角质细胞与成纤维细胞混合移植修复裸鼠全层皮肤缺损的初步报告

目的：利用组织工程原理探讨修复全层皮肤缺损的理想方式。方法：以裸鼠为动物模型,在皮肤全层缺损区域分别移植纤维蛋白胶(n=10),纤维蛋白胶角质细胞悬液（n=10）,纤维蛋白胶成纤维细胞悬液（n=10）以及纤维蛋白胶角质细胞成纤维细胞悬液（n=10）,术后每天对伤口进行大体观察,第5,7,10,14,21,35d,分别取材活检行组织学及免疫组织化学检查。结果：移植有角质细胞组（2和4组）的创面愈合快,术后10d组织学提示创面完全上皮化,抗人特异性HLA－1型抗原、抗involucrin染色和抗Ⅶ型胶原染色阳性证明新生上皮由移植的人角质细胞形成,抗involucrin染色阳性又证明角质细胞分化成熟有角质层形成,抗Laminin染色、抗Ⅶ型胶原染色阳性提示早期基底膜形成。组织学检查提示第4组新生上皮有许多类似皮钉样结构。结论：培养的角质细胞,成纤维细胞结合纤维蛋白胶移植到创面上后,可以形成复层分化良好、接近正常结构和功能的新生成肤组织。     

体外构建复合皮移植后对大鼠创面愈合的影响

目的：观察角质形成细胞和成纤维细胞与无细胞异种真皮基质构成的复合皮移植于全层皮肤缺损创面后的效果，寻找一种新的创面覆盖物。方法：54只SD大鼠作背部全层皮肤缺损创面后分为A、B、C三组，分别以A型复合皮（角质形成细胞＋成纤维细胞＋无细胞异种真皮基质）、B型复合皮（角质形成细胞＋无细胞异种真皮基质）和普通敷料进行移植，术后定期观察创面愈合情况并进行创面收缩率的计算，同时切取创面组织进行组织学检测。结果：三组中，A组的创面愈合及外观情况最好；A组创面收缩率明显低于B、C两组（P（0．05）；组织学检测提示A组复合皮上皮分化充分，胶原增生有序，表皮一真皮连接结构重建明显，未见明显的急性期免疫排斥反应。结论：角质形成细胞和成纤维细胞与无细胞异种真皮基质构成的复合皮移植后能改善创面愈合质量，是一种较理想的、可探索的皮肤替代物。     

Different wound healing properties of dermis,adipose, and gingiva mesenchymal stromal cells

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis‐, adipose‐, and gingiva‐derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin‐derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.     

Evaluation of dermal-epidermal skin equivalents ('composite-skin') of human keratinocytes in a collagen-glycosaminoglycan matrix(Integra artificial skin).

Integra artificial skin (Integra LifeSciences Corp., Plainsboro, NJ, USA) is a dermal template consisting of bovine collagen, chondroitin-6-sulphate and a silastic membrane manufactured as Integra. This product has gained widespread use in the clinical treatment of third degree burn wounds and full thickness skin defects of different aetiologies. The product was designed to significantly reduce the time needed to achieve final wound closure in the treatment of major burn wounds, to optimise the sparse autologous donor skin resources and to improve the durable mechanical quality of the skin substitute. The clinical procedure requires two stages. The first step creates a self neodermis, the second creates a self epidermis on the neodermis. However, it is desirable to cover major burn wounds early in a single step by a skin substitute consisting of a dermal equivalent seeded in vitro with autologous keratinocytes ('composite-skin') out of which a full thickness skin develops in vivo.The goal of this experimental study was to develop a method to integrate human keratinocytes in homogeneous distribution and depth into Integra Artificial Skin. The seeded cell-matrix composites were grafted onto athymic mice in order to evaluate their potential to reconstitute a human epidermis in vivo. We were able to demonstrate that the inoculated human keratinocytes reproducibly displayed a homogeneous pattern of distribution, adherence, proliferation and confluence. The cell-matrix composites grafted in this model exhibited good wound adherence, complete healing, minor wound contraction and had the potential to reconstitute an elastic, functional and durable human skin. Histologically we were able to show that the inoculated human keratinocytes in vivo colonised the matrix in a histomorphologically characteristic epidermal pattern (keratomorula, keratinocyte bubbling) and developed a persisting, stratified, keratinising epidermis which immunohistologically proved to be of human origin. These experimental results demonstrate the establishment of an effective cell cultivation process which may be suitable for scale-up production of the epidermal component as large-scale composite-skin grafts. When seeded into Integratrade mark and grafted onto the nude mouse a replacement skin with normal functioning dermal-epidermal components was developed. These results encourage the design of a clinical trial to assess the function of this composite graft in man.     

