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Bioengineered acellular dermal matrix for the repair of full thickness skin wounds in rats.

Introduction

The skin losses can occur due to acute trauma, resection of cutaneous malignancies, chronic wounds or even surgical interventions. The full thickness injuries are characterized by the complete destruction of epithelial regenerative tissues and due to lack of epithelialisation leads to extensive scarring [1]. The large wounds that cannot be corrected by conventional surgical procedures requires substitutes of missing tissue to keep the wound free of infection, to reduce pain and ensure early wound healing [2]. In autologous skin grafting donor site heal with some scarring and may be very painful. A tissue engineered construct is produced by culturing the required cell types on a biocompatible scaffold or extracellular matrix [3]. The scaffolds include synthetic biodegradable polymers, natural polymers and natural matrices derived from decellularised tissue [4]. The natural bio-scaffold have advantages over synthetic material that it mimic natural extracellular matrix (ECM) structure and composition, simulate natural stimulatory effects of ECM on cells and allows the incorporation of growth factors and other matrix proteins to further enhance cell functions. The scaffold allows cell invasion, their proliferation and secretion of own extra cellular matrix for longer duration leading to a complete and natural tissue replacement [5]. Collagen is the most widely used matrix material for the purpose of tissue engineering [6]. Dermal substitute scaffolds promote fibroblast adhesion, growth and infiltration which accelerate and enhance dermal and epidermal regeneration [7]. The porosity, pore size and pore structure of the scaffolds are important for nutrient supply and more infiltration of fibroblast cells. Open interconnected porous network enhances the diffusion rate of nutrients and waste materials [8]. The fibroblasts are common cells present in the connective tissue that synthesizes and continuously secretes precursors of extracellular matrix in mammalian tissues [9]. To prepare an ideal tissue engineered scaffold it is important that seeded cells proliferate and migrate throughout the matrix architecture and organize a homogenously distributed ECM. Therefore, the present study is designed to develop novel biomaterials for the management of full thickness skin wounds in rats.

Materials and Methods

Preparation of acellular dermal matrix and 3-D bioengineered scaffolds

The skin was procured by euthanizing rat and preserved in cold phosphate buffer saline (PBS) containing 0.1% amikacin. After initial washing, the tissue samples were cut into 20 x 20 mm2 pieces. De-epithelialization was done by placing skin pieces in hypertonic solution (605 mg tris base, 4 gram sodium chloride, 202.5 mg EDTA in 100 ml PBS) for 8 hours. After de-epithelialization, the dermis was decellularized using 1% sodium deoxycholate for 48 hours. Tissue samples were subjected to continuous agitation on an orbital shaker (150 rpm) during the deepithelialization and the decellularization process. Macroscopic and microscopic examination was done for confirmation of de-epithelialization and decellularization. Prepared acellular dermal matrix (ADM) was rinsed thrice (2 hours each) in sterile PBS on an orbital shaker and stored in PBS solution containing 0.1% amikacin and 0.1% sodium azide.

Isolation and culture of the primary mouse embryo fibroblasts (P-MEF) was done as per method described by Purohit [10]. After seeding the P-MEF cells on culture plates they were observed twice daily under inverted phase contrast microscope to access the viability and proliferation of cells. The acellular dermal matrix (ADM) was used as scaffold for the culture of P-MEF. The PMEF cells were seeded upon the ADM using the static seeding technique [11].

Animal preparation

Forty five (45) clinically healthy Sprague Dawley rats of either sex, weighing from 250 to 300 g (mean weight 287 [+ or -] 12-34 g), 3 and 4 months of age were used in the present study and were divided into 3 equal groups. The study was approved by Institute Animal Ethics Committee, Indian Veterinary Research Institute, Izatnagar, India. Animals were housed individually in cages, provided with commercial diet and water ad libitum and maintained under uniform conditions. Animals were acclimatised to approaching and handling for a period of 10-15 days before the start of the study.

Wound creation

Wound creation was carried out aseptically under general anaesthesia using xylazine (5 mg/kg intramuscularly) followed, 10 minutes later, by ketamine (50 mg/kg intramuscularly). Anaesthesia was maintained by additional dose of intravenous ketamine, if required. The animals were restrained in sternal recumbency and dorsal thoracic area was prepared for aseptic surgery. Using a sterile plastic template the vertices of the experimental wounds of 2x2 [cm.sup.2] dimensions were outlined on the dorso-thoracic region of the rats. A full thickness skin defect including the panniculus carnosus, was excised with a #11BP blade in each animal. Haemorrhage, if any, was controlled by applying pressure with sterile cotton gauze. The defect in group I was dressed with standard dressing material (Dermafin, a non-adhesive open mesh dressing, marketed by Hospikit health care and allied products, C-4, Benganga Deonar, Mumbai-38, India) and was taken as control. In group II the defect was repaired with ADM and in group III with acellular dermal matrices seeded with P-MEF. In groups II and III the graft was covered with ADM. All the wounds were bandaged properly with paraffin gauze. The antibiotic (cefadroxil) and the analgesic (meloxicam) were administered for three consecutive days after the surgery.

Evaluation of wound healing

Clinical observations

Rectal temperature was recorded upto 14 post-operative days in all the animals. Feeding pattern and general behavioral changes in all the animals was observed daily during the observation period.

Gross observations

The wound area and percent contraction was measured at day 0, 3, 7, 14, 21, 28 or till completion of healing [12].

Planimetry (Colour Digital Image Processing)

Colour photographs were taken at days 0, 3, 7, 14, 21, 28 or till completion of healing with the help of digital camera at a fixed distance. Analysis of shape, irregularity and colour of the lesion was determined.

Estimation of hydroxyproline and glucosamine at the healing site

The hydroxyproline [13] and glucosamine [14] contents in the healing tissue was measured on days 0, 7, 14, 21 and 28.

