糖尿病小鼠理想的胰岛移植部位

背景：胰岛移植到糖尿病小鼠不同部位影响胰岛移植成功率。 目的：比较糖尿病小鼠小网膜、肾被膜和腋窝3个部位胰岛移植效果,探索一个更理想的移植部位。 方法：应用淋巴细胞分离液分离纯化BALB/c小鼠胰岛;将胰岛移植到造模成功的糖尿病C57BL/6小鼠,按照不同的移植部位小网膜、肾被膜和腋窝进行对比观察。 结果与结论：分离纯化后获得高纯度胰岛,每只供体可获得胰岛(102±4)个,并具有良好生物活性;胰岛移植到糖尿病小鼠小网膜、肾被膜和腋窝3个部位后,均能降低血糖至正常范围,移植到小网膜与肾被膜的血糖值差异无显著性意义(P > 0.05),但均低于腋窝血糖值(P < 0.05)。移植后第7天,苏木精-伊红染色见小网膜部位移植胰岛形态基本完整,肾被膜部位移植胰岛形态不规则,腋窝部位移植物胰岛完全被破坏,伴炎症细胞浸润,小网膜和肾被膜降血糖效果优于腋窝。说明小网膜血供充足容量大,能够接纳较大体积的移植物,可能作为胰岛移植的理想部位。     

生物支架材料诱导脂肪来源干细胞成骨分化的最新热点

文题释义： 脂肪干细胞的优势：脂肪来源干细胞具有以下特点：高增殖能力和分泌活性;兼具多向分化潜能;通过其免疫调节能力,能够提高移植后的愈合效果;来源丰富,可在自体或异体上迅速提取,以上优势使其成为骨组织工程的理想种子细胞。 骨组织工程支架材料的分类：主要分为3类,无机材料、天然高分子材料、合成高分子材料,无机材料包括羟基磷灰石、磷酸三钙、生物活性玻璃、钛金属、镁金属,天然高分子材料包括胶原、丝素蛋白、壳聚糖,合成高分子材料包括聚己内酯、聚乳酸、聚乙醇酸及其共聚物-聚乳酸-羟基乙酸共聚物。 背景：脂肪来源干细胞获取便捷且具有显著的成骨分化能力,被认为是骨缺损修复的理想种子细胞。然而骨组织工程学的研究进展揭示,生物支架材料改性能够直接调控干细胞的成骨分化。 目的：综述能够调控脂肪来源干细胞成骨分化效果的各种生物支架材料。 方法：由第一作者通过检索中国知网、万方、维普、PubMed、Embase和Web of Science数据库2016年1月至2019年5月发表的相关文献,检索词为“脂肪干细胞,支架材料,成骨,金属,钛;Adipose derived stem cells,scaffold,osteogenic,metal,Ti”,最终选取符合标准的文献62篇。 结果与结论：用于骨组织工程的支架材料分为无机材料、天然高分子材料、合成高分子材料3类,无机材料包括羟基磷灰石、磷酸三钙、生物活性玻璃、钛金属、镁金属,天然高分子材料包括胶原、丝素蛋白、壳聚糖,合成高分子材料包括聚己内酯、聚乳酸、聚乙醇酸及其共聚物-聚乳酸-羟基乙酸共聚物。设计能与细胞相互作用以指导其生物反应和骨分化的材料研究一直层出不穷,但如何营造更安全、更合理、更贴近生物体内的细胞的生长微环境仍然面临着很多困难。对生物支架材料的改性能够直接调控干细胞的成骨分化,同时成骨诱导之外的血管化及植入后的感染也是需要关注的问题。 ORCID: 0000-0003-4520-3031(张圣敏) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

