Trends Biomater. Artif. Organs, 27(2), 67-80 (2013) http://www.sbaoi.

org/tibao

Original Article

Bioengineered Acellular Dermal Matrix for the Repair

of Full Thickness Skin Wounds in Rats
Anil K. Gangwar1, Naveen Kumar1, Ashok K. Sharma1, Kh. Sangeeta Devi2, Mamta Negi1,
Sameer Shrivastava3, Dayamon D. Mathew1, V. Remya1, Sonal2, Arundeep P.S. 1, Swapan K. Maiti1,
Vineet Kumar1, D. T. Kaarthick1, N.P. Kurade4, Rajendra Singh5
1
Division of Surgery, 3Division of Animal Biotechnology, 4Division of Pathology, 5Centre 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

The rat skin was de-epithelialized using hypertonic saline solution and further decellularized with 1% sodium deoxycholate.
The primary mouse embryonic fibroblast (P-MEF) cells were seeded on acellular dermal matrix (ADM). Fortyfive rats were
randomly divided into 3 groups of 15 animals each. A 20×20mm2 full thickness skin defect was created on dorsum. The defect
in group-I was dressed with standard dressing material, group-II with ADM, and group-III ADM seeded with P-MEF. In group-
III, complete healing was observed at day21. The values of hydroxyproline contents in group III were significantly higher on
days14 and 21 as compared to group-I. Significant decrease in glucosamine content was observed up to day28 in group-III.
Native graft antigen challenged animals showed highest B-cell response. The acellular graft antigen challenged animals showed
no B-cell response. Biopsy specimens showed minimum histopathological scores in group-III. The level of MDA and SOD
increased significantly up to day14 in group-I. On day3, significantly lower values of GSH were observed in group-I whereas,
significantly higher values of catalase were observed in all groups. SEM examination revealed well spread fibroblasts over the
scaffold in group-III. These results indicate that bioengineered grafts can be used clinically to manage full thickness skin
wounds.

Introduction effects of ECM on cells and allows the incorporation of

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

ECM. Therefore, the present study is designed to develop dorso-thoracic region of the rats. A full thickness skin
novel biomaterials for the management of full thickness defect including the panniculus carnosus, was excised
skin wounds in rats. with a #11BP blade in each animal. Haemorrhage, if any,
was controlled by applying pressure with sterile cotton
Materials and Methods gauze. The defect in group I was dressed with standard
dressing material (Dermafin, a non-adhesive open mesh
Preparation of acellular dermal matrix and 3-D dressing, marketed by Hospikit health care and allied
bioengineered scaffolds products, C-4, Benganga Deonar, Mumbai-38, India) and
The skin was procured by euthanizing rat and preserved was taken as control. In group II the defect was repaired
in cold phosphate buffer saline (PBS) containing 0.1% with ADM and in group III with acellular dermal matrices
amikacin. After initial washing, the tissue samples were seeded with P-MEF. In groups II and III the graft was
cut into 20 x 20 mm2 pieces. De-epithelialization was done covered with ADM. All the wounds were bandaged
by placing skin pieces in hypertonic solution (605 mg properly with paraffin gauze. The antibiotic (cefadroxil)
tris base, 4 gram sodium chloride, 202.5 mg EDTA in and the analgesic (meloxicam) were administered for three
100 ml PBS) for 8 hours. After de-epithelialization, the consecutive days after the surgery.
dermis was decellularized using 1% sodium deoxycholate Evaluation of wound healing
for 48 hours. Tissue samples were subjected to continuous
agitation on an orbital shaker (150 rpm) during the de- Clinical observations
epithelialization and the decellularization process.
Macroscopic and microscopic examination was done for Rectal temperature was recorded upto 14 post-operative
confirmation of de-epithelialization and decellularization. days in all the animals. Feeding pattern and general
Prepared acellular dermal matrix (ADM) was rinsed thrice behavioral changes in all the animals was observed daily
(2 hours each) in sterile PBS on an orbital shaker and during the observation period.
stored in PBS solution containing 0.1% amikacin and
Gross observations
0.1% sodium azide.

