脱细胞猪小肠黏膜下层的组织相容性

目的:制备脱细胞猪小肠黏膜下层(SIS),研究其用作组织工程支架材料的组织相容性。方法:0.1%十二烷基磺酸钠(SDS)处理新鲜猪SIS 30min制备脱细胞SIS,H-E、4,6-二脒基-2-苯基吲哚(DAPI)及天狼星红染色观察脱细胞前后SIS的生物学特性,将其植入SD大鼠背部皮下,检测其组织相容性,对照组SD大鼠植入未脱细胞的正常SIS作为阳性对照。结果:脱细胞SIS呈白色、半透明的膜状物,具有良好的弹性及柔韧性;H-E、DAPI及天狼星红染色光学显微镜观察结果显示脱细胞SIS无细胞及DNA残留,组织结构保留完整,胶原纤维间孔隙率增加;皮下埋植术后全部动物存活,各时间段脱细胞SIS植入组大鼠创口周围皮肤愈合良好,无红肿、渗出等炎症反应。组织学观察脱细胞SIS植入早期结构完整;随着植入时间的延长,脱细胞SIS逐渐降解,结构松散。脱细胞SIS植片周围炎症细胞数明显少于正常SIS植入组。结论:脱细胞猪SIS植入SD大鼠体内具有良好的组织相容性,未引起明显的炎症反应,是一种理想的组织工程支架材料。     

植入式葡萄糖传感器膜材料壳聚糖与Nafion的组织相容性比较

背景：壳聚糖是天然高分子多糖,可单独或者与其他材料复合制作敷料、药物、基因载体、生物涂层、组织工程支架、传感器膜材料等。 目的：了解壳聚糖作为植入式葡萄糖传感器膜材料的组织相容性,并与Nafion膜进行对比。 方法：制备壳聚糖膜并对其理化性质进行表征,比较壳聚糖膜皮下植入与肌肉植入、Nafion膜肌肉植入的生物相容性。 结果与结论：壳聚糖膜的厚度、溶胀率、表观密度等理化参数可以通过浓度、铸膜液体积来控制;壳聚糖膜能生物降解,63 d皮下植入的降解率为(17.0±9.9)%,说明壳聚糖的体内降解速度较慢。壳聚糖膜皮下植入引起的炎症反应较肌肉植入重,63 d后形成的纤维包膜比肌肉植入要厚(P < 0.05);肌肉植入Nafion与壳聚糖膜引起材料周围纤维包膜厚度差异无显著性意义(P > 0.05),两者均在15 d以后趋于稳定。证明壳聚糖膜能生物降解,与Nafion膜均有较好的组织相容性。     

冻融联合优化化学法制备粗大去细胞同种异体神经

背景：异体神经移植修复神经缺损需要面对和解决的是宿主免疫排斥反应问题。因此,如何避免、减轻免疫排斥反应是同种异体神经移植获得成功的关键因素。 目的：探索新的异体神经预处理方法,清除犬周围神经中的许旺细胞和髓鞘,保留完整的基底膜,建立粗大异体神经移植物的预处理方法,获得去细胞异体神经移植物。 方法：取健康成年杂种犬游离双侧坐骨神经,以冻融联合优化化学法对神经进行预处理,光、电镜观察其结构特征,组织学染色及Western blot分析其成分。 结果与结论：预处理后的去细胞神经的延展性和神经外膜的弹韧性良好,许旺细胞和髓鞘被彻底清除,基底膜保留完整,去细胞神经为一没有细胞、髓鞘及其碎片的空的神经基膜管。结果表明该方法有效的清除了周围神经中主要抗原成分许旺细胞及髓鞘,并且保留了促神经再生的重要成分基底膜,可作为制备组织工程化神经较理想的方法。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

羊膜基质/聚丙烯复合补片的制备及其生物相容性评价

制备脱细胞羊膜基质, 运用物理方法将其复合于临床用聚丙烯补片, 以形成具有更好组织相容性的生物补片。通过细胞培养, 以聚丙烯膜为对照, 评价复合生物补片的细胞相容性;通过动物皮下植入实验, 以评价复合补片的组织相容性。结果表明, 复合补片羊膜基质表面上的成纤维细胞生长和形态均好于聚丙烯表面, 细胞增殖较快, 增殖率超过聚丙烯表面的50%。动物实验显示, 复合补片周围增生少, 无明显纤维包膜形成, 初期在植入周围有淋巴细胞等炎性细胞浸润, 随着羊膜逐渐降解, 4周时可见毛细血管新生;而对于聚丙烯材料, 植入处纤维包囊明显, 炎性情况也较严重。经羊膜基质复合的聚丙烯补片具有良好的细胞和组织相容性。     

