**4. Tissue engineering and regenerative medicine**

Every day thousands of clinical procedures are performed to replace or repair tissues in the human body that have been damaged through disease or trauma that use tissue engineering technology. The use of constructs for tissue engineering (TE) and regenerative medicine are promising innovative therapies that can address several clinical situations. These constructs are often combination of cells, scaffolds and biological factors. Although there are only a few commercial products currently in the market for cell/drug delivery, probably because each type of cell requires its own specific encapsulating microenvironment with cell-specific ma‐ terial properties and spatially controlled bioactive features, a vast amount of research is be‐ ing performed worldwide on all aspects of tissue engineering/regenerative medicine exploring polymer materials. To implant cells into defective skeletal muscles, there are two main techniques. The cellular system may be directly injected into the scaffold which is lo‐ calized in the injury site. It can also be performed by pre-adding the cells to the scaffold via injection or co-culture (in most of the cellular systems, cells are allowed to form a monolay‐ er) and then the biomaterial with the cellular system is implanted in the injured muscle. In case of multiple sites of injury, the systemic administration of cells capable of reaching dam‐ aged tissues would be an interesting alternative [64].

#### **4.1. – Scaffolds and Biomaterials**

Scaffolds, which are used to deliver cells, drugs, and genes into the body, can take on vari‐ ous forms from porous solid devices to injectable networks, such as a typical three-dimen‐ sional porous matrix, a nanofibrous matrix, a hydrogel, and microspheres. Although solid scaffold provide a mechanically strong matrix for seeded cells, hydrogel scaffolds and mi‐ crospheres are becoming increasingly popular in TE. The spherical nature maximizes the surface area, and the small volume of beads facilitating biomolecular transport. Regarding hydrogels, they have a similar microstructure to the extracellular matrix (ECM) and allow good physical integration into the defect by the use of minimally invasive approaches for material and cell/drug delivery. The biological, chemical, topography features and mechani‐ cal properties, as well as the degradation kinetics of hydrogels, can be tailored depending on the application [65-68]. Aligned nanoscale and microscale topographic features in scaffolds have been also reported to influence the alignment of cells. For example, this alignment is an important requirement of functional skeletal muscle since it leads to alignment of myoblasts and cytoskeletal proteins and promote myotube assembly along the nanofibres and micro‐ grooves to mimic the myotube organization in muscle fibres [65, 68-70]. Scaffolds are used successfully in various fields of tissue engineering such as bone formation, periodontal re‐ generation, cartilage development, as artificial corneas, in tendon repair and in ligament re‐ placement. In addition, the incorporation of drugs (i.e., inflammatory inhibitors and/or antibiotics) into scaffolds or specific molecules to provide adequate signals to the cells is also possible [71] Depending on the medical applications, scaffolds requirements will depend on its function. Hydrogels can be used as a physical barrier to protect the cells from hostile ex‐ trinsic factors before delivery, or be used as a matrix to drug controlled release or cell adhe‐ sion, growth and differentiation to further improve the secretion of therapeutic proteins from cells. In fact cells are capable of delivering drugs in response to an external stimulus, which is highly advantageous to maintain homeostasis for patients suffering from chronic diseases. For the first application, the scaffold needs:

Very promising results were obtained with chitosan type III membranes and MSCs isolated from the umbilical cord matrix. In addition, we also perform kinematic analysis of the rat walk which is a more sensitive behavioral test. This analysis is increasingly being used to assess functional recovery in peripheral nerve research because of its higher accuracy and better relationship with histological outcome [57-63]. We should bare in mind that locomo‐ tion is also of higher functional relevance since it involves integrated function of both the motor and sensory systems and their respective components, such as skeletal muscles, sen‐ sory endings, efferent and afferent nerve fibers and integrative centers within the central nervous system. Muscles innervated by sciatic nerve branches include both dorsiflexors and plantarflexors and, although in our published studies we focused our kinematic analysis on‐ ly in the stance phase, we now prefer to include analysis of the ankle joint motion also dur‐ ing the swing phase in order to provide additional information [59]. Denervation can be a very useful model of skeletal muscle injury for some experimental studies but some limita‐ tions might be pointed out in studies that attempt to focus exclusively on the muscular re‐ generation process. Nevertheless and as demonstrated by several studies, this muscular regeneration process is highly dependent of the neural supply and the nerve regeneration

Every day thousands of clinical procedures are performed to replace or repair tissues in the human body that have been damaged through disease or trauma that use tissue engineering technology. The use of constructs for tissue engineering (TE) and regenerative medicine are promising innovative therapies that can address several clinical situations. These constructs are often combination of cells, scaffolds and biological factors. Although there are only a few commercial products currently in the market for cell/drug delivery, probably because each type of cell requires its own specific encapsulating microenvironment with cell-specific ma‐ terial properties and spatially controlled bioactive features, a vast amount of research is be‐ ing performed worldwide on all aspects of tissue engineering/regenerative medicine exploring polymer materials. To implant cells into defective skeletal muscles, there are two main techniques. The cellular system may be directly injected into the scaffold which is lo‐ calized in the injury site. It can also be performed by pre-adding the cells to the scaffold via injection or co-culture (in most of the cellular systems, cells are allowed to form a monolay‐ er) and then the biomaterial with the cellular system is implanted in the injured muscle. In case of multiple sites of injury, the systemic administration of cells capable of reaching dam‐

Scaffolds, which are used to deliver cells, drugs, and genes into the body, can take on vari‐ ous forms from porous solid devices to injectable networks, such as a typical three-dimen‐ sional porous matrix, a nanofibrous matrix, a hydrogel, and microspheres. Although solid scaffold provide a mechanically strong matrix for seeded cells, hydrogel scaffolds and mi‐

itself can be influenced by the damaged muscle tissue.

340 Advances in Biomaterials Science and Biomedical Applications

aged tissues would be an interesting alternative [64].

