自体表皮细胞悬液移植修复全层皮肤缺损创面的动物实验研究

目的探讨自体表皮细胞悬液移植技术用于全层皮肤缺损创面修复的适宜密度。方法取健康清洁级成年SD大鼠40只,雌雄不限,体质量210~230 g;根据细胞移植密度不同,随机分为高、中、低细胞密度及空白组(分别为A、B、C、D组,n=10)。取大鼠背部皮肤培养表皮细胞,并制作大鼠全层皮肤缺损创面抗挛缩模型。其中A、B、C组分别将0.2 mL密度为1×10~6、1×10~5、1×10~4个/cm~2的自体表皮细胞悬液移植至创面处,D组给予等量限制性角质形成细胞无血清培养基;取成年Wistar大鼠背部皮肤制备同种异体皮,覆盖各组创面。术后观察大鼠存活情况,于术后7、14、21 d大体观察同种异体皮成活、脱落及创面愈合情况,同种异体皮脱落后计算创面愈合率;21 d时取材行组织学及免疫组织化学染色,观察创面修复情况。结果术后大鼠均存活至实验完成。各组大鼠同种异体皮随时间延长逐渐成活,干燥并开始脱痂;至21 d同种异体皮基本脱落后A、B组创面可见成片上皮,C组创面可见少量菲薄上皮,D组创面无上皮形成。术后21 d同种异体皮脱落后,A、B、C、D组创面愈合率分别为62.9%±9.6%、64.2%±9.1%、38.5%±5.7%、22.7%±5.5%,A、B组创面愈合率显著高于C、D组(P0.05),C组高于D组(P0.05),A、B组间比较差异无统计学意义(P0.05)。组织学观察示,A、B、C组愈合的创面上皮层可见鳞状上皮细胞,A、B组表皮分层明显,C组表皮层薄、可见炎性细胞浸润,D组为肉芽组织。免疫组织化学染色观察示,A、B、C组表皮-真皮连接层Ⅳ型胶原和Ⅶ型胶原表达呈阳性,D组无表皮层呈阴性;A、B组Ⅳ、Ⅶ型胶原表达阳性细胞百分比显著高于C组(P0.05),A、B组间比较差异无统计学意义(P0.05)。结论自体表皮细胞悬液移植技术在大鼠全层皮肤缺损创面修复中可重构皮肤,1.0×10~5个/mL为创面修复的适宜移植密度。     

自体表皮细胞悬液在修复皮肤组织缺损创面中的作用研究

目的:探讨自体表皮细胞悬液在修复皮肤组织缺损创面的作用。方法:在20只大鼠身上分别取邮票大小自体全厚皮片进行消化,分离成单个表皮细胞的悬液;经传代培养后,使用自体表皮细胞悬液分别对20只大鼠背部表皮皮肤缺损创面进行覆盖移植,术后观察移植物成活率和植皮区收缩率,同时取移植物进行组织学观察;并与对照组20只自体愈合创面进行相互比较。结果:表皮细胞悬液移植后1周渐融合成片,伤口愈合;经6周后,伤口平整,轻度瘢痕愈合。而对照组在6周时伤口不平整,呈瘢痕愈合,色素沉着明显。结论:在表皮缺损创面上应用体外培养的自体表皮细胞悬液移植修复可达到重构皮肤的目的。     

脱细胞异体真皮在烧伤后瘢痕修复中的应用

目的：探讨脱细胞异体真皮在烧伤后期瘢痕修复中的应用价值。方法：2006年2月-2007年2月入院治疗的21例烧伤后遗留瘢痕患者（A组）,采用切开并充分松解挛缩的瘢痕组织,利用脱细胞异体真皮加自体刃厚皮移植于继发创面的方法治疗。随机抽取同期21例采用瘢痕松解切除后进行中厚皮移植的患者（B组）及21例进行次全厚皮移植的患者（C组）作对照,比较3组患者的皮片成活情况以及术后1年的随访情况。结果：A组中皮片坏死率较其余两组略高;1年后随访发现,A组在受皮区皮片挛缩程度,外观平整度、色素改变及供皮区瘢痕形成方面均明显优于B组,与C组相当;C组仅适用于较小瘢痕的修复。结论：脱细胞异体真皮加自体皮复合移植是烧伤后期瘢痕修复的有效手段。     

