Changes in VEGF and nitric oxide after deep dermal injury in the female, red Duroc pig-further similarities between female, Duroc scar and human hypertrophic scar

Despite decades of research, our understanding of human hypertrophic scar is limited. A reliable animal model could significantly increase our understanding. We previously confirmed similarities between scarring in the female, red, Duroc pig and human hypertrophic scarring. The purpose of this study was to: (1) measure vascular endothelial growth factor (VEGF) and nitric oxide (NO) levels in wounds on the female Duroc; and (2) to compare the NO levels to those reported for human hypertrophic scar. Shallow and deep wounds were created on four female Durocs. VEGF levels were measured using ELISA and NO levels with the Griess reagent. VEGF and NO levels were increased in deep wounds at 10 days when compared to shallow wounds (p < 0.05). At 15 weeks, VEGF and NO levels had returned to the level of shallow wounds. At 21 weeks, VEGF and NO levels had declined below baseline levels in deep wounds and the NO levels were significantly lower (p < 0.01). We found that VEGF and NO exhibit two distinctly different temporal patterns in shallow and deep wounds on the female Durocs. Furthermore, NO is decreased in female, Duroc scar as it is in human, hypertrophic scar further validating the usefulness of the model.     

皮肤圆锥体结构损伤致增生性瘢痕的病理学观察

目的探讨皮肤圆锥体结构受损与增生性瘢痕(HS)形成的关系,寻找HS形成的原因及适合HS研究的动物模型。方法将2只雌性杜洛克猪(FRDP)背部去毛后,取正常皮肤1块, 行皮肤圆锥体结构观察;然后用气动取皮机造成皮肤缺损深度为0．38、0．76、1．14、1．52 mm的创面, 每个创面面积7．0 cm×7．0 cm,每只猪的背部共8个创面。根据上述创面深度将其分为4组,每组4 个创面。将0．38、0．76 mm组视为浅创面组;1．14、1．52 mm组视为深创面组。伤后不处理,让创面自然愈合。分别在伤后即刻(0)、10、30、60、90、150 d,切取创面组织标本行HE和弹性纤维(VVG)染色。行正常FRDP皮肤组织及受损圆锥体结构观察;大体观察不同深度FRDP创面愈合及瘢痕形成情况;用组织学方法观察HS的形态;VVG染色测定瘢痕组织的厚度并计分。结果正常FRDP背部皮肤存在圆锥体结构,与人类的皮肤圆锥体结构相似。浅创面组伤后3周内愈合,愈合的创面较为平整,无瘢痕组织形成;深创面组伤后4周以上愈合,愈合的创面较厚,无毛发,挛缩,质地坚硬,局部色素变浅或加深。浅创面组皮肤圆锥体结构的上半部分受损,但脂肪穹隆及腺体完好;深创面组皮肤圆锥体结构的下半部分受损,伤及脂肪穹隆及腺体。伤后150 d浅创面组的组织学结构与正常皮肤相似;深创面组圆锥体结构消失,并被大量堆积、没有特定方向、排列紊乱的胶原纤维束充盈。深创面组瘢痕组织厚度评分可见时间越长瘢痕越厚,伤后150 d瘢痕增生达高峰。结论皮肤圆锥体结构受损可导致HS的形成,FRDP可作为HS研究的动物模型。     

Further similarities between cutaneous scarring in the female, red Duroc pig and human hypertrophic scarring

Knowledge of the pathophysiology of hypertrophic scarring following deep dermal injuries is minimal due to the lack of an animal model. We previously confirmed that thick scars in female, red Duroc pigs (FRDP) are similar to human hypertrophic scar. The purpose of this study was to evaluate TGFbeta1, IGF-1, decorin, and versican expression in FRDP wounds. Deep and shallow wounds on the backs of two FRDPs were studied over 5 months. Immunohistochemistry was performed for TGFbeta1, IGF-1, decorin, and versican. TGFbeta1 and IGF-1 mRNA were evaluated by in situ hybridization and RT-PCR. In shallow wounds (1) TGFbeta1 protein was not detectable and IGF-1 protein was seen at 10 days post-wounding. TGFbeta1 and IGF-1 mRNA were elevated for 30 days. (2) Decorin protein was not detected at 10th day, but returned to levels of uninjured skin. (3) Versican protein was not detectable at any time. In deep wounds, (1) TGFbeta1 and IGF-1 protein and mRNA were elevated early, (2) decorin protein was greatly reduced for the first 90 days, and (3) versican protein was present from 30 to 150 days. These findings correlate with findings reported in the literature for human hypertrophic scar and further validate the FRDP model of hypertrophic scarring.     