基于天然高分子材料的组织工程化皮肤支架材料

支架材料在组织工程中具有调控所种植细胞的组织化、细胞增殖和新组织形成的重要作用。文章就目前常用于组织工程化皮肤支架材料的天然高分子材料的分类、研究现状及前沿动态作了概述,对组织工程化皮肤支架材料目前存在的问题、研究的重点和热点作了归纳分析,并对其发展趋势进行了展望,预测了组织工程化皮肤的理想支架材料。     

大鼠胰岛细胞经门静脉肝内移植模型的建立

背景:由于肝脏既是胰岛素的作用部位,又是相对的免疫特惠区,排斥反应小,肝窦及其小静脉结构还有利于胰岛的居留和生长,可供移植容积大,有利于胰岛的生存,因此肝脏是一个较为理想的移植部位。目的:建立一种经门静脉胰岛细胞肝内移植治疗SD大鼠1型糖尿病的动物模型。方法:采用文献方法制备SD大鼠胰岛细胞。以腹腔注射链尿佐菌素诱导建立SD大鼠1型糖尿病模型,随机数字表法分为2组:实验组经门静脉主干穿刺输注SD大鼠胰岛1000胰岛当量1.5mL;对照组经门静脉主干穿刺输注无血清1640液1.5mL。术后未给予免疫抑制剂,观察两组大鼠出血量、血糖、胰岛素水平及肝脏组织形态学变化。结果与结论:所有大鼠均移植成活,门静脉穿刺一次成功率90%,出血量0.5mL。胰岛移植大鼠血糖降为正常的时间1～6(3.7±1.7)d,移植胰岛存活时间为2～15(8.4±4.1)d。术后2周内实验组大鼠空腹血清胰岛素水平明显高于对照组(P0.05,P0.01)。病理结果证实,移植大鼠肝细胞形态、肝小叶结构均正常,肝实质未见坏死灶,血管内无血栓形成;门静脉主干未见狭窄,亦未发生感染,说明胰岛细胞在肝窦内存活并能发挥功能。证实大鼠胰岛经门静脉主干穿刺输注肝内移植是胰岛移植基础研究的理想模型。     

人工骨材料修复骨缺损:多种复合后的生物学与力学特征

背景:当前应用广泛的骨缺损修复方式主要有自体骨移植、异体骨移植和人工骨移植,人工骨移植可避免患者自身取材的痛苦和并发症及同种异体骨免疫排斥的危险。目的:综述近几年来国内外常用的几种人工骨材料的研究进展情况。方法:应用计算机检索2007至2013年中国期刊全文数据库、PubMed数据库、中国生物医学文献数据库、FMJS数据库中有关人工骨材料的研究。中文检索词为"人工骨,材料,骨缺损,骨移植",英文检索词为"artificial bone,material,bone defect,bone graft"。结果与结论:目前常用的人工骨材料有天然高分子材料、人工合成高分子、无机材料、纳米材料。理想的骨移植替代物应该具备以下条件:良好的生物相容性;局部形成微酸性生物环境;利于血管和成骨细胞长入;可完全生物降解;兼具骨生成性、骨传导性和骨诱导性。生物蛋白胶降解及组织相容性较好,且可塑形,但强度较差,以后研究方向主要为加强其强度。人工合成及无机材料强度及支架作用较好,但组织相容性较差,提高其组织相容性很重要。纳米材料为较合适的人工骨材料。目前应用于骨组织工程研究的人工骨材料均有各自的长处和不足,尚不能同时满足理想的骨组织工程细胞外基质材料各方面的要求,而且在生物学和力学特性上与天然骨有差异,故将多种类型材料复合并进行结构和功能修饰,是研制理想骨缺损修复材料的方向。     