皮能快愈敷料修复皮肤软组织缺损的应用研究

目的探讨皮肤软组织缺损,特别是残余创面使用皮能快愈敷料修复的治疗效果。方法两组皮肤软组织缺损的手术效果;A组：皮能快愈敷料修复组10例,采用皮能快愈敷料（双层结构的真皮重建移植物）与自体刃厚皮片复合移植。B组：异体脱细胞真皮支架与自体刃厚皮片复合移植修复患者创面10例。对比评估两组植皮区存活皮片外观,随访近期、远期植皮区皮肤的弹性情况,骨、肌腱外露后皮能快愈敷料及异体脱细胞真皮支架存活情况。结果两组病例植皮区存活皮片均外观好,皮肤的弹性好,与B组相比,A组患者骨、肌腱外露后使用皮能快愈敷料存活。结论采用皮能快愈敷料与自身刃厚皮片复合移植治疗,可在骨、肌腱外露的创面存活。     

异体脱细胞真皮基质与自体皮片复合移植的临床应用

目的:探讨异体脱细胞真皮基质与自体皮片复合移植修复大面积深度烧伤及瘢痕切除后皮肤缺损创面及其愈合后皮肤的外形和功能。方法:应用异体脱细胞真皮基质与自体刃厚皮片组成复合皮移植,以自体刃厚皮片移植作为对照,采用一步移植法治疗切痂后大面积深度烧伤创面及瘢痕切除后皮肤缺损共56例患者60处创面,观察术后皮片的成活情况、外形及功能恢复情况并随访。结果:60处创面全部愈合,移植皮片生长良好,瘢痕增生不明显,未见明显挛缩,皮肤弹性较好。在6～12个月的观察期内,自体刃厚皮片与异体脱细胞真皮基质复合移植后,功能和形态优于单纯自体刃厚皮片移植;随访2年复合移植未发现明显的排异反应。结论:异体脱细胞真皮基质与自体皮片复合移植修复大面积深度烧伤及瘢痕切除后皮肤缺损创面愈合良好,无瘢痕增生,皮肤外观功能满意,无排异反应。     

异体脱细胞真皮基质与自体皮片复合移植的临床应用

目的:探讨异体脱细胞真皮基质与自体皮片复合移植修复大面积深度烧伤及瘢痕切除后皮肤缺损创面及其愈合后皮肤的外形和功能。方法:应用异体脱细胞真皮基质与自体刃厚皮片组成复合皮移植,以自体刃厚皮片移植作为对照,采用一步移植法治疗切痂后大面积深度烧伤创面及瘢痕切除后皮肤缺损共56例患者60处创面,观察术后皮片的成活情况、外形及功能恢复情况并随访。结果:60处创面全部愈合,移植皮片生长良好,瘢痕增生不明显,未见明显挛缩,皮肤弹性较好。在6～12个月的观察期内,自体刃厚皮片与异体脱细胞真皮基质复合移植后,功能和形态优于单纯自体刃厚皮片移植;随访2年复合移植未发现明显的排异反应。结论:异体脱细胞真皮基质与自体皮片复合移植修复大面积深度烧伤及瘢痕切除后皮肤缺损创面愈合良好,无瘢痕增生,皮肤外观功能满意,无排异反应。     

培养的自体角朊细胞膜片与异体真皮重组复合皮移植的实验研究

目的探讨用自体培养的角朊细胞膜片(CKS)及异体真皮重组复合皮移植后的成活机理和组织学变化。方法以30只 SD 大鼠为实验动物,分为自体 CKS 组硬异体复合皮组。术后90天内定期检查并取活检标本作组织学检测,观察移植物存活情况,伤口愈合,表皮-真皮连接区的重建和异体真皮的归宿等。结果自体角朊细胞膜片成活尚好,但其质地及组织学结构欠佳,且太薄易磨损和破溃;胶原纤维增生并排列错乱,创面明显收缩。异体复合皮组不仅移植物成活好,且质地、组织结构、创面收缩及抗磨损等方面均优于 CKS 组,移植后90天仍能看到异体真皮成分,未见明显的免疫排斥反应。结论体外培养的自体角朊细胞膜片不适用于全层皮肤缺损的创面,而异体真皮复合皮有潜在的发展前景。     

