletal-related signaling pathway to determine whether the proteins in the cultured

Fig. 5. On the third day, both the LIPUS-treated and control groups were photographed with a Nikon Plan 20× objective coupled to a video camera. In both LIPUS-treated group (Fig. 5a) and control group (Fig. 5b), there were many neurons with 2-7 processes; some were thick fibers, and some were thin fibers with varicosities. Bar=50 μm. Images of at least 200 neurons for each group were obtained. For each neuron, we measured its longest neurite with the software Image-Pro plus 6.0. Most neurite measured have a length between 50 μm to 80 μm. Data showed that neurites in LIPUS-treated group were significant longer than

**4.2 Changes in protein activity related to the Cytoskeletal-signaling pathway caused** 

To investigate changes in protein activity related to cytoskeletal-signaling pathway caused by LIPUS, total proteins were extracted on the third, seventh, and tenth days following daily LIPUS treatment and their acticity were examined using Western blot analysis (Fig. 6).

To measure the length of neurites to examine whether LIPUS is effective for the elongation of the neuronal processes, we examined the change in the activity and the mRNA expression

that in control group [(73.14 ±8.32) μm vs. (68.18 ±8.96) μm, P<0.01] (Fig. 5c).

**by LIPUS treatment** 

neurons were changed following the LIPUS treatment.

of GSK-3beta to determine the intracellular mechanism of neurite outgrowth following irradiation by LIPUS (Ren, Li et al. 2010).

Fig. 6. Total proteins were extracted on the third, seventh, and tenth days following daily LIPUS treatment and their activity were examined using Western blot analysis. (a) On the third day, there was no significant difference in protein levels between the control and LIPUS groups. (b) On the seventh day, the levels of p-Akt, GSK-3beta, p-GSK-3beta, and p-CRMP-2 were decreased in the LIPUS group compared to the controls. (c) On the tenth day, a remarkable decrease of p-Akt, p-GSK-3beta, and p-CRMP-2 were observed while it appeared that GSK-3beta was slightly decreased. The beta-actin in each lane served as an internal control.

#### **4.2.1 Materials and methods: Western blot analysis**

For Western blot analysis, the treated and untreated cultured cells were harvested at third, seventh, and tenth days. LIPUS group cells were harvested 2 h after the last LIPUS treatment. Whole cell extracts were prepared by boiling the cells in lysis buffer (2% SDS; 10% glycerol; 10 mmol/L Tris, pH 6.8; 100 mmol/L DTT) for 10 min. Proteins were separated by electrophoresis on 4%-12% Bis-Tris gels (Novex; Invitrogen, Carlsbad, CA, USA). Separated proteins were then transferred onto PVDF membranes. The membranes were blocked with 5% nonfat dry milk in PBS, pH 7.4, and 0.1% Tween 20 (PBS-Tween) for 1 h at room temperature. The membranes were incubated with primary antibodies diluted in 5% BSA overnight at 4 ℃. The blots were washed in PBS-Tween and then incubated with diluted secondary antibodies (HRP, 1:10 000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Reactive proteins were visualized with SuperSignal West Pico chemiluminescence's reagent (Pierce Biotechnology, Rockford, IL, USA) followed by exposure to x-ray film.

The primary antibodies used for the Western blot analysis were as follows: rabbit anti-GSK-3beta antibody (21001-1; Signalway Antibody, Pearland, TX, USA), rabbit anti-

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 47

neurite elongation though this pathway, the phosphorylation of GSK-3beta should be upregulated and the activity of GSK-3beta should be inhibited. However, in this research, the activity of GSK-3beta was inhibited by LIPUS and the phosphorylation of GSK-3beta by Akt was inhibited, too. This conflict of results revealed that LIPUS enhances neurite outgrowth through the down-regulation of GSK-3 activity but not through the PI3-kinase/Akt/GSK-3beta pathway. Therefore, we employed semi-quantitative RT-PCR to examine the mRNA of GSK-3beta. The results of the semi-quantitative RT-PCR revealed that the expression of GSK-3beta mRNA decreased after LIPUS irradiation on the seventh day. From these findings, we postulate that when neurons are irradiated by LIPUS, an unknown intracellular mechanism may be activated as a response to this "injury" and, consequently, neurons reduce the mRNA expression of GSK-3. The decrease of GSK-3beta activity comes from reduced expression, but not through the PI3-kinase/Akt/GSK-3 signaling pathway (Ren,

**4.2.3 The expression of GSK-3 mRNA decreased after LIPUS irradiation** 

The mRNA expression of GSK-3beta in the cultured neurons following LIPUS treatment was examined using a semi-quantitative RT-PCR. For this analysis, the LIPUS-treated cultured neurons on the seventh day were selected as they showed a significant decrease in their mRNA levels compared to the control. Data from analysis of the imager indicated mRNA of GSK-3 decreased about 4 folds [(1.001 ±0.017) vs. (0.627 ±0.037), P<0.001] (Fig. 7). As a result, mRNA expression of GSK-3beta was also decreased on the seventh days

Fig. 7. Expression of GSK-3 in neurons was evaluated by semi quantitative RT-PCR. Neurons were irradiated for 7 d and harvested 2 h after the last LIPUS irradiation. The right lane represents the experimental mRNA expression, and the left lane corresponds to the control mRNA expression (Fig. 7a). Data showed that expression of GSK-3 decreased about 4 folds [(1.001 ±0.017) vs. (0.627 ±0.037), P<0.001] (Fig.7b). The -actin in each lane served as

The reduced expression is a kind of global inhibition of GSK-3beta that has a complex effect on neurite elongation. It favors neurite elongation at a low level of inhibition whereas it

Li et al. 2010).

compared to the control.

an internal control.

phospho GSK-3beta (Ser 9) antibody (11002-1; Signalway Antibody), rabbit anti-CRMP-2 antibody (SC-30228, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit antiphospho CRMP-2 (Thr 514) antibody (9397, Cell Signaling Technology, Beverly, MA, USA), goat anti-Akt antibody (SC-1618; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-phospho-Akt (Ser 473) antibody (SC-101629; Santa Cruz Biotechnology, Santa Cruz, CA, USA). All of these primary antibodies were polyclonal and used at a dilution of 1:500. Mouse anti-beta actin polyclonal antibody (SC-81178, 1:1000, Santa Cruz, CA, USA) was used at a dilution of 1:1 000. As secondary antibodies, HRP-conjugated goat anti-rabbit (SC-2004; Santa Cruz Biotechnology, Santa Cruz, CA, USA), donkey antigoat (SC-2020; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-mouse (SC-2005; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a dilution of 1:10 000.

#### **Semi-quantitative RT-PCR analysis**

For RT-PCR analysis of GSK-3 gene expression, neurons were cultured in two 6-well plates. One of the plates was irradiated by LIPUS for 7 d (5 min/day; 10 mW/cm2); the other was the control group without LIPUS treatment. Cultured cells were harvested 2 h after the last irradiation. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the instruction manual, then resuspended in diethylpyrocarbonate (DEPC)-treated water. The extracted RNA was used to synthesize first strand cDNA with the PrimeScript™ RT-PCR Kit (Takara Biotechnology, Dalian, China) according to the kit's manual. Aliquots of synthesized cDNA were added to PCR mixtures containing sense and antisense primers (0.1 μmol/L each) for GSK-3beta, dNTP mixture (0.2 mmol/L of each dNTP), 1.5 mmol/L MgCl2, and rTaq DNA polymerase (1 unit) (Takara Biotechn ology, Dalian, China). The primers for GSK-3 were 5' -AGCCAGTGCAGCAGCCTTCAG C-3' for the sense strand and 5' -TCTCCTCGGACCA GCTGCT TTG-3' for the antisense strand. The primers for -actin were 5' -GAGCTACGAGCTGCC TGACG-3' for the sense strand and 5' - CCTAGAA GCATTTGC GGTGG-3' for the antisense strand. The PCR products were electrophoretically separated in a 2% agarose gel and then visualized and photographed with an imager (Alhpa-imagerTM 2200; Alpha Innotech Corporation, San Leandro, CA, USA).

#### **4.2.2 LIPUS enhances neurite outgrowth through the down-regulation of GSK-3 activity**

On the third day, there was no significant difference in protein levels between the control and LIPUS groups. However, on the seventh and tenth days after irradiation by LIPUS, the levels of p-Akt, GSK-3beta, p-GSK-3beta, and p-CRMP-2 were decreased in the LIPUS group compared to the controls. On the tenth day, a remarkable decrease of p-Akt, p-GSK-3, and p-CRMP-2 were observed while it appeared that GSK-3beta were slightly decreased.

During nerve regeneration, GSK-3 is locally inhibited by some factors at the growth cone through the PI3-kinase/Akt/GSK-3 signaling pathway which favors neurite outgrowth (Chen, Yu et al. 2007). The overexpression of active GSK-3beta blocks neurite growth in cultured neurons (Munoz-Montano, Lim et al. 1999). In the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway, active Akt inhibits GSK-3beta through phosphorylation at Ser 9 and GSK-3beta inhibits CRMP-2 though phosphorylation at Thr 5. If LIPUS enhances

phospho GSK-3beta (Ser 9) antibody (11002-1; Signalway Antibody), rabbit anti-CRMP-2 antibody (SC-30228, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit antiphospho CRMP-2 (Thr 514) antibody (9397, Cell Signaling Technology, Beverly, MA, USA), goat anti-Akt antibody (SC-1618; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-phospho-Akt (Ser 473) antibody (SC-101629; Santa Cruz Biotechnology, Santa Cruz, CA, USA). All of these primary antibodies were polyclonal and used at a dilution of 1:500. Mouse anti-beta actin polyclonal antibody (SC-81178, 1:1000, Santa Cruz, CA, USA) was used at a dilution of 1:1 000. As secondary antibodies, HRP-conjugated goat anti-rabbit (SC-2004; Santa Cruz Biotechnology, Santa Cruz, CA, USA), donkey antigoat (SC-2020; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-mouse (SC-2005; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a dilution of

For RT-PCR analysis of GSK-3 gene expression, neurons were cultured in two 6-well plates. One of the plates was irradiated by LIPUS for 7 d (5 min/day; 10 mW/cm2); the other was the control group without LIPUS treatment. Cultured cells were harvested 2 h after the last irradiation. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the instruction manual, then resuspended in diethylpyrocarbonate (DEPC)-treated water. The extracted RNA was used to synthesize first strand cDNA with the PrimeScript™ RT-PCR Kit (Takara Biotechnology, Dalian, China) according to the kit's manual. Aliquots of synthesized cDNA were added to PCR mixtures containing sense and antisense primers (0.1 μmol/L each) for GSK-3beta, dNTP mixture (0.2 mmol/L of each dNTP), 1.5 mmol/L MgCl2, and rTaq DNA polymerase (1 unit) (Takara Biotechn ology, Dalian, China). The primers for GSK-3 were 5' -AGCCAGTGCAGCAGCCTTCAG C-3' for the sense strand and 5' -TCTCCTCGGACCA GCTGCT TTG-3' for the antisense strand. The primers for -actin were 5' -GAGCTACGAGCTGCC TGACG-3' for the sense strand and 5' - CCTAGAA GCATTTGC GGTGG-3' for the antisense strand. The PCR products were electrophoretically separated in a 2% agarose gel and then visualized and photographed with an imager (Alhpa-imagerTM 2200; Alpha Innotech Corporation, San Leandro, CA,

**4.2.2 LIPUS enhances neurite outgrowth through the down-regulation of** 

On the third day, there was no significant difference in protein levels between the control and LIPUS groups. However, on the seventh and tenth days after irradiation by LIPUS, the levels of p-Akt, GSK-3beta, p-GSK-3beta, and p-CRMP-2 were decreased in the LIPUS group compared to the controls. On the tenth day, a remarkable decrease of p-Akt, p-GSK-3, and

During nerve regeneration, GSK-3 is locally inhibited by some factors at the growth cone through the PI3-kinase/Akt/GSK-3 signaling pathway which favors neurite outgrowth (Chen, Yu et al. 2007). The overexpression of active GSK-3beta blocks neurite growth in cultured neurons (Munoz-Montano, Lim et al. 1999). In the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway, active Akt inhibits GSK-3beta through phosphorylation at Ser 9 and GSK-3beta inhibits CRMP-2 though phosphorylation at Thr 5. If LIPUS enhances

p-CRMP-2 were observed while it appeared that GSK-3beta were slightly decreased.

1:10 000.

USA).

**GSK-3 activity** 

**Semi-quantitative RT-PCR analysis** 

neurite elongation though this pathway, the phosphorylation of GSK-3beta should be upregulated and the activity of GSK-3beta should be inhibited. However, in this research, the activity of GSK-3beta was inhibited by LIPUS and the phosphorylation of GSK-3beta by Akt was inhibited, too. This conflict of results revealed that LIPUS enhances neurite outgrowth through the down-regulation of GSK-3 activity but not through the PI3-kinase/Akt/GSK-3beta pathway. Therefore, we employed semi-quantitative RT-PCR to examine the mRNA of GSK-3beta. The results of the semi-quantitative RT-PCR revealed that the expression of GSK-3beta mRNA decreased after LIPUS irradiation on the seventh day. From these findings, we postulate that when neurons are irradiated by LIPUS, an unknown intracellular mechanism may be activated as a response to this "injury" and, consequently, neurons reduce the mRNA expression of GSK-3. The decrease of GSK-3beta activity comes from reduced expression, but not through the PI3-kinase/Akt/GSK-3 signaling pathway (Ren, Li et al. 2010).

#### **4.2.3 The expression of GSK-3 mRNA decreased after LIPUS irradiation**

The mRNA expression of GSK-3beta in the cultured neurons following LIPUS treatment was examined using a semi-quantitative RT-PCR. For this analysis, the LIPUS-treated cultured neurons on the seventh day were selected as they showed a significant decrease in their mRNA levels compared to the control. Data from analysis of the imager indicated mRNA of GSK-3 decreased about 4 folds [(1.001 ±0.017) vs. (0.627 ±0.037), P<0.001] (Fig. 7). As a result, mRNA expression of GSK-3beta was also decreased on the seventh days compared to the control.

Fig. 7. Expression of GSK-3 in neurons was evaluated by semi quantitative RT-PCR. Neurons were irradiated for 7 d and harvested 2 h after the last LIPUS irradiation. The right lane represents the experimental mRNA expression, and the left lane corresponds to the control mRNA expression (Fig. 7a). Data showed that expression of GSK-3 decreased about 4 folds [(1.001 ±0.017) vs. (0.627 ±0.037), P<0.001] (Fig.7b). The -actin in each lane served as an internal control.

The reduced expression is a kind of global inhibition of GSK-3beta that has a complex effect on neurite elongation. It favors neurite elongation at a low level of inhibition whereas it

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 49

(LV) vectors encoding a functional NT-3 molecule led to the presence of a significantly increased number of axons in the contusion site (Golden, Pearse et al. 2007). The results of Yamauchi(Yamauchi, Miyamoto et al. 2005) et al showed that NT-3 activation of TrkC stimulates Schwann cell migration through two parallel signaling units, Ras/Tiam1/Rac1 and Dbs/Cdc. Poduslo(Poduslo and Curran 1996) et al observed that NT-3 has a higher permeability coefficient across the blood–nerve barrier, and would contact sensory axons soon after reaching the circulation of adult rats. The increase in NT-3 expression might lead to an increase in the number of nerve regeneration in the axons. LIPUS-induced increase in NT-3 expression, as demonstrated in this model, may produce a microenvironment that is permissive for axonal sprouting and Schwann cells migration after peripheral nerve injury. Neurotrophin–neurotrophin interactions are regulated by neurotrophin levels, NT-3 and BDNF in particular can be co-expressed and each can regulate the levels of the other. The relative expression level of the neurotrophins is thought to be mediated through receptor tyrosine kinase (Trk) activity (Mallei, Rabin et al. 2004). NT-3 infusion caused a significant decrease in the level of BDNF proteins in both kindled and non-kindled hippocampus, likely via down-regulation of TrkA (Yamamoto and Hanamura 2005) Furthermore, a study of Karchewski (Karchewski, Gratto et al. 2002) et al showed that NT-3 can act in an antagonistic fashion to NGF in the regulation of BDNF expression in intact neurons, and mitigate BDNF's expression in injured neurons. It is also consistent with a study in which, in contrast, deletion of the NT-3 gene in transgenic mice increased BDNF and TrkB mRNA synthesis, suggesting that decreased NT-3 may disinhibit BDNF expression (Elmer, Kokaia et al. 1997). Similarly, our model has demonstrated an up-regulation of NT-3 mRNA and down-regulation of BDNF mRNA expression after the LIPUS stimulation. Hence, it is possible that NT-3 acts in an opposite fashion result in a down-regulation in BDNF expression in intact Schwann cells. Further investigation is necessary to determine the molecular mechanisms of NT-3 and BDNF signaling pathway by the data presented in our

study (Zhang, Lin et al. 2009).

use.

**5.2 LIPUS enhances neurite elongation in rat cortical neurons** 

LIPUS enhances neurite elongation in rat cortical neurons, indicating that LIPUS could be a potential application for clinical treatment of nerve regeneration in both the central and peripheral nervous systems. The intracellular mechanism also indicates that LIPUS has the same action as the neurotrophic factors, laminin and LiCl. Compared to other pharmacological inhibitors of GSK-3beta, LIPUS has some advantages: (1) LIPUS is considered to be nontoxic, thus it has a wide margin of biologic safety; (2) It directly irradiates target neurons and does not affect other tissues; and (3) The decreased expression comes from a response of neurons and is not affected by the metabolism or blood brain barrier. However, further investigation is required to identify an accurate and continuous application of LIPUS treatment to achieve constant and reproducible results prior to clinical

The results suggest that LIPUS may have several different clinical applications in the improvement of peripheral nerve regeneration. First, given its nontoxicity and a wide margin of biologic safety, it may be used as an effective physical stimulant when engineering peripheral nerve tissue. Schwann cell-based therapies that use transplantation techniques for the treatment of nerve tissue repairing are being widely investigated for their

impairs neurite elongation at a high level of inhibition (Munoz-Montano, Lim et al. 1999). Strong global GSK-3beta inhibition results in excessive microtubule stability all along the neurite shaft due to the inhibition of MAP1B, which eliminates dynamic microtubules, and the abnormal distribution of APC that stabilizes microtubules all along the neurite shaft. In this case, there was no pool of dynamic microtubules at the growth cone, which are necessary for growth cone advancement, and no localization of APC to microtubule plus ends (Kim, Zhou et al. 2006).

