血小板源性生长因子基因修饰的人工复合皮移植大鼠创面效果观察

目的观察血小板源性生长因子B(PDGFB)基因修饰的人工复合皮移植大鼠创面后的效果。方法构建PDGFB真核表达质粒,在脂质体介导下转染大鼠成纤维细胞。分别构建复合皮1(角质形成细胞 猪脱细胞真皮基质 PDGFB基因转染的成纤维细胞)和复合皮2(角质形成细胞 猪脱细胞真皮基质 未转染的成纤维细胞),移植于大鼠背部全层皮肤缺损创面,相应设为转染组、未转染组(各18只)。以不作皮肤移植的8只大鼠全层皮肤缺损创面为对照组。术后2周观察大鼠创面移植皮片存活情况。术后2、4、6周观察大鼠创面大体情况,计算创面收缩率,并取创面组织标本进行组织学观察。结果(1)术后2周,转染组大鼠中皮片完全存活者14只、部分存活者3只、未存活者1只;未转染组大鼠中皮片完全存活者10只、部分存活者4只、未存活者4只。(2)术后2周,对照组创面结痂。术后6周转染组移植皮片表面光滑,有弹性,抗磨擦性强,愈合效果优于其他两组。(3)术后2、4、6周,对照组大鼠创面收缩率均高于其他两组,转染组创面收缩率低于未转染组(P<0.05)。(4)术后2周转染组移植皮片内可见较多毛细血管分布;6周时表皮细胞分化达7～10层,纤维排列致密整齐,毛细血管分布均匀。结论用含PDGFB基因的人工复合皮移植修复创面,可明显提高创面愈合质量。     

脱细胞猪小肠黏膜下层与猪脱细胞真皮基质作为真皮支架的对比研究

目的：应用脱细胞猪小肠黏膜下层与猪脱细胞真皮基质作为真皮替代物与SD大鼠自体刃厚皮片进行复合移植,修复SD大鼠全层皮肤缺损,比较两者优劣性,为临床提供更理想的真皮替代物。方法：以36只SD大鼠为动物模型,随机区组法随机分为两组,每组18只,在其背部造成2.5 cm×2.5 cm的全层皮肤缺损,实验组应用脱细胞猪小肠黏膜下层+自体刃厚皮移植修复,对照组应用猪脱细胞真皮基质+自体刃厚皮移植修复,移植后2周对移植皮片成活率分析研究,移植后4周、8周、12周取材进行一般观察、组织学观察和收缩率的计算。结果：术后2周,实验组植皮存活率大于对照组,差异有统计学意义(P<0.05)。于术后4周、8周、12周动态观察,实验组植皮区收缩率较对照组低,但组间比较差异无统计学意义(P>0.05)。HE染色组织学观察,术后4周,两组植入材料与移植皮片融合度均很好,植入材料内及其周围均有大量的成纤维细胞和新生毛细血管长入,并有炎症细胞浸润;术后8周,两组植入材料内及其周围均有成纤维细胞和新生毛细血管长入,炎症细胞较术后4周时少,两组植入材料的原有胶原纤维结构尚清晰,出现疏松;术后12周,两组植入材料...     