Immunological observations

The ability of biomaterials to stimulate immune system of host was assessed by performing ELISA, wherein the humoral response in the host was judged by detecting the presence of antibodies specific to biomaterials, if any. The sera collected on day 0 and 42 post-implantation from control and bioengineered graft implanted groups were evaluated for antibody titre generated. The level of antibodies present in serum samples collected prior to implantation (day 0) was recorded as base values. Hyperimmune serum raised against the native skin, acellular dermis and acellular dermis seeded with P-MEF were used as standard positive controls. The sera collected from animals were tested in ELISA to assess B-cell immune responses. The graft specific antibodies were expressed as mean [+ or -] SE of absorbance value (ELISA readings) at 492nm wavelength ([OD.sub.492]). The cell mediated immune response was assessed by MTT colorimetric assay. The stimulation of rat spleenocytes with concanavalin A (Con A) and phytohaemagglutinin (PHA) were considered as positive control, where as unstimulated culture cells were taken as negative control. At 0, 21, 28 and 42 days post-implantation, two rats each from each group were sacrificed and spleens were separated aseptically. The spleenocytes were cultured in-vitro [15]. These splenocytes were stimulated with antigen of native skin, acellular dermis, acellular dermis seeded with P-MEF, Con A and PHA.

Estimation of oxidative stress at the healing site

The specimens from the implantation site was collected in chilled PBS (pH 7.4) on day 3, 7, 14, 21 and 28 post-implantation for the estimation of oxidative stress at the healing site. The tissues were placed in iceboxes at below 40C without adding any chemical antioxidant and transported to the laboratory within an hour. Tissue homogenate was prepared in a ratio of 100 mg of wet tissue to 2 ml of 1.15% Tris-potassium chloride buffer, centrifuged at 3000 RPM for 10 minutes and supernatant was collected for further studies. Estimation of antioxidant enzymes included superoxide dismutase (SOD) [16], catalase [17], lipid peroxide (LPO) [18] and reduced glutathione [19].

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Histological observations

The specimens from the implantation site were collected on day 3, 7, 14, 21, and 28 post-implantation for the histopathological evaluation after euthanizing the animals. Tissue samples were obtained by excising the graft with a surrounding rim of normal skin and underlying panniculus carnosus and were fixed in phosphate buffered 10% formalin saline, dehydrated in ethanol, cleared in xylene and embedded in paraffin to get 5 micron thick paraffin sections. The sections were stained with hematoxylin and eosin and evaluated microscopically using histological scoring system [20, 21]. The sections from grafted area were observed under light microscope for epithelialization, inflammation, fibroplasias and neovascularization. The sections were also subjected to Masson's Trichrome staining to observe the collagen fiber density, thickness and their arrangement at the healing site. The group having less histopathological scores considered the better than the other groups.

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Scanning electron microscopic observations

The scanning electron microscopic (SEM) examination of the tissues was performed at 3, 7 and 28 days post-implantation. Scanning electron microscope model Jeol JSM-840 was used for ultra-structure observations at the healing site.

Statistical analysis

The data was analyzed by using the Statistical Program for Social Sciences (SPSS) software version 17.0. One way analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) were used to compare the means at different time intervals among different groups. Student's paired t test was used to compare the mean values at different time intervals with their base values in each group [22].

Results

Treatment with hypertonic saline solution for 8h under constant agitation resulted in complete deepithelialization (Fig.1). The epidermis was separated spontaneously or with slight mechanical assistance (Fig.2). Microscopically, native skin showed cellularity, dermal adenexa and other skin structures (Fig.3). The microscopic observation of the acellular scaffolds prepared by 1% SDC, at 18h, showed thin and compact collagen fibers with mild porosity. At 48h, the collagen fibers were thin, mildly loose with high porosity (Fig.4).

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The method used for isolation and culture of P-MEF was found suitable. After seeding the P-MEF cells showed characteristic growth and adherence pattern in-vitro and proliferated rapidly to complete the monolayer in 72-96 h. The confluent monolayer of P-MEF (a cell type that gives rise to connective tissue) was observed using phase contrast microscopy (Fig.5). Light microscopic examination revealed growth of P-MEF in the 3-D ADM. Fibroblasts cultured on 3-D acellular dermal matrices showed different extent of cell infiltration in to the scaffold at different time intervals. At 96h post-seeding, P-MEF cells proliferated throughout the scaffold and displayed ample infiltration throughout the scaffold with expression of some extracellular matrix (Fig.6). SEM examination of P-MEF cultured ADM revealed growth of these cells on these matrices. Process extension of PMEF cells towards the scaffold were seen indicating effective cell substrate binding (Fig.7).

Clinical observations

The animals of all the groups remained dull on the first day and assumed a haunched back posture while resting in their cages, instead of their natural resting posture of dorsal recumbency. The animals of group III started taking rest in dorsal recumbency from day 12 and in group II on day 21 onwards. However, the animals of group I (control) started taking rest on their back after 21 days. Animals of all the three groups started taking feed and water partially within 24h after surgery and feed and water intake became normal by 3rd post-operative.

Gross observations

Mean [+ or -] SE values of wound area ([mm.sup.2]) and total wound contraction (%) at different time intervals in various groups are presented in table 1. Although the original wound area created was 20x20mm (400[mm.sup.2]), but the wounds were expanded in group I as no graft was used in this group and the wound remained denuded. As the healing progressed, the wound area decreased significantly (P<0.05) at different time intervals in all the groups. On day 7 no significant difference was observed between the groups. However, significant decrease (P>0.05) in wound area was observed in group III on day 14 and wound was healed completely on day 21. On day 21, complete healing was observed in groups I and II. Significant (P<0.05) degree of exudation was observed in animals of group I (control) up to day 5 postoperatively and even mild degree of exudation was observed on 7th postoperative day. However, no exudation was observed in groups II and III after day 5 onwards. Postoperative pain persisted up to day 3 in all the three groups. However, significantly higher (P < 0.05) postoperative pain scores persisted up to day 6 in group I. No pain was observed in groups II and III on day 5 and onwards.

Planimetry

The results of planimetry at different time intervals are presented in fig.8. In group I on day 3, the wound site was found slightly bulged from its surrounding having pinkish red granular surface covered with milky white exudate. On day 7, the site appeared denser and had irregular blackish dried up patches. On day 14, it further dried up forming thick crust having retracted periphery. On day 21, the thick crust fallen off leaving pinkish wound surface. On day 28, the wound completely covered with large scar tissue. In group II on day 3, the top layer of acellular dermal graft appeared yellowish in color with necrotic margins, which shriveled and turned up blacker leaving few yellowish patches on day 7. The dark brown top layer further dried and got detached, leaving some part attached with the underlying tissue on day 14. On day 21, the dried up top layer was completely sloughed off and newly formed granulation tissue within the underlying acellular dermal graft covered the entire surface of the wound. On day 28, the wound healed up completely with no scar. In group III on day 3, the top acellular dermal graft covering became light brown in color and the interface marked with reddened border. On day 7, it got shrink slightly and seemed merged with the granulation tissue which was developed within the underlying mouse embryo fibroblast cultured acellular dermal graft. By day 21, the size of the wound got reduced and healed with no evidence of sloughing of the graft.