In our research, significant morphological changes were found on the third day whereas significant changes in the activity of GSK-3beta were found on the seventh and tenth days. We postulate that daily treatment of LIPUS would result in neurons' response accumulation. Morphological changes were observed on the third day when the inhibition of GSK-3beta is not significant enough to be found. Since overly strong global inhibition of GSK-3beta impairs neurite elongation, whether LIPUS could impair neurite elongation needs further study.

#### **5. Conclusion**

#### **5.1 LIPUS stimulation induced an alteration in Schwann cell function as demonstrated by promoted cell proliferation and NT-3 gene expression**

LIPUS is one of the physical agents that is known to accelerate bone and tissue regeneration following injury (Heckman, Ryaby et al. 1994; Lu, Qin et al. 2006). Consequently, it has been accepted as an effective therapy for nonunion fractures and fresh fracture healing through an easy and non-invasive application (Azuma, Ito et al. 2001; Schortinghuis, Bronckers et al. 2005). Previous studies indicate that LIPUS has positive effects on axonal regeneration by in vivo peripheral nerve injury trials (Crisci and Ferreira 2002; Chang, Hsu et al. 2005). Raso (Raso, Barbieri et al. 2005) et al have demonstrated that the locally applied ultrasound stimuli on the injured sciatic nerve rather than the untreated nerves of rats can effectively enhance the number of Schwann cell nuclei. LIPUS has been used in conjunction with tissue engineered nerves in repairing peripheral nerve defect, Chang (Chang, Hsu et al. 2005) et al demonstrated that applying low-intensity ultrasound on seeded Schwann cells within poly (D, L-lactic acid-co-glycolic acid) conduits have a significantly greater number and area of regenerated axons compared to the sham groups. Although this secondary response by Schwann cells has been well characterized, there is still limited information as to how Schwann cells would directly respond to LIPUS stimulation. Therefore, we cultured Schwann cells in plate as an in vitro model, and applied LIPUS in the model to demonstrate the direct effects of physical stimulation on Schwann cells (Zhang, Lin et al. 2009).

LIPUS stimulation of cultured Schwann cells induced an alteration in cell function as demonstrated by promoted cell proliferation and NT-3 gene expression, which is consistent with that LIPUS enhances peripheral nerve regeneration that was observed from in vivo models (Lowdon, Seaber et al. 1988; Raso, Barbieri et al. 2005). It has been documented that NT-3 has a strong effect on neurite outgrowth (Markus, Patel et al. 2002; Sahenk, Nagaraja et al. 2005). Additionally, some studies using genetically modified Schwann cells to overexpress the NT-3 gene have examined the role of NT-3 in the neuron survival and axonal regeneration/remyelination (Zhang, Zeng et al. 2007; Pettingill, Minter et al. 2008). It has been reported that Schwann cells transduced ex vivo with adenoviral (AdV) or lentiviral

impairs neurite elongation at a high level of inhibition (Munoz-Montano, Lim et al. 1999). Strong global GSK-3beta inhibition results in excessive microtubule stability all along the neurite shaft due to the inhibition of MAP1B, which eliminates dynamic microtubules, and the abnormal distribution of APC that stabilizes microtubules all along the neurite shaft. In this case, there was no pool of dynamic microtubules at the growth cone, which are necessary for growth cone advancement, and no localization of APC to microtubule plus

In our research, significant morphological changes were found on the third day whereas significant changes in the activity of GSK-3beta were found on the seventh and tenth days. We postulate that daily treatment of LIPUS would result in neurons' response accumulation. Morphological changes were observed on the third day when the inhibition of GSK-3beta is not significant enough to be found. Since overly strong global inhibition of GSK-3beta impairs neurite elongation, whether LIPUS could impair neurite elongation needs further

**5.1 LIPUS stimulation induced an alteration in Schwann cell function as demonstrated** 

LIPUS is one of the physical agents that is known to accelerate bone and tissue regeneration following injury (Heckman, Ryaby et al. 1994; Lu, Qin et al. 2006). Consequently, it has been accepted as an effective therapy for nonunion fractures and fresh fracture healing through an easy and non-invasive application (Azuma, Ito et al. 2001; Schortinghuis, Bronckers et al. 2005). Previous studies indicate that LIPUS has positive effects on axonal regeneration by in vivo peripheral nerve injury trials (Crisci and Ferreira 2002; Chang, Hsu et al. 2005). Raso (Raso, Barbieri et al. 2005) et al have demonstrated that the locally applied ultrasound stimuli on the injured sciatic nerve rather than the untreated nerves of rats can effectively enhance the number of Schwann cell nuclei. LIPUS has been used in conjunction with tissue engineered nerves in repairing peripheral nerve defect, Chang (Chang, Hsu et al. 2005) et al demonstrated that applying low-intensity ultrasound on seeded Schwann cells within poly (D, L-lactic acid-co-glycolic acid) conduits have a significantly greater number and area of regenerated axons compared to the sham groups. Although this secondary response by Schwann cells has been well characterized, there is still limited information as to how Schwann cells would directly respond to LIPUS stimulation. Therefore, we cultured Schwann cells in plate as an in vitro model, and applied LIPUS in the model to demonstrate

the direct effects of physical stimulation on Schwann cells (Zhang, Lin et al. 2009).

LIPUS stimulation of cultured Schwann cells induced an alteration in cell function as demonstrated by promoted cell proliferation and NT-3 gene expression, which is consistent with that LIPUS enhances peripheral nerve regeneration that was observed from in vivo models (Lowdon, Seaber et al. 1988; Raso, Barbieri et al. 2005). It has been documented that NT-3 has a strong effect on neurite outgrowth (Markus, Patel et al. 2002; Sahenk, Nagaraja et al. 2005). Additionally, some studies using genetically modified Schwann cells to overexpress the NT-3 gene have examined the role of NT-3 in the neuron survival and axonal regeneration/remyelination (Zhang, Zeng et al. 2007; Pettingill, Minter et al. 2008). It has been reported that Schwann cells transduced ex vivo with adenoviral (AdV) or lentiviral

**by promoted cell proliferation and NT-3 gene expression** 

ends (Kim, Zhou et al. 2006).

study.

**5. Conclusion** 

(LV) vectors encoding a functional NT-3 molecule led to the presence of a significantly increased number of axons in the contusion site (Golden, Pearse et al. 2007). The results of Yamauchi(Yamauchi, Miyamoto et al. 2005) et al showed that NT-3 activation of TrkC stimulates Schwann cell migration through two parallel signaling units, Ras/Tiam1/Rac1 and Dbs/Cdc. Poduslo(Poduslo and Curran 1996) et al observed that NT-3 has a higher permeability coefficient across the blood–nerve barrier, and would contact sensory axons soon after reaching the circulation of adult rats. The increase in NT-3 expression might lead to an increase in the number of nerve regeneration in the axons. LIPUS-induced increase in NT-3 expression, as demonstrated in this model, may produce a microenvironment that is permissive for axonal sprouting and Schwann cells migration after peripheral nerve injury.

Neurotrophin–neurotrophin interactions are regulated by neurotrophin levels, NT-3 and BDNF in particular can be co-expressed and each can regulate the levels of the other. The relative expression level of the neurotrophins is thought to be mediated through receptor tyrosine kinase (Trk) activity (Mallei, Rabin et al. 2004). NT-3 infusion caused a significant decrease in the level of BDNF proteins in both kindled and non-kindled hippocampus, likely via down-regulation of TrkA (Yamamoto and Hanamura 2005) Furthermore, a study of Karchewski (Karchewski, Gratto et al. 2002) et al showed that NT-3 can act in an antagonistic fashion to NGF in the regulation of BDNF expression in intact neurons, and mitigate BDNF's expression in injured neurons. It is also consistent with a study in which, in contrast, deletion of the NT-3 gene in transgenic mice increased BDNF and TrkB mRNA synthesis, suggesting that decreased NT-3 may disinhibit BDNF expression (Elmer, Kokaia et al. 1997). Similarly, our model has demonstrated an up-regulation of NT-3 mRNA and down-regulation of BDNF mRNA expression after the LIPUS stimulation. Hence, it is possible that NT-3 acts in an opposite fashion result in a down-regulation in BDNF expression in intact Schwann cells. Further investigation is necessary to determine the molecular mechanisms of NT-3 and BDNF signaling pathway by the data presented in our study (Zhang, Lin et al. 2009).

#### **5.2 LIPUS enhances neurite elongation in rat cortical neurons**

LIPUS enhances neurite elongation in rat cortical neurons, indicating that LIPUS could be a potential application for clinical treatment of nerve regeneration in both the central and peripheral nervous systems. The intracellular mechanism also indicates that LIPUS has the same action as the neurotrophic factors, laminin and LiCl. Compared to other pharmacological inhibitors of GSK-3beta, LIPUS has some advantages: (1) LIPUS is considered to be nontoxic, thus it has a wide margin of biologic safety; (2) It directly irradiates target neurons and does not affect other tissues; and (3) The decreased expression comes from a response of neurons and is not affected by the metabolism or blood brain barrier. However, further investigation is required to identify an accurate and continuous application of LIPUS treatment to achieve constant and reproducible results prior to clinical use.

The results suggest that LIPUS may have several different clinical applications in the improvement of peripheral nerve regeneration. First, given its nontoxicity and a wide margin of biologic safety, it may be used as an effective physical stimulant when engineering peripheral nerve tissue. Schwann cell-based therapies that use transplantation techniques for the treatment of nerve tissue repairing are being widely investigated for their

Effect of Low-Intensity Pulsed Ultrasound on Nerve Repair 51

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potential as clinical applications (Rochkind, Astachov et al. 2004; Li, Ping et al. 2006; Gravvanis, Lavdas et al. 2007). LIPUS applied in conjunction with other forms of biologic stimulation, is worth considering when optimizing an innovative "multi-level" form of treatment. Second, application of LIPUS in vivo is likely to be considered to stimulate repair of damaged peripheral nerve tissues. Some experimental studies supported the result that both end-to-side and tubulization repair of peripheral nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function (Geuna, Nicolino et al. 2007; Lloyd, Luginbuhl et al. 2007). With the availability of the LIPUS as an activator of Schwann cells, it can be an effective alternative in nerve reconstruction and be of great value in various kinds of peripheral nerve microsurgery. Further investigation is required to identify an accurate and continuous application of LIPUS treatment, in order to achieve constant and reproducible results prior to clinical use.

As demonstrated in the current study, NT-3 and BDNF mRNA expression in Schwann cell response to LIPUS may be independent of the reciprocal regulation between the glial cells and neurons. Normally, during development and axonal injury, this reciprocal relationship between the glial cells and neurons causes a response in the glial cells, which occurs secondary to the neuron. However, data from the in vitro model indicate otherwise. The Schwann cells responded robustly in the absence of neurons, suggesting that Schwann cell responses may be directly elicited through LIPUS stimuli in the model.

#### **6. Acknowledgment**

This research was funded by The Natural Science Foundation of Beijing (Grant number: 5072020), China.

#### **7. References**


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**3** 

*Germany* 

**Disinfection of Human Tissues in** 

*2Department of Orthopedic Surgery, Technische Universitaet Muenchen* 

Liquids show significant temperature-dependent compressibility under high hydrostatic pressure (HHP). For instance, the specific volume of water at atmospheric pressure decreases by 12% when exposed to 400 MPa (1). Self-ionization of water is also promoted by HHP lowering the pH phase transition of water is triggered under excessive HHP; at 1,000 MPa water freezes at room temperature, whereas at 207.5 MPa the freezing point can be lowered to –22°C (1). This allows for pressure shift freezing of foods with instant and small ice crystal formation, storage of food at subzero temperatures without ice formation, or fast thawing of frozen food by pressurization, allowing gentle processing of foods or food

HHP may also cause alterations in biological molecules, associated with a change in their conformation towards of forms which occupy smaller volumes. With increasing pressure, the non-covalent bonds of macromolecules such as proteins are affected leading to changes in their quaternary, tertiary or secondary structure. HHP is presumed to influence the conformational state of lipids as well (2,3) whereas nucleic acids have proved pressureresistant because their secondary structure is mainly stabilized by H-bonds that are almost pressure insensitive (4;5). HHP-induced changes can be reversible, metastable or irreversible, partly depending on the pressure level itself, but also depending on the duration of the pressure treatment, on the temperature during treatment, on the chemical

The growth of eukaryotic and prokaryotic cells can be prevented to a large degree by a number of preservation techniques, most of which act by killing the cells or by slowing down cellular growth. Concerning food products, heating, freezing, drying, vacuum packing, acidifying or the addition of preservatives are the predominant method. At present, however, major trends have emerged towards the use of procedures such as HHP to deliver food products that are less 'heavily' preserved but still with high assurance of no microbiological contamination (6,7). HHP as a means of preserving food, without the

**1. Introduction** 

constituents with minimal structural damage.

conditions and on other conditions of the surroundings.

**Orthopedic Surgical Oncology** 

**by High Hydrostatic Pressure** 

Hans Gollwitzer2 and Wolfram Mittelmeier1 *1Department of Orthopedic Surgery, University of Rostock* 

Peter Diehl1, Johannes Schauwecker2,


## **Disinfection of Human Tissues in Orthopedic Surgical Oncology by High Hydrostatic Pressure**

Peter Diehl1, Johannes Schauwecker2, Hans Gollwitzer2 and Wolfram Mittelmeier1 *1Department of Orthopedic Surgery, University of Rostock 2Department of Orthopedic Surgery, Technische Universitaet Muenchen Germany* 

#### **1. Introduction**

54 Tissue Regeneration – From Basic Biology to Clinical Application

Yamauchi, J., Y. Miyamoto, et al. (2005). "Ras activation of a Rac1 exchange factor, Tiam1,

Zhang, H., X. Lin, et al. (2009). "Effect of low-intensity pulsed ultrasound on the expression

Zhang, J. Y., X. G. Luo, et al. (2000). "Endogenous BDNF is required for myelination and

Zhang, X., Y. Zeng, et al. (2007). "Co-transplantation of neural stem cells and NT-3-

Zhou, S., A. Schmelz, et al. (2004). "Molecular mechanisms of low intensity pulsed

and neuronal polarity." *Cell* 120(1): 137-149.

cells." *Microsurgery* 29(6): 479-485.

12(12): 4171-4180.

24(12): 1863-1877.

54463-54469.

mediates neurotrophin-3-induced Schwann cell migration." *Proceedings of the National Academy of Sciences of the United States of America* 102(41): 14889-14894. Yoshimura, T., Y. Kawano, et al. (2005). "GSK-3beta regulates phosphorylation of CRMP-2

of neurotrophin-3 and brain-derived neurotrophic factor in cultured Schwann

regeneration of injured sciatic nerve in rodents." *The European journal of neuroscience*

overexpressing Schwann cells in transected spinal cord." *Journal of neurotrauma*

ultrasound in human skin fibroblasts." *The Journal of biological chemistry* 279(52):

Liquids show significant temperature-dependent compressibility under high hydrostatic pressure (HHP). For instance, the specific volume of water at atmospheric pressure decreases by 12% when exposed to 400 MPa (1). Self-ionization of water is also promoted by HHP lowering the pH phase transition of water is triggered under excessive HHP; at 1,000 MPa water freezes at room temperature, whereas at 207.5 MPa the freezing point can be lowered to –22°C (1). This allows for pressure shift freezing of foods with instant and small ice crystal formation, storage of food at subzero temperatures without ice formation, or fast thawing of frozen food by pressurization, allowing gentle processing of foods or food constituents with minimal structural damage.

HHP may also cause alterations in biological molecules, associated with a change in their conformation towards of forms which occupy smaller volumes. With increasing pressure, the non-covalent bonds of macromolecules such as proteins are affected leading to changes in their quaternary, tertiary or secondary structure. HHP is presumed to influence the conformational state of lipids as well (2,3) whereas nucleic acids have proved pressureresistant because their secondary structure is mainly stabilized by H-bonds that are almost pressure insensitive (4;5). HHP-induced changes can be reversible, metastable or irreversible, partly depending on the pressure level itself, but also depending on the duration of the pressure treatment, on the temperature during treatment, on the chemical conditions and on other conditions of the surroundings.

The growth of eukaryotic and prokaryotic cells can be prevented to a large degree by a number of preservation techniques, most of which act by killing the cells or by slowing down cellular growth. Concerning food products, heating, freezing, drying, vacuum packing, acidifying or the addition of preservatives are the predominant method. At present, however, major trends have emerged towards the use of procedures such as HHP to deliver food products that are less 'heavily' preserved but still with high assurance of no microbiological contamination (6,7). HHP as a means of preserving food, without the

Disinfection of Human Tissues in Orthopedic Surgical Oncology by High Hydrostatic Pressure 57

pressure medium. In case of smaller specimens, instead of plastic bags, tissue specimens are placed into 15 ml flexible Falcon tubes (Becton-Dickinson, Heidelberg, Germany) (Figure 3a) or 2 ml Nalgene cryogenic vials (Thermo Fisher Scientific, Wiesbaden, Germany) (Figure 3b). The vials filled with Ringer buffer are carefully capped avoiding air bubbles and then sealed tightly with parafilm (American National Can GmbH,

Fig. 1a. High hydrostatic pressure device (Record Maschinenbau, Koenigsee, Germany).

The bags/vials are placed into the central cavity of a water-filled pressure chamber (100 ml) of a custom-made HHP device (Figure 1b). The water is mixed 1:1 with ethylene-glycol to suppress corrosion of the pressure chamber. The temperature of the pressure chamber can

Gelsenkirchen, Germany).

addition of any kind of preservative, has attracted increasing attention (4,6,8), since it has the advantage of leaving covalent molecular bonds intact without impairing flavors, aromas, vitamins and other pharmacologically active molecules (9).

In the medical field, HHP technology is now in preclinical testing with the aim of inactivating both pathological microorganisms and tumor cells in resected tissue segments, such as bone, cartilage and tendon *ex vivo* (10-15). This is a promising clinically relevant approach, especially with respect to rapid killing of tumor cells in bone and the subsequent possibility of re-implantation of the once tumor-bearing bone segment back into the patient.

### **2. HHP and orthopaedic surgery**

In orthopedic surgery, restoration of bone defects caused by malignant solid tumors is achieved by several methods of treatment such as extracorporeal irradiation or autoclaving the affected bone segment, as an alternative approach to synthetic limb reconstruction (16- 19). In contrast, irradiation or autoclaving of osteochondral segments or tendons may lead to severe alteration of their biomechanical and biological properties, a major concern regarding this type of approach (20-22).