Insulin complexed with cyclodextrins stimulates epithelialization and neovascularization of skin wound healing in rats

IntroductionSkin lesions are a significant public health problem, above all that wounds fail to heal properly and become chronic. Due to its reepithelization action, insulin has the potential to heal skin lesions, by stimulating the proliferation and migration of keratinocytes, angiogenic stimulus, and increasing collagen deposition. In the present study insulin was complexed with 2-hydroxypropyl-β-cyclodextrin (HPβCD) and its wound healing effect and inclusion complex (HPβCD-I) were evaluated in excisional wounds in the skin of rats.Material and methodsThree different gel based pharmaceutical forms were created: carbopol 940^® base gel, an insulin gel comprising the base gel plus 50 IU of insulin and a gel complex comprising the base gel plus (HPβCD) complexed with insulin (HPβCD-I) were used to verify wound healing in vitro and in vivo assays.ResultsThe wounds in the skin of rats were treated with gel containing HPβCD-I not cytoxically irritating and cytotoxic. Analysis of cell proliferation and measurement of the length and thickness of the epidermis showed that HPβCD-I prolonged the proliferation and migration of keratinocytes. Revascularization analysis of lesions treated with HPβCD-I compared to those treated with insulin found that angiogenic stimulus was less intense, but more constant and prolonged in the modified release process. There was increased deposition of type I and III collagen fibers in accordance with the treatment time.ConclusionTherefore, the slow release of complexed insulin modulated the reepithelialization process by stimulating cell proliferation and migration of keratinocytes, favoring greater concentration of serum insulin, modulating inflammatory response, matrix remodeling and promoting neovascularization. Angiogenesis extended by the steady release of insulin can be effective in the treatment of chronic wounds.     

Cultured keratinocytes suspended in fibrin glue to cover full thickness wounds on athymic nude mice: comparison of two brands of fibrin glue

 To circumvent well-known drawbacks inherent in cultured epidermal sheet grafts, a new keratinocyte delivery system was experimently investigated. Human subconfluent keratinocytes were labeled with fluorescence cell linker and cultured in two brands of fibrin glue (Tissucol^R and Beriplast^R) in vitro for 5 days. Keratinocyte fibrin glue suspensions were grafted onto full thickness wounds in athymic nude mice for macroscopic and microscopic investigation. Keratinocytes survived in both fibrin glues in vitro for at least 5 days. When grafted onto wounds, cells maintained a high proliferation potential and at 5 days formed small masses or well-organised cell clusters within the fibrin network. After 7 days new epithelia enlarged and became thicker with high keratinization and occasional downward projections. At 10 days, wounds in both fibrin groups were totally epitheliazied but only partially in the control group. Anti-involucrin and anti-laminin immunostainings indicated well-differentiated new epithelium derived from human keratinocytes and early reorganised basement membrane. The fibrin glues seem not only to deliver highly proliferative keratinocytes easily and effectively, but also provide an optimal milieu for their migration, proliferation and differentiation. Received: 7 February 1997 / Accepted: 7 May 1997     

人表皮细胞与无细胞异种真皮复合移植的实验研究

目的 观察人表皮细胞与无细胞异种真皮复合移植全层皮肤缺损创面后的效果,寻找一种新的创面覆盖物。方法 42只裸鼠作背部全层皮肤缺损创面,分别进行复合皮移植（复合皮组）和单纯表皮细胞膜片移植（对照组）,术后定期观察创面愈合情况并进行创面愈合率及创面收缩率的计算,同时留取创面组织进行组织学检查。结果 与对照组相比较,复合皮组的创面愈合及外观情况良好,两组创面愈合率及收缩率的差异有显性意义（P＜0．05）;组织学检测提示复合皮上皮分化充分,胶原增生有序,表皮－真皮连接结构重建明显,未见明显的急性期免疫排斥反应。结论 人表皮细胞与无细胞异种真皮复合移植能改善创面愈合质量,可作为一种新的皮肤代用品。     