Estimation of hydroxyproline and glucosamine

Mean [+ or -] SE values of hydroxyproline and glucosamine in the tissue collected from different groups at different time intervals are presented in table 2. The hydroxyproline contents in healing tissue showed a gradual nonsignificant increase up to 14 days in groups I and II followed by gradual decrease up to 42 days. The values in group III were significantly higher (P<0.05) n day 14 as compared to groups I and II. The peak value of hydroxyproline contents (3.397 [+ or -] 0.593ig/ml) was recorded in this group on day 14. Glucosamine contents decreased non-significantly upto day 21. Thereafter, the values increased in all the group upto day 42.

Immunological observations

On day 42, antibody titre in response to native graft antigen increased in all the groups. The native graft antigen challenged (hyper-immunized) animals showed highest B cell response. The antibody titre of group III was found slightly higher as compared to group II. The antibody titre in response to acellular dermis antigen showed relatively rise in groups II and III, but decrease in OD was observed in group I. The acellular graft antigen challenged (hyper-immunized) animals showed no B cell response. There was less B cell response in animals implanted with acellular dermis seeded with P-MEF and acellular grafts. The P-MEF seeded graft antigen challenged (hyper-immunized) animals showed less B cell response.

Stimulation index (SI) of groups stimulated with native antigen

At day 21, the SI values were highest in different groups stimulated with native graft antigen as compared to stimulation with other antigens. The stimulation was lowest with acellular dermis antigen followed by P-MEF seeded acellular graft antigen.

Stimulation index (SI) of groups stimulated with acellular dermis antigen

On day 21, bioengineered graft implanted group (III) have higher SI value (1.0205 [+ or -] 0.063) as compared to other groups. As compared to the SI values of PHA all groups exhibited considerably less SI values. However, SI values of Con A were higher as compared to groups I. The T cell response was higher in groups II and III as evidenced by higher SI values as compared to control animals.

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Stimulation index (SI) of groups stimulated with PMEF seeded antigen

On day 21, group III animals showed minimal SI values (0.9067 [+ or -] 0.019). The T cell response was more in group III. On day 42, the SI values of all the groups were lower as compared to PHA.

Estimation of oxidative stress at the healing site

Mean [+ or -] SE values of lipid peroxide, reduced glutathione, superoxide dismutase and catalase in the tissue collected from different groups at different time intervals are presented in table 3.

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Lipid peroxide (LPO)

The level of malonyldialdehyde increased significantly

(P<0.05) up to day 14 in group I. Thereafter, the values decreased and reached within normal limits on day 28. Significantly higher (P<0.05) values were also observed in groups II and III on day 7. Thereafter, the values decreased and returned to normal values on day 21. Comparison among the groups revealed that the values were sisnificantly higher (P<0.05) in group I at different time intervals.

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Reduced glutathione (GSH)

On day 3, significantly lower (P<0.05) values of GSH were observed in groups I ans II. However, in group III the values showed no significant difference as compared to normal skin. On day 7, GSH values increased and reached to near normal in groups II and III. The values of group I, reached to near normal on day 21.

Superoxide dismutase (SOD)

Significantly higher values (P<0.05) were recorded upto day 14 in group I. Thereafter, the values decreased and reached within normal limits on day 28 and 42. No significant difference was observed in remaining groups.

Catalase

Significantly (P<0.05) higher values were observed on day 3 in all the groups. Thereafter, the values showed decline trends from day 7 onwards and reached to near normal values on day 28. However, significantly (P<0.05) higher values were persisted in group I at different time intervals.

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Histopathological observations

The results of histopathological observations at different time intervals are presented in fig. 9. The histopathological observation scores in different groups at different time intervals are presented in table 4.

In group I on day 3, partial epithelialization was seen and the granulation tissue showed severe inflammatory reaction, fibroplasia and neovascularization. Neutrophils were in abundance. The total histopathological score was 24 at this stage. Collagen fibers were less dense, thin and worst arranged. On day 7, the stroma showed similar appearance as on day 3 however, fibroplasia was moderate and collagen fibers were thick and better arranged. The score was 20. On day 14, stroma and collagen fibers were similar as on day 3 but, necrosis of superficial surface with infiltration of neutrophils and macrophages was observed. On day 21, epithelialization was complete and the score reduced to 17. Collagen fibers were dense, thick and better arranged. On day 28, epithelium and stroma resembled normal skin and the score further reduced to10.

In group II on day 3, epithelialization started from the wound margins and superficial ADM graft showed severe necrotic changes. Inflammation and fibroplasia was moderate with mild neovascularization around the underlying graft. Collagen fibers were dense, thick and better arranged. The total histopathological score was 16 (table 4). On day 7, superficial graft showed sloughing and the underlying acellular graft showed moderate inflammation with macrophages, lymphocytes and few neutrophils. Severe neovascularization within the stroma was noticed. Fibroblastic cells were seen invading the graft. Collagen fibers were similar to day 3 and the score was 18. On day 14, inflammation and neovascularization were less as compared to day 7. On day 21, the epithelialization was still incomplete and underlying stroma had few capillaries and less dense collagen fibers as compared with day 14. The histopathological score further reduced to 16. On day 28, epithelialization was complete and inflammation had subsided. Fibroplasia was similar to day 21. Macrophages laden with golden pigment were present underneath the healing tissue and ADM graft was completely absorbed. Collagen fibers were denser, thick and best arranged and the total histopathological score reduced to 9.

In group III on day 3, there was no evidence of epithelialization, and the wound was covered with necrotic ADM graft. The underlying ADM graft cultured with P-MEF cells showed good infiltration of fibroblasts within the graft. The stroma proliferation with fibroblasts and newer capillaries was very prominent. Collagen fibers were dense, thin and haphazardly arranged. The total histopathological score was 22. On day 7, there was partial epithelialization and fewer amounts of dermal elements as compared to day 3. However, the collagen fibers were best arranged. On day 14, inflammation was moderate with mild neovascularization. Collagen fibers were denser, thicker and better arranged and the total score reduced to 13. The collagen of the graft was under the process of resorption with organization of granulation tissue. On day 21, epithelialization was complete and inflammation was absent. Fibroplasia resembled normal skin with mild neovascularization. Muscle giant cells were present within the granulation tissue and the score further reduced to 10. In this group, the healing was completed at 21 day. On day 28, the changes were similar to day 21 except neovascularization which resembled normal skin and collagen fibers were best arranged. The histopathological score was 9 at this stage (table 4). This group showed lower scores at different stages of healing.