A new technology, the administration of short-term HHP to the resected bone segment immediately after surgery, now offers an alternative to the conventional ways of treating tumor-affected bone. At the high pressure value of 600 MPa applied, the biomechanical properties of bones, tendons and cartilage remain unchanged (10-14). Under these conditions, normal eukaryotic cells, and also malignant cells are irreversibly damaged and outgrowth of cells from tumor-afflicted bone and cartilage segments is efficiently blocked (14,15,23).

With regard to the biological properties of treated bone, cartilage or tendon, no obvious changes in the adhesive or growth promoting properties of the extracellular matrix proteins after HHP treatment of the bone were observed (11), and successful revitalization of HHPtreated bone segments *in vitro* was observed. Also, no enhanced activity of proteases, which might be released after HHP treatment of resected human bone tumor and provoke autolytic bone resorption, could be detected (24). This report reviews the basics and technical potential of HHP in orthopaedic surgery and sheds light on the prospects of HHP for the treatment of neoplastic bone and infected bone tissue, cartilage and tendon.

#### **3. HHP-device and treatment of affected bone, cartilage, or tendon**

The HHP system (Record Maschinenbau, Koenigsee, Germany) consists of a high pressure autoclave, a pressure generation unit, a temperature and pressure control unit and a material handling unit (Figure 1a). HHP treatment of the tissue samples is accomplished by a pressure-transferring medium, usually water, thus allowing uniform and instantaneous transmission of pressure to the biological sample. To treat infected or tumor-afflicted bone, tendon or cartilage, larger specimens are placed into polyethylene bags and sealed by vacuum-packaging (Komet, Plochingen, Germany) (Figure 2). Sealing in buffer is required to assure uniform and instantaneous pressure transmission throughout the biological sample and to prevent contamination while in contact with the

addition of any kind of preservative, has attracted increasing attention (4,6,8), since it has the advantage of leaving covalent molecular bonds intact without impairing flavors, aromas,

In the medical field, HHP technology is now in preclinical testing with the aim of inactivating both pathological microorganisms and tumor cells in resected tissue segments, such as bone, cartilage and tendon *ex vivo* (10-15). This is a promising clinically relevant approach, especially with respect to rapid killing of tumor cells in bone and the subsequent possibility of re-implantation of the once tumor-bearing bone segment back

In orthopedic surgery, restoration of bone defects caused by malignant solid tumors is achieved by several methods of treatment such as extracorporeal irradiation or autoclaving the affected bone segment, as an alternative approach to synthetic limb reconstruction (16- 19). In contrast, irradiation or autoclaving of osteochondral segments or tendons may lead to severe alteration of their biomechanical and biological properties, a major concern regarding

A new technology, the administration of short-term HHP to the resected bone segment immediately after surgery, now offers an alternative to the conventional ways of treating tumor-affected bone. At the high pressure value of 600 MPa applied, the biomechanical properties of bones, tendons and cartilage remain unchanged (10-14). Under these conditions, normal eukaryotic cells, and also malignant cells are irreversibly damaged and outgrowth of cells from tumor-afflicted bone and cartilage segments is efficiently blocked

With regard to the biological properties of treated bone, cartilage or tendon, no obvious changes in the adhesive or growth promoting properties of the extracellular matrix proteins after HHP treatment of the bone were observed (11), and successful revitalization of HHPtreated bone segments *in vitro* was observed. Also, no enhanced activity of proteases, which might be released after HHP treatment of resected human bone tumor and provoke autolytic bone resorption, could be detected (24). This report reviews the basics and technical potential of HHP in orthopaedic surgery and sheds light on the prospects of HHP

The HHP system (Record Maschinenbau, Koenigsee, Germany) consists of a high pressure autoclave, a pressure generation unit, a temperature and pressure control unit and a material handling unit (Figure 1a). HHP treatment of the tissue samples is accomplished by a pressure-transferring medium, usually water, thus allowing uniform and instantaneous transmission of pressure to the biological sample. To treat infected or tumor-afflicted bone, tendon or cartilage, larger specimens are placed into polyethylene bags and sealed by vacuum-packaging (Komet, Plochingen, Germany) (Figure 2). Sealing in buffer is required to assure uniform and instantaneous pressure transmission throughout the biological sample and to prevent contamination while in contact with the

for the treatment of neoplastic bone and infected bone tissue, cartilage and tendon.

**3. HHP-device and treatment of affected bone, cartilage, or tendon** 

vitamins and other pharmacologically active molecules (9).

into the patient.

(14,15,23).

**2. HHP and orthopaedic surgery** 

this type of approach (20-22).

pressure medium. In case of smaller specimens, instead of plastic bags, tissue specimens are placed into 15 ml flexible Falcon tubes (Becton-Dickinson, Heidelberg, Germany) (Figure 3a) or 2 ml Nalgene cryogenic vials (Thermo Fisher Scientific, Wiesbaden, Germany) (Figure 3b). The vials filled with Ringer buffer are carefully capped avoiding air bubbles and then sealed tightly with parafilm (American National Can GmbH, Gelsenkirchen, Germany).

Fig. 1a. High hydrostatic pressure device (Record Maschinenbau, Koenigsee, Germany).

The bags/vials are placed into the central cavity of a water-filled pressure chamber (100 ml) of a custom-made HHP device (Figure 1b). The water is mixed 1:1 with ethylene-glycol to suppress corrosion of the pressure chamber. The temperature of the pressure chamber can

Disinfection of Human Tissues in Orthopedic Surgical Oncology by High Hydrostatic Pressure 59

Fig. 2. Vacuum sealing device (Komet, Plochingen, Germany).

macromolecules irreversibly (25-27).

Specimens in polyethylene bags are placed into the vacuum chamber, positioning the open section onto the sealing bar. Once a vacuum is formed a heated wire on the bar seals the plastic bag. On the control board vacuum and sealing settings can be adjusted individually.

Exposure of tendons and ligaments to HHP (300 and 600 MPa; 10 min, 20 °C) did not significantly change their biomechanical features, their Young`s modulus or tensile strength, indicating retention of functional properties after HHP-sterilization (12). Retention of biomechanical properties of tissues after HHP is mainly based on the fact that HHP does not affect covalent molecular bonds, leaving parts of the molecule unchanged whereas exposure to chemicals or high temperature often unfold

Fig. 1b. The core of the autoclave chamber is made of an amagnetic stainless steel into which a large cylindrical hole has been bored in order to receive the specimen. A metal-on-metal sealing system provides an excellent leak-free closure with minimal mechanical wear. The pressure is transmitted by means of a hydraulic ram and monitored by a pressure gauge. Incubation temperature is monitored by a thermocouple and thermostatic control is ensured by the circulation of water through rubber tubing. On the control panel pressure settings can be adjusted up to 600 MPa).

be adjusted from 0 – 50 °C by a temperature control unit (Thermo Fisher Scientific, Karlsruhe, Germany) (Figure 4). The temperature should be kept constant at any given level since adiabatic compression of water increases the temperature 3 °C per 100 MPa. Pressure levels up to 600 MPa are adjusted manually with a compression / decompression rate of 100-300 MPa/min. The tissue specimens are held under pressure for a defined length of time (plateau phase), then, within a few seconds, pressure is returned to normal.

Fig. 1b. The core of the autoclave chamber is made of an amagnetic stainless steel into which a large cylindrical hole has been bored in order to receive the specimen. A metal-on-metal sealing system provides an excellent leak-free closure with minimal mechanical wear. The pressure is transmitted by means of a hydraulic ram and monitored by a pressure gauge. Incubation temperature is monitored by a thermocouple and thermostatic control is ensured by the circulation of water through rubber tubing. On the control panel pressure settings can

be adjusted from 0 – 50 °C by a temperature control unit (Thermo Fisher Scientific, Karlsruhe, Germany) (Figure 4). The temperature should be kept constant at any given level since adiabatic compression of water increases the temperature 3 °C per 100 MPa. Pressure levels up to 600 MPa are adjusted manually with a compression / decompression rate of 100-300 MPa/min. The tissue specimens are held under pressure for a defined length of

time (plateau phase), then, within a few seconds, pressure is returned to normal.

be adjusted up to 600 MPa).

Fig. 2. Vacuum sealing device (Komet, Plochingen, Germany).

Specimens in polyethylene bags are placed into the vacuum chamber, positioning the open section onto the sealing bar. Once a vacuum is formed a heated wire on the bar seals the plastic bag. On the control board vacuum and sealing settings can be adjusted individually.

Exposure of tendons and ligaments to HHP (300 and 600 MPa; 10 min, 20 °C) did not significantly change their biomechanical features, their Young`s modulus or tensile strength, indicating retention of functional properties after HHP-sterilization (12). Retention of biomechanical properties of tissues after HHP is mainly based on the fact that HHP does not affect covalent molecular bonds, leaving parts of the molecule unchanged whereas exposure to chemicals or high temperature often unfold macromolecules irreversibly (25-27).

Disinfection of Human Tissues in Orthopedic Surgical Oncology by High Hydrostatic Pressure 61

Fig. 4. Temperature control unit (Thermo Fisher Scientific, Karlsruhe, Germany). Open bath circulator with digital settings, bath vessel and bath bridge made of stainless steel and temperature resistant polymer; electronic control using a digital sensor for

bacteria, yeasts, and moulds are generally sensitive to pressures of 600 MPa (7).

As well as tendons, we have also investigated the biomechanical properties of freshly resected human cortical and trabecular bone specimens or cartilage and menisci exposed to HHP as high as 600 MPa (10 min, 20 °C) (13,28). Under these conditions, no significant alterations relating to the stiffness and relaxation behavior of the osteochondral segments were observed. Unfortunately, inactivation of clinically important bacteria, for instance those present in osteomyelitis was not achieved under these conditions (29,30) although in foods vegetative

predetermined reference value, max. temperature 100 °C.

Fig. 3a. Tissue specimens are placed into flexible 15 ml BD Falcon™ conical tubes (Becton-Dickinson, Heidelberg, Germany) or Figure 2

Fig. 3b. 2 ml Nalgene cryogenic vials (Thermo Fisher Scientific, Wiesbaden, Germany) (3b), filled with Ringer buffer and tightly sealed (e. g. with parafilm, American National Can GmbH, Gelsenkirchen, Germany).

Fig. 3a. Tissue specimens are placed into flexible 15 ml BD Falcon™ conical tubes (Becton-

Fig. 3b. 2 ml Nalgene cryogenic vials (Thermo Fisher Scientific, Wiesbaden, Germany) (3b), filled with Ringer buffer and tightly sealed (e. g. with parafilm, American National Can

Dickinson, Heidelberg, Germany) or Figure 2

GmbH, Gelsenkirchen, Germany).

Fig. 4. Temperature control unit (Thermo Fisher Scientific, Karlsruhe, Germany). Open bath circulator with digital settings, bath vessel and bath bridge made of stainless steel and temperature resistant polymer; electronic control using a digital sensor for predetermined reference value, max. temperature 100 °C.

As well as tendons, we have also investigated the biomechanical properties of freshly resected human cortical and trabecular bone specimens or cartilage and menisci exposed to HHP as high as 600 MPa (10 min, 20 °C) (13,28). Under these conditions, no significant alterations relating to the stiffness and relaxation behavior of the osteochondral segments were observed. Unfortunately, inactivation of clinically important bacteria, for instance those present in osteomyelitis was not achieved under these conditions (29,30) although in foods vegetative bacteria, yeasts, and moulds are generally sensitive to pressures of 600 MPa (7).

Disinfection of Human Tissues in Orthopedic Surgical Oncology by High Hydrostatic Pressure 63

temperature) with respect to cell proliferation, spreading and adherence of human osteoblast-like cells and human osteosarcoma cells (Saos-2) (11). These data encourage further exploration of the potential of HHP to sterilize tumor-affected bone segments prior to re-implantation, since during such treatment eukaryotic bone cells including tumor cells would be irreversibly impaired, while the bone's biomechanical properties and the biological properties of the extracellular matrix proteins fibronectin, vitronectin, and

HHP causes a stress response in many types of mammalian cells, including chondrocytes and bone tumor cells (36). Further to this, Kopakkala-Tani *et al.* investigated whether some of the well known transduction pathways are activated in human chondrosarcoma cells under stress by exposure to moderate HHP of 15-30 MPa and demonstrated an increased level of active, phosphorylated forms of the extracellular signal-related kinase ERK and

HHP may not only exert an effect on tumor and normal cells present in the bone, but also on the tumor-associated proteases released by these cells, which are conductive to tumor bone turnover. At a pressure level of 600 MPa the latent activity of the inactive zymogens prothrombin, plasminogen, pro-uPA and trypsinogen, in addition to the proteolytically active forms thrombin, plasmin, HMW-uPA, and trypsin was minimally affected by HHP (24). The variation seen between different enzymes is probably due to differences in molecular structures and the resulting modifications after HHP treatment (24). It is worthwhile to note that at this pressure level normal bone cells and tumor cells are irreversibly impaired. Additionally, HHP also influences the activity of other enzymes. With that in mind, Masson *et al.* reviewed HHP technology and its potential applications in medicine and pharmaceutical science (9). The authors explained that HHP may affect both the activity and specificity of enzymes and that HHP is used for the engineering of proteins to allow enzyme-catalyzed synthesis of fine chemicals and pharmaceuticals and the production of modified proteins of medical or pharmaceutical interest. Such reactions can be used for food functionalization and for producing "nutraceuticals" to be used in complementary therapy (38). Pressure processing was found to be efficient in reducing the

In general, pressures above 300 MPa cause irreversible protein denaturation at room temperature, whereas lower pressures may result in reversible changes in protein structure. The effects of HHP on enzymes have been divided into two classes: moderate pressure values of 100–200 MPa which activate monomeric enzymes and elevated pressures usually inducing enzyme inactivation (1). Investigations of the impact of moderate HHP up to 200 MPa on alpha-amylase have shown a pressure-dependent stabilization of the enzyme against temperature-induced inactivation (3,40). Interestingly, for some proteases, proteolysis enhancement through HHP (up to 400 MPa) depended on substrate changes and not on changes of the enzyme, as investigated for chymotrypsin in the hydrolysis of beta-

So far, the effect of neoantigens generated during HHP-treatment of bone, cartilage and tendon on the host after re-implantation has not been elucidated and is at present subject to

**5. Effect of HHP-treatment on viability of microorganisms in bone** 

phosphoinositide 3-kinase under these pressure conditions (37).

collagen-I would be preserved (11).

allergenic activity of food (39).

lactoglobulin (41).


Table 1. The table presents different fields of application of Hygh Hydostatic Pressure. Save inactivation of bacteria in all kind of materials is not possible at the moment.

HHP has also been employed to investigate pressure-related *in vivo*-effects on chondrocytes since hydrostatic pressure is a significant component of the mechanical loading environment within articular cartilage. Chondrocytes within cartilage of diarthrotic joints experience hydrostatic pressure levels of 0.1-20 MPa (31). In *ex vivo* investigations therefore intermittent high pressure of 10 MPa was applied to investigate mechanisms mediating the response of chondrocytes to joint motion and loading (30,31). Under these conditions, a decreased release of matrix metalloproreinases (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and interleukin-6 by osteoarthritic chondrocytes was observed, suggesting that pressure influences cartilage stability *in vivo* (32).

We have observed that, regarding bone, exposure of normal cells (e.g. osteoblasts) and tumor cells (e.g. osteo-, chondro- and fibrosarcoma cells) to elevated hydrostatic pressure led to irreversibly damaged, non-viable cells, even after short-term exposure to 350 MPa (14,15,23). Under these conditions, eukaryotic cells experience irreversible destruction and permeabilization of cell membranes by HHP causing cell death (33).

Interestingly, suspended tumor cells were more resistant to HHP than adherent tumor cells, yet, normal bone and tissue cells such as fibroblasts and osteoblasts were less resistant to HHP than tumor cells (14,23). We also observed that at 300 MPa *ex vivo* outgrowth of normal or tumor cells from bone ceased concomitant with impairment of the bone-associated cells (15). This finding points to rapid killing of bone-associated tumor cells, potentially allowing re-implantation of the once tumor-bearing bone segment back into the patient.

Looking at other types of cells, Dibb *et al.* investigated the effects of HHP on normal and neoplastic rat cells in culture in the range 0.1 to 150 MPa (34). Morphological changes characterized by cell rounding were observed in secondary fetal brain cells and fibroblasts at about 70 MPa, whereas in the neoplastic neurogenic cell lines tested similar changes occurred at around 100 MPa, again demonstrating that malignant cells may be more resistant to HHP than their normal counterparts. Similar findings were reported by Yamaguchi *et al.* for Ehrlich ascites tumor cells demonstrating that these tumor cells stopped *in vivo* proliferation at HHP above 130 MPa (35).

#### **4. Effect of pressure on extracellular matrix proteins and enzymes**

Little is known on the change of biological functions of proteins or other constituents of bone, cartilage, or tendon after exposure to HHP. Our own studies have demonstrated that the extracellular matrix proteins fibronectin, vitronectin and collagen-I present in the bone matrix have not deteriorated after HHP-treatment up to 600 MPa (10 min, room

inactivation of different virus (e.g. HIV) in blood

Table 1. The table presents different fields of application of Hygh Hydostatic Pressure. Save

HHP has also been employed to investigate pressure-related *in vivo*-effects on chondrocytes since hydrostatic pressure is a significant component of the mechanical loading environment within articular cartilage. Chondrocytes within cartilage of diarthrotic joints experience hydrostatic pressure levels of 0.1-20 MPa (31). In *ex vivo* investigations therefore intermittent high pressure of 10 MPa was applied to investigate mechanisms mediating the response of chondrocytes to joint motion and loading (30,31). Under these conditions, a decreased release of matrix metalloproreinases (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and interleukin-6 by osteoarthritic chondrocytes was observed, suggesting that

We have observed that, regarding bone, exposure of normal cells (e.g. osteoblasts) and tumor cells (e.g. osteo-, chondro- and fibrosarcoma cells) to elevated hydrostatic pressure led to irreversibly damaged, non-viable cells, even after short-term exposure to 350 MPa (14,15,23). Under these conditions, eukaryotic cells experience irreversible destruction and

Interestingly, suspended tumor cells were more resistant to HHP than adherent tumor cells, yet, normal bone and tissue cells such as fibroblasts and osteoblasts were less resistant to HHP than tumor cells (14,23). We also observed that at 300 MPa *ex vivo* outgrowth of normal or tumor cells from bone ceased concomitant with impairment of the bone-associated cells (15). This finding points to rapid killing of bone-associated tumor cells, potentially allowing

Looking at other types of cells, Dibb *et al.* investigated the effects of HHP on normal and neoplastic rat cells in culture in the range 0.1 to 150 MPa (34). Morphological changes characterized by cell rounding were observed in secondary fetal brain cells and fibroblasts at about 70 MPa, whereas in the neoplastic neurogenic cell lines tested similar changes occurred at around 100 MPa, again demonstrating that malignant cells may be more resistant to HHP than their normal counterparts. Similar findings were reported by Yamaguchi *et al.* for Ehrlich ascites tumor cells demonstrating that these tumor cells stopped

Little is known on the change of biological functions of proteins or other constituents of bone, cartilage, or tendon after exposure to HHP. Our own studies have demonstrated that the extracellular matrix proteins fibronectin, vitronectin and collagen-I present in the bone matrix have not deteriorated after HHP-treatment up to 600 MPa (10 min, room

samples

inactivation of bacteria in all kind of materials is not possible at the moment.

clinical HHP application no application in

food preservation disinfection of infected

disinfection of inserts of

Prosthesis

tissue

pre-clinical application of HHP in

pressure influences cartilage stability *in vivo* (32).

