异种脱细胞真皮替代睑板材料重建眼睑的可行性

背景：眼睑后层重建是眼睑重建的重点和难点,其中睑板替代物更是研究的焦点。异种脱细胞真皮作为一种新型的组织工程材料,在国内外烧伤整形领域,正得到广泛的研究和应用。目的：观察异种（猪）脱细胞真皮植入兔眼睑后的组织相容性极其组织病理学变化。方法：剥取健康小白猪全层皮肤20cm×20cm,制备异种（猪）脱细胞真皮基质。同时制备兔睑板全层缺损模型并植入脱细胞真皮基质,观察大体情况,并分别于第1,2,3周取移植交界处眼睑组织光镜下观察组织学的改变。结果与结论：大体观察未见明显排斥反应及眼睑的变形;光镜下1周时可见局部炎症细胞浸润,2周时炎症细胞减少,3周时正常纤维组织长入,逐渐分割代替植入的胶原纤维,炎症反应消失。提示异种脱细胞真皮免疫原性低,并可引导新生胶原的生长,是一种良好的睑板替代物。     

异种(猪)无细胞真皮基质的制备及体外生物相容性

背景:人同种无细胞真皮基质作为一种永久性真皮支架,已成功应用于烧伤创面修复及美容医学等领域,但由于来源有限,限制了其应用.目的;研制异种(猪)无细胞真皮基质,并对其体外生物相容性进行评价.设计、时间及地点:体外细胞学对比观察实验,于2007-08/2008-06在中国辐射防护研究院生物材料与制药技术研究所实验室完成.材料:实验猪由中国辐射防护研究院实验动物中心提供;人成纤维细胞来自武警山西总队医院健康儿童包皮环切术切除的包皮组织.方法:无菌条件下获取健康小白猪猪皮,用高渗盐溶液-去污剂、胰酶消化及超声清洗的方法,制备猪无细胞真皮基质.体外培养人成纤维细胞,用猪无细胞真皮基质浸提液法及人成纤维细胞和猪无细胞真皮基质直接贴附法,评价猪无细胞真皮基质体外生物相容性.主要观察指标:①猪无细胞真皮基质的组织学形态.②猪无细胞真皮基质的体外生物相容性.结果:制备的猪无细胞真皮基质,完全去除了表皮和真皮中的细胞成分,保留了胶原基质.猪无细胞真皮基质浸提液对人成纤维细胞增殖无明显影响.人成纤维细胞可以在猪无细胞真皮基质上贴附、增殖.结论:此种方法制备的无细胞真皮基质完全去除了表皮和真皮中的细胞成分,有较好的体外生物相容性.     

不同方法制备猪脱细胞真皮基质及创面移植的实验研究

目的比较两种方法制备的猪脱细胞真皮基质分别与自体刃厚皮复合移植修复大鼠全层皮肤缺损的效果。方法使用DispaseⅡ／TritonX-100(中性蛋白酶／曲拉通)和高渗盐水／十二烷基硫酸钠(SDS)两种方法去除猪表皮及真皮中的细胞成分，分别得到猪脱细胞真皮基质Ⅰ和Ⅱ。63只SD大鼠背部全层皮肤缺损分别使用猪脱细胞真皮基质Ⅰ 自体刃厚皮及猪脱细胞真皮基质Ⅱ 自体刃厚皮覆盖，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，并与单纯自体刃厚皮移植相比较。结果两种方法制备的猪脱细胞真皮基质分别与自体刃厚皮复合移植的移植物成活率和植皮区收缩率差异无显著性，组织学观察显示复合皮上皮化良好，胶原纤维排列有序，基底膜结构完整。两复合皮组术后第6周移植物成活率与自体刃厚皮组比较差异均无显著性，术后第4周开始两复合皮组移植物收缩率明显降低。结论两种方法制备的异种脱细胞真皮基质与自体皮复合移植都能很好地修复全层皮肤缺损，改善创面愈合质量。     

异种脱细胞真皮替代睑板材料重建眼睑的可行性

背景:眼睑后层重建是眼睑重建的重点和难点,其中睑板替代物更是研究的焦点。异种脱细胞真皮作为一种新型的组织工程材料,在国内外烧伤整形领域,正得到广泛的研究和应用。目的:观察异种(猪)脱细胞真皮植入兔眼睑后的组织相容性极其组织病理学变化。方法:剥取健康小白猪全层皮肤20cm×20cm,制备异种(猪)脱细胞真皮基质。同时制备兔睑板全层缺损模型并植入脱细胞真皮基质,观察大体情况,并分别于第1,2,3周取移植交界处眼睑组织光镜下观察组织学的改变。结果与结论:大体观察未见明显排斥反应及眼睑的变形;光镜下1周时可见局部炎症细胞浸润,2周时炎症细胞减少,3周时正常纤维组织长入,逐渐分割代替植入的胶原纤维,炎症反应消失。提示异种脱细胞真皮免疫原性低,并可引导新生胶原的生长,是一种良好的睑板替代物。     

