Application of Heparinized Selective Acellular Sheepskin in Wound-healing Promotion of Deep Second-degree Burns

Introduction. This study investigated the application of selective sheepskin acellular dermal matrix (ADM), combined with the anticoagulant heparin, in the wound-healing treatment of deep second-degree burns (DSDB). Methods. A DSDB model was established, and the test animals were randomly divided into 4 groups: iodophor gauze (IG), pig ADM (PA), selective sheepskin ADM (SSA), and heparinized selective sheepskin ADM (HSSA).The microcirculation of the wound surface, pathological structures, healing rates, and fibroblast apoptosis rates were observed at different times in each group. Results. At 24, 48, and 72 hours post-burn, the filling degree of microcirculation in the HSSA group was higher than that in all other groups (P < 0.05). On days 14, 21, and 28 post-burn, the wound healing rates in the 3 biological-dressing groups were significantly better than the rate in the IG group (P < 0.05). On days 14 to 28 post-burn, the number of apoptotic fibroblasts in each group had increased significantly, with the number in the HSSA group on day 28 being significantly higher than that in the other groups (P < 0.05). Discussion. When used in treating burn wounds, HSSA, SSA, and PA protect the cellular wound surface and provide an ideal growing environment for the fibroblasts and endothelial cells to promote wound healing and scar inhibition. Conclusion. The selective sheepskin ADM was successfully produced and found to exhibit better effects in protecting microcirculation and promoting wound healing compared with all other analyzed methods.

The skin is the largest and one of the most important organs in the human body. Acting as a natural barrier, the skin possesses a number of physiological functions, such as sensation, physical protection, respiration, absorption, secretion, excretion, body temperature regulation, and metabolism, among others. Trauma, especially large areas of skin discontinuity caused by burns, triggers a series of pathophysiological changes in the body and can even endanger the patient’s life.^1,2 Accordingly, current burn and trauma research efforts are focused on the challenging task of prompt and effective wound closure.

Some of the most commonly used clinical wound dressings include autologous, allogeneic, xenogeneic, and artificial composite skin grafts (Table 1).³ Although autologous skin is currently the most commonly used and effective wound dressing to repair deep burn wounds, the skin donor area of patients with a large burn is very limited. Because large allogeneic skin grafts are also difficult to obtain and artificial composite skin is expensive, the previously mentioned coverings all have limited clinical applications. As a result, xenogeneic skin grafts may be the optimal, or only, choice for large wounds. 

Currently, xenograft pig acellular dermal matrix (ADM) has been successfully applied in would repair; however, xenograft pig ADM is quite expensive, and it is not suitable for treatment of the Muslim population.^4,5 Taking the minority population of individuals of Muslim faith in the study institution’s area into account, the authors studied the application of sheepskin ADM, combined with the anticoagulant heparin which has been demonstrated to improve the microcirculation of burn wounds.^6,7 The approach was used to treat deep second-degree burns (DSDB), and the study authors observed the healing conditions of burn wounds.

Animals
One healthy, locally raised adult female sheep (31 kg) was selected for the preparation of the skin grafts. Wistar rats of clean grade, without gender limitation, weighing 220 g ± 20 g (certificate number: Beijing Animal 8806R011) and Hartley albino guinea pigs of clean grade, without gender limitation, weighing 380 g ± 20 g, were purchased from Beijing Vital River Animal Technology Co, Ltd (License No. SCXK [Beijing] 2006-0009). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Inner Mongolia Medical University, Inner Mongolia, China. 

Preparation of heparinized selective sheepskin acellular dermal matrix dressing
After removal of the wool, the sheepskinskin was cleaned and cut into 15 cm × 20 cm skin grafts. Next, the grafts were rinsed in 0.1 M phosphate buffered saline (PBS) 3 times, soaked in 0.1% benzalkonium bromide solution (Guangzhou Hengjian Pharmaceutical Co, Ltd, Guangzhou, China) for 30 minutes, fixed in 2% glutaraldehyde solution (Tianjin Jingke Fine Chemistry, Tianjing, China) for 2 hours, washed by distilled water 3 times, cut into skin sections 0.3 mm to 0.4 mm thick, and again washed by distilled water 3 times. The skin sections were digested by shaking with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO) at 37°C for 2 hours, followed by shaking-digestion in 0.5% TritonX-100 (KEYGEN Biotech, Nanjing, China) at 37°C for 24 hours. After washing with PBS 3 times, the selective sheepskin ADM dressings were prepared and stored in a freezer (Haier, Qingdao, China) at -80°C for future use. Before use, the grafts were rapidly restored to ambient temperature, and soaked in low-molecular weight heparin (5000 IU/ampule; batch number 0712401, Qilu Pharmaceutical Co, Ltd, Jinan, China) for 20 min (heparinized selective sheepskin ADM dressing only).

