The natural behavior of mononuclear phagocytes in HTS formation

Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS‐like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1‐ and M2‐like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1‐like macrophages were found predominantly at 1–2 weeks postgrafting; whereas, M2‐like macrophages were abundant at later time points, 3–4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2‐like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.     

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.     

A nude mouse model of hypertrophic scar shows morphologic and histologic characteristics of human hypertrophic scar

Hypertrophic scar (HSc) is a fibroproliferative disorder that occurs following deep dermal injury. Lack of a relevant animal model is one barrier toward better understanding its pathophysiology. Our objective is to demonstrate that grafting split‐thickness human skin onto nude mice results in survival of engrafted human skin and murine scars that are morphologically, histologically, and immunohistochemically consistent with human HSc. Twenty nude mice were xenografted with split‐thickness human skin. Animals were euthanized at 30, 60, 120, and 180 days postoperatively. Eighteen controls were autografted with full‐thickness nude mouse skin and euthanized at 30 and 60 days postoperatively. Scar biopsies were harvested at each time point. Blinded scar assessment was performed using a modified Manchester Scar Scale. Histologic analysis included hematoxylin and eosin, Masson's trichrome, toluidine blue, and picrosirius red staining. Immunohistochemistry included anti‐human human leukocyte antigen‐ABC, α‐smooth muscle actin, decorin, and biglycan staining. Xenografted mice developed red, shiny, elevated scars similar to human HSc and supported by blinded scar assessment. Autograft controls appeared morphologically and histologically similar to normal skin. Xenografts survived up to 180 days and showed increased thickness, loss of hair follicles, adnexal structures and rete pegs, hypercellularity, whorled collagen fibers parallel to the surface, myofibroblasts, decreased decorin and increased biglycan expression, and increased mast cell density. Grafting split‐thickness human skin onto nude mice results in persistent scars that show morphologic, histologic, and immunohistochemical consistency with human HSc. Therefore, this model provides a promising technique to study HSc formation and to test novel treatment options.     

Involvement of Keratinocyte Activation Phase in Cutaneous Graft Healing: Comparison of Full-Thickness and Split-Thickness Skin Grafts

BACKGROUND: Little is known about keratinocytic activation in the graft take and healing process. OBJECTIVE: To investigate the clinical and molecular differences between pure epidermal sheet graft (PESG), split-thickness skin graft (STSG), and full-thickness skin graft (FTSG). METHODS: Three different thickness skin grafts (PESG, STSG, and FTSG) were performed onto three kinds of porcine wounds: shallow, deep, and full. Graft take, contraction, and Ki-67 and beta1 integrin expression in epidermis were studied. RESULTS: All grafts took well. As expected, full wounds covered by PESG and STSG contracted more than those covered by FTSG, whereas shallow wounds covered by FTSG contracted more than those covered by STSG. No difference in contracture was observed among deep wounds covered by PESG, STSG, and FTSG. Up-regulation of Ki-67 and beta1 integrin expression was greater in PESG and STSG, compared with little expression in FTSG. CONCLUSION: The keratinocytic activation phase may occur both in the STSG and PESG healing process, as well as serum imbibition, inosculatory, and revascularization phases.     

A Comparison of Full and Split Thickness Skin Grafts in Radial Forearm Donor Sites

To formally evaluate the functional and aesthetic outcomes between full versus split thickness skin graft coverage of radial forearm free flap donor sites. A retrospective chart review of 47 patients who underwent pedicled or free radial forearm free flap reconstruction from May 1997 to August 2004 was performed. Comparisons were made between patients who had donor site coverage with split thickness skin grafts (STSG) or full thickness skin grafts (FTSG). There was no statistically significant difference between the STSG and FTSG in the number of post-operative dressings, incidence of tendon exposure, time to healing at the skin graft donor site, and time to healing at the skin graft recipient site. The questionnaire data showed there was a trend toward higher scores with the radial forearm scar aesthetics and satisfaction in the FTSG group. Full thickness skin graft coverage of radial forearm free flap donor site is superior to split thickness skin graft coverage in terms of aesthetic outcome, and has no statistically significant difference in terms of tendon exposure, time to healing at the skin graft donor site, time to healing at the skin graft recipient site, or post operative pain.     

The therapeutic potential of a C‐X‐C chemokine receptor type 4 (CXCR‐4) antagonist on hypertrophic scarring in vivo

Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell‐derived factor 1/CXCR4 signaling is up‐regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood‐borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell‐derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE‐9908 on dermal fibrosis was determined in vivo using a human HTS‐like nude mouse model, in which split‐thickness human skin is transplanted into full‐thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE‐9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down‐regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.     

