聚二元共聚物多孔支架活性真皮替代物的体外构建

活性皮肤替代物是指含有活细胞成分的皮肤组织工程产品 ,是皮肤组织工程研究的热点。由于人工合成的生物可降解材料具有生物相容性、加工性能良好以及降解速度可控等优点 ,已被广泛用作细胞支架。然而 ,在多孔的人工材料中种植细胞还存在很多问题。本文应用成纤维细胞胶原凝胶 (fibroblastspopulatedcollagenlattice ,FPCL)的制作方法 ,成功地将人成纤维细胞种植于聚二元共聚物 (乙交酯 /丙交酯polylacticacid/ polyglycolicacidcopolymers ,PLGA)多孔真皮支架中 ,体外培养出一种新型的活性真皮替代物。材　料　与　方　法1.胶原和细胞 …     

利用不同支架材料构建人工真皮的研究

目的 探讨脱细胞基质、胶原蛋白基质网架、胶原凝胶的不同支架材料上构建人工真皮的可行性。方法 采用酶消化法分离幼儿包皮成纤维细胞，并进行传代培养；将第3代成纤维细胞种植于三种不同支架上，14天后利用HE染色、透射电镜和扫描电镜对构建的人工真皮结构进行鉴定。结果 ①酶消化法得到皮肤成纤维细胞，可在含有10％小牛血清的DMEM中传代培养；②成纤维细胞在三种不同支架上形成单层细胞，并可向胶原膜和胶原凝胶内部生长；③成纤维细胞在胶原膜和脱细胞基质内增殖不如胶原凝胶内活跃，但人工真皮收缩不明显，而胶原凝胶构建的人工真皮收缩明显。结论 利用脱细胞基质、胶原蛋白基质网架、胶原凝胶支架材料构建的人工真皮具有较好的组织结构，是一种较理想的皮肤替代物，可作为移植物进行深入研究。     

利用胶原构建皮肤组织工程支架的研究

目的利用胶原构建皮肤组织工程支架。方法用Na2S、弹性蛋白酶预处理胎牛皮得到胶原纤维；经蛋白酶M降解胶原纤维后，0．5mol／L醋酸溶液溶解得到酸溶性胶原；再用蛋白酶N处理上述酸溶性胶原，最终得到生物相容性良好的胶原溶液；将胶原溶液构建皮肤组织工程支架。通过SDS-PAGE试验，分析酸溶性胶原分子量及基本结构；用皮肤组织工程支架材料包覆大鼠创面，观察创面愈合情况，研究支架材料对大鼠创伤愈合的影响；在支架材料上种植成纤维细胞，观察细胞扩增情况，以及支架材料对细胞移植影响。结果通过特异性蛋白酶M处理得到的酸溶性胶原，显示典型的1型胶原SDS-PAGE图谱；构建的皮肤组织工程支架呈多孔海绵状，孔径为50～200μm；在支架材料上种植成纤维细胞扩增情况良好；用于修复大鼠创面，具有促进创面愈合作用。结论酸溶性胶原经蛋白酶N处理后，生物相容性良好，适合于皮肤组织工程支架的构建，并具有良好生物活性。     

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.     

人工真皮替代物的构建及其生物相容性评价

目的　构建人工真皮替代物并评价其生物相容性。方法　取自健康小白猪的异种皮肤先用 0 .5 %安多福浸泡消毒后 ,在无菌操作下 ,取厚度为 0 .3～ 0 .4mm ,经胰蛋白酶消化、冷冻干燥及戊二醛等处理后检测其理化性能和生物学相容性。结果　去细胞真皮底物具有良好的弹性、强度和组织学结构 ;人包皮成纤维细胞均能在 5～ 10min内很好地粘附 ,且生长良好 ,说明人工真皮对细胞生长无毒性 ;人工真皮在大鼠体内的免疫排斥反应轻微 ,组织相容性好。结论　本实验所构建的人工真皮具有良好的理化性能 ,生物相容性好 ,免疫排斥反应小 ,且成本低、来源广 ,是构建组织工程皮肤理想的真皮材料。     