Isolation and culture of the primary mouse embryo The wound area and percent contraction was measured
at day 0, 3, 7, 14, 21, 28 or till completion of healing
fibroblasts (P-MEF) was done as per method described
[12].
by Purohit [10]. After seeding the P-MEF cells on culture
plates they were observed twice daily under inverted Planimetry (Colour Digital Image Processing)
phase contrast microscope to access the viability and
proliferation of cells. The acellular dermal matrix (ADM) Colour photographs were taken at days 0, 3, 7, 14, 21, 28
was used as scaffold for the culture of P-MEF. The P- or till completion of healing with the help of digital camera
MEF cells were seeded upon the ADM using the static at a fixed distance. Analysis of shape, irregularity and
seeding technique [11]. colour of the lesion was determined.
Animal preparation Estimation of hydroxyproline and glucosamine at the
healing site
Forty five (45) clinically healthy Sprague Dawley rats of
either sex, weighing from 250 to 300 g (mean weight The hydroxyproline [13] and glucosamine [14] contents
287 ± 12·34 g), 3 and 4 months of age were used in the in the healing tissue was measured on days 0, 7, 14, 21
present study and were divided into 3 equal groups. The and28.
study was approved by Institute Animal Ethics
Committee, Indian Veterinary Research Institute, Immunological observations
Izatnagar, India. Animals were housed individually in
The ability of biomaterials to stimulate immune system
cages, provided with commercial diet and water ad libitum
of host was assessed by performing ELISA, wherein the
and maintained under uniform conditions. Animals were
humoral response in the host was judged by detecting
acclimatised to approaching and handling for a period of
the presence of antibodies specific to biomaterials, if any.
10–15 days before the start of the study.
The sera collected on day 0 and 42 post-implantation from
Wound creation control and bioengineered graft implanted groups were
evaluated for antibody titre generated. The level of
Wound creation was carried out aseptically under general antibodies present in serum samples collected prior to
anaesthesia using xylazine (5 mg/kg intramuscularly) implantation (day 0) was recorded as base values. Hyper-
followed, 10 minutes later, by ketamine (50 mg/kg immune serum raised against the native skin, acellular
intramuscularly). Anaesthesia was maintained by dermis and acellular dermis seeded with P-MEF were used
additional dose of intravenous ketamine, if required. The as standard positive controls. The sera collected from
animals were restrained in sternal recumbency and dorsal animals were tested in ELISA to assess B-cell immune
thoracic area was prepared for aseptic surgery. Using a responses. The graft specific antibodies were expressed
sterile plastic template the vertices of the experimental as mean ±SE of absorbance value (ELISA readings) at
wounds of 2×2 cm2 dimensions were outlined on the 492nm wavelength (OD492). The cell mediated immune
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 Bioengineered Acellular Dermal Matrix for the Repair of Full Thickness Skin Wounds

response was assessed by MTT colorimetric assay. The homogenate was prepared in a ratio of 100 mg of wet
stimulation of rat spleenocytes with concanavalin A (Con tissue to 2 ml of 1.15% Tris-potassium chloride buffer,
A) and phytohaemagglutinin (PHA) were considered as centrifuged at 3000 RPM for 10 minutes and supernatant
positive control, where as unstimulated culture cells were was collected for further studies. Estimation of antioxidant
taken as negative control. At 0, 21, 28 and 42 days post- enzymes included superoxide dismutase (SOD) [16],
implantation, two rats each from each group were catalase [17], lipid peroxide (LPO) [18] and reduced
sacrificed and spleens were separated aseptically. The glutathione [19].
spleenocytes were cultured in-vitro [15]. These
splenocytes were stimulated with antigen of native skin, Histological observations
acellular dermis, acellular dermis seeded with P-MEF,
The specimens from the implantation site were collected
Con A and PHA.
on day 3, 7, 14, 21, and 28 post-implantation for the
Estimation of oxidative stress at the healing site histopathological evaluation after euthanizing the animals.
Tissue samples were obtained by excising the graft with
The specimens from the implantation site was collected a surrounding rim of normal skin and underlying
in chilled PBS (pH 7.4) on day 3, 7, 14, 21 and 28 post- panniculus carnosus and were fixed in phosphate buffered
implantation for the estimation of oxidative stress at the 10% formalin saline, dehydrated in ethanol, cleared in
healing site. The tissues were placed in iceboxes at below xylene and embedded in paraffin to get 5 micron thick
4 0 C without adding any chemical antioxidant and paraffin sections. The sections were stained with
transported to the laboratory within an hour. Tissue hematoxylin and eosin and evaluated microscopically

Fig 1: Showing treatment with hypertonic saline solution Fig 3: Microscopically native skin showing cellularity,
for 8h under constant agitation resulted in complete de- dermal adnexa and other skin structures (H&E stain, 100X)
epithelialization

Fig 4: Treatment with 1% SDC for 48h resulted in thin and

Fig 2: Figure showing epidermis was separated mildly loose collagen fibres with high porosity (H&E stain,
spontaneously or with slight mechanical assistance 100X)

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 A.K. Gangwar et al.