脱细胞同种异体神经移植物的制备及成分分析

目的　探讨脱细胞同种异体神经移植的制备方法及其含有的成分。方法　采用组织工程化低渗脱细胞方法制备神经异体移植物 ,光电镜观察其结构特征 ;用免疫组织化学法和聚丙烯酰胺凝胶电泳法分析神经异体移植物的成分。结果　正常的 (天然的 )周围神经经脱细胞处理后 ,清除了雪旺细胞、神经外膜和束膜的细胞 ,以及神经纤维的髓鞘和轴突 ,保存了由雪旺细胞基底膜管以及神经外膜和束膜的细胞外基质构成的三维支架结构。成分分析结果显示 ,含有LN、FN以及生长相关蛋白GAP 4 3和 6 5kDa蛋白等促进和诱导受体损伤神经再生的重要成分。结论　脱细胞异体神经移植物含有诱导和促进神经再生的相关蛋白 ,有利于受损神经的再生。     

脱细胞膀胱黏膜下层支架材料的生物学评价

背景:脱细胞膀胱黏膜下层是一种天然的细胞外基质生物材料,主要由Ⅰ、Ⅲ型纤维胶原蛋白构成,是一种理想的生物支架材料。目的:脱细胞猪膀胱组织作为组织工程支架材料的生物学评价。方法:取适量健康猪膀胱置于PBS和叠氮钠的混合溶液,浸泡过夜,刮去膀胱黏膜层。用低渗、-80℃反复冻融、DNase和RNase混合液消化和NaOH裂解连续方法制备脱细胞膀胱黏膜下层。通过观察其组织学结构特征、DNA残留、细胞毒性、细胞黏附效果及皮下炎症反应等来综合评价脱细胞膀胱黏膜下层的生物相容性。结果与结论:正常猪的膀胱经改进的脱细胞方法处理后,绝大部分细胞组分被去处,细胞外基质结构保持完好。细胞毒性试验结果显示脱细胞膀胱黏膜下层细胞毒性为1级,DNA提取结果中未见有DNA残留,细胞黏附结果显示猪平滑肌细胞能在脱细胞支架上较好的贴附生长,SD大鼠皮下植入试验显示无明显的炎症反应。结果证实所制备的脱细胞膀胱黏膜下层结构保存完好,有良好的组织相容性,可能成为组织工程修复的替代材料。     

新型脊髓纳米组织工程支架的组织相容性

背景：纳米技术可改善脊髓组织工程生物材料的性能。 目的：分析新型脊髓纳米组织工程支架的组织相容性。 方法：以胶原为原料制备纤维定向排列及非定向排列的纳米纤维膜,培养及鉴定SD大鼠脊髓源性神经干细胞。将两种纳米纤维膜与SD乳鼠脊髓源性神经干细胞共培养,以正常培养的神经干细胞为对照,通过MTT实验检测纳米纤维膜的细胞相容性;以扫描电镜检测细胞在纳米纤维膜表面的黏附及增殖情况;将纳米纤维膜植入SD大鼠体内,通过组织学检查确定其降解情况及组织相容性;通过免疫组织化学实验确定神经干细胞在体内的存活及移动情况。 结果与结论：两种纳米纤维膜表面的神经干细胞黏附及增殖情况良好,MTT实验结果表明纳米纤维膜的细胞相容性佳,电镜结果表明细胞在纳米纤维膜表面黏附良好,增殖情况佳;在体内纳米纤维膜降解情况良好,组织相容性佳;BrdU标定的神经干细胞在SD大鼠体内存活并移动情况良好。结果表明新型纳米组织工程支架具有良好的细胞及组织相容性。     

脱细胞猪小肠黏膜下层的组织相容性

目的:研究脱细胞猪小肠黏膜下层(small intestinal submucosa,SIS)的组织相容性。方法:酶消化联合去垢剂制备脱细胞猪SIS。将其植入SD大鼠脊柱旁肌肉内,组织学检测植入后不同时间(1、2和4周)样品周围组织反应。另取乳胶植入,做为阳性对照。结果:经脱细胞处理后,猪SIS仅存纤维结构,未见细胞残留。植入后1周,脱细胞SIS形态完整,可见宿主细胞(成纤维细胞和炎性细胞)长入。2周时,可见脱细胞SIS断裂。至4周时,脱细胞SIS降解,新生细胞外基质形成,并可见毛细血管样结构。随植入时间的延长,炎性细胞逐渐减少。在不同时间点,乳胶均可完整取出,未见宿主细胞长入,其周围形成纤维囊。结论:提示脱细胞猪SIS具有良好的组织相容性。     