**4.1. – Scaffolds and Biomaterials**

**4. Tissue engineering and regenerative medicine**


For the second applications further requirements are needed, mainly:


#### *4.1.1. Hydrogel scaffolds*

Hydrogels, three-dimensional (3D) networks of hydrophilic polymers, are appealing for bio‐ logical applications because of their high water content, high permeability, biocompatibility, and the ability of be placed into critical defects in a minimally invasive manner [78]. They are being used in a wide range of tissues, including cartilage, bone, muscle, fat, liver, and neurons. For use in drug/cell delivery, hydrogels should be low-viscosity solutions prior to gelling, which is crucial to maintain cell viability during the encapsulation process, and should rapidly gel in the human body. These properties can be fine-tuned through varia‐ tions in the chemical structure and cross-linking density in hydrogels. Injectable hydrogels can be formed *in situ* by either chemical or physical cross-linking methods [79, 80]. Physical cross-linked hydrogels are capable of phase transition in response to external stimuli such as temperature, pH or both [81]. Chemically cross-linked hydrogels are prepared through pho‐ topolymerization, disulfide bond formation, or reaction between thiols and acrylate or sul‐ fone. The latter hydrogels undergo significant volume changes compared to the first ones [81-83]. The pH/temperature-sensitive hydrogels show several advantages over thermo-sen‐ sitive ones, such as the absence of clogging during injection and avoidance of pH decreased caused by degradation. The pH/temperature-sensitive copolymer hydrogels can be pre‐ pared by combining a pH-sensitive moiety with a temperature-sensitive block. For example, if acidic sulfamethazine oligomers (OSMs) are coupled with thermosensitive poly(e-CL-co-LA)-PEG-poly-(e-CL-co-LA) triblock copolymers a pH/temperature-sensitive hydrogels (OSM-PCLA-PEG-PCLA-OSM) is produced. Photopolymerized hydrogel systems have been reported to provide better temporal and spatial control over the gelation process [79, 81, 84].

#### *4.1.2. Biomaterials for scaffold fabrication*

A wide variety of natural and synthetic materials have been used to prepare injectable hy‐ drogels. Natural polymers, which are either components of or have macromolecular proper‐ ties similar to the natural ECMs, are known to often undergo rapid degradation upon contact with body fluids or medium and show batch-to-batch variation. Synthetic hydrogels offer improved control of the matrix architecture and chemical composition, no immunoge‐ nicity, consistent supply of large quantities, but tend to have lower biological activity. Therefore, modification of natural and synthetic derived hydrogels is usually required [79]. A natural biodegradable 3D scaffold can be made of acellular muscle ECM but it's fragile and difficult to handle [85]. Another natural biodegradable scaffold can be created by using fibrin, which leads to a process much similar to wound healing, in which fibrin forms a tem‐ porary scaffold to serve tissue regeneration and then is replaced by the physiological ECM. Fibrin has the additional advantage that it binds growth factors [70].

**iii.** bioadhesion, to allows cells and tissues to adhere to scaffolds. Some hydrogels such

**iv.** the mechanical properties of the scaffold, commonly controlled with the polymer

Hydrogels, three-dimensional (3D) networks of hydrophilic polymers, are appealing for bio‐ logical applications because of their high water content, high permeability, biocompatibility, and the ability of be placed into critical defects in a minimally invasive manner [78]. They are being used in a wide range of tissues, including cartilage, bone, muscle, fat, liver, and neurons. For use in drug/cell delivery, hydrogels should be low-viscosity solutions prior to gelling, which is crucial to maintain cell viability during the encapsulation process, and should rapidly gel in the human body. These properties can be fine-tuned through varia‐ tions in the chemical structure and cross-linking density in hydrogels. Injectable hydrogels can be formed *in situ* by either chemical or physical cross-linking methods [79, 80]. Physical cross-linked hydrogels are capable of phase transition in response to external stimuli such as temperature, pH or both [81]. Chemically cross-linked hydrogels are prepared through pho‐ topolymerization, disulfide bond formation, or reaction between thiols and acrylate or sul‐ fone. The latter hydrogels undergo significant volume changes compared to the first ones [81-83]. The pH/temperature-sensitive hydrogels show several advantages over thermo-sen‐ sitive ones, such as the absence of clogging during injection and avoidance of pH decreased caused by degradation. The pH/temperature-sensitive copolymer hydrogels can be pre‐ pared by combining a pH-sensitive moiety with a temperature-sensitive block. For example, if acidic sulfamethazine oligomers (OSMs) are coupled with thermosensitive poly(e-CL-co-LA)-PEG-poly-(e-CL-co-LA) triblock copolymers a pH/temperature-sensitive hydrogels (OSM-PCLA-PEG-PCLA-OSM) is produced. Photopolymerized hydrogel systems have been reported to provide better temporal and spatial control over the gelation process [79, 81, 84].

A wide variety of natural and synthetic materials have been used to prepare injectable hy‐ drogels. Natural polymers, which are either components of or have macromolecular proper‐ ties similar to the natural ECMs, are known to often undergo rapid degradation upon contact with body fluids or medium and show batch-to-batch variation. Synthetic hydrogels offer improved control of the matrix architecture and chemical composition, no immunoge‐ nicity, consistent supply of large quantities, but tend to have lower biological activity. Therefore, modification of natural and synthetic derived hydrogels is usually required [79]. A natural biodegradable 3D scaffold can be made of acellular muscle ECM but it's fragile and difficult to handle [85]. Another natural biodegradable scaffold can be created by using

teractions between the scaffold and its surroundings are incorporated;

rate that should match the rate of tissue regeneration [73-77].

*4.1.1. Hydrogel scaffolds*

342 Advances in Biomaterials Science and Biomedical Applications

*4.1.2. Biomaterials for scaffold fabrication*

as fibrin or collagen inherently exhibit bioadhesive properties, but others do not and therefore linker molecules that enable covalent or non-covalent molecular in‐

concentration and molar ratio between polymers and cross-linking molecules, should match those of the tissue at the implantation site as well as the degradation Fibrin hydrogels are made from commercially purified allogeneic fibrinogen and purified thrombin, and have been used in a variety of tissue engineering applications. Its main disad‐ vantages reported to be shrinkage of the gel, low mechanical stiffness and its rapid degrada‐ tion can be overcome by incorporating other polymers such as gelatin, hyaluronic acid, and chondroitin-6-sulfate. Fibrin glue is clearly distinguished from fibrin hydrogels that are pre‐ pared from purified fibrinogen and thrombin. Despite the commercial fibrin glue is availa‐ ble in standardized quality; autologous fibrin glue is cheaper and has no viral transmission and prion infection [86]. Tisseel® VH, is a fibrin glue commercialized by Baxter, and consists of a two-component fibrin biomatrix with highly concentrated human fibrinogen to produce fibrin gel from a blood sample and is safe to be used in TE. FloSeal® is another commercial hemostatic matrix with potential in TE, and consists of a cross-linked bovine-derived Gela‐ tin Matrix component and a human-derived Thrombin component. Literature reports that myoblasts seeded on fibrin gels have been shown to differentiate into contracting muscle fi‐ bres and to demonstrate a normal length–tension and force–frequency relationship [87, 88].