Radial forearm free flap donor site outcomes comparison by closure methods.

OBJECTIVE: To compare the functional and aesthetic outcomes of radial forearm free flap (RFFF) donor sites reconstructed with full-thickness skin graft (FTSG), split thickness skin graft (STSG) alone, and STSG overlying an acellular dermal matrix (AlloDerm). STUDY DESIGN AND SETTING: A cross-sectional cohort study at a tertiary care hospital. RESULTS: Twenty-five head and neck cancer patients who underwent reconstruction with RFFF completed the evaluations (STSG = 10, FTSG = 8, STSG with AlloDerm = 7). Subjective evaluations of postoperative function by questionnaires showed no significant differences among the 3 groups (P = 0.93). In blinded evaluations by surgeons, the STSG group obtained the highest aesthetic outcome score (3.39 of 5.0), followed by FTSG (2.89) and STSG with AlloDerm (2.80). However, the difference was not statistically significant (P = 0.32). Objective measurements of postoperative function by certified occupational therapists were comparable among the 3 groups with the exception of a mildly decreased range of wrist flexion (P = 0.036) and ulnar deviation (P = 0.016) in the FTSG group. CONCLUSIONS: The 3 methods of reconstruction have comparable postoperative functional and aesthetic outcomes. SIGNIFICANCE: Each of the 3 methods of reconstruction has low morbidity and satisfactory aesthetic and functional outcomes.     

Late failure of a split‐thickness skin graft in the setting of homozygous factor V Leiden mutation: a case report and correlative animal model from the Wound Etiology and Healing (WE‐HEAL) study

We present the case of a 53‐year‐old Caucasian male smoker with remote history of left lower extremity deep venous thrombosis (DVT) and a strong family history of thrombosis, who presented to the Center for Wound Healing at MedStar Georgetown University Hospital with spontaneous left leg ulceration. Prothrombotic evaluation showed homozygosity for the factor V Leiden (FVL) mutation. Therapeutic anticoagulation was commenced with warfarin (Coumadin®) and the patient underwent successful debridement and Apligraf® followed by split‐thickness skin graft (STSG) of two wounds. He had an uneventful postoperative course and on the 27th postoperative day the grafts were 95% intact. However, by postoperative day 41 there was 10% graft loss, and over the subsequent 2 weeks both grafts necrosed. On further questioning, it transpired that the patient had discontinued his warfarin on postoperative day 37 because he thought that it was no longer necessary. The patient is enrolled in the Wound Etiology and Healing (WE‐HEAL) study, and at the time of the original graft, residual skin fragments from the STSG were transplanted onto a nude mouse for development of an animal model of wound healing. The mouse graft was successful and was harvested at postoperative day 87 for pathological examination. We review the mechanisms by which prothrombotic states, particularly FVL mutation, can contribute to skin graft failure and delayed wound healing. This case highlights the importance of considering prothrombotic conditions in patients with spontaneous leg ulcerations and the impact of therapeutic anticoagulation on healing. It further allows us to demonstrate the efficacy of the animal model in which residual fragments of STSG tissue are utilised for transplant onto nude mice for manipulation in the laboratory.     

Comparative analysis of functional and aesthetic outcomes of retroauricular full thickness versus plantar glabrous split thickness skin grafts in pediatric palmar hand burns

BackgroundOptimal management of palmar hand burns in children is controversial. We aimed to compare function and aesthetics of retroauricular full thickness skin grafts (FTSG) to plantar glabrous split thickness skin grafts (STSG).Methods32 palmar grafts in paediatric burn patients were analysed: 19 retroauricular FTSG (group 1) and 13 thick plantar glabrous STSG (group 2). The latter were harvested at a thickness of 0.5 mm. The resulting plantar donor defects were covered with a STSG from the scalp, a sequential surgical technique we termed the “Zurich move”. Clinical examination, Cutometer and Colorimeter assessment and validated patient and observer questionnaires were used. Donor site complications and subjective complaints were recorded.ResultsColorimeter results were superior in group 2 with an erythema score of 5.73 ± 2.64 (group 1) versus 2.33 ± 1.97 (group 2, p < 0.001) and a pigmentation score of 9.82 ± 5.42 (group 1) and 1.89 ± 1.92 (group 2, p < 0.001). Observers` scar evaluation using VSS and POSAS showed significantly superior results in group 2 for almost all items. Conversely, group 1 grafts were less stiff with mean normalized tissue extension R0 of 0.80 ± 0.21 versus 0.57 ± 0.24 in group 2 grafts (p < 0.05). In both groups donor sites complications were rare.ConclusionPlantar glabrous STSG showed superior functional and aesthetic results when compared to FTSG in pediatric palmar hand burns. In addition, the “Zurich Move” is safe and provides uncomplicated donor site healing on the scalp and the foot allowing rapid restoration of full function.     