Scanning electron microscopic observations

In group II on day 3, the upper ADM graft dried and partially detached leaving some part attached to the underlying ADM graft. Fibroplasia was moderate with few inflammatory cells and spherical fibroblasts in the acellular dermal graft (Fig.10). Collagen fibers were dense thick and better arranged. On day 7, the pores of the underlying graft wire filled with stroma. There was ingrowth of fibroblasts in to acellular matrix at host graft junction (Fig.11).On day 28, stroma had dense collagen fibers which was covered with epidermal keratinocyte cell line. In group III on day 3, the P-MEF cultured graft showed good infiltration of fibroblasts and secreted collagen within the pores of the matrix. Process extension towards the scaffold was seen indicating effective cell binding to the matrix (Fig.12). The tendency of round fibroblast cells to attach with the pores was also evident. Cells were randomly dispersed over the entire matrix and formed a layer over the graft. The fibroblast proliferated further and well distributed within the graft. On day 28, the healing tissue was similar to normal skin tissue (Fig.13).

Discussion

The rat skin was subjected to hypertonic saline for 8 hours and at this time interval the epidermis was separated spontaneously or with slight mechanical assistant. Osmotic shock with hypertonic solution was used to lyse the cells within tissues [23]. The chelating agent, EDTA, forms a ring shaped molecular complex that firmly binds and isolates a central metal ion. It has been shown that divalent Ca2+ and Mg2+ are necessary for cell attachment to collagen and fibronectin at the Arg-Gly-Asp receptor [24]. By binding the divalent cations that are present at the cell adhesions to the ECM, the EDTA facilitates removal of cellular material from the tissue. Deepithelialization and decellularization of human skin can be done after overnight incubation in 10X PBS with antibiotic cocktail and EDTA [25].

Decellularization of donor skin is one method to retain the native collagen configuration and fiber orientation while removing cell components [26]. In the present study the deepithelized skin was subjected to 1% SDC treatment. The time of reaction (18h and 48h) was adjusted to obtain the optimized ADM for seeding of PMEF. The de-epithelialized skin subjected to 1% SDC treatment for 18h resulted in 100% acellularity with mildly thick collagen fibers. The cellular debris was seen in between the void spaces of collagen fibers. Denser tissues such as dermis, tendon and trachea required prolonged agitation protocols lasting from days to months [27]. However, in the present study desired results were achieved after 48h of treatment with 1% SDC. Other workers repoted that cells and cell products cannot be completely removed from dense tissues even with the most rigorous processing methods [28].

The growth and infiltration of P-MEF within the ADM was found to be maximum at 48 hr. Similar findings were also reported after expansion of human fibroblasts on the micronized acellular dermal matrices following 7 days of culture [11]. The SEM examination also revealed growth and adhesion of fibroblasts within the acellular matrices. These findings are in agreement with the earlier findings where human fibroblasts and mouse fibroblasts (L929 cells) were able to grow over the collagen chitosan sponges and carboxyethyl chitosan/polyvinyl alcohol (CECS/PVA) electrospun fiber mats, respectively with good results [29, 30]. The fibroblast cells seeded on collagen matrix emitted distinct patterns of signaling and migration [31].

Dullness, depression and partial anorexia observed in the immediate postoperative period may be attributed to surgical trauma, pain and inflammation at the site of reconstruction [32]. Double layered skin construct in groups II and III showed significantly less wound contraction than the untreated wounds [33]. The collagen scaffolds not only provides mechanical enforcement counteracting dermal contraction and thus prevented shrinkage, but also creates conditions that induce fibroblasts to produce an authentic dermal matrix [34]. Effective inhibition of wound contraction by contractile fibroblasts has long been considered essential for optimal dermal regeneration in full thickness skin wounds [35]. Fibroblasts growth factor-2 (FGF2) may also reduce wound contraction by inhibiting the phenotypic change of fibroblasts to myofibroblasts, which is likely involved in contraction [36]. Random orientation of the pores in the scaffold may prevent the remaining myofibroblasts from contracting the wound area. When cells are randomly bound to the scaffold the sum of the contracting forces are near zero [37]. The cultured fibroblasts particularly with a dermal support do not regress when transplanted to a living tissue [38]. The fibroblasts contribute to the wound healing process reduce the contraction of the wound and support collagen synthesis and neovascularization.

Degree of exudation was significantly higher (P<0.05) in animals of groups I (control) which may be due to acute inflammatory reaction at the site in response to the surgical trauma. The exudate decreased significantly in groups II and III where ADM and P-MEF treated ADM were used. Kin et al. [39] also reported significant decrease of exudation in full thickness wounds of rats treated with small intestinal submucosa as compared to untreated wounds. A reverse correlation has been observed between the survival area of the skin graft and the degree of exudation of the graft bed [40]. The significantly higher (P < 0.05) postoperative pain scores persisted up to day 6 in animals of group I (open wound) might be due to denuded wound and subsequently the extent of inflammation and tissue reaction. No pain was observed in groups II and III on day 5 and onwards. Injury to tissue causes a number of changes in the nociceptive system. The injured nociceptors become highly sensitized to stimuli. Inflammatory mediators released during and after surgery also sensitized the peripheral nociceptors to further stimuli [41].

On day 3the wounds of the animals of group I was completely filled with pink granulation tissue. The colour of the centre portion of the graft was milky white and the host graft junction was pale in animals of groups II and III. On day 14, the graft was in the stage of resorption in the animals of group III. The wound was covered with thick crust in groups I. On day 21, the crusts of the wound were detached from the wound in group I and wound appeared again pinkish in colour. The graft was completely absorbed and newly formed epidermis covered the whole surface of the wound in groups II and III animals. Early healing in group III might be due to the presence of P-MEF within the acellular dermal graft. A dermal component provides an environment that promotes vascularization of the graft and fibroblasts play an active role in the replacement of dermal matrix [42]. Scar tissue was highest in animals of groups I where no graft was used and healing takes place by severe contraction and scar formation. Minimum scar formation was observed in group III animals.