Alginate is the designation given to a natural family of biodegradable, biocompatible, hy‐ drophilic and non-toxic polysaccharides extracted from some marine algae and some micro‐ organisms. Alginates are linear block co-polymers composed of two different monomers, β-D-mannuronic acid (M) and α-L-glucuronic acid (G), which are linked by (1-4) glycosidic bonds. The main property of alginate that potentiates its use in different areas, it is its ability to bind some divalent cations such as Ca2+ in the carboxylic groups which provides the ge‐ lation of the alginate solution. The properties of the gel are dependent of the ratio between M and G monomers (M:G ratio); if the proportion of the G monomer is predominant, a strong brittle gel it obtained, whereas if the proportion of the M monomer is predominant, the formed gel will be weaker, but more flexible, because there are less junction zones be‐ tween the polymer chains. As alginate is a polyelectrolyte, more specifically a polyanion, it can be ionically associated with a polycation existent in the same solution through hydrogen bonding or electrostatic interactions, forming a polyelectrolyte complex [89, 90]. Cell-encap‐ sulating calcium cross-linked alginate hydrogels have been extensively studied because algi‐ nate molecules are anionic polysaccharides and do not associate with many proteins. Since alginate itself is inert for cell attachment and spreading, the cell adhesion properties can be tailored by linking molecules such as RGD peptides to its backbone [91].

Chitosan is a natural and hydrophilic copolymer, and it is composed by two monomeric units, D-glucosamine and N-acetyl-D-glucosamine linked by β(1–4)-glycosidic bond. This linear polysaccharide has been widely studied in medical applications due to its biocompati‐ bility, biodegradability, non-toxicity, fungistaticity, antimicrobial activity, non-carcinogenic‐ ity, notable affinity to proteins, promotion of cell adhesion as well as proliferation and differentiation [67]. Chitosan results from the alkaline deacetylation of the chitin and its sol‐ ubility is mainly influenced by its molecular weight and degree of deacetylation. Some methods have been developed to lower the molecular weight of chitosan by hydrolysis of the polymeric chains, in order to produce chitosan salts which are soluble in water. Chito‐ san-based hydrogels have been gelled via glutaraldehyde cross-linking, UV irradiation, and thermal variations [81].

Hyaluronic Acid (HA) is a natural, hydrophilic and non-sulfated glycosaminoglycan. This polymer is a linear polysaccharide, in which the repeating unity is a disaccharide composed by two monomers, D-glucuronic acid and N-acetyl-D-glucosamine, linked through alternat‐ ing β1,3 and β1,4 glycosidic bonds. HA has been used as a biomaterial in various medical applications, due to its biocompatibility, biodegradability, and non-immunogenicity. HA is the main component existent in the extracellular matrix (ECM) of living tissues, namely in the connective, epithelial and neural. This polymer, due to its structural and biological prop‐ erties, has the ability of mediate the cell signalling and behaviour, and the matrix organiza‐ tion. HA is able of interact with some cell surface receptors, being involved in the tissue hydrodynamics, cell migration and proliferation. Several strategies have been reported to prepare HA-based hydrogels [92, 93].

Among the most widely used synthetic polymers for scaffolds, either alone or copolymer‐ ized with synthetic or natural polymers, as biodegradable polymers are polyglycolide, poly‐ lactide and its copolymer poly(lactide-co-glycolide), polyphosphazene, polyanhydride, poly(propylene fumarate), polycyanoacrylate, polycaprolactone, polydioxanone, and poly‐ urethanes, and as non-biodegradeable polymers are included polyvinyl alcohol (PVA), poly‐ hydroxyethymethacrylate, and poly(N-isopropylacrylamide) [71, 94].

The majority of natural biomaterials used in clinical applications are derived from animal or human cadavers' sources. In spite of thorough purification methods, these materials bear the inherent risk of transfer viral diseases and may cause immunological body reactions while synthetic biomaterials are not associated with these risks. So, a critical issue in this type of cellular transplants is the search for an optimal vehicle to provide the ideal environ‐ ment for cell hosting and for the release and conduction of molecules to the site of injury for cell-host interaction. Taking this into account we evaluated different biomaterials as vehicles for the cellular system intended to be tested for skeletal muscle regeneration using our myectomy injury model in the rat. Plasma derived substances, hemostatic matrix solutions and hydrogels (Figure 2 and Figure 3) were tested and the *in vivo* response was compared histologically according to the International Standard ISO 10993-6 (*see* 5.1.3). The following procedure was done under sterile conditions. For preparation of the spherical hydrogel, the polymer solution is prepared by adding in a ratio of 1:1 (V/V), a sodium alginate aqueous solution 7% (m/V) to a sodium hyaluronate aqueous solution 0.5% (m/V), under magnetic stirring. Afterwards the polymer solution is inserted into an insulin syringe and a droplet is released into an excess of cerium nitrate solution 135mM, in order to obtain a cross-linked polymer sphere of approximately 60 μl of volume. Cerium nitrate and sodium hyaluronate solution were sterilized by microfiltration (0.22 μm membrane) and sodium alginate powder is sterilized in an autoclave (120ºC for 15 minutes) previous to the solution preparation (Fig‐ ure 2 and Figure 3). These tested biomaterials including the spherical hydrogel were not on‐ ly used as vehicles but their properties were also evaluated and optimized to find a suitable matrix for the cellular implants.

**Figure 2.** Hydrogel preparation (alginate, hyaluronic acid and cerium).

**Figure 3.** Application of a spherical hydrogel containing 1x106 MSCs from the Wharton's jelly, in the 5 mm Ø TA myec‐ tomy defect.

#### **4.2. Cells**

the polymeric chains, in order to produce chitosan salts which are soluble in water. Chito‐ san-based hydrogels have been gelled via glutaraldehyde cross-linking, UV irradiation, and

Hyaluronic Acid (HA) is a natural, hydrophilic and non-sulfated glycosaminoglycan. This polymer is a linear polysaccharide, in which the repeating unity is a disaccharide composed by two monomers, D-glucuronic acid and N-acetyl-D-glucosamine, linked through alternat‐ ing β1,3 and β1,4 glycosidic bonds. HA has been used as a biomaterial in various medical applications, due to its biocompatibility, biodegradability, and non-immunogenicity. HA is the main component existent in the extracellular matrix (ECM) of living tissues, namely in the connective, epithelial and neural. This polymer, due to its structural and biological prop‐ erties, has the ability of mediate the cell signalling and behaviour, and the matrix organiza‐ tion. HA is able of interact with some cell surface receptors, being involved in the tissue hydrodynamics, cell migration and proliferation. Several strategies have been reported to

Among the most widely used synthetic polymers for scaffolds, either alone or copolymer‐ ized with synthetic or natural polymers, as biodegradable polymers are polyglycolide, poly‐ lactide and its copolymer poly(lactide-co-glycolide), polyphosphazene, polyanhydride, poly(propylene fumarate), polycyanoacrylate, polycaprolactone, polydioxanone, and poly‐ urethanes, and as non-biodegradeable polymers are included polyvinyl alcohol (PVA), poly‐

