纤维蛋白凝胶移植培养口腔黏膜细胞的研究

目的 为培养上皮细胞寻找良好的移植载体.方法 用含自体血清培养基培养人口腔黏膜角质细胞,实验组细胞置于纤维蛋白凝胶中,对照组细胞置于DMEM:F12细胞培养液中,将悬液细胞以1×106个/ml浓度分别移植于裸鼠皮下和创面,于移植后1、2、3、4、6周取材,分别行苏木素-伊红染色及冰冻切片抗人HLA-Ⅰ免疫荧光染色及抗人Ⅳ型胶原及抗人层黏蛋白免疫组织化学检查.结果 皮下移植时,凝胶组角质细胞逐步增殖分化形成较大上皮管腔(4.62×103个/cm2),组织有多量血管形成(5.72×103个/cm2),对照组中上皮管腔(1.76×103 个/cm2)及血管数量少(0.88×103 个/cm2),两者差异有统计学意义(P＜0.01),管腔上皮抗人-HLA免疫荧光阳性;实验组创面愈合平均时间为11 d,愈合处皮肤粗糙、角化,抗人-HLA免疫荧光强阳性,免疫组织化学染色示基底膜形成完整,对照组愈合时间为18.5 d,愈合创面凹陷、光滑无角化,抗人-HLA免疫荧光弱阳性,两组创面愈合时间差异有统计学意义(P＜0.05).结论 纤维蛋白凝胶可促进移植黏膜上皮细胞的增殖分化并形成功能完备的上皮组织,是上皮细胞移植的良好载体.     

自体血清与胎牛血清培养口腔黏膜角质细胞及上皮组织的实验研究

目的探讨自体血清培养人口腔黏膜角质细胞的可行性,为组织工程口腔黏膜用于临床提供理论和技术依据。方法应用自行制备的含10%、20%和30%自体血清的培养液及10%胎牛血清培养液,对人口腔黏膜角质细胞进行培养。对比观察不同浓度自体血清和胎牛血清培养的口腔黏膜角质细胞及上皮组织生长情况,绘制10%自体血清组及10%胎牛血清组细胞生长曲线及计算其倍增时间。并行抗-HLA免疫荧光检测培养上皮。结果各浓度自体血清组和10%胎牛血清组细胞生长及形态无明显差别,10%自体血清组细胞倍增时间为24.02±1.80h,与10%胎牛血清组20.90±0.79h比较,差异无统计学意义(P>0.05)。各组细胞24h克隆形成率比较,差异均无统计学意义(P>0.05);随自体血清浓度升高获得黏膜上皮面积增大,厚度增加,以20%自体血清组最为明显,与10%胎牛血清组培养黏膜上皮比较,差异有统计学意义(P<0.05)。各组培养上皮经抗-HLA免疫荧光检测为阳性。结论制备的自体血清能完全替代胎牛血清对口腔黏膜角质细胞进行培养,且培养的口腔黏膜上皮组织分化优于胎牛血清。     

人脐血内皮祖细胞体内促进血管重建的实验研究

目的：通过观察人脐血内皮祖细胞（EPC）在裸鼠体内缺氧状态下分化成内皮细胞的现象，初步研究其促进血管重建的作用。方法：根据EPC表面标记CD133，采用免疫磁珠法分离培养EPC。设计EPC裸鼠成瘤实验，观察瘤体内血管增生情况；将荧光标记的EPC注射于裸鼠腹部皮瓣中，观察EPC参与皮瓣血管重建情况；将EPC接种于聚羟基乙酸（PGA）中，移植于裸鼠皮下，观察EPC参与血管重建。结果：原代EPC贴壁后呈梭形，从第7天开始数量明显增加，呈克隆状生长。透射电镜观察到成熟内皮细胞最具特征性的细胞器——weibel-Palade小体。裸鼠成瘤实验：抗人vWF免疫荧光显示大量EPC参与肿瘤血管生成；裸鼠皮瓣实验：荧光标记EPC参与裸鼠皮瓣血管生成；PGA移植实验：抗人vWF免疫酶组织化学染色显示EPC参与血管生成。结论：EPC参与血管重建，具有促进血管新生，加速缺血组织血管化的作用。     