Fig 5: The confluent monolayer of P-MEF observed under Fig 7: SEM examination of P-MEF cultured ADM revealed
phase contrast microscope (20X) growth of these cells on these matrices

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
Fig 6: At 96h post-seeding P-MEF cells proliferated microscopic observation of the acellular scaffolds
throughout the scaffold and displayed ample infiltration
(H&E stain, 100X)
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).

using histological scoring system [20, 21]. The sections The method used for isolation and culture of P-MEF was
from grafted area were observed under light microscope found suitable. After seeding the P-MEF cells showed
for epithelialization, inflammation, fibroplasias and characteristic growth and adherence pattern in-vitro and
neovascularization. The sections were also subjected to proliferated rapidly to complete the monolayer in 72-96
Masson’s Trichrome staining to observe the collagen fiber h. The confluent monolayer of P-MEF (a cell type that
density, thickness and their arrangement at the healing gives rise to connective tissue) was observed using phase
site. The group having less histopathological scores contrast microscopy (Fig.5). Light microscopic
considered the better than the other groups. examination revealed growth of P-MEF in the 3-D ADM.
Fibroblasts cultured on 3-D acellular dermal matrices
Scanning electron microscopic observations showed different extent of cell infiltration in to the
scaffold at different time intervals. At 96h post-seeding,
The scanning electron microscopic (SEM) examination P-MEF cells proliferated throughout the scaffold and
of the tissues was performed at 3, 7 and 28 days post- displayed ample infiltration throughout the scaffold with
implantation. Scanning electron microscope model Jeol expression of some extracellular matrix (Fig.6). SEM
JSM-840 was used for ultra-structure observations at the examination of P-MEF cultured ADM revealed growth
healing site. of these cells on these matrices. Process extension of P-
Statistical analysis MEF cells towards the scaffold were seen indicating
effective cell substrate binding (Fig.7).
The data was analyzed by using the Statistical Program

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 Bioengineered Acellular Dermal Matrix for the Repair of Full Thickness Skin Wounds

Clinical observations color and the interface marked with reddened border. On
day 7, it got shrink slightly and seemed merged with the
The animals of all the groups remained dull on the first granulation tissue which was developed within the
day and assumed a haunched back posture while resting underlying mouse embryo fibroblast cultured acellular
in their cages, instead of their natural resting posture of dermal graft. By day 21, the size of the wound got reduced
dorsal recumbency. The animals of group III started taking and healed with no evidence of sloughing of the graft.
rest in dorsal recumbency from day 12 and in group II on
day 21 onwards. However, the animals of group I Estimation of hydroxyproline and glucosamine
(control) started taking rest on their back after 21 days.
Animals of all the three groups started taking feed and Mean±SE values of hydroxyproline and glucosamine in
water partially within 24h after surgery and feed and water the tissue collected from different groups at different time
intake became normal by 3rd post-operative. intervals are presented in table II. The hydroxyproline
contents in healing tissue showed a gradual non-
Gross observations significant increase up to 14 days in groups I and II
followed by gradual decrease up to 42 days. The values
Mean±SE values of wound area (mm2) and total wound in group III were significantly higher (P<0.05) n day 14
contraction (%) at different time intervals in various as compared to groups I and II. The peak value of
groups are presented in table I. Although the original hydroxyproline contents (3.397±0.593ìg/ml) was
wound area created was 20x20mm (400mm2), but the recorded in this group on day 14. Glucosamine contents
wounds were expanded in group I as no graft was used in decreased non-significantly upto day 21. Thereafter, the
this group and the wound remained denuded. As the values increased in all the group upto day 42.
healing progressed, the wound area decreased
significantly (P<0.05) at different time intervals in all the Immunological observations
groups. On day 7 no significant difference was observed
between the groups. However, significant decrease On day 42, antibody titre in response to native graft
(P>0.05) in wound area was observed in group III on day antigen increased in all the groups. The native graft
14 and wound was healed completely on day 21. On day antigen challenged (hyper-immunized) animals showed
21, complete healing was observed in groups I and II. highest B cell response. The antibody titre of group III
Significant (P<0.05) degree of exudation was observed was found slightly higher as compared to group II. The
in animals of group I (control) up to day 5 postoperatively antibody titre in response to acellular dermis antigen
and even mild degree of exudation was observed on 7th showed relatively rise in groups II and III, but decrease
in OD was observed in group I. The acellular graft antigen
postoperative day. However, no exudation was observed
challenged (hyper-immunized) animals showed no B cell
in groups II and III after day 5 onwards. Postoperative
response. There was less B cell response in animals
pain persisted up to day 3 in all the three groups. However,
implanted with acellular dermis seeded with P-MEF and
significantly higher (P < 0.05) postoperative pain scores
acellular grafts. The P-MEF seeded graft antigen
persisted up to day 6 in group I. No pain was observed in
challenged (hyper-immunized) animals showed less B cell
groups II and III on day 5 and onwards.
response.
Planimetry
Stimulation index (SI) of groups stimulated with
The results of planimetry at different time intervals are native antigen
presented in fig.8. In group I on day 3, the wound site At day 21, the SI values were highest in different groups
was found slightly bulged from its surrounding having stimulated with native graft antigen as compared to
pinkish red granular surface covered with milky white stimulation with other antigens. The stimulation was
exudate. On day 7, the site appeared denser and had lowest with acellular dermis antigen followed by P-MEF
irregular blackish dried up patches. On day 14, it further seeded acellular graft antigen.
dried up forming thick crust having retracted periphery.
On day 21, the thick crust fallen off leaving pinkish wound Stimulation index (SI) of groups stimulated with
surface. On day 28, the wound completely covered with acellular dermis antigen
large scar tissue. In group II on day 3, the top layer of
acellular dermal graft appeared yellowish in color with On day 21, bioengineered graft implanted group (III) have
necrotic margins, which shriveled and turned up blacker higher SI value (1.0205±0.063) as compared to other
leaving few yellowish patches on day 7. The dark brown groups. As compared to the SI values of PHA all groups
top layer further dried and got detached, leaving some exhibited considerably less SI values. However, SI values
part attached with the underlying tissue on day 14. On of Con A were higher as compared to groups I. The T cell
day 21, the dried up top layer was completely sloughed response was higher in groups II and III as evidenced by
off and newly formed granulation tissue within the higher SI values as compared to control animals.
underlying acellular dermal graft covered the entire Stimulation index (SI) of groups stimulated with P-
surface of the wound. On day 28, the wound healed up MEF seeded antigen
completely with no scar. In group III on day 3, the top
acellular dermal graft covering became light brown in On day 21, group III animals showed minimal SI values
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 A.K. Gangwar et al.