Nerve quantification in female red Duroc pig (FRDP) scar compared to human hypertrophic scar

A significant impediment to studying hypertrophic scar is the lack of an animal model. We have confirmed similarities between scarring in the female red Duroc pig (FRDP) and human hypertrophic scar and conclude that this model warrants validation. Reports have suggested that the cutaneous nervous system may play a role in hypertrophic scar development and several studies have shown nerve density in hypertrophic scar to be increased. The purpose of this study was to further validate the FRDP model of hypertrophic scar by quantifying nerves in FRDP tissue and comparing the findings to human hypertrophic scar. Wounds of varying depth were created on the backs of two FRDP and tissue samples were harvested at 10 days, 1 month and 5 months post-wounding. Human specimens were obtained from six burn patients. Immunohistochemistry was performed and digital images were captured. Color subtractive computer-assisted image analysis was used to quantify nerve density and nerve area fraction. The results demonstrate that nerve tissue is increased in FRDP scar tissue and is quite similar to that in human hypertrophic scar and to that described in the literature. These data provide additional evidence that the FRDP model may be useful for studying hypertrophic scarring.     

Scar formation following excisional and burn injuries in a red Duroc pig model

Scar research is challenging because rodents do not naturally form excessive scars, and burn depth, size, and location cannot be controlled in human longitudinal studies. The female, red Duroc pig model has been shown to form robust scars with biological and anatomical similarities to human hypertrophic scars. To more closely mimic the mode of injury, recreate the complex chemical milieu of the burn wound environment and enhance scar development, an animal model of excessive burn‐induced scarring was developed and compared with the more commonly used model, which involves excisional wounds created via dermatome. Standardized, full‐thickness thermal wounds were created on the dorsum of female, red Duroc pigs. Wounds for the dermatome model were created using two different total dermatome settings: ～1.5 mm and ≥ 1.9 mm. Results from analysis over 150 days showed that burn wounds healed at much slower rate and contracted more significantly than dermatome wounds of both settings. The burn scars were hairless, had mixed pigmentation, and displayed fourfold and twofold greater excess erythema values, respectively, compared with ～1.5 mm and ≥ 1.9 mm deep dermatome injuries. Burn scars were less elastic, less pliable, and weaker than scars resulting from excisional injuries. Decorin and versican gene expression levels were elevated in the burn group at day 150 compared with both dermatome groups. In addition, transforming growth factor‐beta 1 was significantly up‐regulated in the burn group vs. the ～1.5 mm deep dermatome group at all time points, and expression remained significantly elevated vs. both dermatome groups at day 150. Compared with scars from dermatome wounds, the burn scar model described here demonstrates greater similarity to human hypertrophic scar. Thus, this burn scar model may provide an improved platform for studying the pathophysiology of burn‐related hypertrophic scarring, investigating current anti‐scar therapies, and development of new strategies with greater clinical benefit.     

