Ⅰ型胶原修饰聚乳酸聚乙醇酸微球支架上骨髓间充质干细胞的黏附和成骨分化

背景：组织工程骨成骨功能终末细胞需要骨髓间充质干细胞在体外加以诱导或在体内以基因转染等技术加以诱导。 目的：研究Ⅰ型胶原修饰的聚乳酸聚乙醇酸微球支架上骨髓间充质干细胞黏附和成骨分化的能力。 方法：制备聚乳酸聚乙醇酸微球支架,分离纯化雌性SD大鼠骨髓间充质干细胞。将培养至第3代骨髓间充质干细胞与未经处理的聚乳酸聚乙醇酸微球及Ⅰ型胶原修饰的聚乳酸聚乙醇酸微球共同培养14 d,观察细胞在不同支架表面的黏附生长。 结果：扫描电镜及FDA-PI染色发现,骨髓间充质干细胞可在聚乳酸聚乙醇酸微球支架上生长,而与未修饰的聚乳酸聚乙醇酸微球相比骨髓间充质干细胞更容易在Ⅰ型胶原修饰的聚乳酸聚乙醇酸微球上黏附增殖。Ⅰ型胶原修饰的聚乳酸聚乙醇酸微球有利于骨髓间充质干细胞的黏附、增殖,并且有一定诱导干细胞成骨分化的能力。     

兔子宫内膜片层体外构建的实验研究

探索以扩增培养的子宫内膜细胞为种子细胞、以液态胶原为支架材料,构建组织工程化子宫内膜片层的可行性。体外分离并规模化扩增子宫内膜上皮细胞与基质细胞,以Ⅰ型液态鼠尾胶原为支架,按自然子宫内膜的结构在体外重建具有两层结构的组织工程化子宫内膜,体外培养14天时进行H.E.染色与免疫组化鉴定。H.E.染色与免疫组化染色结果表明：再造子宫内膜片层在胶原材料中能够较好地维持双层结构,上皮层呈CK18阳性,在某些部位还能够形成极化的柱状上皮。利用体外扩增培养的子宫内膜上皮细胞与基质细胞构建的组织工程化子宫内膜片层具有子宫内膜组织的双层结构与极化上皮特征。     

胶原海绵支架体外构建组织工程膀胱黏膜***◆

背景：组织工程膀胱黏膜层的构建在组织工程膀胱修复中占有重要的地位,但目前并没有最合理的构建方法。 目的：探讨胶原海绵支架复合猪膀胱尿路上皮细胞体外构建膀胱黏膜结构的可行性。 方法：刮取猪膀胱黏膜层后用酶消化方式进行猪膀胱尿路上皮细胞原代培养,并进行尿路上皮细胞标志物免疫荧光和RT-PCR鉴定。制备疏松多孔的胶原海绵支架材料,将第3代尿路上皮细胞接种在胶原海绵支架上,体外培养4~8 d后观察尿路上皮细胞和胶原海绵材料的复合情况。 结果与结论：原代培养的猪膀胱尿路上皮细胞呈“多角形”、“铺路石”样,以克隆团形式生长。免疫荧光鉴定AE1/AE3阳性,RT-RCR检测uroplakin-ⅠA、uroplakin-Ⅱ阳性。胶原海绵复合尿路上皮细胞体外培养4~8 d后,细胞在胶原海绵支架上生长良好,覆盖胶原材料的表面并长入材料内部,保持了尿路上皮细胞的特性。体外培养6 d时,尿路上皮细胞与胶原支架复合效果最好,同时细胞数量也最多。结果初步表明了胶原海绵复合膀胱尿路上皮细胞可以构建组织工程膀胱黏膜,且体外培养6 d为最佳时间点。     