The majority of natural biomaterials used in clinical applications are derived from animal or human cadavers' sources. In spite of thorough purification methods, these materials bear the inherent risk of transfer viral diseases and may cause immunological body reactions while synthetic biomaterials are not associated with these risks. So, a critical issue in this type of cellular transplants is the search for an optimal vehicle to provide the ideal environ‐ ment for cell hosting and for the release and conduction of molecules to the site of injury for cell-host interaction. Taking this into account we evaluated different biomaterials as vehicles for the cellular system intended to be tested for skeletal muscle regeneration using our myectomy injury model in the rat. Plasma derived substances, hemostatic matrix solutions and hydrogels (Figure 2 and Figure 3) were tested and the *in vivo* response was compared histologically according to the International Standard ISO 10993-6 (*see* 5.1.3). The following procedure was done under sterile conditions. For preparation of the spherical hydrogel, the polymer solution is prepared by adding in a ratio of 1:1 (V/V), a sodium alginate aqueous solution 7% (m/V) to a sodium hyaluronate aqueous solution 0.5% (m/V), under magnetic stirring. Afterwards the polymer solution is inserted into an insulin syringe and a droplet is released into an excess of cerium nitrate solution 135mM, in order to obtain a cross-linked polymer sphere of approximately 60 μl of volume. Cerium nitrate and sodium hyaluronate solution were sterilized by microfiltration (0.22 μm membrane) and sodium alginate powder is sterilized in an autoclave (120ºC for 15 minutes) previous to the solution preparation (Fig‐ ure 2 and Figure 3). These tested biomaterials including the spherical hydrogel were not on‐ ly used as vehicles but their properties were also evaluated and optimized to find a suitable

hydroxyethymethacrylate, and poly(N-isopropylacrylamide) [71, 94].

thermal variations [81].

344 Advances in Biomaterials Science and Biomedical Applications

prepare HA-based hydrogels [92, 93].

matrix for the cellular implants.

There is evidence both from animal studies and clinical investigations that cell therapy in‐ volving different types of stem cells application is promising as means to promote regenera‐ tion of skeletal muscles following severe injuries. Technical or/and ethical difficulties in obtaining sufficient and appropriate stem cells from the bone marrow or from embryos (ob‐ tained from assisted reproduction techniques or somatic nuclear transfer - cloning) have limited the application of this type of therapy. Stem cells are known as an undifferentiated population, with endless self-renewal and sustained proliferation *in vitro* and multilineage differentiation ability [95]. The *in vitro* multilineage differentiation is the concept that gives these cells an extreme priority for use in tissue and cell-based therapies. Stem cells can be loosely classified into 3 categories based on their functional role: hematopoietic stem cells, mesenchymal stem cells (MSCs) and embryonic stem cells [95].

MSCs have become one of the most exciting targets for tissue regeneration due to their high plasticity, proliferative and multilineage differentiation capacity. These cells are capable of differentiating into adipose, bone cartilage and muscle. Among all this notable characteris‐ tics, MSCs reveal other properties of great importance, they present low immunogenicity and high immunosuppressive properties due to a decreased or even absence HLA Class II expression [96]. Differentiation potential of MSCs in multilineage end-stage cells has been proven, so as the treatment potential in musculoskeletal disorders [97, 98]. Since their first isolation in 1968, from rat bone marrow [99], MSCs have been isolated with success from al‐ most all tissue sources: skeletal muscle, adipose tissues, synovial membranes, umbilical cord matrix and blood, placental tissue, amniotic fluid among others. Along with differentiation capacity, an increasing amount of data has demonstrated that the MSCs have the capacity of modulating the surrounding environment, by secretion of multiple factors and activation of endogenous progenitor cells [100, 101].

#### *4.2.1. Umbilical cord*

From our data and from previously published experimental work, the development of cell therapies associated to biomaterials is a promising tool for increase skeletal muscle regener‐ ation, avoiding the irreversible loss of function and limit the fibrous scar tissue presence [57-59]. Recent years have witnessed an explosion in the number of adult stem cells popula‐ tions isolated and characterized. While still multipotent, adult stem cells have long been considered restricted, giving rise only to progeny of their resident tissues. Recently, and cur‐ rently controversial studies have challenged this dogma, suggesting that adult stem cells may be far more plastic than previously appreciated [102, 103]. Extra-embryonic tissues as stem cell reservoirs offer many advantages over both embryonic and adult stem cell sources. The umbilical cord matrix is an important and safe source of MSCs with positive effects in nerve and skeletal muscle regeneration, with no ethical or technical issues. MSC isolated from umbilical cord matrix (Wharton's jelly), as well as embryonic stem cells (ESCs) are ori‐ ginated from inner cell mass of blastocyst [104]. Comparing with ESCs, MSCs have shorter population doubling time; can be easily cultured in plastic flasks, are well tolerated by im‐ mune system; therefore transplantation of these cells into non-immune-suppressed animals does not induce acute rejection. Most important, these cells do not originate teratomas [104]. Like bone marrow stromal cells and other MSCs, the MSCs from the Wharton's jelly are plastic adherent, stain positively for markers of the mesenchymal lineage (CD10, CD13, CD29, CD44, CD90, and CD105) and negatively for markers of the hematopoietic lineage. These MSCs are capable of self-renewal with sustained proliferation *in vitro* and can differ‐ entiate into multiple mesodermal cells. The high plasticity and low immunogenicity of these cells turn them into a desirable form of cell therapy for the injured musculoskeletal tissue without requiring the use of immunosuppressive drugs during the treatments. Interestingly, these cells, which are HLA class II negative, not only express both an immuno-privileged and immuno-modulatory phenotype, but their HLA complex class I expression levels can al‐ so be manipulated, making them a potential cell source for MSC-based therapies. In addi‐ tion and as previously referred, these cells represent a non-controversial source of primitive mesenchymal progenitor cells that can be harvested after birth, cryogenically stored, thawed, and expanded for therapeutic uses. MSCs from the Wharton's jelly display a high proliferative rate and plasticity, being able to differentiate into adipocytes, osteoblasts, chon‐ drocytes, cardiomyocites, neurons, and glia. More recently, Conconi et al. [105] demonstrat‐ ed that CD105(+)/CD31(-)/KDR(-) cells are able not only to differentiate *in vivo* towards the myogenic lineage as demonstrated by the co-localization of HLA 1 and sarcomeric tropo‐ myosine antigens, but also to contribute to the muscle regenerative process. These cells were found to differentiate *in vitro* into myoblast-like cells, expressing Myf5 and MyoD after 7 and 11 days of myogenic induction, respectively. The timing of expression of Myf5 and My‐ oD in CD105(+)/CD31(-)/KDR(-) cells is similar to that described during embryonic develop‐ ment and in myoblast cultures [105].