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。     

同基因细胞源组织工程皮肤修复皮肤缺损的实验研究

目的利用近交系大鼠实验模型评价同基因细胞源组织工程皮肤修复大鼠全层皮肤缺损的效果,为其临床应用奠定基础。方法取近交系F344大鼠乳鼠皮肤,采用Dispase-胰蛋白酶两步消化法分离培养表皮细胞;取SD大鼠皮肤,按照高渗盐-SDS-胰蛋白酶化学处理法制备脱细胞真皮基质;依次利用浸没式培养和气液分离培养法将第2代表皮细胞与脱细胞真皮基质复合体外培养构建组织工程皮肤。取4～5周龄近交系F344大鼠36只,于大鼠背部两侧对称制备全层皮肤缺损,缺损面积为预实验确定的1.5 cm×1.5 cm。将72个创面随机分为3组(n=24),两侧移植不同修复材料:实验组移植组织工程皮肤、阴性对照组移植同种异体脱细胞真皮基质、阳性对照组移植自体全层皮肤(非原位移植)。术后行大体观察、皮片成活率、创面收缩率测量及组织学观察,评价修复效果。结果实验组术后4周创面可达到Ⅰ期愈合,与周围组织紧密连接,外观接近周围正常皮肤。术后4周,阴性对照组皮片成活率为0;实验组为62.5%(15/24),与阳性对照组91.7%(22/24)比较差异有统计学意义(χ2=5.779,P=0.016)。术后4周,实验组和阳性对照组创面收缩率显著低于阴性对照组(P<0.05),实验组与阳性对照组间差异无统计学意义(P>0.05)。组织学观察示实验组术后1周有轻微炎性反应;术后2周真皮层小血管和成纤维细胞大量增生,表皮层逐渐分化成熟;术后4周时伴随新生胶原纤维沉积,移植物真皮层出现胶原改建。结论同基因细胞源组织工程皮肤能够有效修复大鼠全层皮肤缺损,在防止创面收缩和促进创面愈合等方面均可达到类似于自体全层皮肤移植的效果。     

脱细胞真皮移植部位血管细胞粘附分子-1的表达

目的 探讨血管细胞粘附分子-1(VCAM-1)在脱细胞真皮移植部位的动态变化和作用.方法 建立猪试验模型,采用同种/异种脱细胞真皮基质,与猪自体薄皮片复合移植于猪创面,猪自体皮移植组作对照组,分别在移植后3、6、9、12、15、30天时相点取得移植组织标本.流式细胞仪(FCM)检测VCAM-1的表达变化.结果 移植术后3天各组VCAM-1表达无明显差别;对照组6天表达明显高于实验组(p＜0.05);9天～12天异种、异体脱细胞真皮基质组与自体皮移植组对照VCAM-1均高表达,具有显著差异性(p＜0.05～0.01);术后30天各组VCAM-1表达显著低于3天时相点(p＜0.01).结论 同种和异种真皮基质移植部位VCAM-1表达高峰迟滞于自体皮移植;VCAM-1表达可能与脱细胞真皮活化、血管再生有关,值得进一步研究.     

毛乳头细胞促进组织工程皮肤血管化的实验研究

目的探讨毛乳头细胞对表皮干细胞与同种异体脱细胞真皮基质构建的组织工程皮肤血管化的影响。方法取门诊包皮环切术患者皮肤,用于表皮细胞培养;取孕19、20周引产胎儿头部皮肤,用于毛乳头细胞培养;患者及家属均知情同意。以中性蛋白酶联合胰蛋白酶消化、分离表皮细胞,IV型胶原黏附法分选表皮于细胞;以Ⅰ型胶原酶消化法分离毛乳头,常规成纤维细胞培养液培养。以同种异体脱细胞真皮基质为支架,在真皮基质的真皮乳头侧种植毛乳头细胞,基底膜侧种植表皮干细胞构建实验组组织工程皮肤替代物;对照组组织工程皮肤替代物不种植毛乳头细胞。取6～8周龄BALB/C-nu裸鼠60只,随机分成实验组和对照组（n=30）;实验动物背部制造1 cm×1 cm大小全层皮肤缺损模型,将两组组织工程皮肤替代物移植修复创面,2周后观察移植皮肤成活率。术后2、4周,取移植皮肤替代物标本,行HE及免疫组织化学染色,观察移植皮肤替代物CD31表达水平,并计算两组标本血管密度。结果Ⅳ型胶原分选后表皮细胞同时表达角蛋白19、β₁整合素,提示为表皮干细胞。由毛乳头培养出的细胞表达α平滑肌肌动蛋白,提示为毛乳头细胞。动物移植术后2周,实验组移植皮肤成活率为93.3%（28/30）,对照组为80.0%（24/30）,比较差异有统计学意义（χ²=2.31,P=-0.00）。HE染色观察示实验组表皮层达12层,表皮细胞体积较大;对照组表皮层达4～6层,表皮细胞体积较小。实验组术后2、4周,血管密度分别为（38.56±2.49）个/mm2和（49.12±2.39）个/mm²,对照组分别为（25.16±3.73）个/mm²和（36.26±3.24）个/mm²,两组比较差异均有统计学意义（P<0.01）。结论毛乳头细胞可促进移植皮肤替代物血管形成,利于表皮层重建,提高组织工程皮肤移植成活率。     