高机能肝细胞培养支架——海藻酸/半乳糖化壳聚糖海绵体的开发

重症肝功能衰竭是导致死亡的主要疾病之一。目前,肝移植是其唯一有效的临床治疗手段,但供体不足及伦理等问题,使不能及时得到治疗而死亡的患者正在逐年增多,随着组织工程学的发展,人们都寄希望于生物型人工肝脏。笔者正在研究开发三维立体肝细胞培养支架材料,探索体外肝组织再构筑技术和方法,实现高效生物型人工肝脏。笔者研究根据半乳糖残基与肝实质细胞表面的脱唾液酸糖蛋白受体的特异性结合作用,利用半乳糖残基化学改性天然高分子多糖壳聚糖,改善壳聚糖的肝实质细胞亲和性,并制成三维多孔海藻酸／半乳糖化壳聚糖海绵体肝细胞培养支架。从培养细胞形态学和肝功能等生化学分析角度考察了用该支架培养肝实质细胞的聚集体形成及肝功能等。     

生物可降解性消化道支架的应用及其生物相容性

背景：生物可降解支架可在消化道管腔内短期成形,具有良好的生物相容性,随后完全降解,并可以根据临床需要调节支架降解时间,避免了永久性支架的并发症。 目的：评价不同材料制成的生物可降解性消化道支架的应用、相容性评价以及研究进展。 方法：以 “生物可降解,消化道,支架,相容性” 为关键词,采用电子检索方式在万方数据库中检索1999-01/2009-12有关生物可降解性消化道支架的研究。排除重复研究、普通综述或Meta分析类文章,筛选纳入22篇文献进行评价。 结果与结论：可降解支架在消化道疾病中的应用已经显示其有效的扩张性及临床安全性等。可降解材料大多是高分子材料,包括天然可降解高分子、微生物合成高分子材料和合成可降解高分子3类。天然可降解高分子大多是多糖类。天然可降解高分子一般生物相容性良好,但是力学性能较差。微生物合成高分子材料目前研究及应用尚较少。合成高分子种类比较多,常见的有聚丙交酯、聚己内酯、聚乙二醇等。合成高分子优点在于可以比较灵活的设计分子结构,通过发展共聚物、共混物来得到不同性质的材料。可降解支架可以解决良恶性狭窄的再通及瘘口的封堵等,但可降解支架在消化道系统中应用的效力还需要未来进行大量的研究工作来评估。     

人工角膜的研究进展

详细介绍了人工角膜（keratoprosthesis,Kps)的研究情况，描述了当前各种类型的人工角膜及其存在的缺点，并讨论了理想的人工角膜及组织工程化人工角膜应达到的要求。近年来的研究主要集中于运用多孔高分子材料作为中央-周边型人工角膜的周边支架材料，改善材料的生物结合能力。     

三种大鼠骨髓间充质干细胞源性人工肝组织的构建与评价

背景:人工肝组织有可能为治疗急性肝衰竭提供一种新的选择,但人工肝组织的构建存在着种子细胞、支架、培养模式及对人工肝组织营养获取调控等各方面的难题。目的:体外构建3种人工肝组织,筛选最佳人工肝组织以供移植用。方法:纯化、扩增并诱导分化骨髓间充质干细胞为肝样细胞,在Transwell培养体系中分别与去细胞羊膜、DACRON PATCH心血管外科补片、生物外科补片3种支架结合构建3种人工肝组织,培养3 d后进行形态学和功能学检测。结果与结论:密度梯度离心结合贴壁法可获得较高纯度的大鼠骨髓间充质干细胞,并在体外转分化为肝样细胞。在构建的3种人工肝组织中,肝样细胞与生物外科补片支架材料可获得最大程度结合,并具有更强的尿素合成能力和白蛋白分泌功能,为治疗急性肝衰竭提供了实验基础。     

几丁聚糖在组织工程中的应用

支架材料作为组织工程的生物学植入替代物,对细胞移植与引导新组织生长有重要的作用.几丁聚糖可制成无毒性、无刺激性、生物相容性和生物可降解性良好的生物医用材料,在人工皮肤、骨修复材料、手术缝线等方面已广泛应用.本文分析了纯几丁聚糖支架结构和它与其他天然或合成材料复合后的支架结构的物理、化学性质及其独特的生物学功能,同时还进一步介绍了其应用的范例并探讨了发展前景.     