population, with endless self-renewal and sustained proliferation *in vitro* and multilineage differentiation ability [95]. The *in vitro* multilineage differentiation is the concept that gives these cells an extreme priority for use in tissue and cell-based therapies. Stem cells can be loosely classified into 3 categories based on their functional role: hematopoietic stem cells,

MSCs have become one of the most exciting targets for tissue regeneration due to their high plasticity, proliferative and multilineage differentiation capacity. These cells are capable of differentiating into adipose, bone cartilage and muscle. Among all this notable characteris‐ tics, MSCs reveal other properties of great importance, they present low immunogenicity and high immunosuppressive properties due to a decreased or even absence HLA Class II expression [96]. Differentiation potential of MSCs in multilineage end-stage cells has been proven, so as the treatment potential in musculoskeletal disorders [97, 98]. Since their first isolation in 1968, from rat bone marrow [99], MSCs have been isolated with success from al‐ most all tissue sources: skeletal muscle, adipose tissues, synovial membranes, umbilical cord matrix and blood, placental tissue, amniotic fluid among others. Along with differentiation capacity, an increasing amount of data has demonstrated that the MSCs have the capacity of modulating the surrounding environment, by secretion of multiple factors and activation of

From our data and from previously published experimental work, the development of cell therapies associated to biomaterials is a promising tool for increase skeletal muscle regener‐ ation, avoiding the irreversible loss of function and limit the fibrous scar tissue presence [57-59]. Recent years have witnessed an explosion in the number of adult stem cells popula‐ tions isolated and characterized. While still multipotent, adult stem cells have long been considered restricted, giving rise only to progeny of their resident tissues. Recently, and cur‐ rently controversial studies have challenged this dogma, suggesting that adult stem cells may be far more plastic than previously appreciated [102, 103]. Extra-embryonic tissues as stem cell reservoirs offer many advantages over both embryonic and adult stem cell sources. The umbilical cord matrix is an important and safe source of MSCs with positive effects in nerve and skeletal muscle regeneration, with no ethical or technical issues. MSC isolated from umbilical cord matrix (Wharton's jelly), as well as embryonic stem cells (ESCs) are ori‐ ginated from inner cell mass of blastocyst [104]. Comparing with ESCs, MSCs have shorter population doubling time; can be easily cultured in plastic flasks, are well tolerated by im‐ mune system; therefore transplantation of these cells into non-immune-suppressed animals does not induce acute rejection. Most important, these cells do not originate teratomas [104]. Like bone marrow stromal cells and other MSCs, the MSCs from the Wharton's jelly are plastic adherent, stain positively for markers of the mesenchymal lineage (CD10, CD13, CD29, CD44, CD90, and CD105) and negatively for markers of the hematopoietic lineage. These MSCs are capable of self-renewal with sustained proliferation *in vitro* and can differ‐ entiate into multiple mesodermal cells. The high plasticity and low immunogenicity of these cells turn them into a desirable form of cell therapy for the injured musculoskeletal tissue

mesenchymal stem cells (MSCs) and embryonic stem cells [95].

346 Advances in Biomaterials Science and Biomedical Applications

endogenous progenitor cells [100, 101].

*4.2.1. Umbilical cord*

Using the myectomy model we tested the use of Human MSCs isolated from the Wharton's jelly in order to improve skeletal muscle regeneration. The cells were directly infiltrated into the lesion or delivered by different vehicles including Floseal®, Tisseel®, carboximetilcellu‐ lose (Sigma) and spherical hydrogel (own fabrication). MSC from Wharton's jelly were pur‐ chased from PromoCell GmbH (C-12971, lot-number: 8082606.7). The MSCs are cultured and maintained in a humidified atmosphere with 5% CO2 at 37ºC. Mesenchymal Stem Cell Medium, PromoCell (C-28010) is replaced every 48 hours. At 90% confluence, cells are har‐ vested with 0.25% trypsin with EDTA (GIBCO) and passed into a new flask for further ex‐ pansion. MSCs at a concentration of 2 x 105 cells are cultured exhibiting a 90% confluence after 3-4 days. The application of human MSCs in rats is possible without inducing any im‐ munossupression in the experimental animals. The MSCs exhibited a normal star-like shape with a flat morphology in culture (Figure 4). A total of 20 Giemsa-stained metaphases of these cells, were analyzed for numerical aberrations. Sporadic, non-clonal aneuploidy was found in 3 cells (41-45 chromosomes) the other 17 metaphases had 46 chromosomes. The karyotype was determined in a completely analyzed G-banding metaphase and no structur‐ al alterations were found [57]. The karyotype analysis to the MSCs cell line derived from Human Wharton jelly demonstrated that this cell line hasn't neoplasic characteristics and is stable during the cell culture procedures in terms of number and structure of the somatic and sexual chromosomes. Also, the morphologic characteristics of these cells in culture, ob‐ served in an inverted microscope, are normal. These cells presented a star-like shape with a flat morphology, characteristic of the MSCs been adequate to be used in *in vivo* rat experi‐ mental model [57]. The MSCs karyotype was studied in order to be sure that these cells did not present any number or structure chromosome abnormalities due to isolation and cell culture procedures before *in vivo* application. This concern was due to the negative effects that some cellular systems, like the ESCs present, inducing the production of teratomas. The cellular systems implanted into the injured skeletal muscle improved the skeletal muscle re‐ generation since these cells produce growth factors, ECM molecules, and even modulate the inflammatory process.

**Figure 4.** Undifferentiated MSCs from Wharton's jelly, exhibiting a star-like shape with a flat morphology (100x mag‐ nification).

### **5. Evaluation of muscle regeneration**

Muscle biopsies should be considered in order to obtain careful clinical assessment or for in‐ vestigation purposes. After the collection of the muscle samples, they should be immediate‐ ly equally divided in three. One sample should be placed into formalin (for hematoxilin and eosin - HE), another sample should be fixed into 2.5% purified glutaraldehyde in 0.1M Sor‐ ensen phosphate buffer (for electron microscopy - EM) and the other sample should remain unfixed and refrigerated (for histochemistry, biochemistry/genetics analysis).