真皮替代物移植后的血管化过程及组织学变化的实验研究

目的了解不同种类真皮替代物移植后的血管化过程及组织学变化。方法将21只SD大鼠根据其皮下埋植不同的真皮替代物分为猪脱细胞真皮基质(sADM)组、人脱细胞真皮基质(hADM)组及人工真皮(Integra)组。于埋植后2、3、4、7、10、14、21、30、60、90、120、150、180d行移植物大体观察,采用免疫组织化学法观察移植物的血管化过程及组织学变化。结果大体观察术后各组大鼠创口周围皮肤无明显红肿及炎性反应,切口愈合良好,移植物与创面接触紧密。90d后各组移植物不易从体表触及。180d时,部分移植物面积缩小、厚度变薄甚至难以辨认。组织学观察移植术后2d起可见成纤维细胞、中性粒细胞、淋巴细胞等侵入移植物内,3d时与受床组织连接处可见长入的新生毛细血管芽。30—60d,移植物内形成丰富的血管网。150d后近似正常真皮结构。180d后部分移植物有不同程度吸收退化。结论3种真皮替代物移植后均能很快建立与受床组织的血液循环,并长时间存留于创面,但有一定程度的吸收退化。     

角质形成细胞在脱细胞异种真皮上培养的实验研究

目的 在脱细胞异种(猪)真皮上培养角质形成细胞探讨体外复合皮的构建。方法 取出生24h内的SD大鼠全厚皮肤，采用低温酶消化法和密度梯度离心法分离获得纯角质形成细胞；以不用任何滋养层的角质形成细胞培养作为空白对照组，脱细胞异种真皮作为支架的角质形成细胞培养为实验组，原代培养后行气——液面培养。形态学，常规组织学HE染色观察，免疫组化染色(SABC法)检测复合皮肤中的Pancyrtokeratin和层粘连蛋白(Laminin)。结果 HE染色显示有4层以上的角质形成细胞和基底膜层形成，并有轻度角质化；免疫组化染色显示：Pancytokeratin( )，提示在脱细胞猪真皮上生长的为角质形成细胞；Laminin( )，提示培养的角质形成细胞产生了新的基底膜。结论 体外培养的角质形成细胞能在脱细胞异种真皮上良好生长、存活，并有基底膜形成，在体外成功构建了具有表皮和真皮的复合皮，可以作为一种新的组织工程化皮肤。     

真皮模板对烧伤患者创面修复过程中细胞凋亡和p53基因表达的影响

目的动态观察应用真皮模板对烧伤创面修复过程中细胞凋亡和p53基因表达的影响。方法将20例烧伤患者分为试验组11例，采用真皮模板(异体脱细胞真皮基质)加自体薄皮复合移植修复创面；对照组9例，以单纯自体薄皮移植修复创面。两组患者均于术后1、2、3、4、5周取组织标本。采用原位缺口末端标记法(TUNEL)和免疫组织化学法分别检测标本中细胞凋亡和p53基因的表达。HE染色观察细胞数量的变化。结果随着创面的逐渐修复，试验组p53基因表达逐渐增强，在术后第2、3、4周与对照组比较差异有显著性意义(P＜0．05)，第4周达高峰。试验组TUNEL结果显示，术后1周，成纤维细胞开始凋亡，血管内皮细胞凋亡主要发生在第2、3周。两组凋亡细胞面积百分比比较，在第3、4周差异有显著性意义(P＜0．05)。4周后，试验组成纤维细胞和血管内皮细胞数量较对照组减少。结论真皮模板与自体薄皮复合移植，可诱导p53基因表达和促进细胞凋亡，并减少瘢痕增生，从而改善创面修复质量。     