Fig 8: Planimetry observations of different groups at different time intervals

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 Bioengineered Acellular Dermal Matrix for the Repair of Full Thickness Skin Wounds

Fig 9: Histopathological observations of different groups at different time intervals

(0.9067±0.019). The T cell response was more in group Estimation of oxidative stress at the healing site
III. On day 42, the SI values of all the groups were lower
as compared to PHA. Mean ±SE values of lipid peroxide, reduced glutathione,
superoxide dismutase and catalase in the tissue collected

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 A.K. Gangwar et al.

Fig 10: SEM observation showing moderate fibroplasia Fig 12: Scanning electron microscopic observations
with few inflammatory cells and spherical fibroblasts in showing process extension towards the scaffold indicating
the acellular dermal graft effective cell binding to the matrix

Fig 11: SEM observations showing in growth of fibroblasts Fig 13: SEM observations on day 28 showing the healing
in to acellular matrix at host graft junction tissue was similar to normal skin tissue

from different groups at different time intervals are Superoxide dismutase (SOD)
presented in table III.
Significantly higher values (P<0.05) were recorded upto
Lipid peroxide (LPO) day 14 in group I. Thereafter, the values decreased and
reached within normal limits on day 28 and 42. No
The level of malonyldialdehyde increased significantly significant difference was observed in remaining groups.
(P<0.05) up to day 14 in group I. Thereafter, the values
decreased and reached within normal limits on day 28. Catalase
Significantly higher (P<0.05) values were also observed
in groups II and III on day 7. Thereafter, the values Significantly (P<0.05) higher values were observed on
decreased and returned to normal values on day 21. day 3 in all the groups. Thereafter, the values showed
Comparison among the groups revealed that the values decline trends from day 7 onwards and reached to near
were sisnificantly higher (P<0.05) in group I at different normal values on day 28. However, significantly (P<0.05)
time intervals. higher values were persisted in group I at different time
intervals.
Reduced glutathione (GSH)
Histopathological observations
On day 3, significantly lower (P<0.05) values of GSH
were observed in groups I ans II. However, in group III The results of histopathological observations at different
the values showed no significant difference as compared time intervals are presented in fig. 9. The histopathological
to normal skin. On day 7, GSH values increased and observation scores in different groups at different time
reached to near normal in groups II and III. The values of intervals are presented in table V.
group I, reached to near normal on day 21.

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 Bioengineered Acellular Dermal Matrix for the Repair of Full Thickness Skin Wounds