The female, red Duroc pig as an animal model of hypertrophic scarring and the potential role of the cones of skin

Hypertrophic scarring occurs after deep dermal wounds. Our understanding of the etiology is poor; one reason is the lack of an animal model. In 1972, Silverstein described scarring in the Duroc pig but the model was never confirmed nor disproved. Another reason, as we previously suggested, is that hypertrophic scarring only occurs within regions of human skin that contain cones and the cones have not been studied in relation to hypertrophic scarring. We, therefore (i) explored healing in the female, red Duroc model for similarities to human hypertrophic scarring, studying wound thickness, appearance, healing status at 3 weeks, histology, and immunocytochemical localization of decorin, versican, TGFbeta1 and IGF-1; and (ii) examined Duroc skin for cones. We found that healing after deep wounds in Duroc pigs is similar, but not identical, to human hypertrophic scarring. We also found that Duroc skin contains cones. Healing in the female, red Duroc pig is sufficiently similar to human hypertrophic scarring to warrant further study so that it can be accepted or rejected as a model of human hypertrophic scarring. In addition, the relationship of the cones to hypertrophic scarring needs further detail and can be studied in this model.     

Histology of the thick scar on the female, red Duroc pig: final similarities to human hypertrophic scar

The etiology and treatment of hypertrophic scar remain puzzles even after decades of research. A significant reason is the lack of an accepted animal model of the process. The female, red Duroc pig model was described long ago. Since the skin of the pig is similar to that of humans, we are attempting to validate this model and found it to be encouraging. In this project we quantified myofibroblasts, mast cells and collagen nodules in the thick scar of the Duroc pig and compared these to the values for human hypertrophic scar. We found the results to be quite similar and so further validated the model. In addition, we observed that soon after wounding an inflammatory cell layer forms. The thickness of the inflammatory layer approaches the thickness of the skin removed as if the remaining dermis "knows" how much dermis is gone. In deep wounds this inflammatory layer thickens and this thickness is predictive of the thickness of the ultimate scar.     

Deep dermal fibroblast profibrotic characteristics are enhanced by bone marrow–derived mesenchymal stem cells

Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow–derived mesenchymal stem cells (BM‐MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM‐MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell–derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co‐culturing layered fibroblasts with BM‐MSCs in a transwell insert system, BM‐MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM‐MSCs in the pathogenesis of hypertrophic scarring.     

Effect of chymase activity on skin thickness in the Clawn miniature pig hypertrophic scarring model

Objective: The female red Duroc pig, a heavy and cumbersome animal, is routinely used as an animal model for hypertrophic scarring. Chymase, a chymotrypsin-like serine protease, plays an important role in skin fibrosis. This study aimed to create a lightweight pig hypertrophic scarring model using Clawn miniature pigs, and to investigate the role of chymase in hypertrophic scarring.

Methods: After creating four skin wounds (7.5?×?7.5?cm, depth?=?0.15?cm) in each pig, skin biopsies were performed after 15, 30, 60, 90, 120, and 150?days. Skin thickness, water content, hydroxyproline percentage, chymase activity, and transforming growth factor-beta 1 concentration were measured, and pathological analyses were performed.

Results and conclusions: Both tissue thickness and chymase activity were increased in scar tissue, peaked on day 90 after injury, and then gradually decreased. Peripheral scar tissue showed higher chymase activity than central scar tissue. Neither chymase activity nor transforming growth factor-beta 1 was detected in the surrounding normal skin, whereas central scar tissue showed a high transforming growth factor-beta 1 concentration, peaking on day 15, and decreasing to normal by day 120. We found the Clawn miniature pig to be a useful model for hypertrophic scarring. Chymase activity and skin thickness were well-correlated, suggesting that scars thicken when chymase activity is high.     

Wound healing in oral mucosa results in reduced scar formation as compared with skin: Evidence from the red Duroc pig model and humans

Scar formation is a common, unwanted result of wound healing in skin, but the mechanisms that regulate it are still largely unknown. Interestingly, wound healing in the oral mucosa proceeds faster than in skin and clinical observations have suggested that mucosal wounds rarely scar. To test this concept, we created identical experimental wounds in the oral mucosa and skin in red Duroc pigs and compared wound healing and scar development over time. We also compared the pig oral mucosal wound healing to similar experimental wounds created in human subjects. The findings showed significantly reduced scar formation at both clinical and histological level in the pig oral mucosa as compared with skin 49 days after wounding. Additionally, the skin scars contained a significantly increased number of type I procollagen immunopositive cells and an increased fibronectin content, while the oral mucosal wounds demonstrated a prolonged accumulation of tenascin-C. Furthermore, the pig oral mucosal wounds showed similar molecular composition and clinical and histological scar scores to human oral mucosal wounds. Thus, the reduced scar formation in the pig oral mucosa provides a model to study the biological processes that regulate scarless wound healing to find novel approaches to prevent scar formation in skin.     