活性真皮替代物的体内外实验

背景:真皮替代物是构建组织工程皮肤替代物的基础。目的:构建具有活性的组织工程真皮替代物,并进行体内外实验。方法:胶原凝胶和成纤维细胞制成活性真皮替代物,并进行组织学观察。将活性真皮替代物移植于BALB/c裸鼠背部全层皮肤缺损模型,4周后取材观察其组织学变化。结果与结论:①体外实验:随培养时间延长,活性真皮替代物直径逐渐减小,韧性增加,至14d时为原直径18.2%,可夹持提起而不破碎。成纤维细胞在胶原凝胶内成活良好,保持良好的细胞外基质分泌活性,Ⅰ型胶原蛋白、纤维连接蛋白、血管内皮生长因子均阳性表达。②体内实验:活性真皮替代物覆盖创面4周后被表皮覆盖,表皮分层清晰,但较正常表皮层厚;真皮层中成纤维细胞仍存活并发挥作用,胶原纤维排列有序,呈匀质性,无分层,真皮内炎性细胞少见,有成熟血管,缺乏毛囊和皮脂腺。表明应用胶原凝胶三维培养成纤维细胞能构建出具有活性的组织工程真皮替代物,移植创面后可促进其表皮化。     

异种无细胞真皮活性复合皮的制备

目的体外重建异种无细胞真皮活性复合皮.方法将表皮细胞、成纤维细胞种植于异种无细胞真皮基质表面,体外培养约1周,定时取材行真皮表面HE染色、常规组织切片HE染色及扫描电镜观察.结果表皮细胞、成纤维细胞种植后24h即贴附于真皮表面,电镜观察可见有伪足形成.培养5～7d可融合为致密单层细胞膜.结论以无细胞真皮为支架,采用细胞培养技术可于体外重建复合皮.     

经VEGF165转染的成纤维细胞-脱细胞异种真皮替代物的构建与临床应用

目的构建经血管内皮生长因子165(VEGF165)转染的人成纤维细胞-脱细胞异种真皮替代物,并对其在促进深度烧伤患者创面愈合过程中的作用进行评价。方法将30例深Ⅱ度或Ⅲ度烧伤患者随机分为2组,每组15例。移植转染有VEGF165成纤维细胞-脱细胞异种真皮替代物组为实验组,单纯脱细胞异种真皮替代物移植组为对照组。采用分子生物学方法将扩增的VEGF165基因克隆入真核表达载体pcDNA3.1(+),在脂质体介导下将重组质粒转染至经传代培养的人成纤维细胞,再将转染后的人成纤维细胞接种于猪脱细胞真皮表面得到成纤维细胞-脱细胞异种真皮替代物;而后将此替代物与自体薄皮片移植于深度烧伤患者切痂后创面,对创面愈合情况进行观察并与单纯脱细胞真皮移植组进行比较。结果构建的真核表达载体pcDNA3.1(+)-VEGF165可成功转染人成纤维细胞,经转染的人成纤维细胞可顺利接种于脱细胞真皮形成成纤维细胞-脱细胞异种真皮替代物,替代物移植后实验组患者皮片成活率(90.25±5.39)%,对照组患者皮片成活率(83.98±3.63)%,两者有显著性差异(t=3.737,P<0.01)。实验组新生的毛细血管数量要明显多于对照组。结论经VEGF165转染的人成纤维细胞-脱细胞异种真皮替代物具有显著的促进深度烧伤患者创面愈合的作用。     

