**2.3. The biological effect of subcutaneously implanted hydroxyapatite-coated cellulose**

The bone defect study showed that HA-coated cellulose favoured rapid fibrous tissue proliferation instead of bone formation 11. Therefore, it was considered to have no value as a bone replacement material but might be useful in other applications in which accelerated granulation tissue formation is needed. Subcutaneously (figure 5 a, b) implanted silica rich HA-implants showed a massive inflammatory reaction with an intense foreign body reaction and increased invasion of fibrovascular tissue already 1-3 days after implantation. Such strong tissue reaction was not seen with any other subcutaneously implanted cellulose sponge. Tissue growth into uncoated regenerated cellulose was much slower and took place mainly on their surface (figure 6). 38

Subcutaneously implanted HA-sponges activate the inflammatory response and the secretion of cytokines and growth factors important to wound healing, such as TGF-1, TNF-, vascular endothelial growth factor (VEGF) and platelet derived growth factor A (PDGF-A) The long-term study revealed, however, that the excessive connective tissue

stain; equal magnifications; scale bar 200 m).

mainly on their surface (figure 6). 38

**cellulose** 

**Figure 4.** Tissue reactions of carbamino cellulose two weeks after implantation. Subcutaneously implanted 6%-cellulose pearls (p) stayed intact and showed only modest degradation (a), whereas b) 4%-cellulose pearls were degraded and infiltrated with new granulation tissue (gf). Similar behaviour was observed in bone implants: c) 6%-cellulose pearls were surrounded by a thin connective tissue capsule (arrow) whereas about half of the b) 4%-cellulose pearls were partially degraded and surrounded by bone (nb) or a thin osteoid layer (ol) even in the bone marrow (bm) area. (van Gieson

**2.3. The biological effect of subcutaneously implanted hydroxyapatite-coated** 

The bone defect study showed that HA-coated cellulose favoured rapid fibrous tissue proliferation instead of bone formation 11. Therefore, it was considered to have no value as a bone replacement material but might be useful in other applications in which accelerated granulation tissue formation is needed. Subcutaneously (figure 5 a, b) implanted silica rich HA-implants showed a massive inflammatory reaction with an intense foreign body reaction and increased invasion of fibrovascular tissue already 1-3 days after implantation. Such strong tissue reaction was not seen with any other subcutaneously implanted cellulose sponge. Tissue growth into uncoated regenerated cellulose was much slower and took place

Subcutaneously implanted HA-sponges activate the inflammatory response and the secretion of cytokines and growth factors important to wound healing, such as TGF-1, TNF-, vascular endothelial growth factor (VEGF) and platelet derived growth factor A (PDGF-A) The long-term study revealed, however, that the excessive connective tissue

**Figure 5.** a). A schematic presentation of the subcutaneous implantation model used in our studies. Two midline incisions were made on the back of the rats, and sterilised, moistened sponge implants (10 x 5 mm) were inserted bilaterally into subcutaneous pockets under general anaesthesia. b). Subcutaneously implanted cellulose sponges 7 days after implantation. HA-coated implants are darker in colour as a sign of high cellularity and rich neovascularisation, whereas the uncoated implants are pale.

**Figure 6.** The HA-coating accelerated tissue growth into subcutaneously implanted cellulose sponges as well as the inflammatory response and blood vessel formation. a) Haematoxylin-eosin–stained sections 1 (upper), 3 (middle), and 7 (lower) days after implantation. The arrows in HA-coated sponges point at the border between the implant and the surrounding capsule (scale bar = 100 m). b) HAcoated sponges contain large clusters (arrows) of accumulated macrophages (brownish coloured cells). Macrophages favour gathering near to cellulose fibres (arrow head) (day 5; scale bar = 50 m). c) More blood vessels, as indicated by CD31-staining, can bee seen in 5-day-old HA-coated sponge compared to uncoated one (scale bar = 50 m).

formation, which is histologically normal, does not disturb the animals in any way. After 14 days postoperatively, the foreign body reaction in HA-coated sponges starts to diminish. At one month, the difference between the HA-coated and uncoated cellulose had levelled off and at the end of the study, at one year no obvious histological difference between the coated and uncoated were detected (figure 7). 38