Study of heparin carrying and releasing by selective acellular sheepskinskin dressing
The selective sheepskin ADM was first trimmed into 15 samples measuring 5 cm × 6.8 cm to study heparin carrying and then soaked in low-molecular weight heparin solutions (with the same concentrations and the total volumes) for 20 minutes to determine the rest volumes of the low-molecular weight heparin solutions using the following formula:

Adsorption volume (mL) = total volume of the low-molecular weight heparin solution – rest volume of the low-molecular weight heparin solution

The 96 Wistar rats were randomly divided into the DSDB group and the normal group; and the heparinized selective sheepskin acellular dermal matrix (ADM) was then used to cover the wound, but the wound of the normal group was only defurred and covered. The blood concentrations of heparin were detected at 2, 4, 6, 12, 24, and 36 hours after the surgery.

Histological observation
The 0.1 cm × 1 cm selective sheepskin ADM (SSA group) dressing specimens were fixed in 4% paraformaldehyde (Tianjin Jingke Fine Chemistry, Tianjing, China) for 3 days. After preparing the paraffin sections, the specimens were cut into 5 µm slices and stained with conventional hematoxylin and eosin (H&E) (Beijing Cell Biochip Co, Ltd, Beijing, China).

Bacteria and fungi detection
Each of the 5 established SSA specimens was cut into 3 pieces and sent for bacterial and fungal cultures.

Cell growth activity
The 1 cm × 1 cm SSA tissue pieces were added to 1 mL of 10% fetal bovine serum-containing Dulbecco’s Modified Eagle Medium (DMEM, Gibco Co, CA) and soaked for 24 hours at 37°C to prepare the extract solution. The fifth-generation fibroblasts were seeded into a 96-well plate at a concentration of 1 × 104/mL, and cultured at 37°C and a 5% CO₂ atmosphere (Heal Force Biomedical Tech Holdings Ltd, Hong Kong, China) for 24 hours. Subsequently, the media inside the wells were discarded, and 100 µL of extract solution was added into each well. For the control group, culture medium was added instead. The plates were cultivated at 37°C and 5% CO₂. A total of 20 µL MTT (4.5-dimethyl-2-thiazolyl-2.5-diphenyl-2-H-tetrazolium bromide, thiazolyl blue) (5 mg/mL, AMRESCO, Solon, OH) was added to each well after days 3 and 7, respectively, followed by cultivation for 4 hours. Next, the media inside the wells were removed, and 150 µL dimethyl sulfoxide (DMSO, Sigma-Aldrich St. Louis, MO) was added to each well, followed by shaking the solution for 10 minutes. The absorbtion value of each well was detected at 490 nm using a microplate reader (Model 680, Bio-Rad Laboratories, Hercules, CA).

Guinea pigs deep second-degree burns model
According to the reported methods⁸ with some modifications, Hartley albino guinea pigs (n = 120) underwent intraperitoneal anesthesia with 3.6% (100 g/mL) chloral hydrate.^9,10 After shaving the back fur, the guinea pigs were immersed into 80°C hot water for 10 seconds to produce DSDB in 5% total body surface area (TBSA), which was confirmed by biopsy. After injury, the guinea pigs were immediately intraperitoneally injected with 10 mL of Ringer’s solution for recovery. The animals were randomly divided into the 4 groups — iodophor gauze (IG), pig ADM (PA), SSA, and heparinized selective sheepskin ADM (HSSA) — with 30 guinea pigs in each group. After resuscitation, the animals were maintained in individual cages.