Hydroxyproline contents in healing tissue showed a gradual non-significant increase (P>0.05) upto 7 days in group I. In group III the hydroxyproline value was found significantly higher (P<0.05) on day 14. Increased fibroblast proliferation activates the production of collagen. The increased level of hydroxyproline is indicative of increased amount of collagen deposition. The significantly high hydroxyproline content in group III indicates more collagen deposition in this group as compared to group I. The increased hydroxyproline content in the wounds treated with biotinylated GHK incorporated collagen matrices was predominantly due to enhanced collagen synthesis [43]. The higher concentration of glucosamine on day 3 was observed in all the groups. Significant (P<0.05) decrease in glucosamine content was observed on day 7 in the animal of group I. However, the values decreased significantly up to day 28 in groups II and III. The young fibroblasts are responsible for the secretion of mucopolysaccharides which accumulates in the granulation tissue in large quantities in the beginning of healing process. As the healing progresses the concentration of glucosamine gradually decreases. It may be due to the fact that glucosamine being an amino sugar was utilized by granulation tissue for their further growth. The gradual decrease in the glucosamine contents of healing tissues up to 30 postoperative days correlate to the similar observations reported in normal wound healing [44]. The increase in glucosamine contents in early stage of wound healing could also be due to the dilation of capillaries immediately after injury. The dilation of capillaries had resulted in seepage of plasma and lymph rich in mucopolysaccharides.

The nature and degree of immunological response to a graft material is a crucial variable affecting the acceptance or failure of implanted biomaterial. The antibody titre in response to native graft antigen increased in all the groups. The major antigenic determinants are situated in the telopeptide regions of the molecule. The other two types of determinants are composed of the triple helix and of the amino acid sequence of the a-chains. The latter type is accessible only when the collagen is denatured [45]. When the antigenic determinants are exposed due to collagen degeneration, it results in severe immune response [28]. MHC class II antigens are present as surface molecule in the transplanted cells which is responsible for graft rejection. Fibroblasts lack these surface molecules [46]. This makes them immunologically relatively inert. This is fortunate from a tissue engineering point of view, as dermal regeneration matrices can be seeded with allogenic fibroblasts without risking an immune response [42]. There are certain reports supporting the hypothesis that allogenic fibroblasts are tolerated by the host [47].

The antigen prepared from native tissue showed highest SI in MTT assay. The nucleated cell of the body is able to present antigen, first by virtue of a constitutive MHC class I expression, and second by a de novo expression of MHC class II molecules on the surface of the cell. The SI recorded for group II was lower in comparison to the values of group I. The acellular grafts showed minimum antigenicity. The ability of this antigen to stimulate the lymphocytes in-vitro may be attributed to the fact that on treatment with biological detergent, the bonds between protein molecules are broken and results into a change from quaternary and tertiary structure to primary and secondary structures. Therefore, the acellular antigen had ability to trigger CMI response in host because of presence of shorter peptide fragments which can be presented to the immune system by MHC class II pathway and stimulate the [CD4.sup.+] lymphocytes.

On day 3, newly formed ECM was well spread out over the entire scaffold area in groups II and III as compared to group I which might be due to preformed collagen matrix secreted by the mouse embryo fibroblasts as evidenced by histological observation of the P-MEF cultured scaffold. The fibroblasts proliferated more of in the acellular matrices and the bioengineered acellular matrices [48]. Acute inflammatory immune response resulted from polymorphonucleocyte infiltration and subsequent loss of the graft material following implantation [49]. On day 7, although mild to severe inflammation and fibroplasia was present in all the three groups, but inflammation was minimum in group III. The early reduction of inflammation, as in case of group III, might facilitate the progress to the next phase of wound healing. The P-MEF seeded graft of group III was well tolerated by the host and partial epithelialization was observed at this time interval. Epithelialization process had started by 2 weeks in full thickness skin wounds treated with small intestinal submucosal sponges [39]. The acellular and bioengineered grafts (group II and III) were invaded by the fibroblasts and underwent neovascularization without any evidence of rejection. The underlying graft was under the process of resorption. Extracellular collagen degradation occurs by the action of enzymes including matrix metalloproteinases (MMPs) and through phagocytosis by macrophages and fibroblasts [50]. Intracellular collagen processing via phagocytosis occurs in some fibroblasts by cell receptor clustering followed by invagination of the cell membrane [51].

On day 14, least histological score was observed in group III. Delayed healing in group I might be due to more inflammation as compared to other groups. The release of growth factors and cytokines including TNF-a, IL-12 and MIP1- a by the neutrophils, mast cells and macrophages increases the process of inflammation at the site which may slow the repair process [52]. Sloughing of the upper layer of graft was observed in groups II and III. It may be either due to the desiccation of the graft in high environmental temperature or due to an impaired formation of new blood vessels [53]. Meanwhile, the underlying granulation tissue increased in mass that pushed up the superficial acellular dermal graft upwards. The animals of groups II and III showed partial epithelialization, well formed collagen and neovascularization but these processes were faster and earlier in group III. The proliferation and migration of epithelial cells are dependent on an adequate supply of oxygen [54]. Vascularization of the scaffolds from the patient's wound bed normally takes about 3 weeks [55]. The enhanced rate of wound contraction and significant reduction in healing time might be due to enhanced epithelialization. The underlying acellular dermal graft and P-MEF cultured grafts showed good infiltration of fibroblasts within the graft.