Level of Fibronectin mRNA Is Markedly Increased in Human Chronic Wounds

BACKGROUND: Acute wound healing has been extensively investigated over the years, however, little is known about possible healing defects in chronic wounds. Fibronectin (FN) plays a critical role in different phases of wound healing and has been demonstrated to be degraded in chronic wounds by proteases. Fibroblasts cultured from chronic leg ulcers showed a higher level of FN compared to normal fibroblasts. OBJECTIVE: We explored whether the increase in FN protein in chronic wounds is due to increased FN mRNA. In addition, the level of alpha5beta1 integrin FN cell surface receptor was also examined. METHOD: Skin biopsies were taken from normal skin within a few hours of Mohs surgery and from the edge of chronic venous leg ulcers. In situ hybridization was performed to determine the level of FN mRNA. The level of integrin alpha5beta1 was determined by immunohistochemistry. RESULTS: The level of FN mRNA in normal skin and acute wounds was undetectable. In contrast, FN mRNA was heavily induced throughout the dermis of chronic wounds. Immunostaining using a monoclonal antibody against the alpha5 subunit of integrin revealed that chronic wounds and normal skin showed undetectable levels of alpha5beta1 integrin. A large induction of alpha5 was observed in acute wounds. CONCLUSION: For reepithelization to occur, epidermal keratinocytes need to migrate over the wound surface, a process requiring an interaction between FN and its cell surface receptor integrin alpha5beta1. These findings suggest that although FN mRNA is increased in chronic wounds, lack of FN cell surface receptor may prevent migration of epidermal keratinocytes in chronic wounds.     

An in vitro wound healing model for evaluation of dermal substitutes

Reepithelialization of skin wounds is essential to restore barrier function and prevent infection. This process requires coordination of keratinocyte proliferation, migration, and differentiation, which may be impeded by various extrinsic and host‐dependent factors. Deep, full‐thickness wounds, e.g., burns, are often grafted with dermal matrices before transplantation of split‐skin grafts. These dermal matrices need to be integrated in the host skin and serve as a substrate for neoepidermis formation. Systematic preclinical analysis of keratinocyte migration on established and experimental matrices has been hampered by the lack of suitable in vitro model systems. Here, we developed an in vitro full‐thickness wound healing model in tissue‐engineered human skin that allowed analysis of the reepithelialization process across different grafted dermal substitutes. We observed strong differences between porous and nonporous matrices, the latter being superior for reepithelialization. This finding was corroborated in rodent wound healing models. The model was optimized using lentivirus‐transduced keratinocytes expressing enhanced green fluorescent protein and by the addition of human blood, which accelerated keratinocyte migration underneath the clot. Our model shows great potential for preclinical evaluation of tissue‐engineered dermal substitutes in a medium‐throughput format, thereby obviating the use of large numbers of experimental animals.     

UV fluorescence excitation imaging of healing of wounds in skin: Evaluation of wound closure in organ culture model

Background and Objective

Molecules native to tissue that fluoresce upon light excitation can serve as reporters of cellular activity and protein structure. In skin, the fluorescence ascribed to tryptophan is a marker of cellular proliferation, whereas the fluorescence ascribed to cross‐links of collagen is a structural marker. In this work, we introduce and demonstrate a simple but robust optical method to image the functional process of epithelialization and the exposed dermal collagen in wound healing of human skin in an organ culture model.

Materials and Methods

Non‐closing non‐grafted, partial closing non‐grafted, and grafted wounds were created in ex vivo human skin and kept in culture. A wide‐field UV fluorescence excitation imaging system was used to visualize epithelialization of the exposed dermis and quantitate wound area, closure, and gap. Histology (H&E staining) was also used to evaluate epithelialization.