Cellulose - A Biomaterial with Cell-Guiding Property 93

**Figure 8.** HA-coated cellulose contain large amount of CXCR4-positive cells. Numerous clusters (arrow heads) of and individual CXCR4-positive (brownish coloured) cells are detected throughout the HA-

Haematopoietic stem cells seem to be the first stem cells to invade the empty centres of the HA-coated cellulose implants (figure 9 a-c). The more abundant occurrence of HSCs is most probably responsible for the augmented blood vessel formation in HA-coated cellulose. The earliest haematopoietic precursor, the haemangioblast, is namely the precursor for both haematopoietic and endothelial cell lineages, not only during embryogenesis but also in adults 14, 46. The haematopoietic progenitors, especially in the HA-coated implants, were located in close contact with the cellulose fragments (figure 10 a). Hence, the coating of cellulose with HA creates an environment that facilitates stem cell homing more efficiently than uncoated cellulose. In the bone marrow, undifferentiated HSCs are detected near the inner surface of the medullary cavity, i.e. the endosteum, in the so-called endosteal stem cell niche. At this site, the bone is in constant turnover: bone is formed by the osteoblasts and removed by specific macrophages, the osteoclasts. Due to bone degradation, soluble calcium ions (Ca2+) are released into the bone marrow fluid. Various cells, including primitive HSCs, respond to extracellular ionic calcium concentrations through a calcium sensing receptor, CaSR. This receptor seems to have a function of holding HSCs in close physical nearness to the endosteal surface 47. The mineral layer on the cellulose resembles that of bone. When the numerous foreign body giant cells/macrophages gathered around the mineralised cellulose try to get rid of the foreign material, Ca2+ is released generating a beneficial milieu for the primitive HSCs as it resembles the endosteal stem cell niche in the bone marrow. This theory is supported by the numerous CaSR-positive cells near the mineralised cellulose fibres, in the same areas as cells positive for CD34, a common marker for endothelial cells, are observed. These cells are not only found in the granulation tissue but also in the central parts of the implant. Similar cells are seen in uncoated cells, but in remarkably less quantity

coated sponge at day 7.

(figure 9 d-e).

**Figure 7.** Histology of subcutaneous cellulose implants. a) At 14 days HA-coated sponge is filled with granulation tissue (van Gieson-stained whole implants, scale bar = 1000 m). b) Haematoxylin-eosinstained sections one and three months after implantation, scale bar 100 m. c) At one year no significant difference can be observed between HA-coated and uncoated sponges (van Gieson-stained whole implants, scale bar = 1000 m. Modified from 38).

## *2.3.1. Cell trafficking and homing to regenerated cellulose*

Cellular movement and re-localisation are essential for many fundamental physiologic properties, not only during embryonic development, but also during wound healing and organ repair. At the wound site, local and infiltrated cells release chemokines that recruit blood-circulating stem and progenitor cells. These bioactive agents also increase bone marrow cell mobility, thus facilitating cell mobilisation into the peripheral blood and consequently into the sites of wound healing 39. Stromal-derived factor-1 (SDF-1) is one powerful chemokine in stem cell trafficking that regulates both haematopoietic, endothelial and mesenchymal progenitor cells. The biological effects of SDF-1 are mediated by the chemokine receptor CXCR4 40-43. During the early stages of wound healing, SDF-1 seems to be up-regulated by the influence of pro-inflammatory factors like TNF- which creates a SDF-1 concentration gradient that triggers the recruitment of CXCR4-expressing cells from the blood stream to the site of injury, where these cells further differentiate into other functional repair cells 44.

Mineralised cellulose implant not only attracts more inflammatory cells than uncoated cellulose but also circulating bone marrow-derived stem cells of both haematopoietic and mesenchymal origin 45. SDF-1 expression (GEO series accession no. GSE19748 and GSE19749; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSExxx) is upregulated in HA-sponges together with its receptor CXCR4 (figure 8). This strongly indicates that the HA-coated implant has a better homing capacity of circulating bone marrow-derived stem cells than the uncoated one.

coated and uncoated were detected (figure 7). 38

implants, scale bar = 1000 m. Modified from 38).

functional repair cells 44.

cells than the uncoated one.