On day 21, histopathological score was least (score 10) in group III followed by group II (score 16). Epithelialization was also more similar to the normal skin in group III. Presence of fibroblasts in a dermal equivalent stimulates epidermal differentiation and accelerates the healing process by reducing the time need for the fibroblasts to invade the wound tissue and by early synthesis of new skin tissue, because the fibroblasts on the artificial dermis can release biologically active substances such as cytokines [26]. The early epithelialization in the P-MEF cultured group confirmed the accelerated epidermal differentiation and production of an organized dense collagen matrix without a prolonged inflammatory response. Early maturation of the wounds in group III as compared to control group wounds (group I) might be due to presence of pre-seeded fibroblasts in the scaffold. The superficial acellular dermal graft which acts as protective covering for the underlying graft tissue was detached from the wound after day 21. This may be due to the desiccation of the graft in high environmental temperature. Meanwhile, the underlying granulation tissue increased in mass that pushed up the graft upwards and after day 21 the grafted materials were detached. The bilayer concept of wound coverage in which both epidermal and dermal analogs are used is widely accepted [56]. The outer layer of such construct has to have a barrier function to protect the wound not only from bacterial contamination, fluid loss, but also, overheating and accumulation of tissue fluids. On day 28, all the wounds were completely covered with epidermis. Collagen fibers were best arranged and oriented parallel to the skin surface in group III suggested a better repair of the damaged tissue in this groups. The full-thickness skin wounds in rats treated with small intestinal submucosal sponges were completely covered with thin layer of epidermis by 4 weeks [39]. However, incomplete closure of the wound was observed even after 28 days post-implantation [33].

ROS scavenging enzymes plays an important role in the detoxification of ROS during cutaneous wound repair. Oxidative stress supervenes when generated free radicals exceeds the capacity of antioxidants defense of the body [57]. In present study the significantly higher MDA concentration in all the groups upto day 7 might be due to more tissue injury and inflammation and hence more oxidative stress. On day 14, the oxidative stress reduced in groups II and III as compared to group I might be due to the reduction of lipid peroxidation in these groups due to reduced inflammation which persisted in group I due to denuded surface of wound [58]. Reduced MDA concentration has also been recorded in tissue after application of a PDGF containing novel gel for cutaneous wound healing in rats [59].

Reduced glutathione (GSH) plays an important role in the protection of cells against oxidative damage caused by ROS. Oxidation-reduction coupling of GSH is central to the cellular response to oxidative stress. Earlier return of GSH values on day 7 in groups II and III may be due to lower level of oxidative stress in these groups. In group I the values reached to near normal on day 21 might be due to denuded wound surface resulted in more oxidative stress. It is authenticated that oxidative stress reduces GSH level by depleting--SH group [60]. Lower level of GSH in group I might be due to more utilization for detoxification of free radicals [61].

Superoxide dismutase (SOD) is a metallo enzyme that catalyses the dismutation of [O.sub.2]y - to [H.sub.2][O.sub.2] with remarkably high reaction rates. The level of SOD increased significantly (P<0.05) up to day 14 in groups I. Superoxide dismutase level increased on day 7 after wounding and highest levels of SOD mRNAs were found at the early stage of wound repair, when the oxidative burst occurs [62]. Increased concentrations of SOD have been observed in the treated groups by different workers [43, 59]. The results of the present study were in contrast to earlier findings [63] where reduced SOD activity in wound tissue between days 2 and 7 after cutaneous injury in rats. The discrepancy between results may be explained by inhibition of the enzymatic activities of this enzyme at the wound site by the high levels of ROS [64].

Catalase activity increased significantly up to day 3 in all the groups. The scavenged superoxide radicals were converted in to hydrogen peroxide, which stimulates the expression of catalase [65]. Thereafter, the values showed decline trend from day 7 onwards and reached to near normal values on day 21. However, significantly higher values persisted in group I at different time intervals. Increased concentrations of catalase have been observed in the treated groups by different workers [43, 59]. Reduced levels of ROS-detoxifying enzymes result in healing impairments which was supported by the observation of reduced activities of SOD and catalase in wounds of aged rats compared to young rats [66].

Scanning electron microscopic observations were found useful in understanding the healing process and 3-D growth of the cells within the graft tissue. On day 3, the SEM specimen of groups II showed fibroblasts growth and penetration within the scaffolds. The scaffold was covered with dense cell layer and attachment of the fibroblasts cells within the pores in group III as compared to group II. This might be due to migration of P-MEF cells throughout the 3-D ADM in this group as also observed in histopathological findings. On day 14, there was better interaction between P-MEF cultured scaffold and surrounding cellular mass in group III. Biodegradable electrospun scaffold supported the cellular growth and scaffolds becoming infiltrated by granulation tissue including fibroblasts and endothelial cells and the phenomenon was demonstrated by scanning electron microscopy [67]. On day 28, The ECM was well distributed within the scaffold and collagen fibers were dense thick and better arranged in group II. Porosity, mean pore size and orientation of scaffold affect the migration and distribution of cells within the scaffold [51]. Penetration of the cells within the pores of the graft might be due to good interconnected porosity of acellular dermis [68].

It was concluded that P-MEF cells seeded ADM facilitated early and better healing than the normal saline. Histological and electron microscopic observations showed that the bioengineered ADM augmented wound healing activity significantly by increasing cellular proliferation, formation of granulation tissue, neovascularisation, synthesis of collagen, epithelialization and early histological maturation in excisional wounds. P-MEF cells seeded ADM showed minimum immunological response and can be used clinically in the management of large skin wounds.

Acknowledgement

The authors acknowledge the financial assistance received from the Department of Biotechnology, Ministry of Science and Technology, New Delhi, India to carry out this work.

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Anil K. Gangwar (1), Naveen Kumar (1), Ashok K. Sharma (1), Kh. Sangeeta Devi (2), Mamta Negi (1), Sameer Shrivastava (3), Dayamon D. Mathew (1), V. Remya (1), Sonal (2), Arundeep P.S. (1), Swapan K. Maiti (1), Vineet Kumar (1), D. T. Kaarthick (1), N.P. Kurade (4), Rajendra Singh (5)

(1) Division of Surgery, (3) Division of Animal Biotechnology, (4) Division of Pathology, (5) Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India. 243122 (2) Department of Surgery and Radiology, College of Veterinary Science and Animal Husbandry, Faizabad, India. 224229

Received 28 January 2013; Accepted 1 March 2013; Available online 8 June 2013
Table 1: Mean [+ or -] SE of wound area ([mm.sup.2]) and percent
contraction (%) in different groups at different time intervals

Parameters           Groups   Time intervals (Days)
                                          0

                     I        630.33 [+ or -] 17.99 (b)
Wound area           II       400.83 [+ or -] 1.40 (a)
                     III      403.00 [+ or -] 1.57 (a)
                     I                   --
Percentcontraction   II                  --
                     III                 --

Parameters           Groups   Time intervals (Days)
                                           3