*in vivo* proliferation at HHP above 130 MPa (35).

permeabilization of cell membranes by HHP causing cell death (33).

re-implantation of the once tumor-bearing bone segment back into the patient.

**4. Effect of pressure on extracellular matrix proteins and enzymes** 

disinfection of tumor afflicted bone, cartilage, tendons

studies

temperature) with respect to cell proliferation, spreading and adherence of human osteoblast-like cells and human osteosarcoma cells (Saos-2) (11). These data encourage further exploration of the potential of HHP to sterilize tumor-affected bone segments prior to re-implantation, since during such treatment eukaryotic bone cells including tumor cells would be irreversibly impaired, while the bone's biomechanical properties and the biological properties of the extracellular matrix proteins fibronectin, vitronectin, and collagen-I would be preserved (11).

HHP causes a stress response in many types of mammalian cells, including chondrocytes and bone tumor cells (36). Further to this, Kopakkala-Tani *et al.* investigated whether some of the well known transduction pathways are activated in human chondrosarcoma cells under stress by exposure to moderate HHP of 15-30 MPa and demonstrated an increased level of active, phosphorylated forms of the extracellular signal-related kinase ERK and phosphoinositide 3-kinase under these pressure conditions (37).

HHP may not only exert an effect on tumor and normal cells present in the bone, but also on the tumor-associated proteases released by these cells, which are conductive to tumor bone turnover. At a pressure level of 600 MPa the latent activity of the inactive zymogens prothrombin, plasminogen, pro-uPA and trypsinogen, in addition to the proteolytically active forms thrombin, plasmin, HMW-uPA, and trypsin was minimally affected by HHP (24). The variation seen between different enzymes is probably due to differences in molecular structures and the resulting modifications after HHP treatment (24). It is worthwhile to note that at this pressure level normal bone cells and tumor cells are irreversibly impaired. Additionally, HHP also influences the activity of other enzymes. With that in mind, Masson *et al.* reviewed HHP technology and its potential applications in medicine and pharmaceutical science (9). The authors explained that HHP may affect both the activity and specificity of enzymes and that HHP is used for the engineering of proteins to allow enzyme-catalyzed synthesis of fine chemicals and pharmaceuticals and the production of modified proteins of medical or pharmaceutical interest. Such reactions can be used for food functionalization and for producing "nutraceuticals" to be used in complementary therapy (38). Pressure processing was found to be efficient in reducing the allergenic activity of food (39).

In general, pressures above 300 MPa cause irreversible protein denaturation at room temperature, whereas lower pressures may result in reversible changes in protein structure. The effects of HHP on enzymes have been divided into two classes: moderate pressure values of 100–200 MPa which activate monomeric enzymes and elevated pressures usually inducing enzyme inactivation (1). Investigations of the impact of moderate HHP up to 200 MPa on alpha-amylase have shown a pressure-dependent stabilization of the enzyme against temperature-induced inactivation (3,40). Interestingly, for some proteases, proteolysis enhancement through HHP (up to 400 MPa) depended on substrate changes and not on changes of the enzyme, as investigated for chymotrypsin in the hydrolysis of betalactoglobulin (41).

#### **5. Effect of HHP-treatment on viability of microorganisms in bone**

So far, the effect of neoantigens generated during HHP-treatment of bone, cartilage and tendon on the host after re-implantation has not been elucidated and is at present subject to

Disinfection of Human Tissues in Orthopedic Surgical Oncology by High Hydrostatic Pressure 65

[2] Abe F, Kato C and Horikoshi K: Pressure-regulated metabolism in microorganisms.

[3] Yamaguchi T, Kawamura H, Kimoto E and Tanaka M: Effects of temperature and pH on

[4] Hoover DG, Metrick C, Papineau AM, Farkas DF, and Knorr D: Biological effects of high hydrostatic-pressure on food microorganisms. Food Tech *43*: 99-107, 1989.

[6] Gould GW: Biodeterioration of foods and an overview of preservation in the food and

[8] Cheftel JC: Review: High-pressure, microbial inactivation and food preservation. Food

[9] Masson P, Tonello C, and Balny C: High-Pressure Biotechnology in Medicine and

[10] Naal FD, Schauwecker J, Steinhauser E, Milz S, KnochF, Mittelmeier W and Diehl P:

[11] Diehl P, Schmitt M, Schauwecker J, Eichelberg K, Gollwitzer H, Gradinger R, Goebel M,

[12] Diehl P, Steinhauser E, Gollwitzer H, Heister C, Schauwecker J, Milz S, Mittelmeier W

[14] Naal FD, Mengele K, Schauwecker J, Gollwitzer H, Gerdesmeyer L, Reuning U,

[15] Schauwecker J, Wirthmann L, Schmitt M, Tuebel J, Magdolen U, Gradinger R,

[17] Bohm P, Springfeld R and Springer H: Re-implantation of autoclaved bone segments in

bone grafts in tumor surgery. Clin Orthop 368: 196-206, 1999.

Biomechanical and immunohistochemical properties of meniscal cartilage after high hydrostatic pressure treatment. J Biomed Mater Res B Appl Biomater 2008. in

Preissner KT, Mittelmeier W and Magdolen U: Effect of high hydrostatic pressure on biological properties of extracellular bone matrix proteins. Int J Mol Med *16*:

and Schmitt M: Biomechanical and immunohistochemical analysis of high hydrostatic pressure-treated Achilles tendons. J Orthop Sci *11*: 380-385, 2006. [13] Diehl P, Naal FD, Schauwecker J, Steinhauser E, Milz S, Gollwitzer H and Mittelmeier

W: [Biomechanical properties of articular cartilage after high hydrostatic pressure

Mittelmeier W, Gradinger R, Schmitt M and Diehl P: High hydrostatic pressureinduced cell death in human chondrocytes and chondrosarcoma cells. Anticancer

Mittelmeier W and Diehl P: Effect of extracorporeal high hydrostatic pressure on cellular outgrowth from tumor-afflicted bone. Anticancer Res *26*: 85-89, 2006. [16] Araki N, Myoui A, Kuratsu S, Hashimoto N, Inoue T, Kudawara I, Ueda T, Yoshikawa

H, Masaki N and Uchida A: Intraoperative extracorporeal autogenous irradiated

musculoskeletal tumor surgery. Clinical experience in 9 patients followed for 1.1- 8.4 years and review of the literature. Arch Orthop Trauma Surg *118*: 57-65, 1998. [18] Bohm P, Fritz J, Thiede S and Budach W: Reimplantation of extracorporeal irradiated

bone segments in musculoskeletal tumor surgery: clinical experience in eight patients and review of the literature. Langenbecks Arch Surg *387*: 355-365, 2003.

[5] Hoover DG: Pressure effects on biological-systems. Food Tech *47*: 150-155, 1993.

[7] Gould GW: Preservation: past, present and future. Br Med Bull *56*: 84-96, 2000.

Pharmaceutical Science. J Biomed Biotechnol *1*: 85-88, 2001.

treatment]. Biomed Tech (Berl) *51*: 8-14, 2006.

dairy industries. Int Biodeter Biodeg *36*: 267-277, 1995.

hemoglobin release from hydrostatic pressure-treated erythrocytes. J Biochem

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(Tokyo) *106*: 1080-1085, 1989.

Sci Tech Int *1*: 75-90, 1995.

press

285-289, 2005.

Res *25*: 1977-1982, 2005.

preclinical animal experiments. In spite of that, such physically modified proteins may be new innovative tools in the development of vaccines by making use of the changed immunogenicity of pressure-treated proteins or pressure-killed bacteria, viruses or normal and tumor cells (39,42-44).

Also of importance for HHP-teatment of bone is the fact that viruses are very sensitive to HHP, being inactivated at pressures as low as 100 to 300 MPa. Inactivation of numerous viruses such as herpes viruses, rotaviruses, influenza, picornaviruses as well as immunodeficiency viruses by pressure treatment has been successful in blood (45,46). The use of high pressure in decreasing virus concentration in the blood of patients suffering severe virus infections by *ex vivo* pressure treatment of blood has been proposed (47), but studies on HHP inactivation of viruses present in bone, cartilage or tendon have not been reported yet.

Likewise, different procedures are available to inactivate bacteria and fungi, including their spores, in human bone transplants (48). The most efficient methods of inactivation are gamma irradiation and thermal inactivation as well as chemical sterilization methods such as the peracetic acid-ethanol treatment of bone (49). The direct effect of HHP to achieve killing of vegetative bacterial, yeast and mould cells, has been documented as well (50,51), although much higher pressure values of 500 – 700 MPa are needed than for the inactivation of viruses (52,53). Interestingly, Gram-positive bacteria are more resistant to HHP than Gram-negative bacteria (54). A major advantage of HHP processing over gamma irradiation, thermal inactivation or the use of peracetic acid-ethanol treatment is that it preserves the initial mechanical properties of the bone, cartilage and tendon, a prerequisite for reimplantation of the *ex vivo*-treated tissues.

### **6. Conclusion**

HHP technology has found broad application in the food industry, for instance in activating vegetative microorganisms in meat products, milk, juice, etc.

While viruses and bacteria can be inactivated by moderate to high HHP, outgrowth of tumor cells from tumor-afflicted bone and cartilage segments can be efficiently blocked by extracorporeal HHP, while leaving their biomechanical and key biological properties intact.

These findings raise the hope that HHP can eventually be used in orthopaedic surgery as an alternative technique over other established physical or chemical methods of sterilizing resected bone, cartilage or tendon in order to kill viruses, bacteria and cancer cells to allow autologous re-implantation. Still, before that goal is reached, further pre-clinical studies are required.

#### **7. Acknowledgments**

This work was supported in part by the Bavarian Research Foundation (Bayerische Forschungsstiftung, Bayerischer Forschungsverbund for Tissue Engineering and Rapid Prototyping TE3) and by the German Research Association (DFG, SPP 1100).

#### **8. References**

[1] Knorr D: Novel approaches in food-processing technology: new technologies for preserving foods and modifying function. Curr Opin Biotechnol *10*: 485-491, 1999.

preclinical animal experiments. In spite of that, such physically modified proteins may be new innovative tools in the development of vaccines by making use of the changed immunogenicity of pressure-treated proteins or pressure-killed bacteria, viruses or normal

Also of importance for HHP-teatment of bone is the fact that viruses are very sensitive to HHP, being inactivated at pressures as low as 100 to 300 MPa. Inactivation of numerous viruses such as herpes viruses, rotaviruses, influenza, picornaviruses as well as immunodeficiency viruses by pressure treatment has been successful in blood (45,46). The use of high pressure in decreasing virus concentration in the blood of patients suffering severe virus infections by *ex vivo* pressure treatment of blood has been proposed (47), but studies on HHP inactivation of viruses present in bone, cartilage or tendon have not been reported yet. Likewise, different procedures are available to inactivate bacteria and fungi, including their spores, in human bone transplants (48). The most efficient methods of inactivation are gamma irradiation and thermal inactivation as well as chemical sterilization methods such as the peracetic acid-ethanol treatment of bone (49). The direct effect of HHP to achieve killing of vegetative bacterial, yeast and mould cells, has been documented as well (50,51), although much higher pressure values of 500 – 700 MPa are needed than for the inactivation of viruses (52,53). Interestingly, Gram-positive bacteria are more resistant to HHP than Gram-negative bacteria (54). A major advantage of HHP processing over gamma irradiation, thermal inactivation or the use of peracetic acid-ethanol treatment is that it preserves the initial mechanical properties of the bone, cartilage and tendon, a prerequisite for re-

HHP technology has found broad application in the food industry, for instance in activating

While viruses and bacteria can be inactivated by moderate to high HHP, outgrowth of tumor cells from tumor-afflicted bone and cartilage segments can be efficiently blocked by extracorporeal HHP, while leaving their biomechanical and key biological properties intact. These findings raise the hope that HHP can eventually be used in orthopaedic surgery as an alternative technique over other established physical or chemical methods of sterilizing resected bone, cartilage or tendon in order to kill viruses, bacteria and cancer cells to allow autologous re-implantation. Still, before that goal is reached, further pre-clinical studies are

This work was supported in part by the Bavarian Research Foundation (Bayerische Forschungsstiftung, Bayerischer Forschungsverbund for Tissue Engineering and Rapid

[1] Knorr D: Novel approaches in food-processing technology: new technologies for preserving foods and modifying function. Curr Opin Biotechnol *10*: 485-491, 1999.

Prototyping TE3) and by the German Research Association (DFG, SPP 1100).

and tumor cells (39,42-44).

implantation of the *ex vivo*-treated tissues.

vegetative microorganisms in meat products, milk, juice, etc.

**6. Conclusion** 

required.

**7. Acknowledgments** 

**8. References** 


Disinfection of Human Tissues in Orthopedic Surgical Oncology by High Hydrostatic Pressure 67

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[39] Poms RE and Anklam E: Effects of chemical, physical, and technological processes on

[40] Buckow R, Weiss U, Heinz V and Knorr D: Stability and catalytic activity of alpha-

[41] Chicon R, Lopez-Fandino R, Quiros A and Belloque J: Changes in chymotrypsin

[42] Goldman Y, Peled A and Shinitzky M: Effective elimination of lung metastases induced

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[46] Silva JL, Foguel D, Da Poian AT and Prevelige PE: The use of hydrostatic pressure as a

[47] Bradley DW, Hess RA, Tao F, Sciaba-Lentz L, Remaley AT, Laugharn JA and Manak M:

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R und Gollwitzer H: Extracorporeal high hydrostatic pressure as a new technology for the disinfection of infected bone specimens. Biomed Tech (Berl) 53: 190-198,

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Mittelmeier W and Schmitt M: Quantitative analysis of the impact of short-time high hydrostatic pressure on bone tumor-associated proteases. Int J Mol Med *19*: hydrostatic pressure on tumor cell adherence and viability. Oncol Rep *12*: 369-373, 2004.


**4** 

**Heparan Sulfate Proteoglycan Mimetics** 

*1Department of Plastic and Reconstructive Surgery, Erasmus MC,* 

*University Medical Center, Rotterdam,* 

*1The Netherlands* 

*2France* 

*2Laboratoire CRRET, Université Paris Est, Créteil,* 

**Promote Tissue Regeneration: An Overview** 

Wound healing is a complex and dynamic process that requires the coordinated completion of a variety of cellular activities, including phagocytosis, chemotaxis, mitogenesis, and synthesis of components of the extracellular matrix. These activities occur in a cascade that correlates with the appearance of multiple cell types and is regulated by soluble mediators such as growth factors and cytokines. In the wound healing process, three phases can be recognized: hemostasis and inflammation, proliferation, and tissue remodeling. These three phases are distinct but overlap in time (Singer and Clark 1999; Diegelmann and Evans 2004;

Hemostasis and inflammation occur immediately after tissue injury. They prevent ongoing blood and fluid loss and establish an immune barrier against invading micro-organisms. Hemostasis is achieved by vasoconstriction and blood clotting. Platelets initiate the clotting cascade, initially by forming a platelet plug. This platelet plug is followed by a fibrin clot, which provides a provisional matrix scaffold for cell migration. Platelets also secrete a variety of growth factors and cytokines such as fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). These growth factors are located in the fibrin clot and act as promoters in the wound healing process by recruiting inflammatory cells to the wound site and initiating

Once the bleeding is controlled, inflammatory cells migrate into the wound area. This is the start of the inflammatory phase, which is characterized by the sequential infiltration of

Neutrophils are recruited to the wound site within 24-36 h after wounding. They are attracted by the growth factors released by degranulating platelets and by the products of

neutrophils, macrophages, and lymphocytes (Broughton, Janis et al. 2006).

**1. Introduction** 

**1.1 Normal wound healing** 

Broughton, Janis et al. 2006).

angiogenesis (Martin 1997).

**1.2 Hemostasis and inflammation** 

Johan van Neck1, Bastiaan Tuk1, Denis Barritault2 and Miao Tong1


### **Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview**

Johan van Neck1, Bastiaan Tuk1, Denis Barritault2 and Miao Tong1 *1Department of Plastic and Reconstructive Surgery, Erasmus MC, University Medical Center, Rotterdam, 2Laboratoire CRRET, Université Paris Est, Créteil, 1The Netherlands* 

*2France* 

#### **1. Introduction**

68 Tissue Regeneration – From Basic Biology to Clinical Application

[49] Pruss A, Baumann B, Seibold M, Kao M, Tintelnot K, von Versen R, Radtke H, Dorner

[52] Hayakawa I, Kanno T, Tomita M and Fujio Y: Application of high-pressure for spore inactivation and protein denaturation. Journal of Food Science *59*: 159-163, 1994. [53] Wuytack EY, Boven S and Michiels CW: Comparative study of pressure-induced

[54] Shigehisa T, Ohmori T, Saito A, Taji S and Hayashi R: Effects of high hydrostatic-

Microbiol *83*: 181-188, 1997.

Microbiol *64*: 3220-3224, 1998.

T, Pauli G and Gobel UB: Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol. Biologicals *29*: 59-66, 2001. [50] Arroyo G, Sanz PD and Prestamo G: Effect of high pressure on the reduction of microbial populations in vegetables. J Appl Microbiol *82*: 735-742, 1997. [51] Simpson RK and Gilmour A: The effect of high hydrostatic pressure on *Listeria*

monocytogenes in phosphate-buffered saline and model food systems. J Appl

germination of *Bacillus subtilis* spores at low and high pressures. Appl Environ

pressure on characteristics of pork slurries and inactivation of microorganisms associated with meat and meat-products. Int Food Microbiol *12*: 207-216, 1991.