Results

The endogenous fluorescence excitation of cross‐links of collagen at 335 nm clearly shows the dermis missing epithelium, while the endogenous fluorescence excitation of tryptophan at 295 nm shows keratinocytes in higher proliferating state. The size of the non‐closing wound was 11.4 ± 1.8 mm and remained constant during the observation period, while the partial‐close wound reached 65.5 ± 4.9% closure by day 16. Evaluations of wound gaps using fluorescence excitation images and histology images are in agreement.

Conclusions

We have established a fluorescence imaging method for studying epithelialization processes, evaluating keratinocyte proliferation, and quantitating closure during wound healing of skin in an organ culture model: the dermal fluorescence of pepsin‐digestible collagen cross‐links can be used to quantitate wound size, closure extents, and gaps; and, the epidermal fluorescence ascribed to tryptophan can be used to monitor and quantitate functional states of epithelialization. UV fluorescence excitation imaging has the potential to become a valuable tool for research, diagnostic and educational purposes on evaluating the healing of wounds. Lasers Surg. Med. 48:678–685, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.     

Autologous keratinocyte suspensions accelerate epidermal wound healing in pigs

BACKGROUND: Tissue culture techniques enable in vitro expansion of keratinocytes that can be used to treat burns and chronic wounds. These keratinocytes are commonly grafted onto the wounds as differentiated sheets of mature epithelium. Less is however known about the effects of transplanting the cells as suspensions. This study evaluated epidermal regeneration in fluid-treated skin wounds treated with suspensions of cultured and noncultured autologous keratinocytes. MATERIALS AND METHODS: Eighty-seven full-thickness excisional skin wounds were created on the back of 6 pigs and then transplanted with either cultured or noncultured autologous keratinocytes. The wounds were enclosed with liquid-tight chambers containing saline to provide a hydrated and standardized environment. RESULTS: Keratinocyte transplantation resulted in several cell colonies within the granulation tissue of the wound. These colonies progressively coalesced and contributed to a new epithelium. The origin of the transplanted keratinocytes was confirmed by histochemical staining of wounds transplanted with transfected keratinocytes expressing beta-galactosidase. Transplantation of 0.125 x 10(6), 0.5 x 10(6), and 2.0 x 10(6) cultured keratinocytes, and 0.5 x 10(6) and 5.0 x 10(6) noncultured keratinocytes, increased reepithelialization dose dependently over saline-treated controls. The epithelial barrier function recovered faster in transplanted wounds as demonstrated by less protein leakage over the wound surface on Days 7-10 as compared to control wounds. Wound reepithelialization and the number of keratinocyte colonies observed in granulation tissue were significantly less in wounds transplanted with noncultured keratinocytes compared to wounds seeded with cultured keratinocytes. CONCLUSION: Our study demonstrates successful transplantation of keratinocyte suspensions and their dose-dependent acceleration of wound repair. Selection of proliferative cells during culture and higher colony-forming efficiency may explain the greater effects observed with cultured keratinocytes.     

Initial experience with a composite autologous skin substitute.

Patients with large burns are surviving in increasing numbers, but there remains no durable and reliable permanent skin replacement. After initial favorable small animal experiments, a pilot trial of a composite skin replacement was performed in patients with massive burns. A composite skin replacement (CSR) was developed by culturing autologous keratinocytes on acellular allogenic dermis. This material was engrafted in patients with massive burns and compared to a matched wound covered with split thickness autograft. With human studies committee approval, 12 wounds in 7 patients were grafted with CSR while a matched control wound was covered with split thickness autograft. These 7 children had an average age of 6.4+/-1.4 yr and burn size of 75.9+/-5.0% of the body surface. Nine wounds were acute burns and three were reconstructive releases. Successful vascularization at 14 days averaged 45.7+/-14.2% (range 0-100%) in the study wounds and 98+/-1% (range 90-100%) in the control sites (P<0.05). Reduced CSR take seemed to correlate with wound colonization. All children survived. While CSR did not engraft with the reliability of standard autograft, this pilot experience is encouraging in that successful wound closure with this material is possible, if not yet dependable. It is hoped that a more mature epidermal layer may facilitate engraftment, and trials to explore this possibility are in progress.     