荷载角质细胞生长因子纳米微囊组织工程皮肤修复裸鼠皮肤缺损

目的 研究荷载角质细胞生长因子(keratinocyte growth factor,KGF)纳米微囊的新型组织工程皮肤对裸鼠皮肤缺损的修复效果及特点.方法 采用超声乳化一溶剂挥发法及低温干燥法,制备KGF纳米微囊,并构建KGF-脱细胞真皮基质(acellular dermal matrix,ADM);分离培养和鉴定人表皮干细胞群和成纤维细胞;接种表皮干细胞群于KGF-ADM之上,观察其生长情况;将荷载KGF纳米微囊的组织工程皮肤移植于裸鼠皮肤缺损处,以无KGF纳米微囊的组织工程皮肤为空白组,以其自体皮肤移植作对照组.于术后2,6周时分别观察修复区组织学愈合及皮片挛缩情况,并应用抗人角蛋白10及β1-整合素免疫荧光检测修复区表皮和真皮层细胞来源、分化及生长情况.结果 表皮干细胞群在KGF-ADM表面生长良好,粘贴紧密,可见到多角形的终末表皮细胞及小圆形的表皮干细胞,活性良好,有连接成片的趋势,部分形成克隆团块.以荷载KGF纳米微囊组织工程皮肤修复裸鼠皮肤缺损,2、6周时修复效果均优于空白组及对照组,移植的组织工程皮肤边缘可与邻近皮肤完全融合,但存在一定的挛缩.镜下可见修复区组织工程皮肤表皮细胞分层良好,与ADM紧密结合,能产生正常角质层.6周时实验组修复区组织工程皮肤切片免疫荧光检测,基底层仍存有少量β1-整合素阳性的表皮干细胞或短暂扩充细胞.结论 所构建的荷载KGF纳米微囊组织工程皮肤修复裸鼠皮肤缺损的效果,优于无KGF纳米微囊的普通组织工程皮肤及裸鼠自体全厚皮片移植修复效果.     

组织工程人口腔黏膜的制备及异体移植的临床应用

目的观察自行制备的复层组织工程人口腔黏膜移植后生长情况.方法取3月龄患儿唇裂术中切取的多余口腔黏膜组织,分离成纤维细胞与上皮细胞,分别接种于聚乳酸/聚羟基乙酸共聚物(polylactic/glycolic acid copolymer,PLGA)胶原复合膜上培养,后将其移至气液面进行复合,制成复层组织工程口腔黏膜.7例行口腔内良性肿瘤切除后遗留黏膜缺损的患者,缺损范围为1.8 cm×1.6 cm～3.2 cm×2.6 cm.采用组织工程口腔黏膜修复,术后观察其生长情况.1例志愿者在移植术后18和30 d分别取移植部位黏膜行组织学观察.结果制备的组织工程口腔黏膜生长良好,具有上皮层和上皮下层双层结构,之间为PLGA膜;上皮层5～6层细胞,角蛋白染色阳性,上皮下层3～7层细胞,角蛋白染色阴性.7例患者移植修复术后10 d,组织工程口腔黏膜与创面生长良好,颜色较正常黏膜深;18 d后仍可辨出移植区与正常黏膜;30 d后无法区别界限,创面均Ⅰ期愈合,无明显瘢痕组织.组织学观察:18 d创面上皮层及下方肉芽组织生长良好,毛细血管增生有上皮钉突形成,成纤维细胞卵圆形;30 d胶原化更明显,移植区结构与周边正常组织相似.结论应用组织工程口腔黏膜异体移植修复后,黏膜生长良好,但愈合组织上皮的来源还需进一步研究.     