#### **1.1 Normal wound healing**

Wound healing is a complex and dynamic process that requires the coordinated completion of a variety of cellular activities, including phagocytosis, chemotaxis, mitogenesis, and synthesis of components of the extracellular matrix. These activities occur in a cascade that correlates with the appearance of multiple cell types and is regulated by soluble mediators such as growth factors and cytokines. In the wound healing process, three phases can be recognized: hemostasis and inflammation, proliferation, and tissue remodeling. These three phases are distinct but overlap in time (Singer and Clark 1999; Diegelmann and Evans 2004; Broughton, Janis et al. 2006).

#### **1.2 Hemostasis and inflammation**

Hemostasis and inflammation occur immediately after tissue injury. They prevent ongoing blood and fluid loss and establish an immune barrier against invading micro-organisms. Hemostasis is achieved by vasoconstriction and blood clotting. Platelets initiate the clotting cascade, initially by forming a platelet plug. This platelet plug is followed by a fibrin clot, which provides a provisional matrix scaffold for cell migration. Platelets also secrete a variety of growth factors and cytokines such as fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). These growth factors are located in the fibrin clot and act as promoters in the wound healing process by recruiting inflammatory cells to the wound site and initiating angiogenesis (Martin 1997).

Once the bleeding is controlled, inflammatory cells migrate into the wound area. This is the start of the inflammatory phase, which is characterized by the sequential infiltration of neutrophils, macrophages, and lymphocytes (Broughton, Janis et al. 2006).

Neutrophils are recruited to the wound site within 24-36 h after wounding. They are attracted by the growth factors released by degranulating platelets and by the products of

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 71

TNF-α, which are released by activated platelets and/or macrophages (Grotendorst, Soma et al. 1989; Lawrence and Diegelmann 1994). Keratinocyte growth factors (KGFs) and IL-6, which are released by fibroblasts, play a role in attracting neighboring keratinocytes to migrate, proliferate, and differentiate into epithelium (Smola, Thiekotter et al. 1993; Xia,

Fibroblast migration occurs two to four days after injury. Fibroblasts are attracted to the wound area by a number of factors, such as PDGF and TGF-β (Goldman 2004), and dominate the wound cell population in the first week. Within the wound area, fibroblasts proliferate and produce multiple structural molecules, including fibrin, fibronectin, glycosaminoglycans (GAGs), later followed by collagen (Witte and Barbul 1997; Robson, Steed et al. 2001; Ramasastry 2005). Together, these components construct the fibrin/fibronectin based provisional matrix (Clark, Lanigan et al. 1982), which contributes to

Granulation tissue formation starts three to five days after injury and is characterized by angiogenesis. The numerous angiogenic factors that are secreted during the hemostatic phase, such as FGF, VEGF, TGF-β, and PDGF, promote angiogenesis (Servold 1991). Four steps can be recognized in this process: (1) proteolytic degradation of the basement membrane of the parent vessels, allowing the formation of "capillary sprouts"; (2) migration of the endothelial cells towards the angiogenic stimulus; (3) proliferation; (4) maturation and remodeling of endothelial cells into capillary tubes (Velnar, Bailey et al. 2009). Capillary sprouts invade the fibrin/fibronectin based provisional matrix within a few days and organize into a dense microvascular network. This is so called vascularized stroma, together with macrophages and proliferating fibroblasts, constitute the acute granulation tissue that replaces the fibrin/fibronectin based provisional matrix (Witte and Barbul 1997; Baum and Arpey 2005). With collagen accumulation, angiogenesis ceases and the density of the microvascular network diminishes. When homeostasis between collagen synthesis and

Tissue remodeling phase is the final phase of wound healing. It starts one week after injury and lasts over a year or more. The main feature of this phase is the deposition of collagen in an organized network. During this phase, all short term events that were activated after injury cease: most macrophages, endothelial cells, fibroblasts, and myofibroblasts undergo apoptosis or exit from the wound. They leave a mass that consists mostly of collagen and other matrix proteins. Without an increase in collagen content, this largely acellular matrix subsequently is reorganized from a disorganized, mainly type III collagen fibers containing temporary matrix, into a lattice structure which is predominantly composed of type I collagen (Madden and Peacock 1968; Gurtner, Werner et al. 2008). A process that is dependent on collagen synthesis, which in part is the net result of the interaction between matrix metalloproteinases and tissue inhibitors of

Zhao et al. 1999).

**1.3.2 Fibroblast migration** 

the formation of granulation tissue.

**1.3.3 Granulation tissue formation** 

degradation is achieved, tissue remodeling begins.

**1.4 Tissue remodeling** 

complement activation and bacteria degradation (Gurtner, Werner et al. 2008). Infiltrating neutrophils phagocytose contaminating bacteria and release pro-inflammatory cytokines to activate local fibroblasts and keratinocytes (Hubner, Brauchle et al. 1996). Within a few days after injury, neutrophils are extruded as eschar or as a result of apoptosis and finally are replaced by macrophages (Witte and Barbul 1997).

Macrophages migrate into the wound within two or four days after injury and become the predominant cell type. Macrophages are derived from blood monocytes and act as the "orchestra conductor" of wound healing (Lawrence and Diegelmann 1994). In the early stages of wound healing, macrophages phagocytose the remaining debris, bacteria, and apoptotic cells, including neutrophils, thus paving the way for the resolution of inflammation (Guo and Dipietro 2010). Macrophages also secrete a battery of cytokines (e.g., tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6), growth factors (e.g., VEGF, FGF, PDGF,TGF-β, and EGF), and different types of metalloproteinases that degrade the collagen matrix (Henry and Garner 2003). This way macrophages influence cellular recruitment, cellular activation, angiogenesis, fibroplasia and also regulate the synthesis and formation of the provisional extracellular matrix, which serves as a scaffold for dermal regeneration and epidermal proliferation.

Subsequently, T-lymphocytes enter the wound area and peak during the lateproliferative/early remodeling phage. T-lymphocytes likely are involved in controlling the proliferation phase of wound healing. However, their exact role is not completely understood and is a current area of intensive investigation (Broughton, Janis et al. 2006; Guo and Dipietro 2010).

#### **1.3 Proliferation**

The proliferation phase follows and partly overlaps the inflammatory phase. The proliferation phase starts on the third day after injury and lasts for about 2-4 weeks. This phase is characterized by epithelial proliferation and migration over a provisional matrix within the wound (reepithelialization), fibroblast migration, and formation of granulation tissue. With the progression of proliferation, the provisional fibrin/fibronectin based provisional matrix is replaced by newly formed granulation tissue (Broughton, Janis et al. 2006; Velnar, Bailey et al. 2009).

#### **1.3.1 Reepithelialization**

Reepithelialization of the wound area starts within hours after injury. Epidermal cells at the wound margin undergo a marked phenotypic alteration and begin to migrate into the wound area (Paladini, Takahashi et al. 1996). Migrating epidermal cells dissect under the fibrin clot across the wound, separating the desiccated eschar from viable tissue. Epidermal cells behind the leading migrating edge proliferate, mature and finally restore the barrier function of the epithelium. The stimulus for the migration and proliferation of epidermal cells during reepithelialization has not been clearly determined, but several possibilities are well documented. The absence of neighbor cells at the margin of wound (i.e., the "free edge" effect) may induce both migration and proliferation of epidermal cells. Local release of growth factors and cytokines also stimulate these processes. The initial stimulus for proliferation and migration of epidermal cells includes the action of EGF, TGF-α, IL-1 and TNF-α, which are released by activated platelets and/or macrophages (Grotendorst, Soma et al. 1989; Lawrence and Diegelmann 1994). Keratinocyte growth factors (KGFs) and IL-6, which are released by fibroblasts, play a role in attracting neighboring keratinocytes to migrate, proliferate, and differentiate into epithelium (Smola, Thiekotter et al. 1993; Xia, Zhao et al. 1999).

#### **1.3.2 Fibroblast migration**

70 Tissue Regeneration – From Basic Biology to Clinical Application

complement activation and bacteria degradation (Gurtner, Werner et al. 2008). Infiltrating neutrophils phagocytose contaminating bacteria and release pro-inflammatory cytokines to activate local fibroblasts and keratinocytes (Hubner, Brauchle et al. 1996). Within a few days after injury, neutrophils are extruded as eschar or as a result of apoptosis and finally are

Macrophages migrate into the wound within two or four days after injury and become the predominant cell type. Macrophages are derived from blood monocytes and act as the "orchestra conductor" of wound healing (Lawrence and Diegelmann 1994). In the early stages of wound healing, macrophages phagocytose the remaining debris, bacteria, and apoptotic cells, including neutrophils, thus paving the way for the resolution of inflammation (Guo and Dipietro 2010). Macrophages also secrete a battery of cytokines (e.g., tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6), growth factors (e.g., VEGF, FGF, PDGF,TGF-β, and EGF), and different types of metalloproteinases that degrade the collagen matrix (Henry and Garner 2003). This way macrophages influence cellular recruitment, cellular activation, angiogenesis, fibroplasia and also regulate the synthesis and formation of the provisional extracellular matrix, which serves as a scaffold for dermal

Subsequently, T-lymphocytes enter the wound area and peak during the lateproliferative/early remodeling phage. T-lymphocytes likely are involved in controlling the proliferation phase of wound healing. However, their exact role is not completely understood and is a current area of intensive investigation (Broughton, Janis et al. 2006; Guo

The proliferation phase follows and partly overlaps the inflammatory phase. The proliferation phase starts on the third day after injury and lasts for about 2-4 weeks. This phase is characterized by epithelial proliferation and migration over a provisional matrix within the wound (reepithelialization), fibroblast migration, and formation of granulation tissue. With the progression of proliferation, the provisional fibrin/fibronectin based provisional matrix is replaced by newly formed granulation tissue (Broughton, Janis et al.

Reepithelialization of the wound area starts within hours after injury. Epidermal cells at the wound margin undergo a marked phenotypic alteration and begin to migrate into the wound area (Paladini, Takahashi et al. 1996). Migrating epidermal cells dissect under the fibrin clot across the wound, separating the desiccated eschar from viable tissue. Epidermal cells behind the leading migrating edge proliferate, mature and finally restore the barrier function of the epithelium. The stimulus for the migration and proliferation of epidermal cells during reepithelialization has not been clearly determined, but several possibilities are well documented. The absence of neighbor cells at the margin of wound (i.e., the "free edge" effect) may induce both migration and proliferation of epidermal cells. Local release of growth factors and cytokines also stimulate these processes. The initial stimulus for proliferation and migration of epidermal cells includes the action of EGF, TGF-α, IL-1 and

replaced by macrophages (Witte and Barbul 1997).

regeneration and epidermal proliferation.

and Dipietro 2010).

**1.3 Proliferation** 

2006; Velnar, Bailey et al. 2009).

**1.3.1 Reepithelialization** 

Fibroblast migration occurs two to four days after injury. Fibroblasts are attracted to the wound area by a number of factors, such as PDGF and TGF-β (Goldman 2004), and dominate the wound cell population in the first week. Within the wound area, fibroblasts proliferate and produce multiple structural molecules, including fibrin, fibronectin, glycosaminoglycans (GAGs), later followed by collagen (Witte and Barbul 1997; Robson, Steed et al. 2001; Ramasastry 2005). Together, these components construct the fibrin/fibronectin based provisional matrix (Clark, Lanigan et al. 1982), which contributes to the formation of granulation tissue.

#### **1.3.3 Granulation tissue formation**

Granulation tissue formation starts three to five days after injury and is characterized by angiogenesis. The numerous angiogenic factors that are secreted during the hemostatic phase, such as FGF, VEGF, TGF-β, and PDGF, promote angiogenesis (Servold 1991). Four steps can be recognized in this process: (1) proteolytic degradation of the basement membrane of the parent vessels, allowing the formation of "capillary sprouts"; (2) migration of the endothelial cells towards the angiogenic stimulus; (3) proliferation; (4) maturation and remodeling of endothelial cells into capillary tubes (Velnar, Bailey et al. 2009). Capillary sprouts invade the fibrin/fibronectin based provisional matrix within a few days and organize into a dense microvascular network. This is so called vascularized stroma, together with macrophages and proliferating fibroblasts, constitute the acute granulation tissue that replaces the fibrin/fibronectin based provisional matrix (Witte and Barbul 1997; Baum and Arpey 2005). With collagen accumulation, angiogenesis ceases and the density of the microvascular network diminishes. When homeostasis between collagen synthesis and degradation is achieved, tissue remodeling begins.

#### **1.4 Tissue remodeling**

Tissue remodeling phase is the final phase of wound healing. It starts one week after injury and lasts over a year or more. The main feature of this phase is the deposition of collagen in an organized network. During this phase, all short term events that were activated after injury cease: most macrophages, endothelial cells, fibroblasts, and myofibroblasts undergo apoptosis or exit from the wound. They leave a mass that consists mostly of collagen and other matrix proteins. Without an increase in collagen content, this largely acellular matrix subsequently is reorganized from a disorganized, mainly type III collagen fibers containing temporary matrix, into a lattice structure which is predominantly composed of type I collagen (Madden and Peacock 1968; Gurtner, Werner et al. 2008). A process that is dependent on collagen synthesis, which in part is the net result of the interaction between matrix metalloproteinases and tissue inhibitors of

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 73

The differences in physiology and healing dynamics between acute and chronic wounds are numerous (Figure 1) (Schultz and Wysocki 2009). Excessive inflammation and abnormalities in cell-extracellular matrix interaction are considered important mechanisms responsible for the failure of chronic wounds to heal (Eming, Krieg et al. 2007; Menke, Ward et al. 2007;

Fig. 1. Comparison of a chronic wound in which repair is arrested and an acute wound in which repair proceeds in an orderly, sequential fashion. Differences between these wounds

Copyright 2009 by the Wound Healing Society

extracellular matrix, keratinocyte migration, scar formation, bacterial colonization/infection,

Impaired wounds exhibit an out-of-control prolonged inflammatory response that is selfsustaining (Menke, Ward et al. 2007). An over-abundant neutrophil infiltration is responsible for this chronic inflammation (Diegelmann 2003; Diegelmann and Evans 2004). Neutrophils release significant amounts of enzymes such as metalloproteinases (Yager, Zhang et al. 1996; Nwomeh, Liang et al. 1998; Nwomeh, Liang et al. 1999; Lobmann, Ambrosch et al. 2002), which are not balanced by their respective inhibitors. As a result, the balance between matrix degradation and synthesis shifts towards degradation (Bullen, Longaker et al. 1995). In addition, neutrophils release elastase, an enzyme that is capable to destroy growth factors such as PDGF and TGF-β (Yager, Zhang et al. 1996). This prolonged

are seen in clot formation, inflammation, capillary migration, granulation tissue,

and biofilm formation (Schultz and Wysocki 2009).

**2.2 Pathophysiology of impaired wounds** 

Schultz and Wysocki 2009).

metalloproteinases (Madlener, Parks et al. 1998). During this phase, the wound progressively continues to increase in tensile strength. Nevertheless, wounds never regain the original strength. At maximal strength, healed wounds are 80% as strong as normal skin (Madden and Peacock 1968).

### **2. Impaired wound healing**

Impaired healing wounds generally failed to progress through the normal stages of wound healing. Impaired healing wounds can be arrested in any of the different healing stages, however, frequently enter a state of pathologic inflammation. As a result, the wounds cannot be repaired in an orderly and timely manner, subsequently resulting in poor anatomical and functional outcome (Lazarus, Cooper et al. 1994). Both acute wounds and chronic wounds can exhibit impaired healing.

#### **2.1 Factors affecting wound healing**

Wound healing can be impaired by multiple factors in any of the healing phases. These factors are categorized into local and systemic factors. Local factors are those that directly influence the characteristics of the wound. Systemic factors concern the overall health or disease state of the individual which affects the ability to heal (Table 1). However, these factors often are interrelated so their influences are not mutually exclusive. Single or multiple factors may, therefore, play a role in any one or more individual phases, contributing to the overall outcome of the healing process (Guo and Dipietro 2010).


Table 1. Factors that affect wound healing.

In view of the large variety of factors that can be involved in impaired healing wounds, the types of chronic wounds are numerous. However, the most common chronic wounds are pressure ulcers, diabetic ulcers, and venous ulcers. Together, they constitute approximately 70% of all chronic wounds (Eaglstein and Falanga 1997).

#### **2.2 Pathophysiology of impaired wounds**

72 Tissue Regeneration – From Basic Biology to Clinical Application

metalloproteinases (Madlener, Parks et al. 1998). During this phase, the wound progressively continues to increase in tensile strength. Nevertheless, wounds never regain the original strength. At maximal strength, healed wounds are 80% as strong as normal

Impaired healing wounds generally failed to progress through the normal stages of wound healing. Impaired healing wounds can be arrested in any of the different healing stages, however, frequently enter a state of pathologic inflammation. As a result, the wounds cannot be repaired in an orderly and timely manner, subsequently resulting in poor anatomical and functional outcome (Lazarus, Cooper et al. 1994). Both acute wounds and

Wound healing can be impaired by multiple factors in any of the healing phases. These factors are categorized into local and systemic factors. Local factors are those that directly influence the characteristics of the wound. Systemic factors concern the overall health or disease state of the individual which affects the ability to heal (Table 1). However, these factors often are interrelated so their influences are not mutually exclusive. Single or multiple factors may, therefore, play a role in any one or more individual phases,

Danlos syndrome, Marfan's syndrome

In view of the large variety of factors that can be involved in impaired healing wounds, the types of chronic wounds are numerous. However, the most common chronic wounds are pressure ulcers, diabetic ulcers, and venous ulcers. Together, they constitute approximately

Diseases: diabetes , artery disease, peripheral vascular disease Immunocompromised conditions: AIDS, radiation therapy,

Congenital healing disorders: epidermolysis bullosa, Ehlers-

contributing to the overall outcome of the healing process (Guo and Dipietro 2010).

skin (Madden and Peacock 1968).

**2. Impaired wound healing** 

chronic wounds can exhibit impaired healing.

**Local Factors Systemic Factors** 

Table 1. Factors that affect wound healing.

70% of all chronic wounds (Eaglstein and Falanga 1997).