Control of hypertrophic scar from inception by using xenogenic (porcine) acellular dermal matrix (ADM) to cover deep second degree burn

OBJECTIVE: We have spent 7 years to investigate the method of applying porcine acellular dermal matrix (ADM) on deep partial thickness burn wound until the wound heals without dressing change. Known as "Feng's pig skin method" by our hospital, the method appears to encourage rapid re-epithilization with minimum scarring. METHOD: The deep partial thickness burn wound was rinsed cleanly under anesthesia when the patient admitted. ADM was applied on the wound after the detached epidermis was thoroughly removed, wrapped and fixed by sterile gauze and bandages. The dressing was removed within two weeks and the wound completely healed. The outcome of the treatment was analyzed by using the modified Vancouver Burn Scar Assessment Scale. RESULT: All the wounds healed with one dressing within 2 weeks, and the time of wound re-epithelialization shortened to 7-12 days. Scar hyperplasia did not occur, or it was greatly ameliorated compared with traditional treatment after a followed-up period of 3 months to 2 years. The Scar Index was significant lower than that of the traditional exposure method. CONCLUSION: Using ADM to cover deep second degree burn can preserve maximally residual dermal tissue and epithelium, help accelerate the regeneration of epithelial and stem cells, thus shorten the healing time, remodel the skin structure, and consequently has the effect of controlling hypertrophic scar at inception.     

Effects of nitric oxide releasing poly(vinyl alcohol) hydrogel dressings on dermal wound healing in diabetic mice

Healing of chronic wounds such as diabetic foot ulcers is a significant clinical problem. Methods of accelerating healing in these difficult lower extremity sites include use of growth factor-loaded gels, hyperbaric oxygen, grafts, and artificial skin replacements. Nitric oxide (NO) has been proposed as a possible active agent for enhancing wound healing. This study examines the in vitro and in vivo responses to a novel hydrogel that produces therapeutic levels of NO. A hydrogel wound dressing was fabricated using ultraviolet light-initiated polymerization from poly(vinyl alcohol) with a NO donor covalently coupled to the polymer backbone. NO release from the NO-modified hydrogel was shown to occur over a time period of up to 48 hours, and there was no associated decrease in fibroblast growth or viability in vitro associated with NO hydrogels. Fibroblasts in culture with NO hydrogels had an increased production of extracellular matrix compared with cells cultured without the NO hydrogels. Preliminary animal studies in a diabetic mouse, impaired wound healing model were conducted comparing low (0.5 mM) and high (5 mM) doses of NO. Time to complete closure was similar in control wounds and NO-treated wounds; however, at 8 days control wounds were significantly smaller than NO-treated wounds. By days 10 to 13 this delay was no longer apparent. Granulation tissue thickness within the wounds at days 8 and 15 and scar tissue thickness after wound closure were increased in animals exposed to higher dose NO hydrogels. The results of this study suggest that exogenous NO released from a hydrogel wound dressing has potential to modulate wound healing.     

Skin wound healing in the first generation (F1) offspring of Yorkshire and red Duroc pigs: evidence for genetic inheritance of wound phenotype