猪复方壳多糖组织工程皮肤替代物的构建

目的：采用组织工程方法，在鼠尾胶原中加入壳多糖及培养的贵州小型香猪成纤维细胞与角质形成细胞，进行猪复合壳多糖组织工程皮肤替代物的构建。 方法：实验于2001-09／2002-12在解放军第三军医大学大坪医院完成。①实验所需皮肤标本取自3月龄雌性贵州小型香猪4只，取新鲜全层皮肤于D—Hanks平衡盐液浸泡，将标本剪成0．3cm&;#215;2．0cm皮条，4℃消化14-16h后备用。②消化后的标本分离表皮和真皮，分别用于角质形成细胞和成纤维细胞的分离培养。以传代培养的猪角质形成细胞和成纤维细胞作为种子细胞，以加入壳多糖、糖胺聚糖等天然细胞外基质的鼠尾胶原作为真皮基质，于钢丝网上进行气液界面培养，苏木精-伊红染色观察培养物的结构。 结果：①角质形成细胞的培养情况：接种的猪角质形成细胞于培养第2天可见细胞贴壁生长，形成细胞集落，15d左右融合成片，呈“铺路石样”。②真皮成纤维细胞的培养情况：消化法接种的猪成纤维细胞培养两三天后即见细胞贴壁生长，10d左右基本融合，呈旋涡状生长。③猪真皮基质浸没培养的变化情况：刚制备的真皮基质呈红色，倒置显微镜下凝胶各层面均可见尚未恢复形态的成纤维细胞。3d后随着凝胶的收缩，人工真皮逐渐与培养板壁和底分离，透光度下降，不易见到凝胶中的细胞。真皮基质的收缩发生于第3天，第6-12天真皮基质的直径最大收缩到45％左右，以后逐渐减慢。④猪的复合皮肤替代物的培养情况：培养第3天在真皮基质上加入传代后的猪角质形成细胞，其上可见薄的角质形成细胞层。浸没培养6d后角质形成细胞层色素逐渐加深。气液界面培养14d后的人工皮肤有一定的弹性、韧性以及抗拉力和抗挤压力。⑤复方壳多糖组织工程皮肤的光镜观察结果：镜下表皮可明显区分基底层、棘层和角化不全的角质层，共有7-12层细胞。真、表皮连接有起伏。真皮中可见较多的成纤维细胞，真皮支架结构紧密。 结论：成功构建具有真、表皮结构的双层猪组织工程皮肤替代物。与正常猪皮肤的形态结构相似，真皮结构致密，表皮分化良好，为组织工程皮肤的临床应用奠定实验基础。     

改良法制备脱细胞真皮基质的实验研究

目的：检测用低含量胰蛋白酶消化加反复冻融法来制备的脱细胞异种真皮，并观察大鼠皮下移植后的结果。方法：将断层猪皮浸入0．5g／L胰蛋白酶溶液中4℃过夜消化，去除表皮和部分真皮内细胞成分，然后分别行4℃预冷，-70℃冷冻和37℃下融化，如此反复冻融3次。进行大体观察和组织学检测、细菌培养试验、细胞毒性试验。将真皮基质移植于大鼠皮下，术后2，4，6，8周行大体和组织学观察。结果：脱细胞猪真皮基质平整光滑，质地柔软，有弹性，韧性好。镜下观察基底膜存在，胶原结构完整，无细胞存在，无细菌生长，无细胞毒性。大鼠皮下移植后愈合良好，无明显炎症反应。结论：低含量胰蛋白酶消化加反复冻融法制备的脱细胞异种真皮能完全去除细胞成分，且保持胶原结构和基底膜的完整，为一种简便有效的方法。     

选择性去细胞异种(猪)皮与自体微粒皮复合移植对创面愈合的影响

目的:观察选择性去细胞异种(猪)皮与自体微粒皮复合移植对创面愈合的影响。方法:将异种(猪)皮经甲醛、胰蛋白酶、去污剂TritonX-100处理得到一种选择性去细胞异种(猪)皮。以SD大鼠为动物模型,在背部作全层皮肤缺损,使用微粒皮与选择性去细胞异种(猪)皮、异体皮、戊二醛皮及油纱覆盖皮肤缺损,观察创面愈合情况。结果:经组织学,免疫组化染色及电镜观察,选择性去细胞异种(猪)皮表皮层连续、完整,真皮中三大纤维排列有序且无细胞成分,基底膜结构完整。以选择性去细胞异种(猪)皮为覆盖物的创面愈合效果略差于异体皮覆盖创面的效果,但明显优于戊二醛皮及油纱覆盖创面的效果。结论:选择性去细胞异种(猪)皮易于制备,来源广泛,价格低廉,具有无细胞真皮支架和失活表皮,有望取代异体皮成为覆盖烧伤创面的新型敷料。     