Age

Sex hormones Obesity Stress

chemotherapy

Alcoholism Smoking Distant cancer Uremia

Nutritional deficiencies

Infection

Desiccation Necrosis Pressure Trauma Edema Local cancer Radiation Toxins

Tissue maceration Foreign bodies Ischemia

Venous insufficiency

Iatrogenic factors

**2.1 Factors affecting wound healing** 

The differences in physiology and healing dynamics between acute and chronic wounds are numerous (Figure 1) (Schultz and Wysocki 2009). Excessive inflammation and abnormalities in cell-extracellular matrix interaction are considered important mechanisms responsible for the failure of chronic wounds to heal (Eming, Krieg et al. 2007; Menke, Ward et al. 2007; Schultz and Wysocki 2009).

Fig. 1. Comparison of a chronic wound in which repair is arrested and an acute wound in which repair proceeds in an orderly, sequential fashion. Differences between these wounds are seen in clot formation, inflammation, capillary migration, granulation tissue, extracellular matrix, keratinocyte migration, scar formation, bacterial colonization/infection, and biofilm formation (Schultz and Wysocki 2009).

Impaired wounds exhibit an out-of-control prolonged inflammatory response that is selfsustaining (Menke, Ward et al. 2007). An over-abundant neutrophil infiltration is responsible for this chronic inflammation (Diegelmann 2003; Diegelmann and Evans 2004). Neutrophils release significant amounts of enzymes such as metalloproteinases (Yager, Zhang et al. 1996; Nwomeh, Liang et al. 1998; Nwomeh, Liang et al. 1999; Lobmann, Ambrosch et al. 2002), which are not balanced by their respective inhibitors. As a result, the balance between matrix degradation and synthesis shifts towards degradation (Bullen, Longaker et al. 1995). In addition, neutrophils release elastase, an enzyme that is capable to destroy growth factors such as PDGF and TGF-β (Yager, Zhang et al. 1996). This prolonged

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 75

Healing-arrested (chronic) wounds seriously lower the patient's quality of life and their treatments are extremely resource consuming. In the USA the costs related to chronic wounds are estimated over \$25 billion a year (Fan, Tang et al. 2011). In the European Union the costs related to pressure ulcers and venous ulcers are estimated around €20 billion per year. The severity of the problem, likely accompanied by the substantial financial gain that can be envisioned, triggers the development of a great variety of advanced treatment options. Many are described in literature, although mostly with minimal of proof of efficacy. Also many reviews exist on this topic (recent examples are e.g. (Rizzi, Upton et al. 2010; Fan,

In the context of this chapter, only a limited discussion will follow that covers the following

Gene therapy clearly holds promise in the repair of soft tissue disorders like wounds (reviews on this topic e.g. Branski, Pereira et al. 2007; Eming, Krieg et al. 2007). Skin is easily accessible for genetic manipulations and has cellular constituents with a high turnover that can be an effective target for the transfer of genetic material. Especially a temporal delivery of growth factors by gene transfer may be helpful in transforming chronic wounds into

July 2011, gene therapy trials are listed at www.clinicaltrials.gov concerning patients with diabetes-related, and lower limb ischemia-related ulcers. However, the application of

The alpha granules of platelets are rich in growth factors that are considered important for wound healing, amongst them EGF, FGF, PDGF, TGF-β and VEGF. Therefore, the use of platelet rich plasma is considered superior to any single growth factor application. Topical use of platelet rich plasma is described over 2 decades in several case series. E.g. following a weekly application to patients with cutaneous wounds of variable origin, Crovetti et al. (2004) described a complete response in 9 of 24 patients and a partial response in an additional 9 patients (Crovetti, Martinelli et al. 2004). Schade and Roukis (2008) reported beneficial effects of platelet-rich plasma to the healing of split-thickness skin grafts (Schade and Roukis 2008). Kazakos et al. compared the use of platelet rich plasma versus conventional therapy in 59 patients suffering from acute trauma wounds. And reported a beneficial effect on wound size reduction and pain perception over a three week period (Kazakos, Lyras et al. 2009). Steed et al. (1992) performed a randomised controlled trial in 13 diabetic foot patients and observed, over a period of 15 weeks, increased healing in the

genetic manipulation to the treatment of chronic wounds is still in its infancy.

**4. Advanced treatment options for wounds** 

Tang et al. 2011).

1. Gene therapy

**4.1 Gene therapy** 

healing wounds.

'state of art' strategies:

3. Stem cell therapy

2. Platelet rich plasma therapy

**4.2 Platelet rich plasma therapy** 

platelet rich plasma group (Steed, Goslen et al. 1992).

4. Biological dressings and skin substitutes

inflammatory environment also contains excessive reactive oxygen species that further damage cells, growth factors and healing tissues (Wenk, Foitzik et al. 2001). Abnormalities in extracellular matrix - growth factor interactions characterize impaired wound healing (Schultz and Wysocki 2009). Impaired wounds are difficult to heal until the delayed inflammation is reduced and the interactions between extracellular matrix and growth factors are restored. Wound treatment strategies, with a focus on regulating these disrupted interactions, may benefit the treatment of impaired wound healing.

### **3. 'Standard' management options for wounds**

The final goal of any wound management is to achieve wound healing. In the eyes of a cell biologist, these strategies might be seen as attempts to assist the injured tissue in recreating an extracellular matrix and cellular content, to enable tissue regeneration. In a somewhat simplistic view, most of the current routine clinical strategies to improve wound healing, therefore, can be classified into one or more of the categories that are depicted in Table 2.



#### **4. Advanced treatment options for wounds**

Healing-arrested (chronic) wounds seriously lower the patient's quality of life and their treatments are extremely resource consuming. In the USA the costs related to chronic wounds are estimated over \$25 billion a year (Fan, Tang et al. 2011). In the European Union the costs related to pressure ulcers and venous ulcers are estimated around €20 billion per year. The severity of the problem, likely accompanied by the substantial financial gain that can be envisioned, triggers the development of a great variety of advanced treatment options. Many are described in literature, although mostly with minimal of proof of efficacy. Also many reviews exist on this topic (recent examples are e.g. (Rizzi, Upton et al. 2010; Fan, Tang et al. 2011).

In the context of this chapter, only a limited discussion will follow that covers the following 'state of art' strategies:

1. Gene therapy

74 Tissue Regeneration – From Basic Biology to Clinical Application

inflammatory environment also contains excessive reactive oxygen species that further damage cells, growth factors and healing tissues (Wenk, Foitzik et al. 2001). Abnormalities in extracellular matrix - growth factor interactions characterize impaired wound healing (Schultz and Wysocki 2009). Impaired wounds are difficult to heal until the delayed inflammation is reduced and the interactions between extracellular matrix and growth factors are restored. Wound treatment strategies, with a focus on regulating these disrupted

The final goal of any wound management is to achieve wound healing. In the eyes of a cell biologist, these strategies might be seen as attempts to assist the injured tissue in recreating an extracellular matrix and cellular content, to enable tissue regeneration. In a somewhat simplistic view, most of the current routine clinical strategies to improve wound healing, therefore, can be classified into one or more of the categories that are depicted in Table 2.

**BENEFIT TO WOUND** 

and differentiation

the wound area

Bring the wound edges into viable tissue in order to allow cells to deposit the right extracellular matrix needed for their migration

in propagating cell migration into

Draining the wound fluid to clear extracellular matrix degrading enzymes and bacterial toxins

Create a sterile environment by clearing the wound from bacteria and secreted harmful substances, followed by creating a bacterial

Reduce the severity and duration of the immune reaction, to limit the production of soluble growth factors by immune cells that attract infiltrating, scar tissue producing, fibroblasts

Replenish the wound area, extracellular matrix and its cellular components with growth **LITERATURE (REVIEWS)** 

2003)

al. 2011)

2003)

(Attinger and Bulan 2001; Hess and Kirsner

(Okan, Woo et al. 2007; Korting, Schollmann et

(Vowden and Vowden

(Fung, Chang et al. 2003)

Diegelmann and Evans

(Nathan 2002;

(Krishnamoorthy, Morris et al. 2001; Barrientos, Stojadinovic

et al. 2008)

2004)

**HEALING** 

2. Moisture management Create an environment that assists

balance

factors

Table 2. Cell biological effects of clinical treatment modalities

interactions, may benefit the treatment of impaired wound healing.

**3. 'Standard' management options for wounds** 

**TREATMENT / STRATEGY** 

3. Exudate management

4. Local infection management

5. Inflammation management

6. Growth factor management

(e.g. by the application of foams, sponges or vacuum therapy)

(e.g. surgical, enzymatic,

1. Debridement

chemical)


#### **4.1 Gene therapy**

Gene therapy clearly holds promise in the repair of soft tissue disorders like wounds (reviews on this topic e.g. Branski, Pereira et al. 2007; Eming, Krieg et al. 2007). Skin is easily accessible for genetic manipulations and has cellular constituents with a high turnover that can be an effective target for the transfer of genetic material. Especially a temporal delivery of growth factors by gene transfer may be helpful in transforming chronic wounds into healing wounds.

July 2011, gene therapy trials are listed at www.clinicaltrials.gov concerning patients with diabetes-related, and lower limb ischemia-related ulcers. However, the application of genetic manipulation to the treatment of chronic wounds is still in its infancy.

#### **4.2 Platelet rich plasma therapy**

The alpha granules of platelets are rich in growth factors that are considered important for wound healing, amongst them EGF, FGF, PDGF, TGF-β and VEGF. Therefore, the use of platelet rich plasma is considered superior to any single growth factor application. Topical use of platelet rich plasma is described over 2 decades in several case series. E.g. following a weekly application to patients with cutaneous wounds of variable origin, Crovetti et al. (2004) described a complete response in 9 of 24 patients and a partial response in an additional 9 patients (Crovetti, Martinelli et al. 2004). Schade and Roukis (2008) reported beneficial effects of platelet-rich plasma to the healing of split-thickness skin grafts (Schade and Roukis 2008). Kazakos et al. compared the use of platelet rich plasma versus conventional therapy in 59 patients suffering from acute trauma wounds. And reported a beneficial effect on wound size reduction and pain perception over a three week period (Kazakos, Lyras et al. 2009). Steed et al. (1992) performed a randomised controlled trial in 13 diabetic foot patients and observed, over a period of 15 weeks, increased healing in the platelet rich plasma group (Steed, Goslen et al. 1992).

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 77

Table 3. Major growth factors and cytokines that participate in wound healing with cell types and their respective roles in both acute and chronic wounds are listed (Barrientos,

Copyright 2008 by the Wound Healing Society

Although some of the new techniques mentioned above are promising in small-scale trials,

on non-randomised prospective trials, small-scale trials, or single case studies.

The efficacy of most biological products is currently not approved in large trials.

The current evidence for many biological products based treatment is generally based

Stojadinovic et al. 2008). Reference numbers refer to their original publication.

only a minority are evidence based. Important issues that need to be addressed:


**4.5 Concerns about biological products based treatment** 


July 2011, platelet rich plasma trials are listed at www.clinicaltrials.gov concerning patients with burns, skin grafts, and leg ulcers.

#### **4.3 Stem cell therapy**

Literature on the use of bone marrow-derived stems cells, adipogenic stem cells and cutaneous mesenchymal stem cells, that are reported around the hair follicles, is manifold (reviews on this topic e.g. (Sellheyer and Krahl 2010; Wu, Zhao et al. 2010). At the preclinical level, evidence in wound areas is accumulating regarding the differentiation of bone marrow-derived stem cells into dermal fibroblasts, fibrocytes and endothelial progenitor cells. However, at the clinical level, reports of sufficient quality are scarce. Dash et al. (2009) reported beneficial effects on ulcer healing and ulcer pain of the addition of bone marrowderived mesenchymal stem cells to a total of 24 patients with lower limb non-healing ulcers that were randomly allocated to the placebo or stem cell therapy group (Dash, Dash et al. 2009). Walther et al. (2011) reported a pilot on the intraarterial administration of bone marrow derived mononuclear cells to patients with critical ischemia in a phase II randomised-start open label study (Walter, Krankenberg et al. 2011). Forty patients enrolled over a period of 3 years and a significantly improved ulcer healing and reduction of rest pain were found in the bone marrow group.

July 2011, over ten stem cell trials are listed at www.clinicaltrials.gov concerning patients with burn, pressure, diabetes-related and lower limb ischemia-related ulcers.

#### **4.4 Biological dressings and skin substitutes**

The complexity of the skin: its cellular constitution, extracellular matrix characteristics and the many growth factors involved in maintaining functional skin layers, let alone their involvement in wound healing (Table 3), largely determined research efforts.

Reports on dermal replacement and dermal addition strategies are manifold (recent reviews on this topic e.g. (Rizzi, Upton et al. 2010; Fan, Tang et al. 2011)). Briefly, research efforts to establish novel treatments can be categorized in tissue engineered skin based therapies, growth-factor based therapies, extracellular matrix based therapies and combinations thereof.

Dermal replacements, e.g. to cover large ulcers or burn injuries, are described using autografts, allografts or tissue engineered skin substitutes. Over a dozen of tissue engineered dermal replacements are on the market (Rizzi, Upton et al. 2010).

Dermal addition strategies are described using collagen, chondroitin-6-sulfate and hyaluronic acid.

Growth factor addition into the wound bed aims to reestablish or accelerate the natural healing process of chronic and acute wounds. Predominantly via preclinical research efforts, with currently PDGF and bFGF on the clinical market (Rizzi, Upton et al. 2010).

Hodde and Johnson (2007) elaborate on the role of the extracellular matrix to stimulate, direct and coordinate healing by storing a variety of growth factors at physiological levels (Hodde and Johnson 2007).

July 2011, platelet rich plasma trials are listed at www.clinicaltrials.gov concerning patients

Literature on the use of bone marrow-derived stems cells, adipogenic stem cells and cutaneous mesenchymal stem cells, that are reported around the hair follicles, is manifold (reviews on this topic e.g. (Sellheyer and Krahl 2010; Wu, Zhao et al. 2010). At the preclinical level, evidence in wound areas is accumulating regarding the differentiation of bone marrow-derived stem cells into dermal fibroblasts, fibrocytes and endothelial progenitor cells. However, at the clinical level, reports of sufficient quality are scarce. Dash et al. (2009) reported beneficial effects on ulcer healing and ulcer pain of the addition of bone marrowderived mesenchymal stem cells to a total of 24 patients with lower limb non-healing ulcers that were randomly allocated to the placebo or stem cell therapy group (Dash, Dash et al. 2009). Walther et al. (2011) reported a pilot on the intraarterial administration of bone marrow derived mononuclear cells to patients with critical ischemia in a phase II randomised-start open label study (Walter, Krankenberg et al. 2011). Forty patients enrolled over a period of 3 years and a significantly improved ulcer healing and reduction of rest

July 2011, over ten stem cell trials are listed at www.clinicaltrials.gov concerning patients

The complexity of the skin: its cellular constitution, extracellular matrix characteristics and the many growth factors involved in maintaining functional skin layers, let alone their

Reports on dermal replacement and dermal addition strategies are manifold (recent reviews on this topic e.g. (Rizzi, Upton et al. 2010; Fan, Tang et al. 2011)). Briefly, research efforts to establish novel treatments can be categorized in tissue engineered skin based therapies, growth-factor based therapies, extracellular matrix based therapies and combinations

Dermal replacements, e.g. to cover large ulcers or burn injuries, are described using autografts, allografts or tissue engineered skin substitutes. Over a dozen of tissue

Dermal addition strategies are described using collagen, chondroitin-6-sulfate and

Growth factor addition into the wound bed aims to reestablish or accelerate the natural healing process of chronic and acute wounds. Predominantly via preclinical research efforts,

Hodde and Johnson (2007) elaborate on the role of the extracellular matrix to stimulate, direct and coordinate healing by storing a variety of growth factors at physiological levels

with burn, pressure, diabetes-related and lower limb ischemia-related ulcers.

involvement in wound healing (Table 3), largely determined research efforts.

engineered dermal replacements are on the market (Rizzi, Upton et al. 2010).

with currently PDGF and bFGF on the clinical market (Rizzi, Upton et al. 2010).

with burns, skin grafts, and leg ulcers.

pain were found in the bone marrow group.

**4.4 Biological dressings and skin substitutes** 

**4.3 Stem cell therapy** 

thereof.

hyaluronic acid.

(Hodde and Johnson 2007).


Copyright 2008 by the Wound Healing Society

Table 3. Major growth factors and cytokines that participate in wound healing with cell types and their respective roles in both acute and chronic wounds are listed (Barrientos, Stojadinovic et al. 2008). Reference numbers refer to their original publication.

#### **4.5 Concerns about biological products based treatment**

Although some of the new techniques mentioned above are promising in small-scale trials, only a minority are evidence based. Important issues that need to be addressed:


Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 79

et al. 2000) and do therapeutic initiatives to restore the defective extracellular matrix and to

ReGeneraTing Agents (RGTAs) are synthetic heparan sulfate mimics, resistant to glycanase digestion (Figure 2) (Barbier-Chassefiere, Garcia-Filipe et al. 2009; Ikeda, Charef et al. 2011).

reposition heparan sulfates (Agren and Werthen 2007; Gandhi and Mancera 2010).

Fig. 2. RGTA OTR4120 is a structural analogue of glycosaminoglycans.

In wound areas, RGTAs can replace heparan sulfates by binding the free heparan binding sites that become available following heparan sulfate degradation. This way, RGTAs can regulate the bioavailability of the large variety of, locally synthesized, heparin binding proteins which allow the cellular tissue components to re-unfold their natural mechanism to

Copyright 2011 by OTR3,

RGTA OTR4120 is a RGTA member specifically designed to treat chronic wounds and marketed as CACIPLIQ20® (OTR3, Paris, France). The affinity constant of RGTA OTR4120 towards the vacant heparan sulfate binding sites of the extracellular matrix proteins allows a tight binding. This makes a short-term exposure to RGTA OTR4120 sufficient. Once RGTA OTR4120 is in place in the matrix scaffold, the growth factors, cytokines and other heparin binding signaling peptides can be repositioned through RGTA OTR4120 binding in this restored micro-environment. In this way, RGTA OTR4120 is thought to offer a matrix therapy that restores the natural cellular microenvironment. This allows the endogenous signaling of cell communications needed for tissue regeneration to resume their original function thereby halting the self-perpetuating cycles, particularly in impaired healing

**6.2 Working concept of RGTA / RGTA OTR4120** 

achieve wound regeneration.

wounds (Figure 3).

**6. ReGeneraTing Agents** 

**6.1 Structure of RGTA** 


Both standard and advanced wound healing strategies frequently aim to recreate a bioactive extracellular matrix. In the remainder of this overview, the focus is on the constituents of the extracellular matrix that secures the stability of growth factors in the matrix. Their role in enabling tissue homeostasis and tissue regeneration is discussed.