不同接种方式对组织工程牙龈种子细胞黏附效果的影响

背景：如何在体外将支架材料和种子细胞高效地复合以构建组织工程牙周组织是目前牙周病治疗及牙周缺损修复研究的重要方向。 目的：比较传统沉淀接种法和胶原包裹接种法的细胞黏附状况,优化细胞接种方式。 方法：将一定浓度的犬牙龈成纤维细胞,分别采用传统沉淀接种法和胶原包裹接种法接种到聚乳酸-壳聚糖-明胶梯度孔径和均匀孔径支架上,通过细胞计数测定支架上贴附的细胞数量,计算其接种率,并进行对比分析。 结果与结论：采用胶原凝胶包裹接种法将细胞接种至均匀孔径支架和梯度孔径支架上,其接种率均明显高于传统的沉淀接种法(P < 0.01)。用胶原凝胶包裹种子细胞行细胞接种可以有效提高种子细胞的接种率,增加支架上的细胞初始浓度,可以选用胶原凝胶包裹细胞接种方式用于牙龈工程组织构建。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：     

京尼平交联Ⅰ型胶原蛋白材料与人脂肪间充质干细胞的生物相容性

背景:京尼平的低毒性具有一定的种属和细胞特异性,人脂肪间充质干细胞与京尼平交联的Ⅰ型胶原蛋白支架材料的生物相容性对于应用两者构建组织工程脂肪至关重要。目的:评估人脂肪间充质干细胞与京尼平交联的Ⅰ型胶原蛋白支架材料的生物相容性。方法:分离培养人脂肪间充质干细胞,传代培养至第3代,接种于京尼平交联的Ⅰ型胶原蛋白支架材料上。MTT法评估人脂肪间充质干细胞在支架材料上的黏附和增殖情况,支架材料对细胞的毒性作用;光镜和电镜分别观察人脂肪间充质干细胞在支架材料上的黏附和生长过程,以及细胞形态学变化。结果与结论:人脂肪间充质干细胞接种于支架材料后,能够迅速在材料上黏附、增殖,其黏附率平均为86.5%;光镜和扫描电镜显示人脂肪间充质干细胞在支架材料上黏附性良好,随着时间的推移,细胞逐渐增多,可以迁移进入支架内部并均匀分布。结果表明京尼平对细胞的毒性低,交联后的支架材料与人脂肪间充质干细胞具有良好的体外生物相容性。     

聚乙烯醇纺丝纤维编织在组织工程前交叉韧带支架材料中的应用

探讨聚乙烯醇(PVA)纺丝纤维编织用I型胶原胶(COL-I)表面修饰后,构建组织工程前交叉韧带(ACL)支架材料的可行性。用PVA纺丝编织成条束状支架材料,用NIH-3T3细胞株和人前交叉韧带(HACL)细胞分别种植到PVA和经COL-I修饰过的PVA纺丝纤维编织(PVA/COL)支架材料上,立体培养。通过扫描电子显微镜对比评价材料经I型胶原胶表面修饰前后NIH-3T3细胞株和HACL细胞在纺丝编织材料上细胞附增殖情况;在电子拉力机上测试PVA纺丝纤维编织支架材料的力学性能。结果表明:NIH-3T3细胞株和HACL细胞在PVA和PVA/COL支架材料表面和孔隙内黏附增殖并分泌细胞外基质,在PVA/COL支架材料上细胞黏附数量明显增多;COL-I可促进NIH-3T3细胞株分泌细胞外基质,但对HACL细胞作用不明显。拉力测试该编织材料柔韧性强,最大负荷、极限应力和弹性模量分别为52.61 N、14.96 Mpa和202.08 Mpa。说明I型胶原可促进NIH-3T3细胞株和HACL细胞在聚乙烯醇纺丝纤维编织支架材料表面和孔隙内黏附、增殖,可促进NIH-3T3细胞株分泌细胞外基质。聚乙烯醇纺丝纤维编织材料具有一定的...     