                     I        472.25 [+ or -] 23.80 * (c)
Wound area           II           338.33t11.81 * (ab)
                     III      378.50 [+ or -] 3.86 (b) *
                     I         24.824 [+ or -] 3.76 (c)
Percentcontraction   II        15.596 [+ or -] 2.92 (b)
                     III        6.07 [+ or -] 1.00 (a)

Parameters           Groups   Time intervals (Days)
                                            7

                     I         266.90 [+ or -] 31.11 * (b)
Wound area           II       288.50 [+ or -] 15.90 * (bc)
                     III       236.40 [+ or -] 6.86 (c) *
                     I         58.313 [+ or -] 4.30 ** (c)
Percentcontraction   II        28.073 [+ or -] 3.93 ** (a)
                     III       56.62 [+ or -] 1.84 (a) **

Parameters           Groups   Time intervals (Days)
                                          14

                     I        133.75 [+ or -] 17.08 * (a)
Wound area           II           139.36t19.83 * (b)
                     III      84.38 [+ or -] 19.18 (b) *
                     I        87.229 [+ or -] 2.30 ** (b)
Percentcontraction   II       70.168 [+ or -] 5.05 ** (a)
                     III      81.43 [+ or -] 4.67 (a) **

Parameters           Groups   Time intervals (Days)
                                        21                28

                     I         65.67 [+ or -] 9.76 *    Healed
Wound area           II        65.17 [+ or -] 2.93 *    Healed
                     III              Healed            Healed
                     I        89.581 [+ or -] 1.37 **   Healed
Percentcontraction   II       83.741 [+ or -] 0.77 **   Healed
                     III              Healed            Healed

(abc) Values with different alphabets differ significantly (P< 0.05)
between the groups at particular time interval

* Differ significantly (P< 0.05) from day 3 value

Table 2: Mean [+ or -] SE of level of Hydroxyproline ([micro]g/ml)
and glucosamine ([micro]g/ml) in the healing tissue of different
groups at different time intervals

Parameters       Groups   Time Intervals (Days)
                                     3

Hydroxyproline   I        1.237 [+ or -] 0.124 (a)
                 II       1.191 [+ or -] 0.362 (a)
                 III       2.98 [+ or -] 0.14 (c)
Glucosamine      I        0.136 [+ or -] 0.01 (c)
                 II       0.257 [+ or -] 0.02 (b)
                 III      0.167 [+ or -] 0.02 (ac)

Parameters       Groups   Time Intervals (Days)
                                      7

Hydroxyproline   I        1.253 [+ or -] 0.140 (a)
                 II       1.495 [+ or -] 0.113 (ab)
                 III       3.33 [+ or -] 0.37 (b)
Glucosamine      I         0.091 [+ or -] 0.01 (c)
                 II       0.095 [+ or -] 0.01 * (c)
                 III      0.136 [+ or -] 0.02 (bc)

Parameters       Groups   Time Intervals (Days)
                                   14

Hydroxyproline   I        2.237 [+ or -] 0.363
                 II       2.186 [+ or -] 0.361
                 III      3.40 [+ or -] 0.19 *
Glucosamine      I        0.064 [+ or -] 0.01
                 II       0.145 [+ or -] 0.03
                 III      0.083 [+ or -] 0.01

Parameters       Groups   Time Intervals (Days)
                                     21

Hydroxyproline   I        1.655 [+ or -] 0.211 (ab)
                 II       1.907 [+ or -] 0.454 (ab)
                 III      2.67 [+ or -] 0.13 (b) *
Glucosamine      I           0.062 [+ or -] 0.01
                 II          0.137 [+ or -] 0.04
                 III         0.088 [+ or -] 0.00

Parameters       Groups   Time Intervals (Days)
                                   28

Hydroxyproline   I        1.402 [+ or -] 0.299
                 II       1 .634 [+ or -] 0.294
                 III       1.99 [+ or -] 0.36
Glucosamine      I         0.084 [+ or -] 0.01
                 II        0.108 [+ or -] 0.03
                 III      0.0950 [+ or -] 0.03 *

Parameters       Groups   Time Intervals (Days)
                                   42

Hydroxyproline   I        1.608 [+ or -] 0.155
                 II       1.603 [+ or -] 0.310
                 III      1.995 [+ or -] 0.314
Glucosamine      I        0.1 14 [+ or -] 0.01
                 II       0.1 08 [+ or -] 0.01
                 III       0.01 [+ or -] 0.00

(abc) Values with different alphabets differ significantly (P< 0.05)
between the groups at particular time interval

* Differ significantly (P< 0.05) from day 3 value

Table 3: Mean [+ or -] SE of level of LPO (nmole MDA/mg protein), GSH
([micro]mole/mg protein), SOD (units/mg protein) and Catalase
(units/mg protein) in the healing tissue of different groups
at different time

Parameters      Groups   Time intervals (Days)
                                  0

LPO             I        0.628 [+ or -] 0.028
(nmole          II       0.628 [+ or -] 0.028
MDA/mg          III      0.628 [+ or -] 0.028
protein)

GSH             I        0.724 [+ or -] 0.04
([micro]mole/   II       0.724 [+ or -] 0.04
mg protein)     III      0.724 [+ or -] 0.04

SOD             I        3.569 [+ or -] 0.416
(units/mg       II       3.569 [+ or -] 0.416
protein)        III      3.569 [+ or -] 0.416

Catalase        I        4.344 [+ or -] 0.362
(units/mg       II       4.344 [+ or -] 0.362
protein)        III      4.344 [+ or -] 0.362

Parameters      Groups   Time intervals (Days)
                                      3

LPO             I         2.078 [+ or -] 0.551 (b)
(nmole          II        1.479 [+ or -] 0.815 (a)
MDA/mg          III       0.779 [+ or -] 0.052 (a)
protein)

GSH             I          0.111 [+ or -] 0.014 *
([micro]mole/   II         0.200 [+ or -] 0.015 *
mg protein)     III         0.631 [+ or -] 0.012

SOD             I          8.005 [+ or -] 0.384 *
(units/mg       II          3.438 [+ or -] 0.452
protein)        III         3.590 [+ or -] 0.050

Catalase        I        25.024 [+ or -] 0.780 * (a)
(units/mg       II       15.642 [+ or -] 0.654 * (b)
protein)        III      8.814 [+ or -] 0.582 (c) *

Parameters      Groups   Time intervals (Days)
                                       7