Combination of a new composite biocampatible skin graft on the neodermis of artificial skin in an animal model

Introduction: There have been very limited and inconsistent attempts at combining the cultured epidermal autograft (CEA) with the neodermis of artificial skin (Integra). The reasons for this remain unknown. The basement membrane proteins of conventional CEA sheets are easily damaged by the dispase treatment during the harvesting of the CEA from the culture flask. The damage of the basement membrane proteins may affect the anchorage of CEA onto the neodermis of Integra. A new Composite Biocompatible Skin Graft (CBSG) was recently developed. Methods: Composite biocompatible skin graft consists of autologous keratinocytes cultivated on a pliable hyaluronate‐derived membrane (Laserskin) which has been pre‐seeded with allogenic dermal fibroblasts. Basement membrane proteins of CBSG are protected from the dispase treatment because the keratinocytes are directly seeded onto Laserskin. The engraftment of CBSG was evaluated on 20 wounds of 10 rats. Integra was grafted on two freshly excised full‐thickness wounds (3 cm in diameter) in the dorsum of each animal. A polypropylene ring was applied to each wound to prevent the migration of epithelium from the edges. Composite Biocompatible Skin Graft was used to cover the neodermis of Integra after the silicone membrane was removed 14?21 days postgrafting. Results: Fourteen (70%) of 20 skin biopsies taken at day 21 from the centre of the grafted wounds revealed regenerated epithelium. Conclusion: A feasible delivery system of cultured keratinocytes onto the neodermis of Integra is demonstrated in this animal ­experiment.     

用含活性细胞的胶原支架复合皮修复小鼠全层皮肤缺损的研究

目的构建具有活性细胞成分的复合皮，观察其在修复小鼠全层皮肤缺损中的作用。方法将异种成纤维细胞种植于脱软骨细胞胶原支架上培养3d，形成真皮替代物。再将其放置于培养的上皮细胞膜上共同培养10d，组成含活性细胞成分的复合皮。将复合皮移植于小鼠的全层皮肤缺损处，记录其生长情况并于术后定期活检，进行组织学观察。结果胶原支架中可见生长良好的成纤维细胞和复层表皮细胞。移植术后1周，复合皮与小鼠缺损创面粘连紧密，可见明显的血管化。术后6周，创面愈合良好，移植物与创缘融合，未见明显排斥反应。结论以脱软骨细胞胶原为支架构建的复合皮，可作为皮肤替代物修复全层皮肤组织缺损。     

Health‐related quality of life and patient burden in patients with split‐thickness skin graft donor site wounds

Split‐thickness skin grafting is a common procedure to treat different kinds of wounds. This systematic, multicentre, observational, cross‐sectional study of adult patients with split‐thickness skin graft (STSG) donor site wounds was conducted to evaluate quality of life (QoL) impairments caused by donor site wounds following split‐thickness skin grafting. Therefore, 112 patients from 12 wound centres in Germany were examined based on patient and physician questionnaires as well as a physical examination of the donor site wound. Most indications for skin grafting were postsurgical treatment (n = 51; 42.5%) and chronic wounds (n = 47; 39.2%). European QoL visual analoque scale (EQ VAS) averaged 64.7 ± 23.3, European QoL 5 dimensions (EQ‐5D) averaged 77.4 ± 30.0. Wound‐QoL (range: 0‐4) was rated 0.8 ± 0.8 post‐surgery and 0.4 ± 0.6 at the time of survey (on average 21 weeks between the time points). Compared to averaged Wound‐QoL scores of chronic wounds donor site‐related QoL impairments in split‐thickness skin‐graft patients were less pronounced. There were significant differences in patient burden immediately after surgery compared to the time of the survey, with medium effect sizes. This supports the hypothesis that faster healing of the donor site wound leads to more favourable patient‐reported outcomes.     