### **5. Tissue homeostasis and the extracellular matrix**

Proteoglycans consist of a core protein with one or more covalently linked glycosaminoglycan chains. Glycosaminoglycans are long chain, high molecular weight carbohydrates. Some of these are sulfated (chondroitin sulfate, dermatan sulfate and heparan sulfate), other are non-sulfated (hyaluronic acid) (McGrath and Eady, 1997). When combined with water glycosaminoglycans form a gel and contribute to the viscoeleastic properties of connective tissue. In addition to this mechanical role, proteoglycans may also have regulatory roles of which heparan sulfate is a prominent example.

Heparan sulfates are linear polysaccharides with variable degrees of sulfation, N-sulfation and N-acetylation (Dreyfuss, Regatieri et al. 2009) (Tumova, Woods et al. 2000). Heparan sulfates are widely spread throughout the animal kingdom ranging from invertebrates to mammals. In organs and tissues, they are a ubiquitous part of the extracellular matrix since many of the matrix scaffold proteins, such as collagens, fibronectin and laminin, possess heparan sulfate binding sites (Dreyfuss, Regatieri et al. 2009).

A large variety of proteins can bind heparan sulfates. Amongst these are cell surface proteins, extracellular matrix proteins, growth factors, cytokines, chemokines and morphogens. Heparan sulfates protect these proteins from degradation and secure their presence in the extracellular matrix. Due to the ubiquitous nature of heparan sulfates, the large amount of proteins they sequester and their fine-tuning effect in growth factor bioavailability, heparan sulfates participate in many physiological activities (e.g. cell proliferation, -migration, -differentiation and cell - cell interaction). And, therefore, play a prominent role in enabling tissue homeostasis, the process in which the tissues and organs pursue a constant internal environment and cellular composition (Watt and Fujiwara 2011).

However, the tissues response to 'stress', with stress being any form of integrity disturbance such as injury, inflammation, overuse, auto-immune response, is in releasing cleavage enzymes that degrade the proteins and glycosaminoglycans of the extracellular matrix, including heparan sulfates. Through this degradation, the orchestrating role of heparan sulfate in growth factor and cytokine sequestration is lost which ends tissue homeostasis (Barritault, Garcia-Filipe et al. 2010).

In wound healing, particularly in chronic wounds, dysregulation within the extracellular matrix and between cells and the extracellular matrix has gained importance (Cook, Davies et al. 2000) and do therapeutic initiatives to restore the defective extracellular matrix and to reposition heparan sulfates (Agren and Werthen 2007; Gandhi and Mancera 2010).

### **6. ReGeneraTing Agents**

#### **6.1 Structure of RGTA**

78 Tissue Regeneration – From Basic Biology to Clinical Application

As most biological products are expensive the lack of cost-effectiveness studies may

Many sources of the biological products are from human or animal tissues, which hold

Both standard and advanced wound healing strategies frequently aim to recreate a bioactive extracellular matrix. In the remainder of this overview, the focus is on the constituents of the extracellular matrix that secures the stability of growth factors in the matrix. Their role in

Proteoglycans consist of a core protein with one or more covalently linked glycosaminoglycan chains. Glycosaminoglycans are long chain, high molecular weight carbohydrates. Some of these are sulfated (chondroitin sulfate, dermatan sulfate and heparan sulfate), other are non-sulfated (hyaluronic acid) (McGrath and Eady, 1997). When combined with water glycosaminoglycans form a gel and contribute to the viscoeleastic properties of connective tissue. In addition to this mechanical role, proteoglycans may also

Heparan sulfates are linear polysaccharides with variable degrees of sulfation, N-sulfation and N-acetylation (Dreyfuss, Regatieri et al. 2009) (Tumova, Woods et al. 2000). Heparan sulfates are widely spread throughout the animal kingdom ranging from invertebrates to mammals. In organs and tissues, they are a ubiquitous part of the extracellular matrix since many of the matrix scaffold proteins, such as collagens, fibronectin and laminin, possess

A large variety of proteins can bind heparan sulfates. Amongst these are cell surface proteins, extracellular matrix proteins, growth factors, cytokines, chemokines and morphogens. Heparan sulfates protect these proteins from degradation and secure their presence in the extracellular matrix. Due to the ubiquitous nature of heparan sulfates, the large amount of proteins they sequester and their fine-tuning effect in growth factor bioavailability, heparan sulfates participate in many physiological activities (e.g. cell proliferation, -migration, -differentiation and cell - cell interaction). And, therefore, play a prominent role in enabling tissue homeostasis, the process in which the tissues and organs pursue a constant internal environment and cellular composition (Watt and Fujiwara

However, the tissues response to 'stress', with stress being any form of integrity disturbance such as injury, inflammation, overuse, auto-immune response, is in releasing cleavage enzymes that degrade the proteins and glycosaminoglycans of the extracellular matrix, including heparan sulfates. Through this degradation, the orchestrating role of heparan sulfate in growth factor and cytokine sequestration is lost which ends tissue homeostasis

In wound healing, particularly in chronic wounds, dysregulation within the extracellular matrix and between cells and the extracellular matrix has gained importance (Cook, Davies



2011).

(Barritault, Garcia-Filipe et al. 2010).

limit their widespread application.

the, theoretical, risk of transmitting infection and diseases.

enabling tissue homeostasis and tissue regeneration is discussed.

have regulatory roles of which heparan sulfate is a prominent example.

**5. Tissue homeostasis and the extracellular matrix** 

heparan sulfate binding sites (Dreyfuss, Regatieri et al. 2009).

ReGeneraTing Agents (RGTAs) are synthetic heparan sulfate mimics, resistant to glycanase digestion (Figure 2) (Barbier-Chassefiere, Garcia-Filipe et al. 2009; Ikeda, Charef et al. 2011).

Fig. 2. RGTA OTR4120 is a structural analogue of glycosaminoglycans.

#### **6.2 Working concept of RGTA / RGTA OTR4120**

In wound areas, RGTAs can replace heparan sulfates by binding the free heparan binding sites that become available following heparan sulfate degradation. This way, RGTAs can regulate the bioavailability of the large variety of, locally synthesized, heparin binding proteins which allow the cellular tissue components to re-unfold their natural mechanism to achieve wound regeneration.

RGTA OTR4120 is a RGTA member specifically designed to treat chronic wounds and marketed as CACIPLIQ20® (OTR3, Paris, France). The affinity constant of RGTA OTR4120 towards the vacant heparan sulfate binding sites of the extracellular matrix proteins allows a tight binding. This makes a short-term exposure to RGTA OTR4120 sufficient. Once RGTA OTR4120 is in place in the matrix scaffold, the growth factors, cytokines and other heparin binding signaling peptides can be repositioned through RGTA OTR4120 binding in this restored micro-environment. In this way, RGTA OTR4120 is thought to offer a matrix therapy that restores the natural cellular microenvironment. This allows the endogenous signaling of cell communications needed for tissue regeneration to resume their original function thereby halting the self-perpetuating cycles, particularly in impaired healing wounds (Figure 3).

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 81

applications should be timed as the number of free heparan binding sites are limited in wound tissue. RGTA OTR4120 administration every 3 days is proven sufficient to maintain the healing effect in the early phases. Excess RGTA OTR4120 may compete with sites on the matrix-bound RGTA for heparan binding growth factors. In following phases of wound regeneration a weekly administration also proofed to be effective (Tong, Zbinden et al. 2008; Barbier-Chassefiere, Garcia-Filipe et al. 2009). No specific studies are reported to further

Table 4. Polysaccharide digestion by endoglycanases. HM4120 = RGTA OTR4120

Effects of RGTA OTR4120 administration on tissue regeneration at the preclinical level are numerous and reported in over 70 scientific reports in close to 10 animal species. Dermal effects of RGTA OTR4120 administration are described in necrotic skin ulcers in mice (Barbier-Chassefiere, Garcia-Filipe et al. 2009), incisional dermal wounds in rats (Barritault, Garcia-Filipe et al. 2010), second degree burns in rats (Garcia-Filipe, Barbier-Chassefiere et al. 2007) (Zakine, Barbier et al. 2011), a rat surgical excision model (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009; Tong, Tuk et al. 2011) and in rat dermal ischemia ulcers (Tong, Tuk et al. 2011).

Copyright 2010 Elsevier, Ltd.

Barbier-Chassefiere et al. (2009) determined the effects of RGTA OTR4120 in a necrotic skin ulcer model in mice following doxorubicin administration. In the short term, the necrotic surface area was found to be decreased by 40% with an almost 50% reduction in leukocyte count, as a measure for the strength of the inflammatory reaction. RGTA OTR4120 administration increased type I and decreased type III collagen which restoring these values to those found in normal skin (Barbier-Chassefiere, Garcia-Filipe et al. 2009). The results obtained from this ulcer model indicate that RGTA OTR4120 matrix therapy can initiate tissue regeneration. This finding illustrates that matrix contains the proper information to regenerate and confirms the central role for a good-quality extracellular matrix in tissue

**6.4 Preclinical evidence for dermal RGTA OTR4120 actions** 

regeneration (Barbier-Chassefiere, Garcia-Filipe et al. 2009).

optimize the timing of RGTA OTR4120 application.

(Ikeda, Charef et al. 2011)

**6.4.1 Necrotic skin ulcers** 

Fig. 3. RGTA OTR4120, mode of action.

#### **6.3 Synthesis, dosing and timing**

RGTA OTR4120 is prepared as a 85kD molecular weight fraction from T40 dextran by carboxymethylation and O-sulfonation (Barbier-Chassefiere, 2009; Ikeda, Charef et al. 2011). RGTA OTR4120 was proven completely resistant to digestion with multiple endoglycanases: heparanase, chondroitinase, hyaluronidase, dextranase (Table 4) (Ikeda, Charef et al. 2011).

Following a single i.v. injection in mice at a dose of 5 mg/kg the half life of unbound OTR4120 in plasma was less than 60 minutes. A i.p. bolus injection of 50 mg/kg in this same model created a peak plasma concentration of 88 µg/ml after 90 min (Charef, Papy-Garcia et al. 2010). A study on the acute and subacute toxicity, following i.p. administration, revealed no significant toxicological changes for doses up to 50 mg/kg (Charef, Tulliez et al. 2010). Injected doses used in a preclinical setting routinely are in the range of 1 – 2 mg/kg.

When topically administered on dermal wounds, a bell-shaped dose effect-curve was found for RGTA OTR4120 with an optimal dose of 0.1 mg/ml. A similar dose was found when treating skull defects (Colombier, Lafont et al. 1999) and parodontitis (Lallam-Laroye, Escartin et al. 2006).

RGTA OTR4120 is by itself a non acting molecule that enables the cascade of signals, that propagate wound regeneration, to resume with proper timing. However, the frequency of applications should be timed as the number of free heparan binding sites are limited in wound tissue. RGTA OTR4120 administration every 3 days is proven sufficient to maintain the healing effect in the early phases. Excess RGTA OTR4120 may compete with sites on the matrix-bound RGTA for heparan binding growth factors. In following phases of wound regeneration a weekly administration also proofed to be effective (Tong, Zbinden et al. 2008; Barbier-Chassefiere, Garcia-Filipe et al. 2009). No specific studies are reported to further optimize the timing of RGTA OTR4120 application.


Copyright 2010 Elsevier, Ltd.

Table 4. Polysaccharide digestion by endoglycanases. HM4120 = RGTA OTR4120 (Ikeda, Charef et al. 2011)

#### **6.4 Preclinical evidence for dermal RGTA OTR4120 actions**

Effects of RGTA OTR4120 administration on tissue regeneration at the preclinical level are numerous and reported in over 70 scientific reports in close to 10 animal species. Dermal effects of RGTA OTR4120 administration are described in necrotic skin ulcers in mice (Barbier-Chassefiere, Garcia-Filipe et al. 2009), incisional dermal wounds in rats (Barritault, Garcia-Filipe et al. 2010), second degree burns in rats (Garcia-Filipe, Barbier-Chassefiere et al. 2007) (Zakine, Barbier et al. 2011), a rat surgical excision model (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009; Tong, Tuk et al. 2011) and in rat dermal ischemia ulcers (Tong, Tuk et al. 2011).

#### **6.4.1 Necrotic skin ulcers**

80 Tissue Regeneration – From Basic Biology to Clinical Application

RGTA OTR4120 is prepared as a 85kD molecular weight fraction from T40 dextran by carboxymethylation and O-sulfonation (Barbier-Chassefiere, 2009; Ikeda, Charef et al. 2011). RGTA OTR4120 was proven completely resistant to digestion with multiple endoglycanases: heparanase, chondroitinase, hyaluronidase, dextranase (Table 4) (Ikeda,

Following a single i.v. injection in mice at a dose of 5 mg/kg the half life of unbound OTR4120 in plasma was less than 60 minutes. A i.p. bolus injection of 50 mg/kg in this same model created a peak plasma concentration of 88 µg/ml after 90 min (Charef, Papy-Garcia et al. 2010). A study on the acute and subacute toxicity, following i.p. administration, revealed no significant toxicological changes for doses up to 50 mg/kg (Charef, Tulliez et al. 2010).

When topically administered on dermal wounds, a bell-shaped dose effect-curve was found for RGTA OTR4120 with an optimal dose of 0.1 mg/ml. A similar dose was found when treating skull defects (Colombier, Lafont et al. 1999) and parodontitis (Lallam-Laroye,

RGTA OTR4120 is by itself a non acting molecule that enables the cascade of signals, that propagate wound regeneration, to resume with proper timing. However, the frequency of

Injected doses used in a preclinical setting routinely are in the range of 1 – 2 mg/kg.

Fig. 3. RGTA OTR4120, mode of action.

**6.3 Synthesis, dosing and timing** 

Copyright 2011 by OTR3, SAS

Charef et al. 2011).

Escartin et al. 2006).

Barbier-Chassefiere et al. (2009) determined the effects of RGTA OTR4120 in a necrotic skin ulcer model in mice following doxorubicin administration. In the short term, the necrotic surface area was found to be decreased by 40% with an almost 50% reduction in leukocyte count, as a measure for the strength of the inflammatory reaction. RGTA OTR4120 administration increased type I and decreased type III collagen which restoring these values to those found in normal skin (Barbier-Chassefiere, Garcia-Filipe et al. 2009). The results obtained from this ulcer model indicate that RGTA OTR4120 matrix therapy can initiate tissue regeneration. This finding illustrates that matrix contains the proper information to regenerate and confirms the central role for a good-quality extracellular matrix in tissue regeneration (Barbier-Chassefiere, Garcia-Filipe et al. 2009).

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 83

Fig. 5. Effect of OTR4120 on the relative proportions of collagen I and collagen III in control skin and burn sites. Three experimental groups were studied: control (healthy skin from untreated rats), burn sites from untreated rats, and burn sites from rats treated with OTR4120. Figure shows the ratio of collagen III over collagen I (fibrotic index) computed using the data in Figures 1 (A,B). Differences compared with control were evaluated using Student's paired t test; \*p values < 0.05 were considered significant (Garcia-Filipe, Barbier-

Copyright 2006 Wiley Periodicals, Inc.

Fig. 6. Evolution of the mean number of keratinocyte layers. RGTA administration increases the mean number of keratinocyte layers between day 3 and day 7 (*above*) in comparison with the control group (*below*). At day 1 and day 2, this number was 0; on and after day 60, this

Copyright 2011 by the American Society of Plastic Surgeons.

Chassefiere et al. 2007).

number was 6. (Zakine, Barbier et al. 2011)

#### **6.4.2 Anti scar effects of RGTA**

Barritault et al. (2010) described the effects of RGTA OTR4120 administration to a dermal incisional wound model in hairless rats (Barritault, Garcia-Filipe et al. 2010). Topical administration of RGTA OTR4120 to the incision at days 0, 3 and 6 revealed a scar free healing at day 10 whereas in the placebo control scar formation was clearly present (Figure 4).

Copyright 2010 Elsevier Masson SAS.

Fig. 4. Macroscopic view of the scar, treated or not treated with RGTA OTR4120 after skin incision. The incision is induced at day 0. RGTA OTR4120 treatment is by topical application (0.1 mg/ml), using a cotton swab, at day 0 before closure by suture and at days 3 and 6. a: untreated skin incision (saline) at day 0; b: untreated skin incision (saline) at day 9; c: RGTA OTR4120 treated skin incision at day 0; d: RGTA OTR4120 treated skin incision at day 9. (Barritault, Garcia-Filipe et al. 2010)

Also severe dermal burns are characterized fibrosis and excessive scarring. On a cellular level aberrant proliferation, inflammation and a changed extracellular matrix architecture are important characteristics for dermal fibrosis. Especially the increased presence of type III collagen is thought to link to the extend of fibrosis (Ulrich, Noah et al. 2002). Ulrich et al. (2003) demonstrated a long term increased type III collagen in fibrous tissue (Ulrich, Noah et al. 2003). In this view, treatments that normalize type III collagen expression without compromising wound healing are of utmost importance. Garcia-Filipe et al. (2007) determined the effects of RGTA OTR4120 administration to a second degree experimental burn on the skin of a hairless rat (Garcia-Filipe, Barbier-Chassefiere et al. 2007). They observed a profoundly improved fibrotic index: this is the ratio of type III collagen over type I collagen. Normalization of the type III collagen / type I collagen ratio also lasted at their final experimental endpoint at 10 months and was caused by a decreased type III collagen production and created a collagen balance that resembled normal skin (Figure 5).

Barritault et al. (2010) described the effects of RGTA OTR4120 administration to a dermal incisional wound model in hairless rats (Barritault, Garcia-Filipe et al. 2010). Topical administration of RGTA OTR4120 to the incision at days 0, 3 and 6 revealed a scar free healing

at day 10 whereas in the placebo control scar formation was clearly present (Figure 4).

Fig. 4. Macroscopic view of the scar, treated or not treated with RGTA OTR4120 after skin incision. The incision is induced at day 0. RGTA OTR4120 treatment is by topical application (0.1 mg/ml), using a cotton swab, at day 0 before closure by suture and at days 3 and 6. a: untreated skin incision (saline) at day 0; b: untreated skin incision (saline) at day 9; c: RGTA OTR4120 treated skin incision at day 0; d: RGTA OTR4120 treated skin incision at

Copyright 2010 Elsevier Masson SAS.