组织工程肌腱种子细胞的比较研究

目的 探讨猪肌腱细胞、真皮成纤维细胞、骨髓问充质干细胞中何种细胞最适宜作为体外组织工程化肌腱构建的种子细胞.方法 收集猪肌腱细胞、真皮成纤维细胞及骨髓间充质干细胞,以50×106个细胞密度均匀接种于圆柱状聚羟基乙酸(Polyglycolic acids,PGA)上,按细胞种类分为三组,每组n=3,体外培养,并于1、2、6周取材,进行组织学检测、免疫组化检测、胶原定量测定和大体观察.结果 细胞-PGA复合物体外培养时有细胞外基质产生,六周时肌腱细胞组产生胶原量最多,明显优于真皮成纤维细胞和骨髓间充质干细胞(p＜0.01).免疫组化显示形成的主要为Ⅰ型胶原.结论 体外构建组织工程肌腱时肌腱细胞合成胶原能力最强,在现有条件下是体外构建组织工程肌腱的最佳种子细胞.     

混合接种法构建组织工程皮肤

目的 探讨混合接种法体外构建复合皮的可行性.方法 在异种猪脱细胞真皮的真皮面接种人成纤维细胞7 d后,将其分两组进行实验.实验组:将表皮细胞按5×105/cm2与成纤维细胞0.2 ×105/cm2混合后接种于表皮面,培养液用K-SFM与成纤维细胞上清的1:1混合液.对照组:仅接种表皮细胞5×105/cm2,培养液用K-SFM.培养1、3周后取材观察其形态变化,并行免疫组化鉴定.结果 培养3周后,实验组可见表皮层连续,细胞层数3～4层,与真皮连接紧密,有表皮突形成;对照组表皮细胞层仅1～2层,且与真皮分离.实验组Laminin强阳性提示基底膜形成充分,并经透射电镜也可观察到完整的基底膜.结论 将表皮细胞与少量成纤维细胞混合接种,可促进表皮细胞在脱细胞真皮上黏附增殖,并有助于基底膜充分形成.     

应用负载碱性成纤维细胞生长因子的人脱细胞羊膜构建人工活性真皮

目的 探讨构建一种新型人工活性真皮的可行性.方法 组织块法培养幼儿包皮成纤维细胞;采用酶-去垢剂法制备人脱细胞羊膜(HAAM);双相法制备碱性成纤维细胞生长因子(bFGF)-明胶-壳聚糖缓释微球;缓释微球黏附于HAAM;bFGF基体式负载于HAAM,绘制药物缓释曲线;将第3～4代成纤维细胞培养于负载缓释微球的HAAM;扫描电镜观察HAAM、缓释微球的表面特征及缓释微球与HAAM的粘附情况;Western印迹法检测成纤维细胞中层粘连蛋白的表达.结果 制备的HAAM为白色半透明状薄膜,有较高的孔隙率,空隙不规则,孔径大小为10～100 nm,无细胞毒性;bFGF-明胶壳聚糖缓释微球分散较均匀,呈球形,粒径均匀,球体表面比较光滑,载药率为20 ng/g,包封率为80.5％,体外药物缓释曲线显示药物控释效果良好;成纤维细胞在支架表面爬行生长良好,层粘连蛋白表达较对照组高.结论 通过将成纤维细胞种植于负载bFGF-明胶-壳聚糖缓释微球的HAAM,有望制备出一种新型的人工活性真皮.     

血管平滑肌细胞在几种基质膜片上生长情况及三种接种培养方法的比较

目的筛选适合于组织工程血管构建的可降解基质材料及最佳的接种培养方式.方法将血管平滑肌细胞接种于几种现有的可降解基质材料上,采用MTT法观察细胞在几种基质材料上的生长情况;比较静态、水平摇床、旋转接种三种接种培养方式.结果细胞相容性比较好的材料有PGA、胶原加黏多糖、脱钙骨等.三种接种方法的接种率为静态1.5%,水平摇床7.2%,旋转接种培养53.5%.结论PGA、胶原加黏多糖、脱钙骨三种材料适合于组织工程血管再造的应用.而三种接种培养方式以旋转接种培养的效率最高.     