#### **5.1. Routine histological evaluation**

Routine evaluation of the muscle biopsy sample involves the examination of formalin-fixed, paraffin processed sections and unfixed frozen sections with standard histological and en‐ zyme histochemical stains at the light microscopic level. HE is the routine histological stain used for evaluation of basic tissue organization and cellular structure. For HE, the whole piece of tissue should be fixed in a clamp and after the tissue is infiltrated with wax, both longitudinal and cross sections must be cut before embedding. Five levels should be ob‐ tained, especially in cases suspected of vasculitis. The parameters that can be evaluated are: the type of inflammatory infiltrate present; examination of the structure of vessels walls (vasculitis and/or fibrinoid necrosis); presence of endomysial and perimysial fibrosis/fatty infiltration; the range of fiber caliber; presence of angulated fibers; increase in number of centrally located nuclei; central capillary migration; split fiber; group atrophy; necrotic/ myopathic (degenerating) fibers; atrophic fibers; regenerating fibers; target fibers; whorl fi‐ bers and ring fibers. The rounding of fiber contour and the variation of fiber diameter should also be analyzed, however they are better evaluated with frozen sections (Figure 5).

**Figure 5.** HE staining of TA muscles 15 days post myectomy (A - control) and application of fibrin (B), hydrogel (C), Floseal® (D).

#### *5.1.1. Morphological analysis*

generation since these cells produce growth factors, ECM molecules, and even modulate the

**Figure 4.** Undifferentiated MSCs from Wharton's jelly, exhibiting a star-like shape with a flat morphology (100x mag‐

Muscle biopsies should be considered in order to obtain careful clinical assessment or for in‐ vestigation purposes. After the collection of the muscle samples, they should be immediate‐ ly equally divided in three. One sample should be placed into formalin (for hematoxilin and eosin - HE), another sample should be fixed into 2.5% purified glutaraldehyde in 0.1M Sor‐ ensen phosphate buffer (for electron microscopy - EM) and the other sample should remain

Routine evaluation of the muscle biopsy sample involves the examination of formalin-fixed, paraffin processed sections and unfixed frozen sections with standard histological and en‐ zyme histochemical stains at the light microscopic level. HE is the routine histological stain used for evaluation of basic tissue organization and cellular structure. For HE, the whole piece of tissue should be fixed in a clamp and after the tissue is infiltrated with wax, both longitudinal and cross sections must be cut before embedding. Five levels should be ob‐ tained, especially in cases suspected of vasculitis. The parameters that can be evaluated are: the type of inflammatory infiltrate present; examination of the structure of vessels walls (vasculitis and/or fibrinoid necrosis); presence of endomysial and perimysial fibrosis/fatty

unfixed and refrigerated (for histochemistry, biochemistry/genetics analysis).

inflammatory process.

348 Advances in Biomaterials Science and Biomedical Applications

nification).

**5. Evaluation of muscle regeneration**

**5.1. Routine histological evaluation**

Long-standing histological characteristics are still used to identify the mammalian skeletal muscle regeneration process. On muscle cross-sections, these fundamental morphological characteristics are newly formed myofibers of small caliber and with centrally located myo‐ nuclei. Newly formed myofibers are often basophilic (reflecting high protein synthesis) and express embryonic/developmental forms of MHC (reflecting *de novo* fiber formation). On muscle longitudinal sections and in isolated single muscle fibers, central myonuclei are ob‐ served in discrete portions of regenerating fibers or along the entire new fiber, suggesting that cell fusion is not diffuse during regeneration but rather focal to the site of injury [1]. Cross-sectional area (CSA) analysis is one of the features that can be assessed. This can be achieved with imaging software processing (Scio Image, ImageJ) of HE-stained muscle sec‐ tions. A predefined number of fibers is traced per sample and should be determined as ap‐ propriate by the examination of no additional changes in standard deviation. The classification of small and large fibers can be determined for example by setting three stand‐ ard deviations from the mean CSA for the uninjured group at different time points [14]. The CSA and number of myotubes can be used to estimate the development degree of muscle regeneration following injury [36].

#### *5.1.2. Collagen quantification*

Collagen content in the wound bed can be calculated by image analysis of Masson's Tri‐ chrome-stained histological images taken at a predefined image magnification. Color sepa‐ rations must be performed and an analysis threshold must be established for each image series collected using the same brightness and white balance settings. Output images show‐ ing only computed blue coverage must be compared to the color images to ensure the repre‐ sentation of truly blue color due to collagen staining. As a control the analysis must be performed on uninjured (control) skeletal muscle tissue sections stained with Masson's Tri‐ chrome and collected using the same camera and threshold settings to confirm a collagen content of zero for control tissue. The ratio of blue pixels above the threshold to total pixels in the image is used to calculate the collagen content for each image [40].

#### *5.1.3. International Standard (ISO 10993-6)*

The International Standard (ISO 10993-6) specifies test methods for the assessment of the lo‐ cal effects after implantation of biomaterials intended for use in medical devices. These im‐ plantation tests are not intended to evaluate or determine the performance of the test specimen in terms of mechanical or functional loading. The local effects are evaluated by a comparison of the tissue response caused by the tested implant to that caused by the control. The objective of the test methods is to characterize the history and evolution of the tissue response after implantation of a medical device/biomaterial including final integration or re‐ sorption/degradation of the material. The test sample shall be implanted into the tissues most relevant to the intended clinical use of the material. For short-term testing, animals such as rodents or rabbits are commonly used. During the first two weeks after implantation the reaction due to the surgical procedure itself may be difficult to distinguish from the tis‐ sue reaction evoked by the implant and for that reason in our study we collected the muscle samples 15 days after implantation. For degradable/resorbable materials the test period shall be related to the estimated degradation time of the test product. In our case the majority of the vehicles/matrices tested the degradation time is less than 4 days. In the absence of com‐ plete degradation, absorption, or restoration to normal tissue structure and function, the overall data collected may be sufficient to allow characterization of the local effects after im‐ plantation. A sufficient number of implants shall be inserted to ensure that the final number of specimens to be evaluated will give valid results. The evaluation of the biological re‐ sponse must be accomplished by documenting the macroscopic and histopathological re‐ sponses as a function of time. The responses to the test sample must be compared to the responses obtained at the control sample or sham operated sites. The scoring system used for the histological evaluation shall take into account the extent of the area affected, either quantitatively (e.g. in micrometres) or semi-quantitatively (Annex E of this Standard) [44, 106]. The biological response parameters, which shall be assessed and recorded, include:

**i.** the extent of fibrosis/fibrous capsule (layer in μm) and inflammation;

**ii.** the degeneration as determined by changes in tissue morphology;


*5.1.2. Collagen quantification*

350 Advances in Biomaterials Science and Biomedical Applications

*5.1.3. International Standard (ISO 10993-6)*

Collagen content in the wound bed can be calculated by image analysis of Masson's Tri‐ chrome-stained histological images taken at a predefined image magnification. Color sepa‐ rations must be performed and an analysis threshold must be established for each image series collected using the same brightness and white balance settings. Output images show‐ ing only computed blue coverage must be compared to the color images to ensure the repre‐ sentation of truly blue color due to collagen staining. As a control the analysis must be performed on uninjured (control) skeletal muscle tissue sections stained with Masson's Tri‐ chrome and collected using the same camera and threshold settings to confirm a collagen content of zero for control tissue. The ratio of blue pixels above the threshold to total pixels

The International Standard (ISO 10993-6) specifies test methods for the assessment of the lo‐ cal effects after implantation of biomaterials intended for use in medical devices. These im‐ plantation tests are not intended to evaluate or determine the performance of the test specimen in terms of mechanical or functional loading. The local effects are evaluated by a comparison of the tissue response caused by the tested implant to that caused by the control. The objective of the test methods is to characterize the history and evolution of the tissue response after implantation of a medical device/biomaterial including final integration or re‐ sorption/degradation of the material. The test sample shall be implanted into the tissues most relevant to the intended clinical use of the material. For short-term testing, animals such as rodents or rabbits are commonly used. During the first two weeks after implantation the reaction due to the surgical procedure itself may be difficult to distinguish from the tis‐ sue reaction evoked by the implant and for that reason in our study we collected the muscle samples 15 days after implantation. For degradable/resorbable materials the test period shall be related to the estimated degradation time of the test product. In our case the majority of the vehicles/matrices tested the degradation time is less than 4 days. In the absence of com‐ plete degradation, absorption, or restoration to normal tissue structure and function, the overall data collected may be sufficient to allow characterization of the local effects after im‐ plantation. A sufficient number of implants shall be inserted to ensure that the final number of specimens to be evaluated will give valid results. The evaluation of the biological re‐ sponse must be accomplished by documenting the macroscopic and histopathological re‐ sponses as a function of time. The responses to the test sample must be compared to the responses obtained at the control sample or sham operated sites. The scoring system used for the histological evaluation shall take into account the extent of the area affected, either quantitatively (e.g. in micrometres) or semi-quantitatively (Annex E of this Standard) [44, 106]. The biological response parameters, which shall be assessed and recorded, include:

in the image is used to calculate the collagen content for each image [40].

**i.** the extent of fibrosis/fibrous capsule (layer in μm) and inflammation;

**ii.** the degeneration as determined by changes in tissue morphology;


Under the conditions of the study and following the results for the mentioned parameters in the semi-quantitative scoring system (Annex E of this Sandard), the test sample is consid‐ ered as non-irritant (0,0 up to 2,9), slight irritant (3,0 up to 8,9), moderate irritant (9,0 up to 15,0), severe irritant (> 15) to the tissue as compared to the negative control sample [106]. This test method is used for assessing the biological response of muscle tissue to an implant‐ ed material (Annex C of this Standard). As already mentioned, the method compares the bi‐ ological response to implants of test specimens with the biological response to implants of control specimens. The control materials are those used in medical devices of which the clin‐ ical acceptability and biocompatibility characteristics have been established [106]. In our study we developed an adaptation of this Standard by considering the control as the group where the surgical procedure (myectomy) was performed without any biomaterial or cell implantation (Figure 6). Although the surgical technique may profoundly influence the re‐ sult of any implantation procedure, we assumed that our standardized myectomy lesion could be considered as the *Control* group since we were able to determine the local effects of the different implants by their comparison to the minor effects of the surgical procedure. The surgeries were executed under general anesthesia with a xylazine (1.25 mg/100 g BW im) and ketamine (9 mg/100 g BW im) combination [38].

**Figure 6.** ISO 10993-6 scoring for the groups tested. The *Control* group obtained a score of 14.7 (in blue). Scorings above the *Control* group were considered as non-irritant (in yellow), slight irritant (in orange) and moderate irritant (in red).

#### **5.2. Histochemistry**

For histochemistry a basic panel should be performed, preferentially in frozen sections. De‐ pending on the objectives, an extended panel can be done concerning the study of some molecules like the already mentioned Masson's Trichrome-stain or enzyme processes such as ATPase; NADH-TR or Esterase (Bancroft&Stevens). When necessary, other special stains can be performed on paraffin sections.

#### **5.3. Immunohistochemistry**

In general, the immunohistochemical stains are utilized for the diagnosis of various muscu‐ lar dystrophies. They also may help to determine the subtypes of inflammatory cells within an infiltrate or for other investigation purposes. Specific skeletal muscle markers such as myosin heavy chain and desmin can be applied in order to clearly identify this tissue. The distinction of SCs, considered as the reservoir of myogenic precursor cells, from other cells must be made (like plasma cells, which may be occasionally seen under the basal lamina in pathologic conditions). SCs can be easily demonstrated by immunostaining for N-CAM; they also express vimentin. Activated SCs generally express Myo-D and myogenin [107]. The regenerating fibers express N-CAM, MyoD and myogenin, and also embryonic and ne‐ onatal isoforms of myosin heavy chain. In contrast to mature fibers, MCH class I histocom‐ patibility complex is expressed in regenerating fibers.

#### **5.4. Immunofluorescence**

For the preparation of TA muscles for immunofluorescence they should be embedded in Tissue-Tek OCT compound. Sections are cut at 10 μm using a Leica CM1850 cryostat and placed onto Surgipath microscope slides. Laminin-α2 chain is detected with a 1:500 dilu‐ tion of rabbit anti-laminin-α2 (2G) polyclonal antibody. The laminin-α1 chain is detected with a rat anti-laminin-α1 monoclonal antibody. Primary rabbit antibodies are detected with a 1:500 dilution of fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody and the rat monoclonal antibody is detected with 1:500 dilution of fluorescein isothiocyanateconjugated anti-rat secondary antibody. In all immunofluorescence experiments, secon‐ dary only antibody controls are included to test for specificity. For mouse monoclonal antibodies, endogenous mouse immunoglobulin is blocked with a mouse-on-mouse (MOM) kit. A 1-μg/ml concentration of tetramethylrhodamine-conjugated wheat germ agglutinin (WGA) is used to define muscle fibers. To examine immune response, cytotoxic T cells are detected with fluorescein isothiocyanate-labeled rat anti-mouse CD8a and macrophages are detected with fluorescein isothiocyanateconjugated anti-mouse F4/80 at 1:1000 (Figure 7 and Figure 8) [50].

**Figure 7.** Double imunofluorescence staining for laminin and CD31.

**Figure 8.** Pax7+ SC counterstained with DAPI.

#### **5.5. Electron microscopy**

**5.2. Histochemistry**

can be performed on paraffin sections.