In group I on day 3, partial epithelialization was seen reduced to 10. In this group, the healing was completed
and the granulation tissue showed severe inflammatory at 21 day. On day 28, the changes were similar to day 21
reaction, fibroplasia and neovascularization. Neutrophils except neovascularization which resembled normal skin
were in abundance. The total histopathological score was and collagen fibers were best arranged. The
24 at this stage. Collagen fibers were less dense, thin and histopathological score was 9 at this stage (table V). This
worst arranged. On day 7, the stroma showed similar group showed lower scores at different stages of healing.
appearance as on day 3 however, fibroplasia was moderate
and collagen fibers were thick and better arranged. The Scanning electron microscopic observations
score was 20. On day 14, stroma and collagen fibers were
In group II on day 3, the upper ADM graft dried and
similar as on day 3 but, necrosis of superficial surface
partially detached leaving some part attached to the
with infiltration of neutrophils and macrophages was
underlying ADM graft. Fibroplasia was moderate with
observed. On day 21, epithelialization was complete and
few inflammatory cells and spherical fibroblasts in the
the score reduced to 17. Collagen fibers were dense, thick
acellular dermal graft (Fig.10). Collagen fibers were dense
and better arranged. On day 28, epithelium and stroma
thick and better arranged. On day 7, the pores of the
resembled normal skin and the score further reduced to10.
underlying graft wire filled with stroma. There was
In group II on day 3, epithelialization started from the ingrowth of fibroblasts in to acellular matrix at host graft
wound margins and superficial ADM graft showed severe junction (Fig.11).On day 28, stroma had dense collagen
necrotic changes. Inflammation and fibroplasia was fibers which was covered with epidermal keratinocyte
moderate with mild neovascularization around the cell line. In group III on day 3, the P-MEF cultured graft
underlying graft. Collagen fibers were dense, thick and showed good infiltration of fibroblasts and secreted
better arranged. The total histopathological score was 16 collagen within the pores of the matrix. Process extension
(table V). On day 7, superficial graft showed sloughing towards the scaffold was seen indicating effective cell
and the underlying acellular graft showed moderate binding to the matrix (Fig.12). The tendency of round
inflammation with macrophages, lymphocytes and few fibroblast cells to attach with the pores was also evident.
neutrophils. Severe neovascularization within the stroma Cells were randomly dispersed over the entire matrix and
was noticed. Fibroblastic cells were seen invading the formed a layer over the graft. The fibroblast proliferated
graft. Collagen fibers were similar to day 3 and the score further and well distributed within the graft. On day 28,
was 18. On day 14, inflammation and neovascularization the healing tissue was similar to normal skin tissue
were less as compared to day 7. On day 21, the (Fig.13).
epithelialization was still incomplete and underlying
stroma had few capillaries and less dense collagen fibers Discussion
as compared with day 14. The histopathological score The rat skin was subjected to hypertonic saline for 8 hours
further reduced to 16. On day 28, epithelialization was and at this time interval the epidermis was separated
complete and inflammation had subsided. Fibroplasia was spontaneously or with slight mechanical assistant.
similar to day 21. Macrophages laden with golden Osmotic shock with hypertonic solution was used to lyse
pigment were present underneath the healing tissue and the cells within tissues [23]. The chelating agent, EDTA,
ADM graft was completely absorbed. Collagen fibers forms a ring shaped molecular complex that firmly binds
were denser, thick and best arranged and the total and isolates a central metal ion. It has been shown that
histopathological score reduced to 9. divalent Ca2+ and Mg2+ are necessary for cell attachment
to collagen and fibronectin at the Arg-Gly-Asp receptor
In group III on day 3, there was no evidence of [24]. By binding the divalent cations that are present at
epithelialization, and the wound was covered with the cell adhesions to the ECM, the EDTA facilitates
necrotic ADM graft. The underlying ADM graft cultured removal of cellular material from the tissue.
with P-MEF cells showed good infiltration of fibroblasts Deepithelialization and decellularization of human skin
within the graft. The stroma proliferation with fibroblasts can be done after overnight incubation in 10X PBS with
and newer capillaries was very prominent. Collagen fibers antibiotic cocktail and EDTA [25].
were dense, thin and haphazardly arranged. The total
histopathological score was 22. On day 7, there was partial Decellularization of donor skin is one method to retain
epithelialization and fewer amounts of dermal elements the native collagen configuration and fiber orientation
as compared to day 3. However, the collagen fibers were while removing cell components [26]. In the present study
best arranged. On day 14, inflammation was moderate the deepithelized skin was subjected to 1% SDC
with mild neovascularization. Collagen fibers were treatment. The time of reaction (18h and 48h) was
denser, thicker and better arranged and the total score adjusted to obtain the optimized ADM for seeding of P-
reduced to 13. The collagen of the graft was under the MEF. The de-epithelialized skin subjected to 1% SDC
process of resorption with organization of granulation treatment for 18h resulted in 100% acellularity with mildly
tissue. On day 21, epithelialization was complete and thick collagen fibers. The cellular debris was seen in
inflammation was absent. Fibroplasia resembled normal between the void spaces of collagen fibers. Denser tissues
skin with mild neovascularization. Muscle giant cells were such as dermis, tendon and trachea required prolonged
present within the granulation tissue and the score further agitation protocols lasting from days to months [27].
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 A.K. Gangwar et al.