Perfusion of India ink
India ink perfusion was performed through the carotid artery at 0, 6, 12, 24, 48, and 72 hours in 5 guinea pigs from each group. The guinea pigs were then anesthetized using 3.6% chloral hydrate with 10ml/kg, and fixed in the supine position. A 4 cm to 6 cm transverse incision was made under the lower edge of the thyroid cartilage to expose the right common carotid artery, and 50 mL India ink (Beijing ZHONGXI YUANDA Technology Co, Ltd, Beijing, China) was bolus-injected through a No. 5 vein puncture needle, which was inserted approximately 1.5 cm deep, with an injection rate of 20 mL/min. After the perfusion, the normal skin gradually stained black, while the burned area appeared grey. The guinea pigs died instantly without pain.¹¹

Determination of filling degrees
After successful perfusion of the India ink, the specimens were immediately prepared for H&E staining. Five low-magnification fields of each slice were randomly selected for the determination of the linear thicknesses of the India ink perfusion area and whole dermis using the TKC1381EG pathological imaging analyzing system (Leica Co, Ltd, Wetzlar, Germany), and the length ratio was calculated to understand the wound microcirculation.

Rat model for deep second-degree burns and experimental grouping
Next, healthy adult Wistar rats (n = 160) were used for the DSDB model (4% TBSA) by the same method already described, and randomly divided into the same 4 groups — IG PA, SSA, and HSSA — with 40 rats in each group. After resuscitation, the animals were maintained in individual cages. After the surgery, the wound of 1 rat was sampled for Masson staining to observe whether the wound tissue damage had reached the standards of a DSDB.

Observation of wound healing rate
On days 3, 7, 14, 21, and 28 postinjury, the animals were anesthetized for the observation of wound healing, and the unhealed parts were traced onto a transparent gummed paper with a 1:1 ratio for the measurement of the local wound area, according to the following formula: 

Wound healing rate = [(original wound area - unhealed wound area) / original wound area] × 100%. 

The wound healing rates of the different postinjury time points were calculated.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
On postoperative days 3, 7, 14, 21, and 28, the rats were humanely euthanized using 3.6% chloral hydrate with 10 mL/kg, and the wound tissues were removed and fixed in 4% paraformaldehyde. The TUNEL assay was performed on the paraffin-embedded sections according to the Roche TUNEL kit instructions (Roche, Basel, Switzerland). The reaction mixture without terminal deoxynucleotidyl transferase enzyme was set as the negative control. Lastly, staining with 3,3’-diaminobenzidine (Fuzhou Mixim Biotech Development Co, Ltd, Fuzhou, China) was carried out according to the manufacturer’s instructions. The specimens were observed under a light microscope, with brown or yellow expression inside the nuclei considered as positive signals. Ten random visual fields of positive signals for each tissue section at the different time points of the different experimental groups and the control group, were observed with the naked eye under a 400× optical microscope. The positive signals of each field were counted, and the average for each tissue section was used for the subsequent calculations.

Masson staining of the wound
On postoperative days 3, 7, 14, 21, and 28, the rats were humanely euthanized, and the wound tissues were removed and fixed in 4% paraformaldehyde to prepare the paraffin sections, followed by 5-minute Weigert iron hematoxylin staining (Fuzhou Maixin Biotechnology Company, Fujian, China), acid ethanol differentiation, water rinsing, blue staining again by Masson blue staining solution, water rinsing, 1 minute of distilled water rinsing, 5-minute Ponceau-fuchsin staining, 1 to 2 minutes of phosphomolybdic acid solution rinsing, 1 to 2 minutes of aniline blue staining, fast dehydration with 95% ethanol, 3-time dehydration with dehydrated alcohol (5 to 10 seconds each time), xylene hyalinization (three, 1 to 2 minute intervals), and closure with neutral gum.

Statistical analysis
The data were processed using SPSS 15.0 statistical software (SPSS Inc, Chicago, IL). The single-factor analysis of variance and t-test were used for the statistical analyses, with P < 0.05 considered statistically significant. All data are expressed as x– ± s.

General observations
The SSA dressing appeared porcelain white. The epidermis was integrated, soft, and elastic, and the flexibility was good, with a certain tensile strength, while not easily broken. The thickness of the overall dressing was uniform and smooth, without wool attaching to the epidermis, while the dermis was rough (Figure 1). 

Dynamic changes of heparin carrying and releasing by selective sheepskin acellular dermal matrix
The carrying amount of low-molecular weight heparin by selective sheepskin ADM was 58.82 ± 14.73 iu/cm². After the wounds in both groups were covered with the SSA, the low-molecular weight heparin could enter the blood regardless of the method of percutaneous absorption or transwound absorption, so its plasma concentration was increased within 12 hours but significantly decreased 12 hours later (Table 2). 