352 Advances in Biomaterials Science and Biomedical Applications

patibility complex is expressed in regenerating fibers.

**5.3. Immunohistochemistry**

**5.4. Immunofluorescence**

Figure 8) [50].

For histochemistry a basic panel should be performed, preferentially in frozen sections. De‐ pending on the objectives, an extended panel can be done concerning the study of some molecules like the already mentioned Masson's Trichrome-stain or enzyme processes such as ATPase; NADH-TR or Esterase (Bancroft&Stevens). When necessary, other special stains

In general, the immunohistochemical stains are utilized for the diagnosis of various muscu‐ lar dystrophies. They also may help to determine the subtypes of inflammatory cells within an infiltrate or for other investigation purposes. Specific skeletal muscle markers such as myosin heavy chain and desmin can be applied in order to clearly identify this tissue. The distinction of SCs, considered as the reservoir of myogenic precursor cells, from other cells must be made (like plasma cells, which may be occasionally seen under the basal lamina in pathologic conditions). SCs can be easily demonstrated by immunostaining for N-CAM; they also express vimentin. Activated SCs generally express Myo-D and myogenin [107]. The regenerating fibers express N-CAM, MyoD and myogenin, and also embryonic and ne‐ onatal isoforms of myosin heavy chain. In contrast to mature fibers, MCH class I histocom‐

For the preparation of TA muscles for immunofluorescence they should be embedded in Tissue-Tek OCT compound. Sections are cut at 10 μm using a Leica CM1850 cryostat and placed onto Surgipath microscope slides. Laminin-α2 chain is detected with a 1:500 dilu‐ tion of rabbit anti-laminin-α2 (2G) polyclonal antibody. The laminin-α1 chain is detected with a rat anti-laminin-α1 monoclonal antibody. Primary rabbit antibodies are detected with a 1:500 dilution of fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody and the rat monoclonal antibody is detected with 1:500 dilution of fluorescein isothiocyanateconjugated anti-rat secondary antibody. In all immunofluorescence experiments, secon‐ dary only antibody controls are included to test for specificity. For mouse monoclonal antibodies, endogenous mouse immunoglobulin is blocked with a mouse-on-mouse (MOM) kit. A 1-μg/ml concentration of tetramethylrhodamine-conjugated wheat germ agglutinin (WGA) is used to define muscle fibers. To examine immune response, cytotoxic T cells are detected with fluorescein isothiocyanate-labeled rat anti-mouse CD8a and macrophages are detected with fluorescein isothiocyanateconjugated anti-mouse F4/80 at 1:1000 (Figure 7 and

Electron microscopic (EM) examination of the glutaraldehyde-fixed portion of the biopsy is performed when the light microscopic studies are inconclusive. Thus, it is reserved for se‐ lected circumstances in which the pathologist determines that EM has the potential of con‐ tributing significantly to determining a specific diagnosis. A specimen placed in glutaraldehyde must be small, approximately 1-2 mm in width and depth, allowing the complete tissue penetration by this fixative. Glutaraldehyde makes tissue brittle and inter‐ feres with immunohistochemical studies, so it is not appropriate for the paraffin specimen. With EM, other muscle cell parameters can be analyzed in detail: the myofibril architecture; the plasma and sarcolemmal membrane; the mitochondria (size, density and shape); T-tu‐ bule; amount of lipid; nucleus; phagocytic granules and amount of glycogen. EM is extreme‐ ly useful in some cases: to identify inclusions primarily found by light microscopy; to help in the characterization of stored material found on light microscopy and define its intracel‐ lular localization; to analyze structural abnormalities found by light microscopy; can assist in the diagnosis of mitochondrial myopathy or seeking evidence to support a diagnosis of dermatomyositis (EM can be used to look for tubuloreticular inclusion in endothelial cells when light microscopic fails to reveal it).

#### **5.6. Contraction force measurement**

To obtain an estimate of total TA muscle strength reduced by the injury and possibly recov‐ ered by the cell/vehicle implants, contractile force due to electrical stimulation can be meas‐ ured before injury, after injury and at the time of sacrifice (at different time points) for nonimplanted and implanted animals. This can be accomplished with the animals under general anesthesia and by anchoring the knee joint using a custom clamping system anch‐ ored to the floor of the surgical stereomicroscope stand and attaching a silk ligature to the cleft between digits 1 and 2 that must be anchored to a transducer at the other end. This can also be executed by cutting the TA tendon just before the insertion at the ankle and tying it with a 4.0 nylon suture attached to the isometric transducer. The exposed muscle is stimulat‐ ed using 2 custom needle electrodes placed at the proximal muscle surface. Electrical stimu‐ lation of the TA muscle is applied at 5 volts, 4 ms pulse duration, at 500 ms intervals and the resultant tetanic force recorded (200 points(s) using a BioPac MP-100 (Harvard Apparatus) and accompanying software (AcknowledgeTM). The muscle must be kept hydrated during the procedure using sterile saline. Maximum tetanic force is measured by reducing the stim‐ ulation interval to 20 ms, generating continuous stimulation simulating tetanus condition. Another method of applying the electrical stimulation can be obtained by exposing the sciat‐ ic nerve with an incision in the hamstring region The tibial nerve is cut just after the sciatic nerve splits into the tibial and peroneal nerves to eliminate any contraction from the *gastro‐ cnemius* muscle causing background in the force data. The exposed sciatic nerve is then laid over two electrodes with a small piece of parafilm and should also be kept moist with peri‐ odic treatment of mineral oil. Stimulation is made using a supra-maximal square-wave pulse of 0.1 ms duration. Measurements are performed at the length at which maximal ex‐ tension is obtained during the twitch and the data should be recorded for sub-maximal and maximal isometric force. Specific maximal force should be quantified by correcting for mus‐ cle mass [40, 98].

### **Acknowledgements**

The authors would like to thank the support by Dr. José Manuel Correia Costa, from INSRJ, Porto, Portugal; and Biosckin, Molecular and Cell Therapies SA for the umbilical cord units supply and access to the GMP cell culture room (Scientific Protocol between Porto Universi‐ ty and Biosckin, Molecular and Cell Therapies SA). This work was supported by Fundação para a Ciência e Tecnologia (FCT), Ministério da Ciência e Ensino Superior (MCES), Portu‐ gal, through the financed research project PTDC/DES/104036/2008, and by QREN Nº 1372 para Criação de um Núcleo I&DT para Desenvolvimento de Produtos nas Áreas de Medici‐ na Regenerativa e de Terapias Celulares – Núcleo Biomat & Cell. A Gärtner (SFRH/BD/ 70211/2010) and I Amorim (SFRH/BD/76237/2011) acknowledge FCT for financial support.