Membrane cell grafts , fresh and frozen, to cover full thickness wounds in athymic nude mice

Biobrane, an adherent, flexible temporary wound dressing was incubated with cultured human keratinocytes. The cells adhered quickly, forming ”membrane-cell-grafts” (MCG). Some of the grafts were frozen and, after thawing, viability was verified with an XTT colorimetric assay. MCGs, fresh and cryopreserved, were transplanted on full thickness wounds created on athymic nude mice and resulted in a differentiated epithelium of human phenotype. For further investigation immunohistochemistry, immunofluorescence and electronmicroscopy were performed. Conventional cultured epidermal grafts (CEG) and wounds without cell grafts served as control. Compared with CEG-grafted sites a reduced wound contraction was noticed and complete remodelling of the basement membrane zone was found. The effectiveness of the easy, uncomplicated production, cryopreservation and use, as well as the short culture period could lead to a new approach in the treatment of burns and chronic wounds. Received: 12 February 1998 / Accepted: 2 February 1999     

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。     

Use of a composite skin graft composed of cultured human keratinocytes and fibroblasts and a collagen-GAG matrix to cover full-thickness wounds on athymic mice

In patients with extensive full-thickness burns, wound coverage may be accelerated if skin can be expanded to produce a skin replacement that reproducibly supplies blood to the wound and has good structural qualities. In addition, development of skin replacements may benefit patients who require reconstruction or replacement of large areas of abnormal skin. We have developed a composite skin replacement composed of cultured human keratinocytes (HK) and fibroblasts. Cultured human fibroblasts are seeded into the interstices, and cultured HKs are applied to the surface of a matrix composed of type I collagen crosslinked with a glycosaminoglycan, which has a defined physical structure. After HKs reach confluence on the matrix surface, the composite grafts are placed on full-thickness wounds on the dorsum of athymic mice. Graft acceptance, confirmed by positive staining with antibodies specific for human HLA-ABC antigens on HKs, is approximately 90%. A defined skin structure is present histologically by day 10 after grafting, with a differentiated epithelium and a subepidermal layer densely populated by fibroblasts and capillaries without evidence of inflammation. Fluorescent light microscopy to identify laminin and type IV collagen and electron microscopy confirm the presence of basement membrane components by 10 days after grafting. Attachment of the graft to the wound is similar with and without the addition of human basic fibroblast growth factor, a potent angiogenic agent, to the skin replacement before graft placement on wounds.     

Effect of artificial dermal substitute, cultured keratinocytes and split thickness skin graft on wound contraction

In this study, the effect of different wound treatments on contraction was evaluated in an established porcine model. In two separately conducted experiments full thickness wounds treated with artificial dermal substitute, split thickness skin graft (STSG), meshed STSG applied in combination with cultured keratinocytes or meshed STSG alone were compared with untreated wounds. The surface area of all wounds was quantified at regular time intervals. After 20 days wounds from some groups were subjected to histological analysis to establish the degree of epithelialization. Wounds treated with STSG contracted more than with artificial dermal substitute until day 21. From day 21 to day 35 wounds treated with STSG showed the least contraction. Wounds sprayed with cultured keratinocytes demonstrated a slower rate of contraction than those with meshed STSG alone after 20 days. The untreated control wounds showed a greater rate of contraction and had almost closed by day 20. This study demonstrates that there is a significant difference in contraction between wounds treated with artificial dermal substitute and control wounds and between wounds treated with STSG with cultured keratinocytes and meshed STSG alone. STSG with cultured keratinocytes, unmeshed STSG, and artificial dermal substitute all reduced wound contraction significantly.     

In vivo cultured skin composed of two-layer collagen sponges with preconfluent cells.

Although various kinds of cultured skin substitutes have been developed, it takes several weeks to produce them before grafting. In their previous study, the authors succeeded in producing cultured skin easily in a short period of time by layering two collagen sponges. In the current study, to shorten this period even further, they grafted the cell-preconfluent artificial skin immediately after seeding the cells. They used two collagen sponges with different pore sizes and crosslink densities. They seeded 1,000,000 cells per square centimeter of fibroblasts and 1,000,000 cells per square centimeter of keratinocytes on the respective collagen sponges and grafted them on a full-thickness, excised wound on the back of severe combined immunodeficient mice. Two weeks after grafting, epithelium and dermislike tissue were formed. They then decreased the number of keratinocytes and grafted them. Four weeks after grafting, at seeding densities of 50,000 to 1,000,000 cells per square centimeter of keratinocytes, the preconfluent artificial skin took histologically, and human type IV and type VII collagen were stained immunohistochemically. This cell-preconfluent artificial skin composed of two-layer collagen sponges seems promising for widespread clinical use.