Histological observations of the selective sheepskin acellular matrix dressing
Examination of the H&E-stained SSA dressing under light microscope revealed integration of the epidermis, with an obvious basement membrane. The dermis appeared pink, with gaps caused by eluted blood vessels and cells, and the collagens were arranged in neat rows, without cellular components, blood vessels, and skin appendages (Figure 1). The Masson staining was performed to detect the selective sheepskinskin, and blue collagens were seen under the microscope, indicating the successful preparation of the SSA (Figure 2) and confirming the successful establishment of the SSA dressing. 

Bacterial and fungal detection
The cultivation results of all SSA dressing samples were negative.

Cell growth activity
After 24-hour cultivation, the fifth-generation fibroblasts seeded in the 96-well plate appeared spindle-shaped and adhered to the well walls. After 3 and 7 days of cultivation, there were no significant differences between cellular MTT values in the experimental groups and the control group (P > 0.05; Table 3). 

Wound area microcirculation
The comparisons of the India ink filling degrees in the wound areas at 0, 6, and 12 hours posttreatment exhibited no significant differences among the DSDB guinea pigs (P > 0.05). The guinea pigs of all groups exhibited damaged epidermis as well as swelling and fusion of the necrotic dermal collagen fibers at 48 hours postinjury, while the India ink-stained part was the survived dermis (Figure 3). At 24, 48, and 72 hours postburn, the comparison of the India ink filling degrees between the HSSA group and the other 3 groups was statistically significant (P < 0.05). The India ink filling conditions of each group at the different time points are shown in Table 4.

Histological observation of rat deep second-degree burn wounds
Twelve hours after the injury, Masson staining showed brown-red regions representing the coagulated necrotic tissues deep into the reticular dermis, which corresponded with the pathological changes of DSDB (Figure 4). 

Wound healing rates
On postoperative day 3, the wound areas of the test animals in each group were found to be clean, without exudate and secretion. The skin surfaces of the necrotic tissues of a subset of rats in the HSSA, SSA, and PA groups separated from the wound basement, although no bleeding was observed. On day 7, the necrotic tissues of the 3 biological dressing treatment groups began to dissolve and fall off, while the basements of the wounds were fresh without accumulation of exudates or secretion. On days 14, 21, and 28, the wounds had narrowed, and the wound healing rates in the treatment groups were significantly higher than the rate in the conventional IG group (P < 0.05). Simultaneously, formation of skin islands was observed. On day 21, the wound healing rate in the HSSA group was significantly better than that in the PA group (P < 0.05). Additionally, on days 21 and 28, the wound-healing rates in the HSSA group were significantly better than those in the SSA group (P < 0.05) (Table 5). 

Apoptosis
The apoptotic cells in the wound tissues were widely distributed, exhibiting brownish-yellow color (Figure 5). The mean of the positive signals (Table 6) reflected the apoptosis degree after therapy using the different biological dressings. On day 3 post-DSDB, the wound tissues of all groups exhibited a small amount of fibroblast apoptosis (P > 0.05). On day 7 postinjury, the fibroblast apoptosis of all groups was significantly increased compared to that on day 3, while there were still no significant differences between the groups (P > 0.05). Corresponding with the gradual healing of the wounds, the fibroblast apoptosis rate of the wound tissues in each group increased significantly on days 14 to 28, with the apoptosis in the PA, SSA, and HSSA groups being significantly higher than that in the IG group (P < 0.05), especially the HSSA group, which reached a peak on day 28. This indicates that during the wound healing process, the major cellular components of scar tissues, namely the fibroblasts, continuously underwent apoptosis, which increased with time. In the late period of wound healing, more apoptotic fibroblasts corresponded with fewer residues of wound scar tissues, thereby resulting in better wound healing.

Masson staining
Wound tissue detection using Masson staining revealed that in the early postburn stage between days 3 and 7, the wounds in each group exhibited damaged epidermis and partially necrotic dermis, but the growth conditions of collagen fibers showed no significant difference. Fourteen days after the injury, the wounds in the HSSA group were basically covered by epidermal tissues; 21 days later, the wounded epidermis in the HSSA groups could be seen covered by certain structural stratifications, and the collagen fibers inside the wound dermis showed certain directional arrangements. However, the epidermis in the IG group did not show obvious structural stratifications 21 days after the injury, and although the collagen fibers inside the wound tissue were larger than those on days 7 and 14, the arrangement was still not orderly. At 28 days after DSDB, the anatomic structural stratifications of the wound tissues in the HSSA group were much more clear, and microscopic observation revealed the collagen fibers within the dermis were arranged in order and close to the anatomic structures of normal tissues. However, the effects of wound recoveries in the SSA and PA groups were less than the HSSA group. The intergroup difference was significant, but their effects were better than those in the IG group (Figure 6). 