Extrahepatic islet transplantation with microporous polymer scaffolds in syngeneic mouse and allogeneic porcine models

Intraportal transplantation of islets has successfully treated select patients with type 1 diabetes. However, intravascular infusion and the intrahepatic site contribute to significant early and late islet loss, yet a clinical alternative has remained elusive. We investigated non-encapsulating, porous, biodegradable polymer scaffolds as a vehicle for islet transplantation into extrahepatic sites, using syngeneic mouse and allogeneic porcine models. Scaffold architecture was modified to enhance cell infiltration leading to revascularization of the islets with minimal inflammatory response. In the diabetic mouse model, 125 islets seeded on scaffolds implanted into the epididymal fat pad restored normoglycemia within an average of 1.95 days and transplantation of only 75 islets required 12.1 days. Increasing the pore size to increase islet-islet interactions did not significantly impact islet function. The porcine model was used to investigate early islet engraftment. Increasing the islet seeding density led to a greater mass of engrafted islets, though the efficiency of islet survival decreased. Transplantation into the porcine omentum provided greater islet engraftment than the gastric submucosa. These results demonstrate scaffolds support murine islet transplantation with high efficiency, and feasibility studies in large animals support continued pre-clinical studies with scaffolds as a platform to control the transplant microenvironment.     

Development of an ectopic site for islet transplantation, using biodegradable scaffolds

Clinical islet transplantation in liver has achieved normoglycemia. However, this site may not be ideal for islet survival. To create a more optimal site for islet transplantation, we have developed a construct with biodegradable scaffolds. Islets were seeded in scaffolds and transplanted into the epididymal fat pad of diabetic BALB/c mice. Controls included islets transplanted underneath the kidney capsule or into the fat pad without scaffolds. All animals with islets in scaffolds or the kidney became normoglycemic and maintained this metabolic state. When islets were transplanted without scaffolds the time to achieve normoglycemia was significantly increased and less than 45% of mice survived. An oral glucose tolerance test was performed on the scaffold and kidney groups with similar blood glucose levels and area under the curve values between the groups. Grafts were removed at more than 100 days posttransplantation and all animals became hyperglycemic. There was no significant difference in insulin content between the grafts and all grafts were well vascularized with insulin-positive beta cells. Therefore, islets in scaffolds function and restore diabetic animals to normoglycemic levels, similar to islets transplanted underneath the kidney capsule, suggesting scaffolds can be used to create a site for islet transplantation.     

Permanent protection of PLG scaffold transplanted allogeneic islet grafts in diabetic mice treated with ECDI-fixed donor splenocyte infusions

Allogeneic islet cell transplantation is a promising treatment for human type 1 diabetes. Currently, human islets are transplanted via intra-portal infusions. While successful, it leads to significant early islet attrition from instant blood-mediated inflammatory reaction. An extra-hepatic site was established by transplanting islet-loaded microporous poly(lactide-co-glycolide) (PLG) scaffolds into the epididymal fat pad in syngeneic islet transplant models. This study examined this technology in allogeneic islet transplantation and determined whether transplant tolerance could be effectively induced to protect PLG scaffold transplanted allogeneic islets. The efficacy of an established tolerance induction strategy using donor splenocytes treated with ethylcarbodiimide(ECDI) was tested. ECDI-fixed donor splenocytes were infused 7 days before and 1 day after islet transplantation. Immediate normoglycemia was restored, and treated mice maintained indefinite normoglycemia whereas untreated mice rejected islet grafts within 20 days of transplantation. Interestingly, efficacy of tolerance induction was superior in PLG scaffold compared with intra-portal transplanted islets. Protection of PLG scaffold islet allografts was associated with several mechanisms of immune regulation. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that does not impair tolerance induction. This approach of combining tolerance induction with scaffold islet transplantation has potential therapeutic implications for human islet transplantation.     