*2.3.1. Cell trafficking and homing to regenerated cellulose* 

formation, which is histologically normal, does not disturb the animals in any way. After 14 days postoperatively, the foreign body reaction in HA-coated sponges starts to diminish. At one month, the difference between the HA-coated and uncoated cellulose had levelled off and at the end of the study, at one year no obvious histological difference between the

**Figure 7.** Histology of subcutaneous cellulose implants. a) At 14 days HA-coated sponge is filled with granulation tissue (van Gieson-stained whole implants, scale bar = 1000 m). b) Haematoxylin-eosinstained sections one and three months after implantation, scale bar 100 m. c) At one year no significant difference can be observed between HA-coated and uncoated sponges (van Gieson-stained whole

Cellular movement and re-localisation are essential for many fundamental physiologic properties, not only during embryonic development, but also during wound healing and organ repair. At the wound site, local and infiltrated cells release chemokines that recruit blood-circulating stem and progenitor cells. These bioactive agents also increase bone marrow cell mobility, thus facilitating cell mobilisation into the peripheral blood and consequently into the sites of wound healing 39. Stromal-derived factor-1 (SDF-1) is one powerful chemokine in stem cell trafficking that regulates both haematopoietic, endothelial and mesenchymal progenitor cells. The biological effects of SDF-1 are mediated by the chemokine receptor CXCR4 40-43. During the early stages of wound healing, SDF-1 seems to be up-regulated by the influence of pro-inflammatory factors like TNF- which creates a SDF-1 concentration gradient that triggers the recruitment of CXCR4-expressing cells from the blood stream to the site of injury, where these cells further differentiate into other

Mineralised cellulose implant not only attracts more inflammatory cells than uncoated cellulose but also circulating bone marrow-derived stem cells of both haematopoietic and mesenchymal origin 45. SDF-1 expression (GEO series accession no. GSE19748 and GSE19749; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSExxx) is upregulated in HA-sponges together with its receptor CXCR4 (figure 8). This strongly indicates that the HA-coated implant has a better homing capacity of circulating bone marrow-derived stem

**Figure 8.** HA-coated cellulose contain large amount of CXCR4-positive cells. Numerous clusters (arrow heads) of and individual CXCR4-positive (brownish coloured) cells are detected throughout the HAcoated sponge at day 7.

Haematopoietic stem cells seem to be the first stem cells to invade the empty centres of the HA-coated cellulose implants (figure 9 a-c). The more abundant occurrence of HSCs is most probably responsible for the augmented blood vessel formation in HA-coated cellulose. The earliest haematopoietic precursor, the haemangioblast, is namely the precursor for both haematopoietic and endothelial cell lineages, not only during embryogenesis but also in adults 14, 46. The haematopoietic progenitors, especially in the HA-coated implants, were located in close contact with the cellulose fragments (figure 10 a). Hence, the coating of cellulose with HA creates an environment that facilitates stem cell homing more efficiently than uncoated cellulose. In the bone marrow, undifferentiated HSCs are detected near the inner surface of the medullary cavity, i.e. the endosteum, in the so-called endosteal stem cell niche. At this site, the bone is in constant turnover: bone is formed by the osteoblasts and removed by specific macrophages, the osteoclasts. Due to bone degradation, soluble calcium ions (Ca2+) are released into the bone marrow fluid. Various cells, including primitive HSCs, respond to extracellular ionic calcium concentrations through a calcium sensing receptor, CaSR. This receptor seems to have a function of holding HSCs in close physical nearness to the endosteal surface 47. The mineral layer on the cellulose resembles that of bone. When the numerous foreign body giant cells/macrophages gathered around the mineralised cellulose try to get rid of the foreign material, Ca2+ is released generating a beneficial milieu for the primitive HSCs as it resembles the endosteal stem cell niche in the bone marrow. This theory is supported by the numerous CaSR-positive cells near the mineralised cellulose fibres, in the same areas as cells positive for CD34, a common marker for endothelial cells, are observed. These cells are not only found in the granulation tissue but also in the central parts of the implant. Similar cells are seen in uncoated cells, but in remarkably less quantity (figure 9 d-e).