吸入性损伤犬口腔黏膜移植于气管后的组织形态学观察

目的探讨吸入性损伤气管内黏膜移植的可行性。方法制备犬蒸汽吸入性损伤模型,共12只,随机分成实验组和对照组,每组6只;制取自体口腔黏膜片,移植于切除损伤黏膜后的气管内创面,对照组不作黏膜移植。术后2周、3周分别取标本,进行大体观察、光镜和透射电镜检查。结果实验组6例移植黏膜均成活,未见感染、坏死或瘢痕形成;对照组术后2例创面感染,4例肉芽或瘢痕形成。术后24 h血白细胞计数实验组和对照组分别为8.65±1.54、13.45±1.65(×109/L),差异有统计学意义(P<0.05),实验组创面愈合时间明显缩短。组织学显示,口腔黏膜移植于气管术后2周均可以成活并扩展,成活后仍表现为鳞状上皮,但结构有适应性变化。术后3周未观察到化生现象。结论局限于气管的吸入性损伤早期进行黏膜移植是可行的,有利于减少感染,促进创面愈合。     

人脂肪来源细胞体内构建组织工程化脂肪的实验研究

目的探讨以组织工程技术，应用脂肪来源细胞（Adipose—derived cells，ADCs）体内构建脂肪组织的可行性。方法吸脂术获得人脂肪组织，一部分直接植入裸鼠体内；另一部分用酶消化法分离、培养ADCs，将第三代细胞接种于纤维凝胶（Fibrin glue）支架中，经成脂肪培养液诱导1周后，植入裸鼠体内，4周后取材，用称重法及油红、HE染色检测结果。实验分为直接注射脂肪组、单纯支架组、细胞-支架复合体脂肪诱导组、非诱导组。结果直接注射组在裸鼠皮下形成脂肪组织，但吸收量大；细胞-支架复合体诱导组有大量脂肪类组织形成，油红染色显示组织有脂滴形成；细胞-支架复合体非诱导组和单纯支架组均未发现脂肪组织形成。结论采用组织下程技术将吸脂术获得脂肪来源细胞接种纤维凝胶支架，体内构建脂肪组织，具有可行性。     

前脂肪细胞组织工程学的初步探讨

目的探讨胶原-透明质酸海绵支架与前脂肪细胞复合培养形成工程化脂肪组织的可行性。方法切取成年女性腹部皮下脂肪组织，经体外分离培养前脂肪细胞，与胶原-透明质酸支架混合，移植到裸鼠体内，未混合细胞的支架作为对照组。移植后4周和8周取材，对所取标本进行大体观察及厚度、高度测量和免疫组织化学检测。结果初步证实复合物植入后能形成黄色脂肪样组织。有新生血管形成，免疫组织化学检测证实脂肪细胞的存在及良好分布。结论胶原一透明质酸海绵支架可以作为组织工程技术应用研究中形成脂肪组织的支架，移植到裸鼠体内，在其皮下能形成脂肪样组织。     

人胆管癌裸鼠移植瘤2号模型的建立

目的 建立人胆管癌裸鼠移植瘤模型。方法 将人胆管癌组织接种于裸鼠皮下和肝脏，逐代观察移植瘤生长情况，进行形态学和生物学特性鉴定。结果 建立了人胆管中分化乳头状腺癌裸鼠移植瘤2号模型，命名为HBDCM2—ZSH。已连续传代7代，皮下移植瘤生长率为100％。潜伏期21d，成瘤时间为6周左右。移植瘤在形态和生物学上仍保持人胆管癌的特点。结论 HBD-CM2-ZSH裸鼠移植瘤模型是一种接近人体的胆管癌模型，可为胆管癌研究提供动物模型。     