Fibroproliferative scars in humans often demonstrate familial inheritance patterns, and genetics may contribute to healing and scarring. Genetic factors may also influence the scarring phenotype in a porcine model. Healing of full thickness excisional skin wounds in Yorkshire pigs closely resembles normal healing in humans, while identical wounds in red Duroc pigs form hypercontracted, hyperpigmented scars. The present study has evaluated the healing process in the first generation cross (F1) of red Duroc and Yorkshire pigs. Gross and histologic analysis revealed that the F1 animals exhibit an intermediate healing phenotype, with some features of each parent breed. F1 full thickness wounds were significantly hypercontracted and fibrotic, but apigmented. Analysis of mRNA expression patterns for a panel of relevant molecules (N=32) in the F1 animals revealed some similarities to each parent breed, as well as unique patterns for other molecules. Furthermore, a depth dependency to the healing response was observed at the gross, histologic, and molecular levels, with deep dermal wounds healing similar to Yorkshire wounds. These findings suggest that the genetic contribution to scar phenotype in this animal model is complex. However, the results indicate that further understanding in this model may provide insights into risk factors for hypertrophic scarring in human burn patients.     

Systemic depletion of macrophages in the subacute phase of wound healing reduces hypertrophic scar formation

Hypertrophic scars are caused by trauma or burn injuries to the deep dermis and can cause cosmetic disfigurement and psychological issues. Studies suggest that M2‐like macrophages are pro‐fibrotic and contribute to hypertrophic scar formation. A previous study from our lab showed that M2 macrophages were present in developing hypertrophic scar tissues in vivo at 3–4 weeks after wounding. In this study, the effect of systemic macrophage depletion on scar formation was explored at subacute phase of wound healing. Thirty‐six athymic nude mice that received human skin transplants were randomly divided into macrophage depletion group and control group. The former received intraperitoneal injections of clodronate liposomes while the controls received sterile saline injections on day 7, 10, and 13 postgrafting. Wound area, scar thickness, collagen abundance and collagen bundle structure, mast cell infiltration, myofibroblast formation, M1, and M2 macrophages together with gene expression of M1 and M2 related factors in the grafted skin were investigated at 2, 4, and 8 weeks postgrafting. The transplanted human skin from the control group developed contracted, elevated, and thickened scars while the grafted skin from the depletion group healed with significant less contraction and elevation. Significant reductions in myofibroblast number, collagen synthesis, and hypertrophic fiber morphology as well as mast cell infiltration were observed in the depletion group compared to the control group. Macrophage depletion significantly reduced M1 and M2 macrophage number in the depletion group 2 weeks postgrafting as compared to the control group. These findings suggest that systemic macrophage depletion in subacute phase of wound healing reduces scar formation, which provides evidence for the pro‐fibrotic role of macrophages in fibrosis of human skin as well as insight into the potential benefits of specifically depleting M2 macrophages in vivo.     

Genetic analysis of skin wound healing and scarring in a porcine model

Contraction is a normal part of skin wound healing and wound closure; however, excessive contraction and severe scarring concern patients and physicians alike. The present study has investigated the degree and kinetics of wound contraction in a porcine model of wound healing, to elucidate the genetic and molecular basis for abnormal skin wound healing and scarring. Healing of excisional skin wounds in juvenile female Yorkshire pigs closely resembled normal healing in humans. In contrast, identical wounds in female red Duroc pigs contracted significantly more, forming hypercontracted, hyperpigmented scars. Yorkshire x red Duroc F1 animals healed without hyperpigmentation, but with significantly greater wound contraction than observed in either parent breed. To examine the genetic transmission of the hypercontractile phenotype, all F1 animals were bred to a single Yorkshire boar, generating 20 backcross animals. All backcross animals healed with significantly less contraction than the normal Yorkshire animals. These findings suggest that the genetic contribution to scar phenotype in this animal model is complex, with a limited number of major genes controlling wound contraction, and an unknown number of minor genes that appear to modulate the impact of the major genes.     