脱细胞血管基质制备及体内外生物相容性

背景:利用脱细胞血管基质作为血管支架具有以下优点:脱细胞血管基质保留了自然血管的复杂三维结构;脱细胞基质表面的生长因子和结构域有利于细胞的黏附和浸润。目的:制备脱细胞血管基质并对其体内外生物相容性进行评价。方法:采用胰蛋白酶、Triton X-100逐步处理猪颈动脉制备脱细胞血管基质。采用皮下植入实验、急性毒性实验和体外细胞毒性实验等评价其生物相容性。结果与结论:脱细胞基质材料具有良好的化学稳定性,未释放对红细胞产生破坏溶解作用的有害元素,未引起急性溶血反应,对细胞的生长无毒性影响。脱细胞基质材料在动物体内植入后早期有较多炎性细胞浸润,到实验观察的后期无明显炎性细胞浸润,脱细胞基质内可见成纤维细胞。另外,脱细胞基质材料对周边组织未产生毒性作用,伤口Ⅰ期愈合。同时组织学切片显示:支架材料与周边组织相容性好,未产生排斥反应。说明脱细胞基质材料在动物体内具有很好的生物相容性。     

IKVAV多肽生物活性水凝胶修复大鼠脑损伤

目的探索利用组织工程技术修复中枢神经损伤的可行性。方法人工合成PHPMA水凝胶材料,在其表面接枝具有神经生长活性的IKVAV肽段,检测分析性状后植入大鼠大脑皮层,于术后不同时期取脑切片,组织化学染色观察植入部位细胞反应;GFAP免疫组化检测星形胶质细胞在材料中的分布;Glees镀银染色显示移植物中神经纤维生长情况,DAB血管染色法观察新生血管的长入。结果制备的三维活性PHPMA水凝胶植入鼠脑皮层后能与周围组织良好整合。术后6周材料周围及内部均有细胞浸润,GFAP阳性细胞在材料内分布均匀;12周后材料内部可见新生血管;18周后材料中心有神经纤维长入。结论活性PHPMA水凝胶具有良好的脑组织相容性,植入脑缺损部位有助于细胞迁移、血管发生及神经轴突生长。     

去细胞坐骨神经天然支架的成分分析

背景：目前周围神经去细胞的方法较多,应用较多的是化学萃取法和液氮冻融法,但是化学萃取法耗时较长,而液氮冻融法则去除细胞不彻底。作者在查阅大量文献和多次实践的基础上,最终确定了冻融+化学萃取+机械振荡的优化法对坐骨神经进行脱细胞处理。 目的：对优化法制备的去细胞坐骨神经天然支架进行形态学分析,并对其成分进行鉴定。 方法：取Wistar大鼠双侧坐骨神经,随机分为2组,对照组不做特殊处理,实验组用冻融+化学萃取+机械振荡的优化法进行脱细胞处理。 结果与结论：用优化法制备的去细胞坐骨神经天然支架呈三维管状立体结构,许旺细胞、髓鞘及轴突去除完全。经免疫组化鉴定,Ⅰ型、Ⅲ型胶原和层粘连蛋白等细胞外基质主要成分得到保留。     

Collagen nerve conduits--assessment of biocompatibility and axonal regeneration

Bridging of nerve gaps is still a major problem in peripheral nerve surgery. Alternatively to autologous nerve grafts tissue engineering of peripheral nerves focuses on biocompatible conduits to reconstruct nerves. Such non-neural conduits fail to support regeneration over larger gaps due to lacking viable Schwann cells that promote regeneration by producing growth factors and cell guiding molecules. This problem may be overcome by implantation of cultivated Schwann cells into suitable scaffolds. In the present experiments we tested a collagen type I/III tube as a potential nerve guiding matrix. Revascularization, tolerance and Schwann cell settlement were evaluated by light, fluorescence and scanning electron microscopy after different implantation times. The conduits were completely revascularized between day 5 and 7 post-operatively and well integrated into the host tissue. Implanted Schwann cells adhered, survived and proliferated on the inner surface of the conduits. Nevertheless, bridging a 2 cm gap of the sciatic nerve of adult Wistar rats with these collagen/Schwann cell conduits led to a disappointing regeneration compared to controls with autologous grafts. From these results, we conclude that a sufficient biocompatibility of bioartificial nerve conduits is a necessary prerequisite, however, it remains only one of several parameters important for peripheral nerve regeneration.     