人原发性胃恶性淋巴瘤裸鼠原位移植高转移模型的建立

目的 建立人原发性胃恶性淋巴瘤裸小鼠原位移植高转移模型.方法 采用人原发性胃恶性淋巴瘤术中新鲜瘤组织块植入裸小鼠胃壁黏膜下层,观察原位移植的成瘤率和移植瘤的侵袭、转移,并进行形态学（光镜、电镜、免疫组织化学）、染色体核型和流式细胞分析.结果在裸小鼠体内建成了一株人原发性胃恶性淋巴瘤原位移植高转移模型（HGBL-0305）.移植瘤的组织病理学为原发性胃弥漫性大B细胞淋巴瘤.免疫组织化学显示,CD19、CD20、CD22、CD79α阳性,CD3、CD7阴性.染色体众数范围56～69条;移植瘤细胞DNA指数为1.47±0.12,均为异倍体.目前该瘤株在裸鼠体内生长4年,已经传至45代,共移植裸鼠156只;肿瘤移植生长率和液氮冻存复苏成活率均为100%.人胃恶性淋巴瘤在裸鼠胃内自主侵袭性生长,浸润破坏胃壁各层组织结构.HGBL-0305模型的肝转移率为69.5%,脾转移率为55.6%,淋巴结转移率为45.7%,腹腔种植转移率为30.5%.结论 HGBL-0305模型是成功的人原发性胃恶性淋巴瘤裸鼠原位移植自发性高转移模型,完整地模拟了人原发性胃恶性淋巴瘤患者的自然临床病理过程,为研究原发性胃恶性淋巴瘤发病机制、转移生物学和抗转移治疗提供了理想的动物模型.     

Tissue-engineered buccal mucosa urethroplasty-clinical outcomes

INTRODUCTION: Whilst buccal mucosa is the most versatile tissue for urethral replacement, the quest continues for an ideal tissue replacement for the urethra when substantial tissue transfer is needed. Previously we described the development of autologous tissue-engineered buccal mucosa (TEBM). Here we report clinical outcomes of the first human series of its use in substitution urethroplasty. METHODOLOGY: Five patients with urethral stricture secondary to lichen sclerosus (LS) awaiting substantial substitution urethroplasty elected to undergo urethroplasty using TEBM, with full ethics committee support. Buccal mucosa biopsies (0.5 cm) were obtained from each patient. Keratinocytes and fibroblasts were isolated and cultured, seeded onto sterilised donor de-epidermised dermis, and maintained at air-liquid interface for 7-10 d to obtain full-thickness grafts. These grafts were used for urethroplasty in a one-stage (n=2) or a two-stage procedure (n=3). Follow-up was performed at 2 and 6 wk, at 3, 6, 9, and 12 mo, and every 6 mo thereafter. RESULTS: Follow-up ranged from 32 to 37 mo (mean, 33.6). The initial graft take was 100%, as assessed by visual inspection. Subsequently, one patient had complete excision of the grafted urethra and one required partial graft excision, for fibrosis and hyperproliferation of tissue, respectively. Three patients have a patent urethra with the TEBM graft in situ, although all three required some form of instrumentation. CONCLUSIONS: Whilst TEBM may in the future offer a clinically useful autologous urethral replacement tissue, in this group of patients with LS urethral strictures, it was not without complications, namely fibrosis and contraction in two of five patients.     

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.     

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。     

Reconstructed human skin produced in vitro and grafted on athymic mice

BACKGROUND: The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. METHODS: The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. RESULTS: After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. CONCLUSIONS: This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.     

Tissue engineering in urethral reconstruction—an update

The field of tissue engineering is rapidly progressing. Much work has gone into developing a tissue engineered urethral graft. Current grafts, when long, can create initial donor site morbidity. In this article, we evaluate the progress made in finding a tissue engineered substitute for the human urethra. Researchers have investigated cell-free and cell-seeded grafts. We discuss different approaches to developing these grafts and review their reported successes in human studies. With further work, tissue engineered grafts may facilitate the management of lengthy urethral strictures requiring oral mucosa substitution urethroplasty.     