培养的自体角朊细胞膜片与异体真皮重组复合皮移植的实验研究

目的探讨用自体培养的角朊细胞膜片（ＣＫＳ）及异体真皮重组复合皮移植后的成活机理和组织学变化。方法以３０只ＳＤ大鼠为实验动物,分为自体ＣＫＳ组及异体复合皮组。术后９０天内定期检查并取活检标本作组织学检测,观察移植物存活情况,伤口愈合,表皮真皮连接区的重建和异体真皮的归宿等。结果自体角朊细胞膜片成活尚好,但其质地及组织学结构欠佳,且太薄易磨损和破溃;胶原纤维增生并排列错乱,创面明显收缩。异体复合皮组不仅移植物成活好,且质地、组织结构、创面收缩及抗磨损等方面均优于ＣＫＳ组,移植后９０天仍能看到异体真皮成分,未见明显的免疫排斥反应。结论体外培养的自体角朊细胞膜片不适用于全层皮肤缺损的创面,而异体真皮复合皮有潜在的发展前景。     

培养的自体角朊细胞膜片与异体真皮重组复合皮移植的实验研究

目的 探讨用自体培养的角朊细胞膜片（CKS）及异体真皮重组复合皮移植后的成活机理和组织学变化。方法 以30只SD大鼠为实验动物,分为自体CKS组及异体复合皮组。术后90天内定期检查并取活检标本作组织学检测,观察移植物存活情况,伤口愈合,表皮－真皮连接区的重建和异体真皮的归宿等。结果 自体角朊细胞膜片成活尚好,但其质地及组织学结构欠佳,且太薄易磨损和破溃;胶原纤维增生并排列错乱,创面明显收缩。异体复合皮组不仅移植物成活好,且质地、组织结构、创面收缩及抗磨损等方面均优于CKS组,移植后90天仍能看到异体真皮成分,未见明显的免疫排斥反应。结论 体外培养的自体角朊细胞膜片不适用于全层皮肤缺损的创面,而异体真皮复合皮有潜在的发展前景。     

猪角脘-真皮模型观察透明质酸膜运载系统对培养的角脘细胞移植成活率的影响

少量表皮细胞经培养扩展为表皮细胞膜片,供临床移植需要。该过程需数周时间,且在将细胞膜片从培养器皿消化下来的过程中,损失了由表皮细胞产生的基底膜蛋白,移植时细胞膜片易碎,致培养的自体表皮细胞膜片在全层皮肤缺损创面上移植成活率不高。预植异体真皮,在异体真皮上移植自体表皮细胞膜片,可在一定程度上提高移植成活率。培养细胞膜片     

Transplantation of tracheal epithelial cells onto a prefabricated capsule pouch with fibrin glue as a delivery vehicle

OBJECTIVE: The purpose of this study was to investigate whether in vitro cultured tracheal epithelial cells can be transplanted onto a prefabricated capsule surface in vivo for possible use in tracheal reconstruction. METHODS: Tracheal epithelial cells from 12 donor inbred rats were harvested for culture and expansion. In 16 recipient inbred rats, 2 sterile cylinders made of silicone rubber were implanted in each rat bilaterally in the folds of both the left and right anterior rectus sheath by wrapping the sheaths around the cylinders to induce a capsule formation. Ten days later, the cell cultures were divided and suspended in 1 of 2 delivery vehicles (standard culture medium or fibrin glue) and implanted onto the capsule surface. To compare the 2 delivery vehicles, we used fibrin glue on one side and the standard culture medium on the other. RESULTS: After 2 (group 1, n = 8) and 4 (group 2, n = 8) weeks, histologic findings, immunohistochemical staining, and electron microscopy demonstrated the capsule to be covered with a tracheal neoepithelium in group 1 and additional ciliated cells and secretory cells in a confluent layer in group 2 but only on the side with fibrin glue as the delivery vehicle. No viable epithelial cells were identified on the side with the standard culture medium in either group. CONCLUSION: We conclude that cultured epithelial cells can be successfully transplanted onto a prefabricated capsule surface with fibrin glue, which will differentiate into morphologic, nearly normal epithelium, showing potential for tracheal reconstruction.     