即刻种植中异体脱细胞真皮基质引导的骨组织再生

背景:脱细胞真皮基质可作为软组织支架使成纤维细胞和上皮细胞在其中增殖,并最终成为宿主组织的一部分.目的:观察异体脱细胞真皮基质引导即刻植入种植体周围骨组织再生的效果.方法:选择口腔修复行即刻种植患者15例,使用异体脱细胞真皮基质行骨组织引导再生,种植体植入后,测量术中、植入后6,12个月唇侧骨壁厚度,同时观察种植体周围软组织愈合情况.结果与结论:6个月后均完成最终修复,无感染及明显并发症.异体脱细胞真皮基质有效关闭了即刻种植位点的软组织创口,6个月内逐渐与种植体周软组织发生整合.植入后6,12个月种植位点唇侧骨壁厚度与唇侧骨量高于术中测量值(P < 0.05),植入后6,12个月组间骨壁厚度及骨量变化不明显(P > 0.05).说明异体脱细胞真皮基质作为屏障膜能有效引导骨组织再生,降低即刻种植术中关闭创口的难度,短期临床结果满意.     

Acellular porcine dermal matrix produced with different methods and an experimental study on its transplantation to skin wound.]

OBJECTIVE: To observed the effect of healing quality of composite skin grafting consisting of acellular porcine dermal matrix combined with autologous split-thickness skin graft. METHODS: Porcine skin was treated with dispase II/triton X-100 or hyperosmotic saline/sodium-dodecyl-sulfate (SDS) respectively, and acellular porcine dermal matrix I (APDMI) and APDM II were obtained. Sixty-three Sprague-Dawley rats with full-thickness skin defects on the back were separately covered with APDMT + split-thickness autologous skin, or APDM II + split-thickness autologous skin. The quality of wound healing was observed, the rates of survival and contraction of the grafts were calculated, the tissue samples were harvested for histological examination, and compared with that of autologous split-thickness skin graft. RESULTS: The wound healing quality of composite skin I, and II was good. There was no significant difference in the rate of survival and contraction of the grafts between the two composite skin grafting groups. It was indicated by histological examination intact basal membrane. There was no significant difference in the survival rate between composite skin grafting groups and autologous split-thickness skin at the 6 th week after operation, but the contraction rates of the grafts in the composite skin groups were lower. CONCLUSION: Full-thickness skin defect can be healed by covering with acellular porcine dermal matrix produced by two methods combined with split-thickness autolograft, and it can help improve the quality of wound healing.     

引导种植牙区骨再生的异种脱细胞真皮基质

背景：异种脱细胞真皮基质属于口腔修复膜材料,因具有良好的生物相容性而被广泛应用。目的：评价异种脱细胞真皮基质在牙区引导牙种植骨再生的效果。方法：以免疫组化染色后显微镜观察异种脱细胞真皮基质的显微结构和细胞相容性,评价应用在引导骨再生技术中的可能性。对应用异种脱细胞真皮基质引导骨再生的牙种植修复患者进行观察随访,评价成骨效果以及对缺损软组织的影响,并与Bio-Gide生物膜以及博特医用胶原膜等其它修复膜引导骨再生的效果进行比较。结果与结论：异种脱细胞真皮基质的显微结构显示其有基底膜面和组织面,基底膜面可见指突结构和毛囊孔,组织面为鳞片状结构,对成骨样细胞的增殖活力和碱性磷酸酶的活性无影响,具有良好的细胞相容性。临床研究显示在牙种植中应用异种脱细胞真皮基质引导骨再生能够获得良好的骨再生效果,与Bio-Gide生物膜以及博特医用胶原膜引导骨再生的效果无明显差异,并且用于修复骨增量后软组织的不足同样能够获得较好的骨再生效果。     

Characterization of chitosan–gelatin scaffolds for dermal tissue engineering

Porous scaffolds for dermal tissue engineering were fabricated by freeze‐drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1‐(3‐dimethylaminopropyl)‐3‐ethyl‐carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well‐distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin‐based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue‐engineered skin. Copyright © 2011 John Wiley & Sons, Ltd.     