骨髓间充质干细胞复合组织工程化脱细胞真皮基质构建组织工程皮肤

目的：体外培养扩增SD大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs），复合组织工程化脱细胞真皮基质构建组织工程皮肤，为进一步临床应用奠定基础。方法：将SD大鼠骨髓间充质干细胞进行体外培养扩增后，以生长状态良好的骨髓间充质干细胞接种于制备好的组织工程化脱细胞真皮支架上，进行体外联合培养，构建组织工程皮肤。观察细胞生长情况及组织工程皮肤结构。结果：体外培养的SD大鼠骨髓间充质干细胞生长良好，传代扩增容易，组织工程化脱细胞真皮基质去细胞完全，骨髓间充质干细胞在脱细胞真皮基质中生长良好，可体外构建组织工程皮肤。结论：利用体外扩增培养的骨髓间充质干细胞及制备的组织工程化脱细胞真皮基质可以体外联合构建组织工程皮肤。     

Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats

In search of an optimal transplantation regime for sufficient dermal and epidermal regeneration after a full-thickness skin injury, wounds on athymic rats were grafted with split-thickness skin grafts or acellular human dermis followed by transplantation with human keratinocytes either in single-cell suspension or cultured on porous biodegradable microcarriers. After 2 weeks, all wounds grafted with acellular human dermis showed a well organised and vascularised dermal component and reepithelialisation on the grafted dermal matrix was complete 21 days after transplantation with human keratinocytes. Wounds grafted with human keratinocytes seeded on biodegradable microcarriers or split-thickness skin grafts displayed over time (i.e. 16-21 days post-transplantation) a significantly thicker epithelial cell layer in comparison to wounds grafted with keratinocytes in single-cell suspensions or microcarriers not seeded with cells. Furthermore, measurements of dermal thickness in the closed wounds 21 days after grafting showed a significantly thicker and well organised neodermal component in wounds transplanted with keratinocytes seeded on microcarriers or split-thickness skin grafts compared to all other wounds. Positive immunostaining towards von Willebrand factor revealed the plausible proangiogenic effects of transplantation with keratinocytes seeded on microcarriers. Analysis of representative tissue sections after fluorescence in situ hybridisation visualised that grafted human keratinocytes were present in the epidermal layers covering the wounds 16 and 21 days after transplantation, strongly indicating preservation of cell viability. These results shows that the use of biodegradable microcarriers in the culture of autologous keratinocytes for treatment of full-thickness wounds not only facilitate the cultivation, transportation and transplantation processes but also enhances the dermal regeneration induced by a dermal scaffold which results in a clinical result that is significantly superior to the one obtained when keratinocytes are transplanted in a single-cell suspension.     

以活性复合真皮基质为载体构建组织工程皮肤的研究

目的 构建含活性真皮基质的组织工程皮肤. 方法将人成纤维细胞(Fb)与Ⅰ型牛胶原混合接种于猪脱细胞真皮基质(PADM)的表面,构建活性真皮替代物.其上接种人表皮细胞进行气-液面培养,获得组织工程皮肤,进行组织学观察. 结果 Fb在胶原内结构完整,与PADM形成复合真皮基质.所构建的组织工程皮肤表皮层结构与人正常皮肤相似,具备基底层、棘层、颗粒层和角质层,细胞之间有桥粒连接,细胞分化良好. 结论 Fb-胶原-PADM真皮替代物可作为较好的构建组织工程皮肤的真皮支架.     