In the initial stage of burning, the microcirculation would be in the ischemic shock period during to vasospasm and blood stasis inside the wound tissues.^12,13 Heparin could prevent or mitigate the progressive wound damages in the early stage, and promote wound healing. Oken et al¹⁴ reported that heparin could improve the local microcirculation of burn-damaged tissues; thus the early wound edema could be reduced, resulting in the reduction of early progressive tissue damages and contributing to wound healing.

Each squared centimeter of skin of the selective acellular sheepskin carried 58.82 ± 14.73 µm heparin. After it was applied to the wound, the carried heparin slowly penetrated and was absorbed by the wound, thus, the authors speculate, improving the pharmacological effects of the low-molecular weight heparin. In this experiment, the India ink filling degrees in the HSSA group at treatment hours 24, 48, and 72 were higher than the IG, PA, and SSA groups (P < 0.05), indicating that HSSA could reduce the progressive degree of the wound. Additionally, the early application of this dressing after the injury could effectively maintain the remnants of dermal tissues cells, avoiding further necrosis among the ecological tissues. The wound healing experiment revealed that the comparison between the HSSA and IG groups showed significantly shortened healing time. The H&E staining observation revealed that on day 21 postinjury, the epidermis of wound tissues of the HSSA group exhibited a certain hierarchical structure, the collagen fibers exhibited a certain arrangement direction inside the dermal layers of wound tissues, a large number of capillaries presented in the dermal tissues under the microscope, and the proliferated fibroblasts exhibited more regular arrangement. However, the relative structural stratifications of the epidermis in the IG group on day 21 postinjury were not clear enough, indicating that the HSSA group significantly shortened healing time and promoted the growth and repair of cells; therefore, heparin played an important role in wound healing.

After the wound injury formed, the appearance of fibroblasts marked the beginning of trauma healing.^16-18 During the wound healing stages, if any link related to the apoptosis regulation was out of control, the healing might develop in a completely contrary direction. On one hand, if the apoptosis was procedurally uncontrolled in the early wound healing stage, various cells involved in wound healing would develop in the direction of procedural apoptosis and result in difficult healing of wound tissues and the ultimate formation of hard-to-heal wounds.^19,20 On the other hand, when the expressions of series of regulatory signals were blocked, the overgrowth fibroblasts promoted the excessive deposition of the secreted collagen fibers, thus forming the pathological hypertrophic scarring. In this case, it would be difficult for the damaged skin tissues to develop into normal skin tissues, affecting partial physiological functions of skin.²¹ The results of this study showed that on post-DSDB days 3 and 7, a small amount of fibroblasts in the wound tissues exhibited apoptosis, and the comparison among different groups showed no statistical significance (P > 0.05). With the healing of rat DSDB on postinjury days 14 to 28, the number of apoptotic fibroblasts in each group increased significantly, with the PA, SSA, and HSSA groups reporting significantly higher numbers than the IG group (P < 0.05). At the same time, the number of apoptotic fibroblasts  in the HSSA group reached their peak on postinjury day 28 (P < 0.05). When used to treat burn wounds, HSSA, SSA, and PA protect the wound surface and provide an ideal growing environment for the fibroblasts and endothelial cells to promote wound healing and scar inhibition.

This study indicates that heparinized SSA has a better effect on DSDB wounds from morphology, but its molecular mechanism of promoting wound healing should be further studied.

The research group believes heparinized SSA slowly releases low-molecular weight heparin, thus improving the microcirculation of wound tissues and promoting the regeneration of blood vessels. The strong biological dressing characteristics of SSA can be used to sterilely close and protect an exposed wound and its related ecological tissues, thus accelerating the repair and healing of DSDB wounds.

Affiliation: The Third Affiliated Hospital of Inner Mongolia Medical University, Baotou, Inner Mongolia, China

Correspondence:
Lingfeng Wang, PhD
Department of Burns, The Third Affiliated Hospital of Inner Mongolia Medical University, Burn Institute of Inner Mongolia
20 Shaoxian Road Kun District
Baotou 014010, China
lingfengwangcn@126.com

Disclosure: This study was supported by the National Natural Science Foundation of China (81060154), Natural Science Foundation of Inner Mongolia (2010Zd27).