组织块HF染色法观察脱细胞真皮基质上接种细胞的生长情况

在组织工程皮肤的体外构建中，及时观察脱细胞真皮基质（accllular dcrmal matrix,ADM）表面接种的表皮细胞或皮纤维细胞的形态和密度，对判断细胞存活情况、生长速度及融合程度具有重要意义。但由于真皮组织不透明，在相差显微镜下无法直接观察接种细胞的生长情况。笔者拟利用组织块HE染色技术进行相应改进。     

023 Development of Tissue Engineering Skin Based on Acellular Dermal Matrix

Aim: In tissue engineering of the skin, the selection of a scaffold or a matrix in which a cultured cells grow is quite important. We have developed a tissue engineering skin composed of human keratinocytes and fibroblasts on an acellular allogenic dermal matrix (ADM), derived from cryopreserved human skin. Methods: ADM was prepared from cryopreserved split‐thickness human skin by treating with Dispase and Triton X‐100. The tissue engineering skin were produced by seeding human keratinocytes and fibroblasts on ADM. Several days after seeding, the tissue engineering skin was exposed to an air‐liquid interface for another 7 days. Then, the histological structure of the skin and the production of growth factors by the skin were investigated. Results: The produced ADM was found to be completely acellular with remaining structure of the basement membrane components, such as type IV collagen and laminin. The developed tissue engineering skin had stratified keratinocytes on the surface of the ADM migrating fibroblasts in the dermal collagen structure, resembling to the normal skin appearance. It was found that several important growth factors in wound healing process, such as TGF‐α, TGF‐β and VEGF, were produced by the tissue engineering skin. Conclusions: It was suggested that ADM is suitable for a scaffold in tissue engineering of the skin.     

Promotion of acellular dermal matrix resolution in vitro by matrix metalloproteinase-2

OBJECTIVE: To determine whether acellular human dermis is degraded by matrix metalloproteinases (MMPs), a large class of matrix-degrading enzymes. METHODS: The degradation of acellular human dermis specimens was evaluated in vitro. Wild-type murine fibroblasts with a broad-spectrum MMP inhibitor, GM6001, and MMP-2-deficient fibroblasts were placed on the basement membrane and dermal surfaces of acellular human dermis. Matrix degradation and fibroblast infiltration into the matrix were assessed after a 20-day incubation period. RESULTS: The basement membrane thickness of the specimens cultured with wild-type fibroblasts was significantly less than that of specimens cultured with GM6001 (P<.001), and the infiltration of fibroblasts into the dermal surface was limited by the addition of GM6001 (P=.002). To determine whether MMP-2 was involved in this in vitro phenotype, MMP-2-deficient fibroblasts were assessed in comparison with wild-type fibroblasts. Wild-type fibroblasts degraded the basement membrane surface (P<.001) and infiltrated the dermal surface (P = .003) more efficiently than did MMP-2-deficient fibroblasts. CONCLUSIONS: The results from our in vitro experiments suggest that MMPs and specifically MMP-2 may play an important role in the resorption of acellular human dermis. Addition of MMP inhibitors to implanted dermal matrices may slow fibroblast infiltration and improve their longevity in vivo.     

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.     

异体脱细胞组织补片修复即刻牙种植软组织创面的临床研究

目的 评价异体脱细胞组织补片（又称异体脱细胞真皮基质补片，简称补片）修复即刻牙种植创面的临床疗效。方法 应用异体脱细胞组织补片修复51例患者、67个牙位的即刻种植后口腔黏膜缺损创面，将补片的基底面向上，覆盖于种植体及牙槽嵴上，并插入黏骨膜瓣下，碘仿纱包或预成基托固定。术后随访4～6个月，对口腔补片修复创面的愈合情况及组织病理结果观察。种植体骨结合情况随访6个月～4年。结果 术后创面完全封闭愈合54个牙位，部分封闭愈合11个牙位，未封闭愈合2个牙位。无排斥反应。经4～6个月后组织病理检查补片已完全黏膜上皮化，67颗种植体情况经1年观察，无一例失败。结论 异体脱细胞组织补片具有良好的即刻种植创面封闭作用，不影响种植体与骨的结合，且具有手术创伤小、修复美学效果好等优点，可以作为牙即刻种植创面的修复材料。     