Islet Mass Plays a Critical Role in Initiation, but not Progression, of the Diabetogenic Process in NOD Mice

Pancreatectomy (90%) at a preinsulitis age (7 weeks) protects NOD mice from diabetes while pancreatectomy at a mid-insulitis age (13 weeks) has no such protective effect. The present study examined the effects of islet transplantation in pancreatectomized diabetes-free NOD mice. Transplantation of syngeneic NOD islets as well as allogeneic C3H/He and C57BL/6 islets 3 weeks after pancreatectomy-induced spontaneous diabetes whereas transplantation of xenogeneic Sprague-Dawley rat islets or allogeneic C3H/He skin failed to induce diabetes, demonstrating that the diabetogenic antigen(s) of NOD islets is also expressed by islets of diabetes-resistant mouse strains but not by xenogeneic rat islets. Removal of NOD islet grafts by nephrectomy 7-14 days after transplantation had no effect on the subsequent chronic development of diabetes, while graft removal 3 days after transplantation completely abolished the diabetogenic effect of islet transplantation. Thus, activation of the diabetogenic response by islet isografting takes less than 7 days and the continuous presence of a large islet mass is not required for progression to diabetes. While islet transplantation at 10 and 15 weeks of age caused diabetes, delayed islet transplantation at 23 and 35 weeks of age failed to induce diabetes in pancreatectomized diabetes-free NOD mice, suggesting that initiation of the diabetogenic autoimmune process must take place within a certain window of time. The pancreatectomy/islet transplantation model is excellent for studying the immunological events surrounding activation and progression of the diabetogenic autoimmune process and for identifying the diabetogenic islet antigen(s).     

Islet-encapsulation in ultra-thin layer-by-layer membranes of poly(vinyl alcohol) anchored to poly(ethylene glycol)-lipids in the cell membrane

The microencapsulation of islets of Langerhans (islets) in a semipermeable membrane, i.e., the creation of a bioartificial pancreas, has been studied as a safe and simple technique for islet transplantation without the need for immunosuppressive therapy. The total volume of the implant tends to increase after enclosure of the islets in the semipermeable membrane, which limits transplantation sites. Thus, ultra-thin membranes are required for clinical applications. Here, we propose a novel method to encapsulate islets in an ultra-thin membrane of poly(vinyl alcohol) (PVA) anchored to a poly(ethylene glycol) (PEG)-phospholipid conjugate bearing a maleimide group (Mal-PEG-lipids, PEG Mw: 5000) in the cell membranes of islets. When Mal-PEG-lipids were added to an islet suspension, they spontaneously formed a thin layer on cells of the outer layer of islets. The PEG-lipid layer on the islets was covered by a PVA monolayer, and the PVA membrane was further reinforced by using the layer-by-layer method with thiol/disulfide exchange reactions. No practical volume increase in islets was observed after microencapsulation by this method. In addition, encapsulation of the islet surface in PVA membranes did not impair insulin release in response to glucose stimulation.     

Encapsulation of islets with ultra-thin polyion complex membrane through poly(ethylene glycol)-phospholipids anchored to cell membrane

The microencapsulation of islets of Langerhans (islets) has been studied as a safe and simple technique for islet transplantation without the need for immuno-suppressive therapy. However, thinner membranes are desired, because the increased total volume of the implant led to limited transplantation sites. Here, we propose a novel method for microencapsulation by polyion complex membrane formation on islets. Amino group-terminated poly(ethylene glycol)-conjugated phospholipids (PEG-lipids, M(w): 5000) spontaneously formed a thin layer on cells existing in the outer layer of islets when they were added to islet suspension. This layer-by-layer membrane could be further formed on the PEG-lipid layer through polyion complex formation between amino groups at the end of PEG chains, sodium alginate and poly(l-lysine). Islets could be microencapsulated by this method without volume increase. Encapsulation of the islet surface with PEG-lipids and polyion complex membranes did not impair the insulin release function in response to glucose stimulation. Our method is promising to encapsulate islets without affecting cell viability or increasing volume.     