NaOH消蚀法制备脱细胞真皮基质修补腹壁疝

背景:与其他疝修补材料相比,脱细胞真皮基质具有易血管化、抗感染、从而可用替代传统补片用于感染腹壁缺损重建等特点。目的:观察NaOH消蚀法制备的脱细胞真皮基质作为修补材料应用于腹疝的应用价值。方法:以全厚猪皮制成脱细胞真皮基质,45只SD雄性大鼠制备腹壁疝模型,随机数字表法分为腹壁疝组:直接缝合皮肤,Marlex网组和脱细胞真皮基质组:分别应用大小为3.5cm×4.0cm的Marlex网和脱细胞真皮基质缝合皮肤;观察修复后1周时有无腹壁疝发生,脱细胞真皮基质组修复后1,2,3,5,10周分别取材,其中每周各组取4只用于苏木精-伊红染色,光镜观察。修复后第5周Marlex网组和脱细胞真皮基质组各取6只用于抗张力试验。结果与结论:术后1周脱细胞真皮基质组与Marlex网组腹壁疝发生率均显著低于腹壁疝组(P<0.001)。脱细胞真皮基质的胶原纤维无明显变化,即有少量成纤维细胞植入,术后2周可见新生血管,术后5周脱细胞真皮基质内部血管密度基本稳定。将单独脱细胞真皮基质片与Marlex网行抗张力试验,Marlex网的抗张力显著高于脱细胞真皮基质(P<0.001)。但植入体内5周后,脱细胞真皮基质筋膜组织的抗张力高于Marlex-筋膜组织(P<0.05)。提示复合消蚀制备的脱细胞真皮基质可以作为良好的疝修补材料。     

Clinical regenerative medicine: the skin

Tissue engineering of skin is classified into acellular artificial skin and cellular artificial skin. Acellular artificial skin or artificial dermis, is composed of an inner collagen sponge and an outer silicone film. When placed on wounds, the collagen sponge is spontaneously converted into a dermis-like connective tissue. Addition of bFGF or cultured fibroblasts accelerates synthesis of the dermis-like tissue. Cultured epidermis often fails to take on a full-thickness skin defect because of lack of dermal component. Cultured skin with both epidermal and dermal components seems to be an ideal skin substitute, but its take rate is still low. Regeneration of complete skin with skin appendages, vascular networks, elastic fibers and so on is desired.     

Interface integration of layered collagen scaffolds with defined matrix stiffness: implications for sheet‐based tissue engineering

Successful application of sheet‐based engineering for complex tissue reconstruction requires optimal integration of construct components. An important regulator of cellular responses (such as migration and collagen deposition) mediating interface integration is matrix stiffness. In this study we developed a sheet‐based 3D model of interface integration that allows control of interface matrix stiffness. Fluid was removed from acellular or fibroblast‐seeded bilayer collagen hydrogel constructs, using plastic compression to increase collagen density and matrix stiffness. Cell‐seeded constructs were either compressed at day 0 and cultured for 7 days (compressed culture, high stiffness) or left uncompressed during culture and compressed on day 7 (compliant‐compressed culture, low stiffness). Constructs were fitted onto a mechanical testing system to measure interface adhesive strength. Analysis of stresses by finite element modelling predicted a sharp rise of stress and rapid failure at the interface. While cell‐seeded constructs showed a six‐fold increase in interface adhesive strength compared to acellular control constructs (p < 0.05), there was no significant difference between low‐ and high‐stiffness cultures after 1 week. Cell migration across the interface was greater in low‐ compared to high‐stiffness constructs at 24 h (p < 0.05); however, no significant difference was observed after 1 week. Visualization of interfaces showed fusion of the two layers in low‐ but not in high‐stiffness constructs after 1 week of culture. The ability to regulate cellular behaviour at an interface by controlling matrix stiffness could provide an important tool for modelling the integration of sheet‐based bioengineered tissues in bioreactor culture or post‐implantation. Copyright © 2009 John Wiley & Sons, Ltd.     

脱细胞枪乌贼神经的制备及其生物相容性

背景:脱细胞哺乳动物神经有有髓纤维直径小、细胞成分不易彻底清除、复合细胞不均匀的缺点。目的:制备脱细胞枪乌贼神经,并探讨其生物相容性。方法:用Hasse化学萃取法制备脱细胞枪乌贼神经,观察其脱细胞的效果、管道及基底膜的完整性,用MTT法检测脱细胞枪乌贼神经与细胞相容性,用皮下植入实验检测其组织相容性。结果与结论:制备的脱细胞枪乌贼神经细胞及髓鞘清除彻底、神经纤维膜管及基底膜保留完整,且膜管粗大。脱细胞枪乌贼神经皮下植入植入后1和2周材料周围有炎症细胞浸润。植入后4周材料周围有薄层纤维结缔组织囊,材料内部及周围只有少量炎症细胞。结果证实,采用Hasse化学萃取法制备的脱细胞枪乌贼神经,细胞清除彻底,神经纤维膜管及基底膜保留完整,与异种细胞及组织相容性良好。