A novel immune competent murine hypertrophic scar contracture model: A tool to elucidate disease mechanism and develop new therapies

Hypertrophic scar (HSc) contraction following burn injury causes contractures. Contractures are painful and disfiguring. Current therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune‐competent murine HSc model. A third‐degree burn was created on dorsum of C57BL/6 mice. Three days postburn, tissue was excised and grafted with ear skin. Graft contraction was analyzed and tissue harvested on different time points. Outcomes were compared with human condition to validate the model. To confirm graft survival, green fluorescent protein (GFP) mice were used, and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus in contraction was analyzed. Cellularity was assessed with 4′,6‐diamidino‐2‐phenylindole. Collagen maturation was assessed with Picro‐sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR). Elastic moduli of skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ～45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild‐type mice, and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed that panniculus carnosus doesn't contribute to contraction. Microscopic analyses showed that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT‐PCR showed up‐regulation of transforming growth factor beta, alpha smooth muscle actin, and rho‐associated protein kinase 2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.     

Comparison of therapeutic effects between artificial dermis combined with autologous split‐thickness skin grafting and autologous intermediate‐thickness skin grafting alone in severely burned patients: A prospective randomised study

The purpose of this study was to evaluate the therapeutic effects of artificial dermis combined with autologous split‐thickness skin grafting (STSG) compared with autologous intermediate‐thickness skin grafting (ITSG) alone in severely burned patients. Fifty‐six severely burned patients admitted to our hospital from December 2017 to January 2019 were enrolled and evenly grouped according to the random number table method [AD‐STSG group: 28 patients, receiving the treatment of artificial dermis (AD) combined with autologous STSG; ITSG group: 28 patients, receiving autologous ITSG treatment alone]. The healing time and Vancouver Scar Scale (VSS) score of the donor area and graft area, survival rate and infection status of the autologous skin, psychological status (determined by Self‐rating Anxiety Scale and Self‐rating Depression Scale), and the activity of functional parts of all enrolled patients were included in the evaluation. General items of patients in AD‐STSG group and ITSG group, including age, sex, and degree of burn, were all comparable. A significantly shortened healing time of donor skin in AD‐STSG group was observed when compared with ITSG group (P < .05) while the recipient skin healed in the same tendency between the two groups. In addition, 21 days after the operation, AD‐STSG group presented with significantly higher survival rate of graft skin than ITSG group (P < .05) while same infection status was observed in the two groups. Significantly lower VSS scores were found in AD‐STSG group than that in ITSG group 3‐, 6‐ and 10‐months after operation (P < .05). Statistical difference regarding psychological status of patients from two groups was unobservable before operation while significantly lower Self‐rating Anxiety Scale (SAS) and Self‐rating Depression Scale (SDS) scores were found in AD‐STSG group than that in ITSG group 3‐, 6‐ and 10‐months after operation (P < .05). Also, AD‐STSG group presented improved mobility of functional part than that in ITSG group 10‐months after operation without statistical difference (P = .051). Artificial dermis combined with autologous split‐thickness skin grafting showed better therapeutic outcomes for the treatment of severely burned patients than autologous intermediate‐thickness skin grafting in terms of graft healing time, scar formation, psychological recovery, and perhaps in functional reconstruction.     

An experimental study on the repair of full skin loss of nude mice with composite graft of epidermal stem cells

This study is to constitute a composite skin substitute with epidermal stem cells (ESCs) and fibroblasts on collagen sponge. ESCs were selected by rapid attachment to collagen IV for 10 min. Collagen was extracted from rat's tail. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde. Fibroblasts were inoculated on collagen sponge and cultured for 1 week prior to inoculation of ESCs. Having cultured for 2 weeks in submerged culture, the bioengineered tissue was raised to the air-liquid interface and cultured for 2 weeks. The artificial skin was then grafted onto full skin loss wounds of nude mice. Collagen sponge membrane lacking cell inoculation and an artificial skin with epidermal cells (ECs) and fibroblasts were used as controls. The wounds were observed daily. Tissue samples were harvested and examined by means of histology, immunohistochemistry and electron microscopy. The wounds in the test group healed at a significantly faster rate than controls, with good skin appearance and minimal scar formation. The control group showed delayed wound healing and intensive wound contraction as compared to the test group. Thus the skin substitute with ESCs seemed to be a good equivalent.     