Fibroblasts improve performance of cultured composite skin substitutes on athymic mice

BACKGROUND: This study investigated the impact of adding human fibroblasts to a cultured composite skin substitute model of cultured human keratinocytes and acellular human dermis. METHODS: Skin substitutes were prepared by seeding human keratinocytes on the papillary side of acellular dermis with or without seeding fibroblasts on the reticular side. Performance of the grafts was compared both in vitro by histology and in vivo on surgically created full-thickness wounds on athymic mice. Graft size and contraction were measured and immunohistochemical stains were done to reveal vascularization. RESULTS: Skin substitutes with fibroblasts formed thicker epidermis than skin substitutes without fibroblasts. When transplanted onto athymic mice, skin substitutes with fibroblasts maintained their original size with only 2% contraction. In contrast, skin substitutes without fibroblasts showed 29% contraction. Vascular basement membrane specific mouse CD31staining and endothelial cell specific mouse collagen type IV staining revealed vascularization as early as 1 week posttransplant in grafts with fibroblasts, and was significantly higher than grafts without fibroblasts at 2 weeks. CONCLUSIONS: Addition of fibroblasts to keratinocyte based composite skin substitutes improves epidermis formation, enhances vascularization and reduces contraction.     

The role of fibroblasts in dermal vascularization and remodeling of reconstructed human skin after transplantation onto the nude mouse.

The vascularization and the dermal remodeling of two different types of human skin reconstructed "in vitro" and grafted onto the nude mouse were studied. They were composed of human keratinocytes grown either on a human acellular deepidermized dermis (DED), or on a lattice composed of human fibroblasts embedded in bovine type I collagen, a living dermal equivalent (LDE). At different stages after grafting, the transplants were harvested and processed for an immunohistological study with species-specific and non-species-specific antibodies. At one month after grafting, the two types of grafted dermis contained blood vessels whose vascular basement membranes were labeled with a mouse-specific anti-type IV collagen antibody. With an antibody specific for human type IV collagen, a constant labeling of the vascular basement membrane was only observed in the LDE containing fibroblasts. In the DED, a constant association of the mouse endothelial cells with human type IV collagen was observed at early stages after grafting. At later stages, the human type IV collagen progressively disappeared. On the other hand, the dermal-epidermal junction underneath the human epidermis contained human type IV collagen in the two types of reconstructed skin. Labeling with the species-specific antibodies directed against human or murine type I collagen showed that the ratio murine type I collagen versus human type I collagen increased with time, suggesting that the DED is progressively invaded by mouse fibroblasts that produce the mouse collagen. On the other hand, in the LDE, the preexisting bovine type I collagen became progressively undetectable while both human type I collagen and elastic fibers were deposited by numerous human fibroblasts. Mouse type I collagen was not detected. Altogether, these observations made by grafting human skin reconstructed "in vitro" onto the nude mouse should be interesting for evaluating the usefulness of grafting a dermal substrate together with the epidermal sheet in the treatment of burns.     

异种(猪)脱细胞真皮基质与自体皮复合移植的临床应用

目的：观察基底良好的深度创面用异种（猪）脱细胞真皮基质与自体皮复合移植的临床疗效。方法：2000年1月-2002年2月用异种（猪）真皮基质与自体薄片复合移植（二步法）修复基底良好的深度创面52例。结果：37例（71.1%）植皮完全成活，15例（28.9%）植皮95%以上成活，无一例补植皮。随访3-6个月复合移植皮肤颜色、质地基本接近于正常皮肤，瘢痕增生不明显。结论：异种（猪）真皮基质可推广应用于修复基底良好的深度创面的复合移植。     

The application of new biosynthetic artificial skin for long-term temporary wound coverage

Temporary dressings protect wounds from desiccation and infection. In our previous study, we used meshed acellular porcine dermis (APD) to enhance wound healing and decrease wound contraction; however, the wounds showed meshed scar [Wang HJ, Chen TM, Cheng TY. Use of a porcine dermis template to enhance widely expanded mesh autologous split-thickness skin graft growth: preliminary report. J Trauma 1997;42(2):177–82]. In this study, we produced an artificial skin composed of a cross-linked silicon sheet on the surface of APD which we have called silicone acellular porcine dermis (SAPD). This new artificial skin can protect the wound long enough to promote wound healing either by second intention or covered long enough until cultured epithelium autograft (CEA) or autologous skin graft can be harvested for permanent coverage.