LPO             I         5.735 [+ or -] 0.463 * (c)
(nmole          II        3.242 [+ or -] 0.898 * (c)
MDA/mg          III         2.955 [+ or -] 0.102b *
protein)

GSH             I           0.345 [+ or -] 0.029 *
([micro]mole/   II           0.657 [+ or -] 0.022
mg protein)     III         0.821 [+ or -] 0.016 *

SOD             I         8.680 [+ or -] 0.655 * (b)
(units/mg       II         6.736 [+ or -] 0.299 (ab)
protein)        III        3.766 [+ or -] 0.465 (a)


Catalase        I         14.361 [+ or -] 0.933 * (b)
(units/mg       II       10.304  [+ or -] 0.158 * (a)
protein)        III        7.901 [+ or -] 0.391 (e)

Parameters      Groups   Time intervals (Days)
                                     14

LPO             I        3.896 [+ or -] 0.240 * (b)
(nmole          II        0.983 [+ or -] 0.130 (a)
MDA/mg          III       0.650 [+ or -] 0.063 (a)
protein)

GSH             I          0.307 [+ or -] 0.045 *
([micro]mole/   II          0.732 [+ or -] 0.030
mg protein)     III         0.733 [+ or -] 0.028

SOD             I          6.017 [+ or -] 0.351 *
(units/mg       II          4.333 [+ or -] 0.074
protein)        III         3.872 [+ or -] 0.226

Catalase        I         12.149 [+ or -] 0.519 *
(units/mg       II          7.884 [+ or -] 0.878
protein)        III         6.151 [+ or -] 0.416

Parameters      Groups   Time intervals (Days)
                                  21

LPO             I        1.122 [+ or -] 0.062
(nmole          II       0.613 [+ or -] 0.003
MDA/mg          III      0.642 [+ or -] 0.006
protein)

GSH             I        0.718 [+ or -] 0.028
([micro]mole/   II       0.729 [+ or -] 0.038
mg protein)     III      0.711 [+ or -] 0.024

SOD             I        5.719 [+ or -] 0.681
(units/mg       II       4.368 [+ or -] 0.090
protein)        III      4.934 [+ or -] 0.827

Catalase        I        5.150 [+ or -] 0.988
(units/mg       II       6.317 [+ or -] 0.355
protein)        III      4.336 [+ or -] 0.376

Parameters      Groups   Time intervals (Days)
                                    28

LPO             I        0.760 [+ or -] 0.113 (b)
(nmole          II       0.617 [+ or -] 0.037 (ab)
MDA/mg          III      0.476 [+ or -] 0.051 (a)
protein)

GSH             I          0.748 [+ or -] 0.025
([micro]mole/   II         0.711 [+ or -] 0.016
mg protein)     III        0.760 [+ or -] 0.013

SOD             I          3.846 [+ or -] 0.227
(units/mg       II         3.806 [+ or -] 0.485
protein)        III        3.456 [+ or -] 0.441

Catalase        I          4.708 [+ or -] 0.225
(units/mg       II         4.704 [+ or -] 0.683
protein)        III        4.359 [+ or -] 0.426

Parameters      Groups   Time intervals (Days)
                                  42

LPO             I        0.556 [+ or -] 0.004
(nmole          II       0.526 [+ or -] 0.046
MDA/mg          III      0.472 [+ or -] 0.013
protein)

GSH             I        0.738 [+ or -] 0.007
([micro]mole/   II       0.785 [+ or -] 0.026
mg protein)     III      0.711 [+ or -] 0.025

SOD             I        3.784 [+ or -] 0.083
(units/mg       II       3.719 [+ or -] 0.577
protein)        III      3.392 [+ or -] 0.186

Catalase        I        3.954 [+ or -] 0.338
(units/mg       II       4.653 [+ or -] 0.360
protein)        III      4.228 [+ or -] 0.066

(abc) Values with different alphabets differ significantly (P< 0.05)
between the groups at particular time interval

* Differ significantly (P< 0.05) from day 3 value

Table 4: Histopathological scores of different groups at
different time intervals

Parameters                           Time intervals (Days)

                                       3                       7

                               I      II      III      I      II

Epithelization                 2       2       3       2       2
Inflammation                   4       3       3       4       3
Fibroplasia                    4       3       4       3       3
Neovascularization             4       2       4       4       4
Collagen fiber Density         3       2       2       3       2
Collagen fiber Thickness       3       2       3       2       2
Collagen fiber arrangement     4       2       3       2       2
Total                         24      16      22      20      18

Parameters                         Time intervals (Days)

                                              14

                              III      I      II      III      I

Epithelization                 2       2       2       2       1
Inflammation                   2       4       2       2       2
Fibroplasia                    3       4       3       3       3
Neovascularization             3       4       3       2       3
Collagen fiber Density         2       3       2       1       3
Collagen fiber Thickness       2       3       2       1       3
Collagen fiber arrangement     1       4       2       2       2
Total                         15      24      16      13      17

Parameters                         Time intervals (Days)

                              21                      28

                              II      III      I      II      III

Epithelization                 2       1       1       1       1
Inflammation                   2       1       1       1       1
Fibroplasia                    3       1       1       2       1
Neovascularization             2       2       1       1       1
Collagen fiber Density         3       1       2       1       2
Collagen fiber Thickness       2       2       2       2       2
Collagen fiber arrangement     2       2       2       1       1
Total                         16      10      10       9       9

Epithelization: 1-Present, 2-Partially present, 3-Absent

Inflammation: 1-Resembling normal skin, 2-Mild, 3-Moderate, 4-Severe

Fibroplasia: 1-Resembling normal skin, 2-Mild, 3-Moderate, 4-Severe

Neovascularization: 1-Resembling normal skin (0-1 new blood vessel),
2-Mild (2-5), 3-Moderate (6-10), 4-Severe (> 10)

Collagen fiber density: 1-Denser, 2-Dense, 3-Less dense

Collagen fiber thickness: 1-Thicker, 2-Thick, 3-Thin

Collagen fiber arrangement: 1- Best arranged, 2-Better arranged,
3-Worse arranged, 4-Worst arranged

The group having less histopathological score was considered the best.
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Title Annotation:Original Article
Author:Gangwar, Anil K.; Kumar, Naveen; Sharma, Ashok K.; Devi, Kh. Sangeeta; Negi, Mamta; Shrivastava, Sam
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Date:Apr 1, 2013
Words:10687
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