Objective evaluation of the effect of autologous platelet concentrate on post-operative scarring in deep burns

Introduction

The healing of grafted areas after surgical treatment of deep burns frequently generates mutilating scars, and rises the risk of subsequent scar hypertrophy. Scar assessment based on clinical evaluation is inherently subjective, which stimulates search for objective means of evaluation.

Objective

The aim of this study was to objectively evaluate the effect of using autologous platelet concentrate (APC) in combination with split thickness skin grafting (STSG) on scarring processes following surgery of deep burns as compared with application of STSG alone.

Method

Selected viscoelastic properties of 38 scars on 23 patients in total were examined using the Cutometer MPA 580 under controlled conditions for long-term outcomes 1, 3, 6 and 12 months after surgery following deep burns.

Results

The findings of this study suggest that the STSG + APC combination reduces the time of scar viscoelastic properties recovery as compared with application of STSG alone. This was statistically significant for viscoelastic parameters R₂ and Q₁.

Conclusion

APC has been advocated to enhance scarring after surgery of deep dermal and full thickness burns. We objectively demonstrated that the viscoelastic properties of scars treated with STSG + APC combination return more rapidly to the plateau state than areas treated with STSG only.     

以胶原海绵为载体培养的人表皮细胞移植

目的：将体外培养的人表皮细胞接种到胶原海绵上,构建一种表皮替代物,移植到鼠创面后研究观察皮肤的再生,方法：将手术切下的包皮经中性蛋白酶分离表皮,真皮,再经胰蛋白酶消化制成表皮细胞悬液,移入培养瓶中培养,将处于对数生长期的人表皮细胞直接接种到培养皿中的胶原海绵 ,再加上表皮细胞液培养3天后,移植到裸鼠全层皮肤缺损的创面上,以单纯无接种细胞的胶原海绵作为对照,并进行组织学,免疫组织化学及电子显微镜观察。结果：这种表皮替代物移植到创面后,表皮细胞继续增殖分化,形成一层新生表皮,与对照组相比,创面闭合早且收缩程度小,表皮成熟早且分层较多,基底膜形成较早,表皮下胶原纤维较少。结论：体外培养的表皮细胞能在胶原海绵上生长,移植到创面上后,该细胞可自动移行到创面并形成多层表皮结构,抑制创面收缩,可用于修复皮肤缺损。     

Interleukin‐33 encourages scar formation in murine fetal skin wounds

The magnitude of the inflammatory response after skin injury is important for determining whether wounds in developing fetal skin will heal scarlessly (minimal inflammation) or with prominent scars (robust inflammation). One class of inflammatory mediators gaining attention for their role in wound inflammation is alarmins. In the current study, the alarmin interleukin‐33 (IL‐33) was examined in a mouse model of fetal wound healing. IL‐33 expression was elevated in scar‐forming embryonic day 18 wounds compared to scarless embryonic day 15 wounds. Furthermore, injection of IL‐33 into embryonic day 15 wounds caused scarring when wounds were analyzed at 7 days postwounding. The introduction of IL‐33 into embryonic day 15 wounds did not induce statistically significant changes in the number of neutrophils, mast cells, or macrophages in vivo. However, IL‐33 treatment enhanced collagen expression in cultured fibroblasts derived from adult and fetal murine skin, suggesting that IL‐33 may directly stimulate fibroblasts. In vitro studies suggested that the stimulation of collagen production by IL‐33 in fibroblasts was partially dependent on NF‐κB activation. Overall, the data suggest an association between IL‐33 and scar formation in fetal wounds.     

Inhibitory effects of local pretreated epidermis on wound scarring: a feasible method to minimize surgical scars

OBJECTIVE: To inhibit surgical scarring by pretreating epidermis at the operation site. METHODS: Eight patients who were to undergo operation through a modified incision incisions technique and other eight subjects presenting for skin grafting were recruited. For the modified incision patients a method to make the site 'epidermis-free' was developed. At the operating site a split thickness rectangular skin flap was raised with a width of one cml transverse to the incision direction. Incision was then made through the exposed dermis. The flap was repositioned onto the incision site after intradermal suturing of the incision line following the subcutaneous operation. When skingrafting the graft was used in extended form by de-epithelialising the margins of the wound by 1cm before graft placement. Then a skin graft with medial full-thickness and marginal split-thickness areas was transplanted onto the extended wound. In the control site-matched groups, surgical skin incision and skin grafting were performed as usual. Clinical observation and immunohistological examination were applied to evaluate the wound healing and scar formation in all subjects. RESULTS: Both epidermis-free incision and extended skin graft sites showed perfect wound healing with short-term subjective scarring disturbance and slight wound scars, different from the control groups. The histological results showed the healing tissues in the experimental groups were more similar to normal dermis than those in the control groups. The immuoreactivities of type I and type II collagen in epidermis-free incision were both much lower than those in the control incision and the ratio of type I to type III collagen in the experimental incision was nearer to normal value. CONCLUSION: Pretreating local epidermis can effectively minimize postoperative scarring by modulating collagen synthesis.     