Also severe dermal burns are characterized fibrosis and excessive scarring. On a cellular level aberrant proliferation, inflammation and a changed extracellular matrix architecture are important characteristics for dermal fibrosis. Especially the increased presence of type III collagen is thought to link to the extend of fibrosis (Ulrich, Noah et al. 2002). Ulrich et al. (2003) demonstrated a long term increased type III collagen in fibrous tissue (Ulrich, Noah et al. 2003). In this view, treatments that normalize type III collagen expression without compromising wound healing are of utmost importance. Garcia-Filipe et al. (2007) determined the effects of RGTA OTR4120 administration to a second degree experimental burn on the skin of a hairless rat (Garcia-Filipe, Barbier-Chassefiere et al. 2007). They observed a profoundly improved fibrotic index: this is the ratio of type III collagen over type I collagen. Normalization of the type III collagen / type I collagen ratio also lasted at their final experimental endpoint at 10 months and was caused by a decreased type III collagen production and created a collagen balance that resembled

**6.4.2 Anti scar effects of RGTA** 

day 9. (Barritault, Garcia-Filipe et al. 2010)

normal skin (Figure 5).

Fig. 5. Effect of OTR4120 on the relative proportions of collagen I and collagen III in control skin and burn sites. Three experimental groups were studied: control (healthy skin from untreated rats), burn sites from untreated rats, and burn sites from rats treated with OTR4120. Figure shows the ratio of collagen III over collagen I (fibrotic index) computed using the data in Figures 1 (A,B). Differences compared with control were evaluated using Student's paired t test; \*p values < 0.05 were considered significant (Garcia-Filipe, Barbier-Chassefiere et al. 2007).

Fig. 6. Evolution of the mean number of keratinocyte layers. RGTA administration increases the mean number of keratinocyte layers between day 3 and day 7 (*above*) in comparison with the control group (*below*). At day 1 and day 2, this number was 0; on and after day 60, this number was 6. (Zakine, Barbier et al. 2011)

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 85

inflammation controlled environment that progressed to the normal stages of wound

Fig. 7b. Wound strength measured as the ratio of the breaking strength of the wounded skin versus that of normal skin on days 14, 21, and 79 after wounding. Error bars represent the standard deviation. \*p < 0.05 and \*\*\*p < 0.001 indicate the significant differences between treated groups and control groups (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009).

Copyright 2008 by the Wound Healing Society

In summary: the ability of RGTA OTR4120 administration to reduce inflammation; increase angiogenesis; improved healing quality, amongst others reflected by the recurrence of normal dermal collagen balance and an increased tissue breaking strength have now been demonstrated in multiple models (Alexakis, Guettoufi et al. 2001; Alexakis, Caruelle et al. 2004; Alexakis, Mestries et al. 2004; Barbier-Chassefiere, Garcia-Filipe et al. 2009; Tong, Tuk et al. 2011). All are signs of matrix reconstruction to a formulation that more closely resembles normal matrix. Specifically, RGTA OTR4120 decreasing collagen III accumulation, will dramatically reduce the fibrosis that frequently accompanies wound healing. These

The first scientific report on the clinical use of RGTA OTR4120 was a study by Chebbi et al. (2008) who described the effects of local RGTA OTR4120 administration for one month to patients with treatment-resistant corneal-ulcers and to patients with treatment-resistant corneal dystrophy (Chebbi, Kichenin et al. 2008). RGTA OTR4120 administration resulted in the majority of cases in the complete healing of the ulcer. The effect on the keratitis was moderate, however, a significant pain reduction was observed that highly improved the

In a within-subject study, Groah et al. (2011) demonstrated the effect of RGTA OTR4120 administration in a patient population of largely persistent pressure ulcers and vascular/venous ulcers (Groah, Libin et al. 2011). The mean duration of the ulcers was 2.5

promising preclinical findings warrant studies on human subjects.

patient's quality of life (Chebbi, Kichenin et al. 2008).

**6.5 Clinical evidence** 

healing.

A study by Zakine et al. (2011), using this same model, revealed an increased reepithelialisation together with an increased number of keratinocyte layers and blood vessels in the RGTA OTR4120 administered group at the early stages of tissue regeneration that returned to control levels after 1 month post wounding (Figure 6) (Zakine, Barbier et al. 2011).

#### **6.4.3 Full-thickness excisional wounds**

Tong et al. studied the effects of RGTA OTR4120 administration to rat full-thickness excisional wounds (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009). RGTA OTR4120 administration to surgical wounds in rapid healing normal rats significantly improved wound regeneration. RGTA OTR4120 administration promotes epidermal proliferation, increased neodermal granulation tissue deposition, inflammation resolution, improved the vascular response to local heating (Figure 7a), improved collagen maturation and improved the wound breaking strength at all measurement times up to 3 months post wounding (Figure 7b) (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009).

Fig. 7a. The wounded skin and normal skin vascular responses to local heating (44°C for 10 minutes), expressed as the percentages of the increase blood flow over baseline flow, measured by laser Doppler flow with local heat provocation on days 13, 20, and 78 after wounding. Error bars represent the standard deviation. \*\**p*<0.01 and \*\*\**p* < 0.001 indicate the significant differences between treated groups and control groups. (Tong, Zbinden et al. 2008)

#### **6.4.4 Ischemia-reperfusion wounds**

Tong et al. (2011) also studied the effects of RGTA OTR4120 administration to ischemiareperfusion wounds (Tong, Tuk et al. 2011). Similar findings as for the excisional wounds were observed in a cutaneous ischemia-reperfusion model obtained by magnet clamping of a skin fold in the neck area of the animal although with a delayed timing due to the clearance of the necrotic tissue (Tong, Tuk et al. 2011). In addition, monocyte/macrophage staining and CD68 detection on Western blots revealed that RGTA OTR4120 administration facilitated an inflammation controlled environment that progressed to the normal stages of wound healing.

Fig. 7b. Wound strength measured as the ratio of the breaking strength of the wounded skin versus that of normal skin on days 14, 21, and 79 after wounding. Error bars represent the standard deviation. \*p < 0.05 and \*\*\*p < 0.001 indicate the significant differences between treated groups and control groups (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009).

In summary: the ability of RGTA OTR4120 administration to reduce inflammation; increase angiogenesis; improved healing quality, amongst others reflected by the recurrence of normal dermal collagen balance and an increased tissue breaking strength have now been demonstrated in multiple models (Alexakis, Guettoufi et al. 2001; Alexakis, Caruelle et al. 2004; Alexakis, Mestries et al. 2004; Barbier-Chassefiere, Garcia-Filipe et al. 2009; Tong, Tuk et al. 2011). All are signs of matrix reconstruction to a formulation that more closely resembles normal matrix. Specifically, RGTA OTR4120 decreasing collagen III accumulation, will dramatically reduce the fibrosis that frequently accompanies wound healing. These promising preclinical findings warrant studies on human subjects.

#### **6.5 Clinical evidence**

84 Tissue Regeneration – From Basic Biology to Clinical Application

A study by Zakine et al. (2011), using this same model, revealed an increased reepithelialisation together with an increased number of keratinocyte layers and blood vessels in the RGTA OTR4120 administered group at the early stages of tissue regeneration that returned to control levels after 1 month post wounding (Figure 6) (Zakine, Barbier et al. 2011).

Tong et al. studied the effects of RGTA OTR4120 administration to rat full-thickness excisional wounds (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009). RGTA OTR4120 administration to surgical wounds in rapid healing normal rats significantly improved wound regeneration. RGTA OTR4120 administration promotes epidermal proliferation, increased neodermal granulation tissue deposition, inflammation resolution, improved the vascular response to local heating (Figure 7a), improved collagen maturation and improved the wound breaking strength at all measurement times up to 3 months post wounding

**6.4.3 Full-thickness excisional wounds** 

(Tong, Zbinden et al. 2008)

**6.4.4 Ischemia-reperfusion wounds** 

(Figure 7b) (Tong, Zbinden et al. 2008; Tong, Tuk et al. 2009).

Fig. 7a. The wounded skin and normal skin vascular responses to local heating

indicate the significant differences between treated groups and control groups.

(44°C for 10 minutes), expressed as the percentages of the increase blood flow over baseline flow, measured by laser Doppler flow with local heat provocation on days 13, 20, and 78 after wounding. Error bars represent the standard deviation. \*\**p*<0.01 and \*\*\**p* < 0.001

Copyright 2008 by the Wound Healing Society

Tong et al. (2011) also studied the effects of RGTA OTR4120 administration to ischemiareperfusion wounds (Tong, Tuk et al. 2011). Similar findings as for the excisional wounds were observed in a cutaneous ischemia-reperfusion model obtained by magnet clamping of a skin fold in the neck area of the animal although with a delayed timing due to the clearance of the necrotic tissue (Tong, Tuk et al. 2011). In addition, monocyte/macrophage staining and CD68 detection on Western blots revealed that RGTA OTR4120 administration facilitated an The first scientific report on the clinical use of RGTA OTR4120 was a study by Chebbi et al. (2008) who described the effects of local RGTA OTR4120 administration for one month to patients with treatment-resistant corneal-ulcers and to patients with treatment-resistant corneal dystrophy (Chebbi, Kichenin et al. 2008). RGTA OTR4120 administration resulted in the majority of cases in the complete healing of the ulcer. The effect on the keratitis was moderate, however, a significant pain reduction was observed that highly improved the patient's quality of life (Chebbi, Kichenin et al. 2008).

In a within-subject study, Groah et al. (2011) demonstrated the effect of RGTA OTR4120 administration in a patient population of largely persistent pressure ulcers and vascular/venous ulcers (Groah, Libin et al. 2011). The mean duration of the ulcers was 2.5

Heparan Sulfate Proteoglycan Mimetics Promote Tissue Regeneration: An Overview 87

(Figure 8B). Healthy granulation tissue formed and the wound closed in the 5th week of treatment (Figure 8C-E). Further documentation of the wound area proved it remained

The findings presented support the use of RGTA OTR4120 (CACIPLIQ20®) in treating

Research to reduce the complexity of the molecule, to facilitate its synthesis and to increase its effectiveness, is ongoing (Ikeda, Charef et al. 2011). Also oral administration, as a patient friendly means of administration with a RGTA OTR4120 delivery to the side of injury via

Matrix therapy restores the natural extracellular microenvironment which allows the local cascade of signals to resume in the proper time and order to trigger tissue regeneration. (Barbier-Chassefiere, Garcia-Filipe et al. 2009). Therefore, matrix therapy with engineered biopolymers such as RGTA OTR4120 is simpler and easier to use than cell or gene therapy

Mr Ronald Lau, Amersfoort, the Netherlands, is thanked for his dedication while applying

Agren, M. S. and M. Werthen (2007). "The extracellular matrix in wound healing: a closer look at therapeutics for chronic wounds." *Int J Low Extrem Wounds* 6(2): 82-97. Alexakis, C., J. P. Caruelle, et al. (2004). "Reversal of abnormal collagen production in

Alexakis, C., A. Guettoufi, et al. (2001). "Heparan mimetic regulates collagen expression and

Alexakis, C., P. Mestries, et al. (2004). "Structurally different RGTAs modulate collagen-type

Attinger, C. E. and E. J. Bulan (2001). "Debridement. The key initial first step in wound

Crohn's disease intestinal biopsies treated with regenerating agents." *Gut* 53(1): 85-

TGF-beta1 distribution in gamma-irradiated human intestinal smooth muscle

expression by cultured aortic smooth muscle cells via different pathways involving fibroblast growth factor-2 or transforming growth factor-beta1." *FASEB J* 18(10):

(chronic) wounds by means of restoring the damaged extracellular matrix.

(micro)circulation, are tested (Charef, Papy-Garcia et al. 2010)

and is a new alternative in regenerative medicine.

cells." *FASEB J* 15(9): 1546-1554.

healing." *Foot Ankle Clin* 6(4): 627-660.

The majority of the results presented here were published.

CACIPLIQ20® to a diabetic foot patient.

completely healed (Figure 8F).

**6.6 Future perspectives** 

**7. Concluding remarks** 

**8. Acknowledgement** 

**9. References** 

90.

1147-1149.

years. RGTA OTR4120 was administered on the debrided wound, twice weekly for 5 minutes each time. After 4 weeks both a significant reduction in the wound size as well as in the pain perception was found (Groah, Libin et al. 2011).

Van Neck et al. (2011) described the complete healing following of recurrent scalp ulcers RGTA OTR4120 administration (Van Neck, Zuidema et al. 2011).

RGTA OTR4120 also was administered to a 60-year old male patient with a complex medical history. He suffered from obesity (BMI 35), developed type II diabetic over a decade ago and was on insulin treatment. Furthermore, he was known with alcohol and nicotine abuse, heart failure, pacemaker, cardiac and vascular disease and kidney and liver failure. In 1992 and 2002 he has had several toe amputations on both his feet likely caused by his poor cardiac and vascular condition. The patient had developed the diabetic pressure ulcer under investigation on the palmar side of his right foot over 8 months ago. So far, he had been unsuccessfully treated with a wide variety of wound dressings (foams, alginate, hydro colloids, silver dressings, foils, impregnated gauzes and collagen) all applied up to three times weekly if needed. At the start of the OTR4120 treatment the wound measured 2.5 cm2 (Figure 8A). The wound displayed an almost immediate response to OTR4120 treatment

Fig. 8. Diabetic foot of a 60 year old male patient in a state of non-healing for over 8 months. A) day 0, the start of the twice weekly RGTA OTR4120 topical application. At this stage the wound measured 2.5 cm2; B) day 7, healthy granulation tissue immediately formed following RGTA OTR4120 treatment; C) day 16; D) day 31, near to complete wound closure; E) day 46; F) day 56, long term closure control. The inner dimensions of the grey shaded square are 30x30mm.

(Figure 8B). Healthy granulation tissue formed and the wound closed in the 5th week of treatment (Figure 8C-E). Further documentation of the wound area proved it remained completely healed (Figure 8F).

#### **6.6 Future perspectives**

86 Tissue Regeneration – From Basic Biology to Clinical Application

years. RGTA OTR4120 was administered on the debrided wound, twice weekly for 5 minutes each time. After 4 weeks both a significant reduction in the wound size as well as in

Van Neck et al. (2011) described the complete healing following of recurrent scalp ulcers

RGTA OTR4120 also was administered to a 60-year old male patient with a complex medical history. He suffered from obesity (BMI 35), developed type II diabetic over a decade ago and was on insulin treatment. Furthermore, he was known with alcohol and nicotine abuse, heart failure, pacemaker, cardiac and vascular disease and kidney and liver failure. In 1992 and 2002 he has had several toe amputations on both his feet likely caused by his poor cardiac and vascular condition. The patient had developed the diabetic pressure ulcer under investigation on the palmar side of his right foot over 8 months ago. So far, he had been unsuccessfully treated with a wide variety of wound dressings (foams, alginate, hydro colloids, silver dressings, foils, impregnated gauzes and collagen) all applied up to three times weekly if needed. At the start of the OTR4120 treatment the wound measured 2.5 cm2 (Figure 8A). The wound displayed an almost immediate response to OTR4120 treatment

Fig. 8. Diabetic foot of a 60 year old male patient in a state of non-healing for over 8 months. A) day 0, the start of the twice weekly RGTA OTR4120 topical application. At this stage the wound measured 2.5 cm2; B) day 7, healthy granulation tissue immediately formed

following RGTA OTR4120 treatment; C) day 16; D) day 31, near to complete wound closure; E) day 46; F) day 56, long term closure control. The inner dimensions of the grey shaded

square are 30x30mm.

the pain perception was found (Groah, Libin et al. 2011).

RGTA OTR4120 administration (Van Neck, Zuidema et al. 2011).

The findings presented support the use of RGTA OTR4120 (CACIPLIQ20®) in treating (chronic) wounds by means of restoring the damaged extracellular matrix.

Research to reduce the complexity of the molecule, to facilitate its synthesis and to increase its effectiveness, is ongoing (Ikeda, Charef et al. 2011). Also oral administration, as a patient friendly means of administration with a RGTA OTR4120 delivery to the side of injury via (micro)circulation, are tested (Charef, Papy-Garcia et al. 2010)

#### **7. Concluding remarks**

Matrix therapy restores the natural extracellular microenvironment which allows the local cascade of signals to resume in the proper time and order to trigger tissue regeneration. (Barbier-Chassefiere, Garcia-Filipe et al. 2009). Therefore, matrix therapy with engineered biopolymers such as RGTA OTR4120 is simpler and easier to use than cell or gene therapy and is a new alternative in regenerative medicine.

#### **8. Acknowledgement**

Mr Ronald Lau, Amersfoort, the Netherlands, is thanked for his dedication while applying CACIPLIQ20® to a diabetic foot patient.

The majority of the results presented here were published.

#### **9. References**


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134.


**5** 

*Brazil* 

**Angiogenesis in Wound Healing** 

Angiogenesis, the formation of new blood vessels from pre-existing, vessels is a crucial process for tumor growth and metastasis (Folkman 1990; Kaafarani, Fernandez-Sauze et al. 2009). The new vessels supply the tumor cells with nutrients and oxygen and ensure efficient drainage of metabolites. Under normal conditions, a tissue or tumor cannot grow beyond 1 to 2 mm in diameter without neovascularization. This distance is defined by limits in the diffusion of oxygen and metabolites, such as glucose and amino acids (Folkman 1971). In addition to supplying nutrients for tumor growth, angiogenesis is also a gateway for tumor cells and signals to the bloodstream. This direct communication with the bloodstream is essential for the dissemination and metastasis of cancer. After their arrival and deployment in distant organs, metastatic cells again induce angiogenesis in order to support

As well as this important role of angiogenesis in tumor growth, the whole process of tissue regeneration depends on a new intake of oxygen and metabolites. Growth of new cells for regeneration involves a large energy demand that occurs for the process of cellular mitosis. Therefore, understanding the biochemical mechanisms involved in angiogenesis is

Since the hypothesis proposed by the surgeon Judah Folkman in the early 70's, which indicated that the inhibition of angiogenesis as a therapeutic target that could halt or even reduce tumor growth (Folkman 1971), intense and successful research on the molecular mechanisms of angiogenesis tumor began. In recent decades, numerous pro- and antiangiogenic molecules, as well as their ligands and intracellular signaling pathways, have

The main aim of wound treatment is achieving a rapid closure of the lesion combined with a functional and aesthetically satisfactory scar. To improve current practice, it is essential to gain a better understanding of the biological processes involved in wound healing and tissue regeneration. Many studies have investigated the complex process of wound repair, and the cell behaviors, chemical signals and extracellular matrices that together lead to

necessary for developing interventions in complex tissue regeneration processes.

**1. Introduction** 

been identified.

scarring.

**2. The wound healing process** 

tumor growth (Eichhorn, Kleespies et al. 2007).

Ricardo José de Mendonça *Department of Biological Sciences Federal University of Triangulo Mineiro* 