兔耳解剖特点与成功建立增生性瘢痕模型的相关性实验研究

目的 观察兔耳不同部位的解剖结构特点,探讨不同手术方式以及术后处理方法对兔耳增生性瘢痕形成的影响,为成功建立增生性瘢痕动物模型提供理论依据.方法 新西兰白兔25只,切取5只兔10只耳60份全层组织标本,进行正常组织学观察;20只兔40只耳,每只兔耳腹侧各建立直径为8mm的全层皮肤缺损6个,总计240个创面.其中10只兔120个创面随机分为4组,手术后7d给予不同处理;另外10只兔120个创面术后不做处理.连续观察创面愈合以及瘢痕增生情况6个月,分别于手术后4、8周留取瘢痕组织行病理学检查和测量瘢痕增生指数.结果 正常兔耳不同部位的解剖结构特点不一致;建立兔耳瘢痕模型,部位宜选择在双侧兔耳腹侧内侧缘中、下侣部位,创伤深度宜破坏软骨膜,瘢痕形成率高,瘢痕增生指数高,持续时间长;术后剥痂可促进创面愈合,不利于瘢痕形成与增生.结论 兔耳自身的解剖结构特点与成功建立增生性瘢痕模型有一定的相关性,选择合适的建模部位、合理的创伤深度、术后恰当的处理均可影响瘢痕的形成和增生程度,可以提高增生性瘢痕建模成功率.     

Lobular capillary hemangioma in a post-burn scar

Hemangioma is a benign proliferation of endothelial cells within the superficial dermis, the deep dermis, internal organs and subcutis, or in both locations. It is classified as “capillary”, “cavernous”, or “lobular” hemangiomas. It is commonly seen in children during birth. It is very rare that hemangioma is seen in post-burn hypertrophic scar. A 42-year-old female who was treated 1 year back for 40 % of second degree superficial to deep burns had come with a history of swelling over the scar in the chin area since 1 month. There was history of occasional bleeding from the lesion. There was no history of trauma or pain. On examination, a 6?×?4 cm compressible, nontender swelling consistent with hemangioma was seen over the chin scar. The patient was taken up for excision of the lesion, and the defect was covered by a split-thickness skin graft. The histopathological examination showed that all features are suggestive of lobular hemangioma. Level of evidence: Level V, diagnostic study.     

早期保守去痂延期植皮治疗颜面深度烧伤

目的：探讨颜面部深度烧伤后,预防局部瘢痕增生和畸形的最好治疗方法。方法：颜面部深度烧伤早期,采用保守削痂,延期植皮和双眼睑早期减张,扩张创面立即植皮的处置方式,结果：本组12例患于伤后3周内创面全部愈合。随诊未发现明显瘢痕增生和畸形,面部表情自然,情绪表达充分。结论：应用早期保守去痂延期植皮的方法,可促进全颜面创面尽快修复,减少瘢痕增生和颜面畸形的发生。     

Effect of skin graft thickness on scar development in a porcine burn model

Animal models provide a way to investigate scar therapies in a controlled environment. It is necessary to produce uniform, reproducible scars with high anatomic and biologic similarity to human scars to better evaluate the efficacy of treatment strategies and to develop new treatments. In this study, scar development and maturation were assessed in a porcine full-thickness burn model with immediate excision and split-thickness autograft coverage. Red Duroc pigs were treated with split-thickness autografts of varying thickness: 0.026 in. (“thin”) or 0.058 in. (“thick”). Additionally, the thin skin grafts were meshed and expanded at 1:1.5 or 1:4 to evaluate the role of skin expansion in scar formation. Overall, the burn-excise-autograft model resulted in thick, raised scars. Treatment with thick split-thickness skin grafts resulted in less contraction and reduced scarring as well as improved biomechanics. Thin skin autograft expansion at a 1:4 ratio tended to result in scars that contracted more with increased scar height compared to the 1:1.5 expansion ratio. All treatment groups showed Matrix Metalloproteinase 2 (MMP2) and Transforming Growth Factor β1 (TGF-β1) expression that increased over time and peaked 4 weeks after grafting. Burns treated with thick split-thickness grafts showed decreased expression of pro-inflammatory genes 1 week after grafting, including insulin-like growth factor 1 (IGF-1) and TGF-β1, compared to wounds treated with thin split-thickness grafts. Overall, the burn-excise-autograft model using split-thickness autograft meshed and expanded to 1:1.5 or 1:4, resulted in thick, raised scars similar in appearance and structure to human hypertrophic scars. This model can be used in future studies to study burn treatment outcomes and new therapies.