We delivered 4 cm × 5 cm full-thickness wound on the back of 350 g Sprague–Dawley rats. Thirty-six rats were divided into two groups. Eighteen rats had SAPD and the other 18 were covered with Biobrane. The wounds were first examined 2 weeks after grafting and followed weekly for an additional 4 weeks to evaluate the wound and study pathological changes by using H.E. and Masson's stains. Wound size was calculated by ruler and analyzed by Student's t-test.

At the 2-week inspection, both SAPD and Biobrane showed tight adherence to the wound with no change of wound size. Both the SAPD and Biobrane dermal templates were pink. In the Biobrane-covered group, the wounds contracted soon after the tie-over dressing was removed. Its dermal layer is a layer of thin porcine dermal substance, which was promptly digested by tissue hyaluronidase and provides no real dermal template. In the SAPD-covered group however, the wound size was maintained significantly from third to sixth week after grafting (p < 0.001). SAPD was designed with thick epidermal silicone and a well-organized porcine dermis so that it incorporates into the recipient wound. Clinically the silicone layer of SAPD dislodged from APD about 6–7 weeks after grafting and was followed by dermal matrix exposure and infection. In pathological examination, much like a human skin graft, new vessels were found in APD about 1 week after grafting with minimal inflammatory cells infiltrated in the graft and wound. Six weeks after grafting, the collagen of APD incorporated into the wound, showing palisade arrangement and no sign of rejection. In the Biobrane group however, the wounds showed severe inflammation, the porcine dermal matrix was digested and disappeared 3 weeks after coverage.

In conclusion, SAPD is a thick biosynthetic artificial skin, which protects the rat wound significantly longer than Biobrane and prevents contraction. We expect that using of SAPD for temporary wound coverage will provide enough time to grow autologous-cultured epithelium or to reharvest skin grafts.     

脱细胞异种(猪)真皮基质作为填充材料的初步研究与应用

目的：为临床修复体表凹陷畸形寻求良好的填充材料。方法：用自制脱细胞异种（猪）真皮基质，植入兔皮下，定时进行测量，并进行光镜及电镜观察。临床应用2例。结果：脱细胞异种（猪）真皮基质植入后观察1年，仅在4周内有极轻微的炎性反应，无排斥反应，2周左右有成纤维细胞及毛细胞血管长入，吸收率低于对照组。2例临床应用填充效果较满意，质地较软，无破溃、外露。结论：脱细胞异种（猪）真皮基质组织相容性好，吸收率低，质地软，是一种有良好前景的软组织填充材料。     

Wound healing effect of acellular artificial dermis containing extracellular matrix secreted by human skin fibroblasts

In this study, an acellular artificial dermis, composed of human collagen and glycosaminoglycan (GAG) secreted by cultured human fibroblasts on a bovine collagen sponge, was developed. Much of the newly secreted extracellular matrix (ECM) remained after the cell removal process. The main theme of this study focused on the matrix, rather than the viable cell components of the skin, as the major dermal deficit in the wound. Both the acellular artificial and bioartificial dermises, containing viable cells with ECM, were significantly less soluble than the collagen sponge, and the relative GAG content in the bioartificial and acellular artificial dermises was approximately 115-120% of the chondroitin-6-sulfate (CS) content found in the collagen sponge. In the group receiving the collagen sponge, the wound area gradually decreased to approximately 10% of its original area, while in the groups receiving the bioartificial and acellular artificial dermises, the wound area also gradually decreased to approximately 60 and 50%, respectively, of the original size over the 5 weeks after grafting. Both the bioartificial and acellular artificial dermises formed thicker, denser collagen fibers; more new blood vessel formation was observed in both cases. The basement membrane of the regenerated epidermal-dermal junction was thicker and more linear in the acellular artificial dermis graft than in the collagen sponge graft. In conclusion, the wound healing effects of acellular artificial dermis are no less than those of the bioartificial dermis, and much better than the collagen sponge graft with respect to wound contraction, angiogenesis, collagen formation, and basement membrane repair.