成纤维细胞-无细胞真皮替代物的生物学活性及移植实验

目的 研究含成纤维细胞的无细胞真皮替代物的生物学活性及真皮支架作用。方法 将成纤维细胞种植于无细胞真皮表面培养,形成活性真皮替代物。采用ELISA法和RIA法测定培养上清中IL－6、IL－8、TGF－β1及细胞外基质层粘连蛋白、透明质酸的分泌。并将真皮替代物植入BALB／c-nu小鼠（裸鼠）全层皮肤缺损创面,观察血管化速度、创面收缩率。结果 成纤维细胞种植于无细胞真皮表面生长良好,可形成单层细胞膜片,并分泌多种细胞因子和细胞外基质成份。活性真皮替代物植入创面后,与单纯无细胞真皮移植相比,血管化速度加快,收缩率减小。结论 无细胞真皮上种植成纤维细胞后具有较强的生物学活性,可作为较好的真皮替代物。     

异种脱细胞真皮与异体巩膜作为HA义眼台包裹材料的实验研究

目的 观察异种脱细胞真皮（acellular xenogenic dermal matrix,X-ADM）和异体巩膜作为羟基磷灰石（hydroxy apatite,HA）义眼台包裹材料,用于实验兔眼的临床表现及病理组织学变化观察.方法 24只纯种新西兰兔,行一只眼的眼球摘除术后,随机平均分为实验组和对照组.于肌锥内分别置入由异种脱细胞真皮及异体巩膜包裹的HA义眼台.术后观察眼部表现,于1、2、4、6、8和12周,连同异种脱细胞真皮或异体巩膜取出义眼台,光镜下观察包裹材料与义眼台的组织病理学改变,包括炎症反应及血管化情况.取4、8和12周标本做透射电镜观察上述组织的超微结构改变.结果 与异体巩膜组相比,异种脱细胞真皮组在同期的成纤维细胞及新生血管的生长更活跃,而且长入较早,新生胶原形成丰富,几乎没有炎症细胞的浸润以及发生排斥反应.结论 异种脱细胞真皮血管化快,免疫原性低,是一种良好的巩膜替代物.     

Use of an acellular dermal allograft for dural replacement: an experimental study.

OBJECTIVE: In this study, a nonimmunogenic, acellular, dermal collagen matrix termed XenoDerm (LifeCell Corp., The Woodlands, TX) was examined for use as a dural replacement material in a porcine model. This model was used to investigate whether AlloDerm (LifeCell), an almost identical material made from human dermis, could be safely used in neurological surgery. METHODS: Bilateral temporoparietal dural defects were surgically created in 12 Yucatan minipigs. One side was repaired with autologous pericranium, and the other was repaired with XenoDerm. The pigs were killed after 1, 3, or 6 months, and the areas of dural repair were collected and examined macroscopically and histologically. XenoDerm is derived from porcine skin collected in thin sheets. It is processed so that the epidermis and all dermal cells are removed without disruption of the collagen matrix, rendering the material immunogenically inert and resistant to calcification. It is packaged as a freeze-dried sheet and is easily rehydrated at the time of surgery. RESULTS: There were no postoperative complications, and all pigs survived. Both grafts performed well as dural replacements in all cases. There was no macroscopic evidence of inflammation or cerebrospinal fluid leakage. The XenoDerm grafts were intact, retained their original dimensions, and resembled the surrounding dura. The autologous pericranial grafts, in contrast, were thicker than when implanted and had bony excrescences firmly adhering to their surfaces. Again, however, there was no evidence of cerebrospinal fluid fistulae. There was no gross adherence to the underlying meninges or brain tissue in any specimen. Repopulation by fibroblasts and neovascularization were evident in the XenoDerm grafts as early as 1 month after surgery; by 3 months, the XenoDerm had been remodeled to assume the connective tissue appearance of the surrounding dura. CONCLUSION: In this porcine model, an allograft of acellular dermis is a nearly ideal dural replacement. AlloDerm, the human equivalent of XenoDerm, would be an allograft of acellular dermis after implantation in human subjects. On the basis of this study and previous work with AlloDerm in other reconstructive applications, it is proposed that this material could be similarly used for duraplasty in human subjects.     