Ultrastructural evidence for blood microvessels devoid of an endothelial cell lining in transplanted pancreatic islets.

The aim of the present study was to investigate, at the ultrastructural level, the process of revascularization of freshly isolated islets or cultured islets after transplantation under the kidney capsule of syngeneic mice. Native islets in adult pancreases from mice, pigs, and humans contained only capillaries with fenestrated endothelium. However, the endothelial cell lining was disrupted in both freshly isolated and cultured mouse islets. Shortly after transplantation (6 weeks) approximately 80% of graft microvessels contained no endothelial cell lining. Similar data on microvessel morphology were found when fetal porcine islet-like cell clusters were implanted into athymic nude mice. Re-endothelialization was a slow process, with 25% of the microvessels still lacking endothelium 6 months after transplantation of cultured mouse islets or islet-like cell cluster. However, when freshly isolated mouse islets are used only 25% of microvessels within the islet graft lacked endothelium 6 weeks after implantation. We suggest that capillaries damaged during islet isolation may provide a preformed channel, serving as a scaffold for newly formed islet graft blood vessels. The presence of non-endothelialized microvessels, with an associated lack of barrier function, might make transplanted islets more prone to thrombosis or an attack by the immune system. This provides a tentative explanation for the increased vulnerability of islet grafts when compared with whole pancreas transplants.     

Islet film cures diabetes

Intraportal transplantation of islets is usually performed by a percutaneous, transhepatic catheterization technique for type 1 diabetic patients. We show here an easier method of islet implantation into the liver. An islet film was made using rat islet and was simply placed on the liver of a diabetic recipient rat. Histologic examinations revealed that the islets passed through the liver capsule within a week after the implantation, entered the liver parenchyma within 3 weeks, and were encircled with hepatocytes by day 222. Recipient rats were cured of diabetes; however nonimplanted control rats had high levels of blood glucose. The film-transplanted islets could provide adequate insulin to maintain stable blood glucose levels. This novel method requires little skill and no special instruments. This islet film approach may prove suitable for simple islet transplantation in clinical cases.     

Neutrophilic granulocytes are the predominant cell type infiltrating pancreatic islets in contact with ABO-compatible blood

The poor outcome of intraportal islet transplantation may be explained by the instant blood-mediated inflammatory reaction (IBMIR), characterized by islet entrapment in blood clots, leucocyte infiltration and disruption of islet morphology. Here we employ a newly developed in vitro system to identify the blood cells involved in this process. Islets were mixed with ABO-compatible blood in heparinized tubes and incubated for various times up to 6 h. Clots were analysed immunohistochemically for detection of platelets (CD41a), leucocytes/lymphocytes (CD11b), granulocytes (CD16, lysozyme), neutrophilic granulocytes (neutrophil elastase), eosinophilic granulocytes (NaCN + H(2)O(2)), macrophages (CD68), dendritic cells (CD209/DC-SIGN), B cells (CD20) and T cells (CD4, CD8). Platelets were rapidly deposited around the islets in contact with the blood, reaching a maximum by 30 min. The first neutrophilic granulocytes appeared in the islets after 15 min, increased at 1 h and peaked at 2 h. Small numbers of macrophages were found infiltrating the islets already after 5 min, with a slight increase over time. However, control stainings of cultured islets and pancreas biopsies identified these cells as being largely of donor origin. No T cells, B cells, dendritic cells or eosinophilic granulocytes were detected during the 6 h observation time. Neutrophilic granulocytes were identified as the main infiltrating blood cell in islets exposed to blood, implying that these cells play a key role in clinical islet transplantation. Because islets are known to be exquisitely susceptible to oxidative stress, development of drugs targeting neutrophilic cytotoxicity could markedly improve the outcome of islet transplantation.