聚乳酸-聚乙醇酸可降解支架构建组织工程食管

目的:观察人食管上皮细胞在聚乳酸-聚乙醇酸(PLGA)支架上的贴附和生长情况,利用组织工程技术培养工程化人工食管.方法:制作PLGA三维细胞支架;分离培养成人食管上皮细胞,体外扩增后种植到PLGA支架上.在体外和裸鼠体内分别培养食管上皮细胞-支架复合物,分期终止培养,进行组织学染色、扫描电镜、细胞角蛋白免疫组织化学检测.结果:体外培养显示,人食管上皮细胞在支架材料上贴附生长良好,长期培养仍保持食管上皮细胞特性,裸鼠体内培养4周后可形成食管黏膜样组织.结论:PLGA支架适合食管上皮细胞黏附生长,可作为食管组织工程的细胞载体.     

静电纺丝纳米纤维膜作为骨骼肌组织工程支架材料的细胞相容性

背景：有报道以生物可降解的胶原盘或聚L-乳酸、聚羟基乙酸、聚L-乳酸/聚羟基乙酸共聚物等作为骨骼肌组织工程的支架材料,各有优缺点,不能完全满足骨骼肌组织工程的需要。 目的：探讨静电纺丝纳米纤维膜作为骨骼肌组织工程支架材料的可行性。 方法：制备7种不同组分的静电纺丝纳米纤维膜,以其浸提液为培养基培养第3代SD乳鼠成肌细胞,以含体积分数20%新生小牛血清的F12培养基培养的为对照。采用MTT法和扫描电镜检测成肌细胞在各组材料的黏附及生长情况。 结果与结论：各组分静电纺丝纳米纤维膜吸光度值与对照组间差异无显著性意义(P > 0.05)。各组分静电纺丝纳米纤维膜组成肌细胞黏附率差异有显著性意义(P < 0.05)。扫描电镜与上述结果一致。含70%聚乳酸+20%蚕丝蛋白+10%胶原组成电纺丝纳米纤维膜组可见大量成肌细胞黏附,呈梭形,两极伸展,排列规律,效果最好。其他各组细胞少,形态不规则,似衰退期成肌细胞。提示静电纺丝纳米纤维膜无细胞毒性,对成肌细胞的增殖无影响,成肌细胞能良好地黏附;以70%聚乳酸+ 20%蚕丝蛋白+10%胶原组分效果最佳。     

一种具有骨诱导活性的羟基磷灰石结晶的制备与研究

目的将骨形成蛋白2（BMP2）活性多肽与Ⅰ型胶原复合煅烧牛松质骨以制备出具有骨诱导活性的矿化羟基磷灰石结晶,并进一步探讨其生物学性能,为组织工程化人工骨提供实验基础。方法将BMP2活性多肽与Ⅰ型胶原复合煅烧牛松质骨作为实验组,以Ⅰ型胶原复合煅烧牛松质骨作为对照组。通过环境扫描电镜和能谱法以及x射线衍射实验观察能否生成羟基磷灰石结晶,同时对2组材料进行体外细胞培养,计算2组材料细胞黏附率,初步评价BMP2活性多肽引导Ⅰ型胶原复合煅烧牛松质骨表面矿化生成羟基磷灰石结晶的作用与能力。结果环境扫描电镜下可见实验组煅烧骨表面有矿化羟基磷灰石结晶生成,而对照组没有：能谱法对实验组矿化骨部分进行钙磷元素检测,质量比分别为16．23％、7．76％．原子百分数分别为6．34％、3．88％,X射线衍射检测证实矿化物的成分为磷灰石。大鼠骨髓基质干细胞分别与2组材料体外复合培养24h,实验组细胞黏附率明显高于对照组（a〈0．05）。结论BMP2活性多肽能引导Ⅰ型胶原复合煅烧骨表面矿化生成羟基磷灰石结晶,可以改善煅烧骨的骨诱导活性,提高细胞黏附性能,与煅烧骨复合后是一种理想的骨组织支架复合材料。     