Cellulose - A Biomaterial with Cell-Guiding Property 95

progenitors mature in association with macrophages 53, 54, the plentiful macrophages, especially in the HA-coated implants, might further back up the erythropoietic differentiation of HSCs in the granulation tissue. Microarray data (GEO series accession no. GSE19748 and GSE19749; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSExxx) revealed many genes related to erythropoiesis like erythropoietin and its receptor EpoR, the transciption factors Hif-1gata-1 and -2, and particularly Alas2, which is exclusively expressed in developing red blood cells called erythroblasts and is required for the

Haemoglobin has traditionally been thought to serve as the main oxygen transporter in erythrocytes. Many studies, including ours 56-65, show, however, that haemoglobin expression is much more versatile than previously has been assumed. During granulation tissue formation in the cellulose sponges, the haemoglobin expression pattern showed a biphasic pattern 52. The first peak appeared during the most intense inflammatory response in the initiation of the healing process before invasion of HSCs, indicating that also another cell type is participating in the haemoglobin expression. Since active macrophages are known to express globin 64, these cells (figure 10 e-g) are most likely responsible for the early globin expression in the granulation tissue. In macrophages, the globins are most probably involved in processes different from oxygen transport and delivery to tissues. There is accumulating evidence that haemoglobin also binds, stores and transports nitric oxide. Nitric oxide is an important gaseous signalling molecule in wound healing 66 involved, among other things, in the formation of granulation tissue and new blood vessels 67-69. While nitric oxide is a prerequisite for successful wound healing, an excess of this signalling molecule may be as harmful as its underproduction 67. The fact that an intense expression of inducible nitric oxidase synthase (iNOS), an enzyme that catalyses the

**Figure 10.** Double staining confirmed different haemoglobin positive cell types in cellulose implants. The granulation tissue in cellulose sponges contains haemoglobin (a) -producing glycophorin Apositive cells (b) implying that haematopoietic precursor cells are able to differentiate into red blood cells. c) Merged image of haemoglobin- and glycophorin A-positive cells. d). Red blood cells in a blood vessel in the capsule area of HA-implant; haemoglobin (upper) positive, glycophorin A (middle) and merge image (lower). e) CD-68 positive cells indicating macrophages. The same cells are also positive for haemoglobin (f). (g) Merged image of CD-68- and glycophorin A-positive cells (scale bar 20 m,

expression of -globin 55.

modified from 52).

**Figure 9.** Stem cells are located near the cellulose fragments. a) General histology showing cells gathering around the cellulose fragments (cf) at day 7. The arrows point at pores (scale bar = 50 m). b). HA-coated implants contains numerous cells (arrowhead) positive for c-kit, a marker for premature cells (day 7; scale bar = 25 m). c) Small rounded cells (arrowhead) positive for CD34, a commonly used marker for HSCs (day 7; scale bar = 25 m). More CaSR-positive cells (red fluorescence) are observed in HA-coated implants at day 7 (d) than in uncoated (e) sample (scale bar = 25 m).

In the cellulose implants, mesenchymal stem cells are mainly found in the forming granulation tissue 45 in line with the fact that these primitive cells home to the wound site and differentiate into connective tissue cells that produce the extracellular matrix of the granulation tissue 48. In addition, MSCs secrete signals that limit systemic and local inflammation, decrease apoptosis in the threatened tissue, stimulate neovascularisation, activate local stem cells, modulate the immune cells, and exhibit direct antimicrobial activity 18, 49, 50. Therefore, the more abundant occurrence of MSCs in the HA-coated cellulose sponge most probably contribute to the enhanced blood vessel formation compared to uncoated cellulose and to the declining of the foreign body reaction during the second week of implantation. MSCs also secrete many cytokines that stimulate haematopoiesis, mainly the myeoloid cell lineage, but MCSs seems to have a supportive effect on erythropoiesis, the process of red blood cell formation, as well 51.