猪脱细胞真皮基质修复兔腹壁缺损的实验研究

目的研究猪脱细胞真皮基质修复兔腹壁缺损的效果,探讨异种脱细胞真皮基质应用的可行性。方法健康小白猪1头,取背部及两侧皮肤制备脱细胞真皮基质。26只日本大耳白兔,雌雄不限,体重2.2～2.3 kg,随机分为对照组(n=6)和实验组(n=20)。对照组制备5.0 cm×0.5 cm腹壁缺损,单纯缝合关闭缺损。实验组制备5.0 cm×2.5 cm腹壁缺损,用同样大小的猪脱细胞真皮基质补片(简称"补片")修复,补片基底膜面朝向肠管。术后观察是否有疝形成,比较两组腹腔内脏器粘连情况,以及对照组腹壁肌筋膜单纯缝合处和实验组补片-腹壁肌筋膜吻合处的最大张力,组织学观察补片是否有纤维血管组织长入及其在体内的生物学转归。结果实验动物均无疝形成。术后5周,实验组补片和腹壁融为一体,补片皮肤面和脏器面均有纤维血管组织长入,补片处于新生组织掺入重建过程。实验组1只动物补片和腹腔内脏器粘连较重(2级),5只发生了轻微粘连(1级),12只无粘连(0级);对照组1只轻微粘连(1级),5只无粘连(0级);两组粘连分级比较差异无统计学意义(Z=—0.798,P=0.425)。术后5周,实验组补片-腹壁肌筋膜吻合处的最大张力为(13.0±5.5)N,对照组腹壁肌筋膜单纯缝合处为(13.6±4.0)N,差异无统计学意义(t=—0.410,P=0.683)。组织学观察显示,术后5周,实验组补片中有大量小血管,并有中性粒细胞及淋巴细胞为主的炎性浸润,补片边缘偶见巨噬细胞,补片-腹壁肌筋膜吻合处由纤维结缔组织连接;术后6个月,补片及周围炎性反应消退,胶原纤维结构发生了改建,补片和肌筋膜层由有纤维结缔组织愈合。结论补片修复兔腹壁缺损取得了较好效果,补片-腹壁肌筋膜层愈合,其吻合处的力学强度达到了自体腹壁单纯缝合吻合的力学强度,补片胶原纤维结构发生了改建。     

激光微孔猪脱细胞真皮基质制备及创面移植的观察

目的 制备激光微孔猪脱细胞真皮基质（PADM），寻找其与自体刃厚皮复合移植的最佳孔间距。方法 采用胰蛋白酶法制备PADM，用激光加工机打孔，制备孔间距为0．8、1．0、1．2、1．5mm的激光微孔PADM。在144只SD大鼠背部制作全层皮肤缺损创面，分为微孔Ⅰ、Ⅱ、Ⅲ、Ⅳ组，依次采用前述4种孔间距的激光微孔 PADM＋自体刃厚皮移植；网状组采用网状PADM＋自体刃厚皮移植；对照组单纯移植自体刃厚皮。每组24只大鼠。观察移植后2、4、6周各组创面愈合情况，计算移植物成活率和创面收缩率，并行组织学观察。结果 移植后2、4周，微孔Ⅰ、Ⅱ组移植物成活率均低于对照组（P〈0．05），但与网状组比较，差异无统计学意义（P〉0．05）；移植后6周，微孔Ⅰ、Ⅱ组成活率明显高于网状组（P〈0．05）。移植后4、6周微孔Ⅰ、Ⅱ组创面收缩率较对照组显著降低（P〈0．05），移植后6周[（16．0±2．6）％、（15．1±2．4）％]明显低于网状组[（19．3±2．4）％，P〈0．05]。组织学观察见各微孔组上皮化良好，胶原纤维排列整齐，基底膜结构完整。结论 孔间距为0．8mm或1．0mm的激光微孔PADM与自体刃厚皮复合移植可提高创面修复质量，其中孔间距1．0mm效果最佳。