Bladder cell culture on small intestinal submucosa as bioscaffold: experimental study on engineered urothelial grafts

OBJECTIVES: To investigate the feasibility to perform primary urothelial cell culture using porcine small intestinal submucosa as a delivery scaffold both in vitro and after in vivo implantation in a rabbit model. MATERIALS AND METHODS: Bladder mucosa samples were aseptically obtained from a group of eight male rabbits. The mucosa was cut into fragments and placed on small intestinal submucosa matrices for selective urothelial cell culture. After complete in vitro epithelization the matrices were shaped into tubes and placed in the subcutaneous tissue and subdartos of donor rabbits. The pattern of cell growth and delivery was evaluated on retrieved grafts using histology and immunostaining at the end of the in vitro phase; then 5, 10 and 20 days after implantation. RESULTS: Histological and immunohistochemical analysis of the in vitro primary culture showed the acellular matrices covered with a thin uninterrupted monolayer of urothelial cells. The implants examined on the day 5 maintained the epithelial configuration of the cultured grafts in all samples retrieved. On the day 10 the urothelium showed increased thickness taking on a bilayer configuration. On day 20, all grafts presented the transitional cells arranged in a double layer closely resembling the natural urothelium. The immunostaining pattern displayed the maintaining of urothelial cell phenotype. No differences in epithelium growth and delivery were noted between the two sites of implantation. Five days after implantation, the histological analysis of small intestinal submucosa showed a medium degree tissue reaction with the presence of acute inflammatory cells. Angiogenesis was demonstrated by the development of several new vessels inside the matrix. After twenty days, small intestinal submucosa was gradually replaced with host tissue. CONCLUSION: The small intestinal submucosa proved to function as a means of delivering of autologous urothelial cells cultured in vitro. After ectopic in vivo implantation the bioscaffold maintained viability and growth of the surrounding cells until its degradation.     

Tissue-Engineered Buccal Mucosa Urethroplasty—Clinical Outcomes

Introduction

Whilst buccal mucosa is the most versatile tissue for urethral replacement, the quest continues for an ideal tissue replacement for the urethra when substantial tissue transfer is needed. Previously we described the development of autologous tissue-engineered buccal mucosa (TEBM). Here we report clinical outcomes of the first human series of its use in substitution urethroplasty.

Methodology

Five patients with urethral stricture secondary to lichen sclerosus (LS) awaiting substantial substitution urethroplasty elected to undergo urethroplasty using TEBM, with full ethics committee support. Buccal mucosa biopsies (0.5 cm) were obtained from each patient. Keratinocytes and fibroblasts were isolated and cultured, seeded onto sterilised donor de-epidermised dermis, and maintained at air–liquid interface for 7–10 d to obtain full-thickness grafts. These grafts were used for urethroplasty in a one-stage (n = 2) or a two-stage procedure (n = 3). Follow-up was performed at 2 and 6 wk, at 3, 6, 9, and 12 mo, and every 6 mo thereafter.

Results

Follow-up ranged from 32 to 37 mo (mean, 33.6). The initial graft take was 100%, as assessed by visual inspection. Subsequently, one patient had complete excision of the grafted urethra and one required partial graft excision, for fibrosis and hyperproliferation of tissue, respectively. Three patients have a patent urethra with the TEBM graft in situ, although all three required some form of instrumentation.

Conclusions

Whilst TEBM may in the future offer a clinically useful autologous urethral replacement tissue, in this group of patients with LS urethral strictures, it was not without complications, namely fibrosis and contraction in two of five patients.     

口腔粘膜游离移植再造尿道

目的：探讨采用口腔粘膜游离移植，对局部缺乏组织的尿道下裂行尿道再造的方法。方法：1998－2001年对25例患者应用口腔粘膜游离移植再造阴茎段尿道，半年后吻合瘘口。结果：1例一期术后并发感染，愈后无尿道狭窄，所有病例二期吻合瘘口后，再造尿道通畅。结论：口腔粘膜丰富的网状毛细血管网、韧厚的上皮层和相对较薄的皮下板层结构是移植成功的关键。以口腔粘膜游离移植行尿道再造是一种可行的方法。