三种不同方法制备去细胞神经支架的比较

背景：去细胞异体神经移植物具有完全仿真的三维结构,为细胞附着提供了较理想的三维空间,是目前所有人工合成材料所无法比拟的。 目的：对比分析液氮冷冻法、化学法及改良法3种方法对大鼠坐骨神经去细胞化的优劣。 方法：切取SD大鼠10 mm坐骨神经,分别以液氮冻融法、TritonX-100处理化学去细胞法、改良法处理的化学去细胞法分别制作去细胞神经支架,行苏木精-伊红染色,并在扫描电镜下观察支架超微结构。 结果与结论：液氮冻融法去细胞支架内有较多许旺细胞残留,有较多轴突及髓鞘,基膜完整,基膜管欠通畅;TritonX-100处理化学法去细胞神经支架内无明显许旺细胞,管内未见明显轴突及髓鞘残留,基膜完整,基膜管通畅;改良法处理的化学去细胞神经支架内许旺细胞及轴突、髓鞘成分几乎完全消失,去细胞效果更好,基膜完整,基膜管更通畅。表明TritonX-100处理化学去细胞法、改良法处理的化学去细胞法脱细胞效果较好,其中以改良法处理的化学去细胞法最好。     

Peripheral Nerve Regeneration and Electrophysiological Recovery with CIP-Treated Allogeneic Acellular Nerves

Acellular nerve grafts are a desirable alternative to autografts, both because the source of acellular nerves is potentially unlimited and because they have the same matrix structure as natural nerves, which would facilitate axon growth from the defective nerve stump. Although some acellular nerves have been developed, most of them were studied in isogenic transplantation models and evaluated only by histological observation. In the present study, novel allogeneic acellular nerves prepared using the cold isostatic pressuring (CIP) method were developed and assessed as a potential substitute for autografts. The host immune response to acellular nerves and fresh nerves was analyzed using Lewis rats as donors and SD rats as recipients, which is the allogeneic transplantation model, by subcutaneous implantation for one month. In addition, sciatic nerve transplantation into a 10-mm nerve gap was carried out using the same model, and the axonal growth in acellular nerve transplantation was evaluated histologically and electrophysiologically, and compared with that of axons in the autograft transplant area. The subcutaneously implanted acellular nerves contained more macrophages and less vasculature than the allogeneic fresh nerves. In spite of these results of the subcutaneous implantation, Schwann cell infiltration in the graft transplanted into the sciatic nerve gap was observed after the short-term transplantation. The myogenic potential, which was measured as an index of electrophysiological function in acellular nerve transplantation, was also recovered in the long-term transplantation. Our results indicate that the acellular nerves developed herein have the potential to support nerve regeneration and might be useful as an alternative to autografts.     

Revascularization of tissue-engineered nerve grafts and invasion of macrophages.

Nonneural derived nerve conduits fail to support regeneration over larger gaps due to lacking viable Schwann cells. Thus, tissue engineering of nerves is focusing on implantation of viable Schwann cells into suitable scaffolds. We established grafts made from acellular muscles and veins, respectively, seeded with cultured Schwann cells. As timing of revascularization is crucial to determine Schwann cell survival and depending axonal regeneration we studied establishment of vascular architecture in a rat sciatic nerve model (2-cm gap) after 3, 5, 7, and 10 days postoperatively, using albumin bound Evans blue. Additionally, macrophage recruitment was immunohistochemically assessed. Engineered grafts showed a delayed revascularization, starting between day 5 and 7 in comparison to normal autografts, that revascularized by day 3. Macrophage recruitment in autologous nerve grafts was evident by day 3. The engineered groups revealed no macrophage invasion until day 7. As Schwann cells survive up to 7 days in autologous grafts without blood supply, depending purely on diffusion, establishment of vascular structure between day 5 and 7 is rapid enough to support Schwann cell survival in engineered grafts. As these grafts are lacking Wallerian degeneration delayed macrophage invasion may not impair degeneration-dependent regeneration, but presence of macrophage derived or induced growth factors may be decreased.     