However, in the present study desired results were persisted up to day 6 in animals of group I (open wound)
achieved after 48h of treatment with 1% SDC. Other might be due to denuded wound and subsequently the
workers repoted that cells and cell products cannot be extent of inflammation and tissue reaction. No pain was
completely removed from dense tissues even with the observed in groups II and III on day 5 and onwards. Injury
most rigorous processing methods [28]. to tissue causes a number of changes in the nociceptive
system. The injured nociceptors become highly sensitized
The growth and infiltration of P-MEF within the ADM to stimuli. Inflammatory mediators released during and
was found to be maximum at 48 hr. Similar findings after surgery also sensitized the peripheral nociceptors
were also reported after expansion of human fibroblasts to further stimuli [41].
on the micronized acellular dermal matrices following 7
days of culture [11]. The SEM examination also revealed On day 3the wounds of the animals of group I was
growth and adhesion of fibroblasts within the acellular completely filled with pink granulation tissue. The colour
matrices. These findings are in agreement with the earlier of the centre portion of the graft was milky white and the
findings where human fibroblasts and mouse fibroblasts host graft junction was pale in animals of groups II and
(L929 cells) were able to grow over the collagen chitosan III. On day 14, the graft was in the stage of resorption in
sponges and carboxyethyl chitosan/polyvinyl alcohol the animals of group III. The wound was covered with
(CECS/PVA) electrospun fiber mats, respectively with thick crust in groups I. On day 21, the crusts of the wound
good results [29, 30]. The fibroblast cells seeded on were detached from the wound in group I and wound
collagen matrix emitted distinct patterns of signaling and appeared again pinkish in colour. The graft was
migration [31]. completely absorbed and newly formed epidermis
covered the whole surface of the wound in groups II and
Dullness, depression and partial anorexia observed in the III animals. Early healing in group III might be due to
immediate postoperative period may be attributed to the presence of P-MEF within the acellular dermal graft.
surgical trauma, pain and inflammation at the site of A dermal component provides an environment that
reconstruction [32]. Double layered skin construct in promotes vascularization of the graft and fibroblasts play
groups II and III showed significantly less wound an active role in the replacement of dermal matrix [42].
contraction than the untreated wounds [33]. The collagen Scar tissue was highest in animals of groups I where no
scaffolds not only provides mechanical enforcement graft was used and healing takes place by severe
counteracting dermal contraction and thus prevented contraction and scar formation. Minimum scar formation
shrinkage, but also creates conditions that induce was observed in group III animals.
fibroblasts to produce an authentic dermal matrix [34].
Effective inhibition of wound contraction by contractile Hydroxyproline contents in healing tissue showed a
fibroblasts has long been considered essential for optimal gradual non-significant increase (P>0.05) upto 7 days in
dermal regeneration in full thickness skin wounds [35]. group I. In group III the hydroxyproline value was found
Fibroblasts growth factor-2 (FGF2) may also reduce significantly higher (P<0.05) on day 14. Increased
wound contraction by inhibiting the phenotypic change fibroblast proliferation activates the production of
of fibroblasts to myofibroblasts, which is likely involved collagen. The increased level of hydroxyproline is
in contraction [36]. Random orientation of the pores in indicative of increased amount of collagen deposition.
the scaffold may prevent the remaining myofibroblasts The significantly high hydroxyproline content in group
from contracting the wound area. When cells are III indicates more collagen deposition in this group as
randomly bound to the scaffold the sum of the contracting compared to group I. The increased hydroxyproline
forces are near zero [37]. The cultured fibroblasts content in the wounds treated with biotinylated GHK
particularly with a dermal support do not regress when incorporated collagen matrices was predominantly due
transplanted to a living tissue [38]. The fibroblasts to enhanced collagen synthesis [43]. The higher
contribute to the wound healing process reduce the concentration of glucosamine on day 3 was observed in
contraction of the wound and support collagen synthesis all the groups. Significant (P<0.05) decrease in
and neovascularization. glucosamine content was observed on day 7 in the animal
of group I. However, the values decreased significantly
Degree of exudation was significantly higher (P<0.05) up to day 28 in groups II and III. The young fibroblasts
in animals of groups I (control) which may be due to are responsible for the secretion of mucopolysaccharides
acute inflammatory reaction at the site in response to the which accumulates in the granulation tissue in large
surgical trauma. The exudate decreased significantly in quantities in the beginning of healing process. As the
groups II and III where ADM and P-MEF treated ADM healing progresses the concentration of glucosamine
were used. Kin et al. [39] also reported significant gradually decreases. It may be due to the fact that
decrease of exudation in full thickness wounds of rats glucosamine being an amino sugar was utilized by
treated with small intestinal submucosa as compared to granulation tissue for their further growth. The gradual
untreated wounds. A reverse correlation has been decrease in the glucosamine contents of healing tissues
observed between the survival area of the skin graft and up to 30 postoperative days correlate to the similar
the degree of exudation of the graft bed [40]. The observations reported in normal wound healing [44]. The
significantly higher (P < 0.05) postoperative pain scores increase in glucosamine contents in early stage of wound