## *2.3.2. Experimental granulation tissue expresses haemoglobin*

An unexpected finding was that the granulation tissue induced by cellulose sponge contains haemoglobin producing glycophorin A-positive cells (figure 10 a-d) indicating that the haematopoietic precursor cells are also able to differentiate into the erythropoietic lineage 52. This, in turn, suggests that this repair tissue is capable of making blood. In healthy adults, globin has been considered to be expressed only in the bone marrow area by immature erythropoietic precursors. When the mature red blood cell or erythrocyte emerges from the bone marrow, it has lost its nucleus, ribosomes and mitochondria, which means that the cell is no longer capable of gene expression. As in bone marrow, where erythroid progenitors mature in association with macrophages 53, 54, the plentiful macrophages, especially in the HA-coated implants, might further back up the erythropoietic differentiation of HSCs in the granulation tissue. Microarray data (GEO series accession no. GSE19748 and GSE19749; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSExxx) revealed many genes related to erythropoiesis like erythropoietin and its receptor EpoR, the transciption factors Hif-1gata-1 and -2, and particularly Alas2, which is exclusively expressed in developing red blood cells called erythroblasts and is required for the expression of -globin 55.

94 Cellulose – Medical, Pharmaceutical and Electronic Applications

**Figure 9.** Stem cells are located near the cellulose fragments. a) General histology showing cells gathering around the cellulose fragments (cf) at day 7. The arrows point at pores (scale bar = 50 m). b). HA-coated implants contains numerous cells (arrowhead) positive for c-kit, a marker for premature cells (day 7; scale bar = 25 m). c) Small rounded cells (arrowhead) positive for CD34, a commonly used marker for HSCs (day 7; scale bar = 25 m). More CaSR-positive cells (red fluorescence) are observed in

In the cellulose implants, mesenchymal stem cells are mainly found in the forming granulation tissue 45 in line with the fact that these primitive cells home to the wound site and differentiate into connective tissue cells that produce the extracellular matrix of the granulation tissue 48. In addition, MSCs secrete signals that limit systemic and local inflammation, decrease apoptosis in the threatened tissue, stimulate neovascularisation, activate local stem cells, modulate the immune cells, and exhibit direct antimicrobial activity 18, 49, 50. Therefore, the more abundant occurrence of MSCs in the HA-coated cellulose sponge most probably contribute to the enhanced blood vessel formation compared to uncoated cellulose and to the declining of the foreign body reaction during the second week of implantation. MSCs also secrete many cytokines that stimulate haematopoiesis, mainly the myeoloid cell lineage, but MCSs seems to have a supportive effect on erythropoiesis, the

An unexpected finding was that the granulation tissue induced by cellulose sponge contains haemoglobin producing glycophorin A-positive cells (figure 10 a-d) indicating that the haematopoietic precursor cells are also able to differentiate into the erythropoietic lineage 52. This, in turn, suggests that this repair tissue is capable of making blood. In healthy adults, globin has been considered to be expressed only in the bone marrow area by immature erythropoietic precursors. When the mature red blood cell or erythrocyte emerges from the bone marrow, it has lost its nucleus, ribosomes and mitochondria, which means that the cell is no longer capable of gene expression. As in bone marrow, where erythroid

HA-coated implants at day 7 (d) than in uncoated (e) sample (scale bar = 25 m).

process of red blood cell formation, as well 51.

*2.3.2. Experimental granulation tissue expresses haemoglobin* 

Haemoglobin has traditionally been thought to serve as the main oxygen transporter in erythrocytes. Many studies, including ours 56-65, show, however, that haemoglobin expression is much more versatile than previously has been assumed. During granulation tissue formation in the cellulose sponges, the haemoglobin expression pattern showed a biphasic pattern 52. The first peak appeared during the most intense inflammatory response in the initiation of the healing process before invasion of HSCs, indicating that also another cell type is participating in the haemoglobin expression. Since active macrophages are known to express globin 64, these cells (figure 10 e-g) are most likely responsible for the early globin expression in the granulation tissue. In macrophages, the globins are most probably involved in processes different from oxygen transport and delivery to tissues. There is accumulating evidence that haemoglobin also binds, stores and transports nitric oxide. Nitric oxide is an important gaseous signalling molecule in wound healing 66 involved, among other things, in the formation of granulation tissue and new blood vessels 67-69. While nitric oxide is a prerequisite for successful wound healing, an excess of this signalling molecule may be as harmful as its underproduction 67. The fact that an intense expression of inducible nitric oxidase synthase (iNOS), an enzyme that catalyses the