用毛囊隆突细胞构建复合皮的实验观察

目的 以人毛囊隆突细胞为种子细胞体外构建组织工程复合皮，在体观察其功能性修复全层皮肤缺损的可行性。方法 胶原酶消化法体外分离培养人毛囊隆突细胞和毛乳头细胞，实验分为A、B两组。A组将毛囊隆突细胞与毛乳头细胞按1：2混合，接种于胶原包被的聚羟基乙酸纤维支架中；B组单纯接种相同数量的毛乳头细胞。而后覆盖角质形成细胞膜片，构成组织工程复合皮，移植于裸鼠全层皮肤缺损创面。观察创面愈合情况，分别于术后2、4、6周在光学显微镜下观察移植物组织学变化。结果 组织工程复合皮能够有效修复A、B两组裸鼠全层皮肤缺损。术后2周，A、B组创面均可见完整的表皮及真皮结构。术后4-6周，A组复合皮表皮层明显增厚并形成基膜的钉突，可见毛囊样结构；B组仅表皮层有所增厚但基膜平整，未见钉突和毛囊结构形成。结论 以聚羟基乙酸真皮基质为支架，用角质形成细胞、毛囊隆突细胞和毛乳头细胞共同构建的组织工程复合皮，可以有效修复裸鼠全层皮肤缺损。其中毛囊隆突细胞参与了创面解剖修复，同时可能引导组织结构和功能的修复。     

Novel method of skin substitution in plastic surgery

A skin substitute has been developed by growing a large number of epidermal cells from a skin biopsy (1 cm2) taken from burn patients. In tissue culture the cells divide and grow quickly to form a monolayer sheet. These sheets were then trypsinized into free cells, which were applied onto the wounds of two patients suffering from full skin thickness burns. The cultured epithelial grafts continued to thicken and expand successfully until they became confluent with the surrounding epidermis. All the wounds healed successfully. Clinically and histologically these cultured epithelial autografts were proven to be of the same quality as split thickness skin grafts.     

Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps

OBJECTIVE: To evaluate urothelium regeneration by grafting viable cultured urothelial cell sheets, harvested from temperature-responsive culture surfaces, on demucosalized gastric flaps in a dog model. MATERIALS AND METHODS: Viable urothelium was obtained from eight beagle dogs by partial cystectomy. Harvested urothelial cells were seeded on temperature-responsive culture dishes modified with the thermally sensitive polymer, poly(N-isopropylacrylamide). Urothelial cells cultured for 3 weeks generated contiguous urothelial cell sheets that were noninvasively harvested with no enzymatic treatment from these dishes, by reducing culture temperature. Urothelial cell sheets were autografted onto surgically demucosalized gastric flaps. Three weeks after autografting the dogs were killed and the gastric flaps with the urothelial cell sheets were examined. Cell and tissue characteristics were compared between these urothelial cell sheet-grafted gastric flaps and native urothelium. Ultrafine structures were also examined by electron microscopy. RESULTS: Five of the eight urothelial cell sheet-grafted flaps showed viable urothelial regeneration. Urothelial cell sheets attached spontaneously to demucosalized tissue surfaces completely, with no suture or fixing, and developed into a stratified viable epithelium very similar to native urothelium. Regenerated urothelium remained unstained by antiproton pump antibody, which typically stains epithelial cells positively in gastric mucosal layers. On three of the eight flaps where there were severe haematomas, grafted cell sheets were not adherent and there was no urothelial regeneration. CONCLUSIONS: Urothelial cell sheets were autografted onto dog demucosalized gastric flaps successfully, with no suture or fixation, generating a multilayered urothelium in vivo. The novel intact cell-sheet grafting method rapidly produces native-like epithelium in vivo. This versatile technology should prove useful in urinary tract tissue engineering and surgical 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.     