76
 Bioengineered Acellular Dermal Matrix for the Repair of Full Thickness Skin Wounds

healing could also be due to the dilation of capillaries healing. The P-MEF seeded graft of group III was well
immediately after injury. The dilation of capillaries had tolerated by the host and partial epithelialization was
resulted in seepage of plasma and lymph rich in observed at this time interval. Epithelialization process
mucopolysaccharides. had started by 2 weeks in full thickness skin wounds
treated with small intestinal submucosal sponges [39].
The nature and degree of immunological response to a The acellular and bioengineered grafts (group II and III)
graft material is a crucial variable affecting the acceptance were invaded by the fibroblasts and underwent
or failure of implanted biomaterial. The antibody titre in neovascularization without any evidence of rejection. The
response to native graft antigen increased in all the groups. underlying graft was under the process of resorption.
The major antigenic determinants are situated in the Extracellular collagen degradation occurs by the action
telopeptide regions of the molecule. The other two types of enzymes including matrix metalloproteinases (MMPs)
of determinants are composed of the triple helix and of and through phagocytosis by macrophages and fibroblasts
the amino acid sequence of the á-chains. The latter type [50]. Intracellular collagen processing via phagocytosis
is accessible only when the collagen is denatured [45]. occurs in some fibroblasts by cell receptor clustering
When the antigenic determinants are exposed due to followed by invagination of the cell membrane [51].
collagen degeneration, it results in severe immune
response [28]. MHC class II antigens are present as On day 14, least histological score was observed in group
surface molecule in the transplanted cells which is III. Delayed healing in group I might be due to more
responsible for graft rejection. Fibroblasts lack these inflammation as compared to other groups. The release
surface molecules [46]. This makes them of growth factors and cytokines including TNF-á, IL-12
immunologically relatively inert. This is fortunate from and MIP1- á by the neutrophils, mast cells and
a tissue engineering point of view, as dermal regeneration macrophages increases the process of inflammation at the
matrices can be seeded with allogenic fibroblasts without site which may slow the repair process [52]. Sloughing
risking an immune response [42]. There are certain reports of the upper layer of graft was observed in groups II and
supporting the hypothesis that allogenic fibroblasts are III. It may be either due to the desiccation of the graft in
tolerated by the host [47]. high environmental temperature or due to an impaired
formation of new blood vessels [53]. Meanwhile, the
The antigen prepared from native tissue showed highest underlying granulation tissue increased in mass that
SI in MTT assay. The nucleated cell of the body is able to pushed up the superficial acellular dermal graft upwards.
present antigen, first by virtue of a constitutive MHC class The animals of groups II and III showed partial
I expression, and second by a de novo expression of MHC epithelialization, well formed collagen and
class II molecules on the surface of the cell. The SI neovascularization but these processes were faster and
recorded for group II was lower in comparison to the earlier in group III. The proliferation and migration of
values of group I. The acellular grafts showed minimum epithelial cells are dependent on an adequate supply of
antigenicity. The ability of this antigen to stimulate the oxygen [54]. Vascularization of the scaffolds from the
lymphocytes in-vitro may be attributed to the fact that on patient’s wound bed normally takes about 3 weeks [55].
treatment with biological detergent, the bonds between The enhanced rate of wound contraction and significant
protein molecules are broken and results into a change reduction in healing time might be due to enhanced
from quaternary and tertiary structure to primary and epithelialization. The underlying acellular dermal graft
secondary structures. Therefore, the acellular antigen had and P-MEF cultured grafts showed good infiltration of
ability to trigger CMI response in host because of presence fibroblasts within the graft.
of shorter peptide fragments which can be presented to
the immune system by MHC class II pathway and On day 21, histopathological score was least (score 10)
stimulate the CD4+ lymphocytes. in group III followed by group II (score 16).
Epithelialization was also more similar to the normal skin
On day 3, newly formed ECM was well spread out over in group III. Presence of fibroblasts in a dermal equivalent
the entire scaffold area in groups II and III as compared stimulates epidermal differentiation and accelerates the
to group I which might be due to preformed collagen healing process by reducing the time need for the
matrix secreted by the mouse embryo fibroblasts as fibroblasts to invade the wound tissue and by early
evidenced by histological observation of the P-MEF synthesis of new skin tissue, because the fibroblasts on
cultured scaffold. The fibroblasts proliferated more of in the artificial dermis can release biologically active
the acellular matrices and the bioengineered acellular substances such as cytokines [26]. The early
matrices [48]. Acute inflammatory immune response epithelialization in the P-MEF cultured group confirmed
resulted from polymorphonucleocyte infiltration and the accelerated epidermal differentiation and production
subsequent loss of the graft material following of an organized dense collagen matrix without a prolonged
implantation [49]. On day 7, although mild to severe inflammatory response. Early maturation of the wounds
inflammation and fibroplasia was present in all the three in group III as compared to control group wounds (group
groups, but inflammation was minimum in group III. The I) might be due to presence of pre-seeded fibroblasts in
early reduction of inflammation, as in case of group III, the scaffold. The superficial acellular dermal graft which
might facilitate the progress to the next phase of wound acts as protective covering for the underlying graft tissue