A Simple Dressing Technique Following Dermofasciectomy and Full Thickness Skin Grafting of the Fingers in the Treatment of Severe Dupuytren’s Contracture

Dupuytren’s disease with severe finger contractures and recurrent contractures following previous surgery often have extensive skin involvement. In these severe cases, excision of the diseased chord along with the involved skin is a good option to reduce the risk of recurrance. The resulting skin defect can be covered with a full thickness skin graft (FTSG) or a cross finger flap. Cross finger flaps have donor finger morbidity and hence a full thickness graft is usually preferred. The FTSG extending to the midlateral margins on both sides of the finger reduces the risk of joint contracture due to graft shrinkage. Once the FTSG is sutured in place, the standard practice is to compress and secure the graft to its recipient bed with a tie-over dressing and this can be time consuming. We present a simple dressing technique to secure the FTSG without the need for a tie-over dressing.     

在胶原海绵膜上构建人表皮细胞和成纤维细胞复合移植物的实验研究

目的将原代培养的人表皮细胞和成纤维细胞，接种到一种新的载体-胶原海绵膜上，构建新的复合皮肤替代物。方法来源于人包皮的表皮细胞和成纤维细胞分别原代培养，成纤维细胞经消化后接种在胶原海绵膜上(1×10     

An immunohistological study of the revascularization process in human skin transplanted onto the nude mouse

The revascularization of human skin transplanted onto the nude mouse was studied by performing double-labeling immunofluorescence microscopy on human skin before transplantation and at different stages, ranging from one week to six months after grafting. With a crossreacting anti-factor-VIII antigen antibody used to identify the endothelial cells, and with human-specific monoclonal antibodies directed against vimentin or HLA-DR antigen, it appeared that the original human endothelial cells of transplanted skin progressively disappear, while murine endothelial cells invade the graft. Moreover, in double-labeling experiments with a crossreacting anti-factor-VIII antibody and a human-specific anti-type-IV-collagen antibody, anastomosis between host and graft vessels and a constant codistribution of graft endothelial cells with human type IV collagen were observed. Finally, double staining with species-specific antibodies directed against murine or human type IV collagen showed that mouse type IV collagen appeared progressively in the graft and was constantly colocalized with human type IV collagen. From these observations, it was concluded that revascularization of human skin transplanted onto the nude mouse proceeds as follows: inoculation; disappearance of human endothelial cells and migration of mouse endothelial cells into the graft over the basement membrane of preexisting human vessels; and production of a new vascular basement membrane by mouse endothelial cells on the original basement membrane of human graft vessels.     

Comparison of biopolymer scaffolds for the fabrication of skin substitutes in a porcine wound model

This study compared three acellular scaffolds as templates for the fabrication of skin substitutes. A collagen-glycosaminoglycan (C-GAG), a biodegradable polyurethane foam (PUR) and a hybrid combination (PUR/C-GAG) were investigated. Scaffolds were prepared for cell inoculation. Fibroblasts and keratinocytes were serially inoculated onto the scaffolds and co-cultured for 14 days before transplantation. Three pigs each received four full-thickness 8 cm × 8 cm surgical wounds, into which a biodegradable temporising matrix (BTM) was implanted. Surface seals were removed after integration (28 days), and three laboratory-generated skin analogues and a control split-thickness skin graft (STSG) were applied for 16 weeks. Punch biopsies confirmed engraftment and re-epithelialisation. Biophysical wound parameters were also measured and analysed. All wounds showed greater than 80% epithelialisation by day 14 post-transplantation. The control STSG displayed 44% contraction over the 16 weeks, and the test scaffolds, C-GAG 64%, Hybrid 66.7% and PUR 67.8%. Immunohistochemistry confirmed positive epidermal keratins and basement membrane components (Integrin alpha-6, collagens IV and VII). Collagen deposition and fibre organisation indicated the degree of fibrosis and scar produced for each graft. All scaffold substitutes re-epithelialised by 4 weeks. The percentage of original wound area for the Hybrid and PUR was significantly different than the STSG and C-GAG, indicating the importance of scaffold retainment within the first 3 months post-transplant. The PUR/C-GAG scaffolds reduced the polymer pore size, assisting cell retention and reducing the contraction of in vitro collagen. Further investigation is required to ensure reproducibility and scale-up feasibility.