The efficacy of collagen dermis membrane and fibrin on cultured epidermal graft using an athymic mouse model

A human skin substitute consisting of human cultured keratinocytes, collagen dermis, and fibrin was evaluated in athymic mice. Eighty athymic mice were divided randomly into four groups. A 1.5x1.5-cm full-thickness wound defect was created on the back of each athymic mouse under anesthesia. These wounds were covered by sheets of cultured epidermal graft (group A), cultured epidermal graft with collagen dermis and fibrin (group B), cultured epidermal graft with collagen dermis (group C), or cultured epidermal graft with fibrin (group D). The grafts were secured and kept moist by specially designed saline gauze chambers. The take rates of the cultured graft with more than 50% of the wound covered were 65%, 15%, 50%, and 45% respectively. Group B had a significantly lower graft take rate, however the difference was not significant among groups A, C, and D. Light microscopy of biopsies of the grafted sites at 12 days showed complete epithelialization. The incidence of discharge from wound beds in groups A, B, C, and D was 0%, 15%, 15%, and 10% respectively. The results suggest that cultured cells are best grafted directly onto the wound bed or in combination with either a thin layer of collagen or fibrin but not both because the collagen dermal membrane and the fibrin together may impose too great a diffusion barrier for the cultured cell graft to become vascularized.     

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。     

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。     

Reversed radial forearm flap: an ad hoc flap for hand reconstruction

Complex wounds of the hand and vital structures are important to reconstruct. They should be covered as soon as possible in order to maintain the function of the hand. The reversed radial forearm flap is a versatile option for hand reconstruction. Reversed radial forearm flaps were harvested in 15 cases. Doppler ultrasound was used in all cases to evaluate the vascular status of the flap. No complications were observed in this series. All skin grafts healed well. The reversed radial forearm flap is a workhorse tool for the coverage of the hand.     

皮肤源祖细胞-透明质酸复合物对糖尿病大鼠创面愈合的影响

目的研究皮肤源祖细胞（SKP）-透明质酸（HA）复合物的构建方法，观察其对糖尿病（DM）大鼠创面愈合的影响。方法分离培养SD大鼠SKP，以HA为载体构建复合物，观察复合物中SKP的分化特性。选取60只SD大鼠腹腔注射链脲菌素诱导成DM模型．背部对称制作2个直径1cm全层皮肤缺损创面，随机分为SKP-HA组，创面涂布100μl SKP-HA；HA组，创面涂布100μl HA；对照组，创面涂布DMEM／F12培养基。每组20只。各组大鼠于伤后1、2、3、4周检测创面愈合率，留取创面组织标本检测羟脯氨酸（Hyp）含量，并观察SKP在创面愈合过程中的迁移。结果大鼠SKP与HA共培养后生长良好，复合物中的SKP可保持其特性：向神经元细胞、神经胶质细胞、脂肪细胞分化。伤后2周SKP-HA组、HA组的创面愈合率分别为（72．1±2．8）％、（53．7±2．9）％，均明显高于对照组的（42．5±1．5）％（P＜0．05）；伤后3周SKP—HA组高于HA组及对照组（P＜0．05或0．01）；伤后4周SKP—HA组、HA组创面完全愈合，与对照组比较，差异有统计学意义（P＜0．01）。伤后1周各组Hyp含量差异无统计学意义（P＞0．05）；伤后2～4周，SKP-HA组、HA组Hyp含量均高于对照组（P＜0．01）；伤后3、4周SKP-HA组高于HA组（P＜0．01）。SKP在创面得以成活并随时间的延长逐渐向真皮层迁移。结论SKP-HA复合物可促进DM大鼠创面愈合。