77
 A.K. Gangwar et al.

was detached from the wound after day 21. This may be in rats. The discrepancy between results may be explained
due to the desiccation of the graft in high environmental by inhibition of the enzymatic activities of this enzyme
temperature. Meanwhile, the underlying granulation at the wound site by the high levels of ROS [64].
tissue increased in mass that pushed up the graft upwards
and after day 21 the grafted materials were detached. The Catalase activity increased significantly up to day 3 in all
bilayer concept of wound coverage in which both the groups. The scavenged superoxide radicals were
epidermal and dermal analogs are used is widely accepted converted in to hydrogen peroxide, which stimulates the
[56]. The outer layer of such construct has to have a barrier expression of catalase [65]. Thereafter, the values showed
function to protect the wound not only from bacterial decline trend from day 7 onwards and reached to near
contamination, fluid loss, but also, overheating and normal values on day 21. However, significantly higher
accumulation of tissue fluids. On day 28, all the wounds values persisted in group I at different time intervals.
were completely covered with epidermis. Collagen fibers Increased concentrations of catalase have been observed
were best arranged and oriented parallel to the skin surface in the treated groups by different workers [43, 59].
in group III suggested a better repair of the damaged tissue Reduced levels of ROS-detoxifying enzymes result in
in this groups. The full-thickness skin wounds in rats healing impairments which was supported by the
treated with small intestinal submucosal sponges were observation of reduced activities of SOD and catalase in
completely covered with thin layer of epidermis by 4 wounds of aged rats compared to young rats [66].
weeks [39]. However, incomplete closure of the wound
Scanning electron microscopic observations were found
was observed even after 28 days post-implantation [33].
useful in understanding the healing process and 3-D
ROS scavenging enzymes plays an important role in the growth of the cells within the graft tissue. On day 3, the
detoxification of ROS during cutaneous wound repair. SEM specimen of groups II showed fibroblasts growth
Oxidative stress supervenes when generated free radicals and penetration within the scaffolds. The scaffold was
exceeds the capacity of antioxidants defense of the body covered with dense cell layer and attachment of the
[57]. In present study the significantly higher MDA fibroblasts cells within the pores in group III as compared
concentration in all the groups upto day 7 might be due to group II. This might be due to migration of P-MEF
to more tissue injury and inflammation and hence more cells throughout the 3-D ADM in this group as also
oxidative stress. On day 14, the oxidative stress reduced observed in histopathological findings. On day 14, there
in groups II and III as compared to group I might be due was better interaction between P-MEF cultured scaffold
to the reduction of lipid peroxidation in these groups due and surrounding cellular mass in group III. Biodegradable
to reduced inflammation which persisted in group I due electrospun scaffold supported the cellular growth and
to denuded surface of wound [58]. Reduced MDA scaffolds becoming infiltrated by granulation tissue
concentration has also been recorded in tissue after including fibroblasts and endothelial cells and the
application of a PDGF containing novel gel for cutaneous phenomenon was demonstrated by scanning electron
wound healing in rats [59]. microscopy [67]. On day 28, The ECM was well
distributed within the scaffold and collagen fibers were
Reduced glutathione (GSH) plays an important role in dense thick and better arranged in group II. Porosity, mean
the protection of cells against oxidative damage caused pore size and orientation of scaffold affect the migration
by ROS. Oxidation-reduction coupling of GSH is central and distribution of cells within the scaffold [51].
to the cellular response to oxidative stress. Earlier return Penetration of the cells within the pores of the graft might
of GSH values on day 7 in groups II and III may be due be due to good interconnected porosity of acellular dermis
to lower level of oxidative stress in these groups. In group [68].
I the values reached to near normal on day 21 might be
due to denuded wound surface resulted in more oxidative It was concluded that P-MEF cells seeded ADM facilitated
stress. It is authenticated that oxidative stress reduces GSH early and better healing than the normal saline.
level by depleting –SH group [60]. Lower level of GSH Histological and electron microscopic observations
in group I might be due to more utilization for showed that the bioengineered ADM augmented wound
detoxification of free radicals [61]. healing activity significantly by increasing cellular
proliferation, formation of granulation tissue,
Superoxide dismutase (SOD) is a metallo enzyme that neovascularisation, synthesis of collagen, epithelialization
catalyses the dismutation of O2ÿ - to H2O2 with remarkably and early histological maturation in excisional wounds.
high reaction rates. The level of SOD increased P-MEF cells seeded ADM showed minimum
significantly (P<0.05) up to day 14 in groups I. immunological response and can be used clinically in the
Superoxide dismutase level increased on day 7 after management of large skin wounds.
wounding and highest levels of SOD mRNAs were found
at the early stage of wound repair, when the oxidative Acknowledgement
burst occurs [62]. Increased concentrations of SOD have
been observed in the treated groups by different workers The authors acknowledge the financial assistance received
[43, 59]. The results of the present study were in contrast from the Department of Biotechnology, Ministry of
to earlier findings [63] where reduced SOD activity in Science and Technology, New Delhi, India to carry out
wound tissue between days 2 and 7 after cutaneous injury this work.
78
 Bioengineered Acellular Dermal Matrix for the Repair of Full Thickness Skin Wounds

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