**Figure 10.** Double staining confirmed different haemoglobin positive cell types in cellulose implants. The granulation tissue in cellulose sponges contains haemoglobin (a) -producing glycophorin Apositive cells (b) implying that haematopoietic precursor cells are able to differentiate into red blood cells. c) Merged image of haemoglobin- and glycophorin A-positive cells. d). Red blood cells in a blood vessel in the capsule area of HA-implant; haemoglobin (upper) positive, glycophorin A (middle) and merge image (lower). e) CD-68 positive cells indicating macrophages. The same cells are also positive for haemoglobin (f). (g) Merged image of CD-68- and glycophorin A-positive cells (scale bar 20 m, modified from 52).

formation of nitric oxide, which reflects the production of nitric oxide observed in 3-day-old HA-implant, but not at day 10 52, coincides with the strong inflammatory reaction that starts to decline during the second week of implantation 38. The production of haemoglobin during this phase might eliminate the excess nitric oxide and prevent its negative effect on matrix deposition, neovascularisation and apoptosis. In uncoated cellulose implants, iNOS is detected at day 10, which supports the observation of slower sequence of events in the granulation tissue formation in these uncoated implants

Cellulose - A Biomaterial with Cell-Guiding Property 97

that attracts more circulating bone marrow-derived progenitor cells whereas the calcium

Due to the cell-guiding property of silica rich HA-coated regenerated oxidised cellulose in combination with the capacity to promote proliferation of richly vascularised connective tissue, this material might have potential in clinical situations when rapid granulation tissue growth is needed as in treatment of poorly healing wounds. The contact with the HAcellulose sponge would be local and temporary, therefore minimizing any possible disadvantages. In addition to safety issues, the manufacturing process of coating cellulose with HA is relatively simple and cheap, and the HA-coated cellulose sponge is easy to

Wound healing is a complex and dynamic process of restoring cellular structures and tissue layers in the body. The physiological and coordinated response to injury is practically similar in all tissues and involves three distinct but overlapping phases that can be divided into inflammation, new tissue formation and remodelling 70. In turn, these three phases comprehend coordinated series of events that includes chemotaxis, phagocytosis, neocollagenesis, collagen degradation, and collagen remodelling. Furthermore, neovascularisation, epithelisation, and the production of new glycosaminoglycans (GAGs) and proteoglycans are vital during wound healing process.

The key initiators of the healing process are the platelets, which within minutes after injury aggregate and form fibrin clot in aim to control bleeding. In addition to their important role in hemostasis, platelets also liberate growth factors that will attract inflammatory and phagocytosing cells. The first cells to arrive are polymorphonuclear leucocytes, i.e. neutrophils that secrete proinflammatory cytokines. Shortly thereafter circulating monocytes will appear, are activated and converted in the tissue to macrophages that kill bacterial pathogens, scavenge tissue debris and destroy remaining neutrophils. Macrophages also mediate the transition from the inflammatory phase to the proliferative phase by releasing a variety of chemotactic agents and growth factors that stimulate cell

The second phase of wound healing is often called the proliferative phase or the granulation tissue formation phase. This stage starts normally two to three days after injury and lasts approximately two to three weeks. During this phase the provisional extracellular matrix is gradually filled with granulation tissue. The phenomenal feature is to diminish the area of tissue loss by contraction and fibroplasia. The infiltrated cells produce a new extracellular matrix, rich in blood vessels, which carry oxygen and nutrients to maintain the metabolic processes. Although new collagen and other extracellular matrix proteins are continuously actively synthesised, the earlier formed

phosphate layer contributes to hastened stem cell homing to the cellulose sponge.

handle, form and sterilise.

**BOX 2. BIOLOGY OF WOUND HEALING** 

migration, proliferation and formation of tissue matrix.