人发角蛋白丝束修复大鼠坐骨神经损伤的实验研究

目的：观察人发角蛋白(HHK)丝束桥接体诱导坐骨神经再生过程中形态字变化：方法：制备坐骨神经损伤SD大鼠动物模型，分别植入HHK或HHK 胶原屏障膜，术后2d、2、3、6、9、12周行组织学观察。结果：术后2d到2周，断端的施万细胞去分化，沿着HHK束表面纵向分裂增殖。术后3周HHK开始降解，施万细胞大量增生：HHK周围有很多巨噬细胞和多核巨细胞，并出现轴突和大量微血管：术后6周，HHK丝周围可见大量新生的神经纤维。术后9周，HHK降解显著，有明显的神经外膜和束膜。术后12周，HHK完全降解，其部位被新的神经纤维取代。HHK丝束 胶原膜屏障膜组与HHK丝束组没有明显区别。结论：HHK具有良好的桥接作用，坐骨神经沿着HHK再生时在其外周就能由微血管和结缔组织形成一屏障膜，无需外加屏障膜。     

Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve

Summary We have conducted experiments in the adult rat visual system to assess the relative importance of an absence of trophic factors versus the presence of putative growth inhibitory molecules for the failure of regeneration of CNS axons after injury. The experiments comprised three groups of animals in which all optic nerves were crushed intra-orbitally: an optic nerve crush group had a sham implant-operation on the eye; the other two groups had peripheral nerve tissue introduced into the vitreous body; in an acellular peripheral nerve group, a frozen/thawed teased sciatic nerve segment was grafted, and in a cellular peripheral nerve group, a predegenerate teased segment of sciatic nerve was implanted. The rats were left for 20 days and their optic nerves and retinae prepared for immunohistochemical examination of both the reaction to injury of axons and glia in the nerve and also the viability of Schwann cells in the grafts. Anterograde axon tracing with rhodamine-B provided unequivocal qualitative evidence of regeneration in each group, and retrograde HRP tracing gave a measure of the numbers of axons growing across the lesion by counting HRP filled retinal ganglion cells in retinal whole mounts after HRP injection into the optic nerve distal to the lesion. No fibres crossed the lesion in the optic nerve crush group and dense scar tissue was formed in the wound site. GAP-43-positive and rhodamine-B filled axons in the acellular peripheral nerve and cellular peripheral nerve groups traversed the lesion and grew distally. There were greater numbers of regenerating fibres in the cellular peripheral nerve compared to the acellular peripheral nerve group. In the former, 0.6–10% of the retinal ganglion cell population regenerated axons at least 3–4 mm into the distal segment. In both the acellular peripheral nerve and cellular peripheral nerve groups, no basal lamina was deposited in the wound. Thus, although astrocyte processes were stacked around the lesion edge, a glia limitans was not formed. These observations suggest that regenerating fibres may interfere with scarring. Viable Schwann cells were found in the vitreal grafts in the cellular peripheral nerve group only, supporting the proposition that Schwann cell derived trophic molecules secreted into the vitreous stimulated retinal ganglion cell axon growth in the severed optic nerve. The regenerative response of acellular peripheral nerve-transplanted animals was probably promoted by residual amounts of these molecules present in the transplants after freezing and thawing. In the optic nerves of all groups the astrocyte, microglia and macrophage reactions were similar. Moreover, oligodendrocytes and myelin debris were also uniformly distributed throughout all nerves. Our results suggest either that none of the above elements inhibit CNS regeneration after perineuronal neurotrophin delivery, or that the latter, in addition to mobilising and maintaining regeneration, also down regulates the expression of axonal growth cone-located receptors, which normally mediate growth arrest by engaging putative growth inhibitory molecules of the CNS neuropil.     

组织工程化人工神经研究进展

利用各种神经导管可成功桥接修复短段周围神经缺损已为许多学者公认 ,但是 ,这些神经导管由于缺乏许旺细胞或内部支架来支持、促进神经再生轴突长距离生长 ,因此不能有效修复长段周围神经缺损[1,2 ] 。应用组织工程技术构建神经导管 ,为修复长段周围神经缺损提供了新的方法和思路。这项研究的核心是模拟周围神经天然结构 ,将许旺细胞与生物支架材料有机结合成为类似Ｂ櫣ｎｇｎｅｒ带的结构 ,为再生神经提供良好的生长环境 ,充分发挥许旺细胞对再生神经的营养 ,诱导作用 ,从而促进神经的再生。组织工程化人工神经的主要内容是将经体外培养扩…