复合胰岛素样生长因子1壳聚糖胶原支架与人牙周膜细胞的增殖

背景：胰岛素样生长因子1具有促进成纤维细胞有丝分裂的作用,同时具有促进牙周细胞生长、分化及合成细胞外基质的作用。 目的：观察负载胰岛素样生长因子1的壳聚糖胶原支架对于人牙周膜细胞增殖的作用。 方法：将人牙周膜细胞分别接种于负载胰岛素样生长因子1的壳聚糖胶原支架与普通胶原支架上,于接种的1 h、24 h及1周检测重组人转化生长因子β1的释放,于第1,7,28天检测两组细胞的黏附和增殖情况。 结果与结论：负载胰岛素样生长因子1的壳聚糖胶原支架组第1,24小时和第1周的重组人转化生长因子β1释放率明显低于普通胶原支架组(P < 0.01)。两组接种第1天的细胞黏附和增殖检测比较差异无显著性意义  (P > 0.05),负载胰岛素样生长因子1的壳聚糖胶原支架组接种第7,28天的细胞黏附和增殖情况优于普通胶原支架组(P < 0.01)。表明负载胰岛素样生长因子1的壳聚糖胶原支架可显著促进人牙周膜细胞的增殖。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：     

Extracellular matrix protein gene expression of bovine chondrocytes cultured on resorbable scaffolds

It has been demonstrated that using cultured chondrocytes that have been seeded onto various biomatrices can enhance the quality of the articular cartilage repair tissue. As tissue-engineering becomes increasingly more complex there is a need to understand how a specific biomaterial may influence gene expression. In this study several commonly used scaffold materials for cartilage tissue engineering were evaluated with respect to their influence on matrix gene expression. Primary cultures of bovine chondrocytes were established in monolayer then seeded onto polylactic acid (PLLA), polyglycolic acid (PGA), collagen matrices. The induction of collagen type I, collagen type II, and aggrecan was observed at various time points on these biomaterials using RT-PCR. The collagen type I gene was upregulated on collagen scaffolds throughout the culture period. PLLA and PGA showed initial induction followed by downregulation. Monolayer culture did not induce collagen I message. Collagen II genes were selectively upregulated after 72 and 96 h post seeding depending the scaffold material. Monolayer culture had strong induction of collagen II. The aggrecan protein was consistently expressed in all scaffold materials cultures and monolayer.     

New ureteral scaffold constructed with composite poly(L-lactic acid)-collagen and urothelial cells by new centrifugal seeding system

A tissue-engineered ureteral scaffold was constructed with composited poly L-lactic acid (PLLA)-collagen endoluminal stent and uroepithelial cells (UECs) using a new seeding system. The electrospun PLLA-collagen nanofibrous mesh was seeded efficiently with human ureteral epithelial cells using a modified centrifugal seeding device. The cellular nanofibrous mesh was then wound around a spiral endoluminal stent to form a cellular composited PLLA-collagen ureteral scaffold. The cellular ureteral scaffold was subcutaneously implanted into nude mice. Cell attachment, distribution, and viability in vitro were investigated along with the cell fate in vivo. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that scaffolds seeded with centrifugal method had higher cellular activity than scaffolds seeded with static method (p < 0.05), and the metabolic activity per cell had no significant differences between the two methods (p > 0.05). Histologic analysis showed that the entrapped UECs remained in the scaffolds after 2 wk of implantation. The results of the study indicated that the composited PLLA-collagen endoluminal stent could serve as alternative cell carrier for tissue engineering ureter. In addition, the new modified centrifugal seeding system allowed rapid homogeneous distribution of cells onto the nanofibrous mesh, which will be useful to ureteral reconstruction.     

组织工程支架材料在泌尿系统损伤修复中的应用

背景：泌尿外科组织工程技术为泌尿道组织、器官的微创修复和功能重建带来了新的希望。近年来,有关泌尿系组织工程支架材料的研究以及组织工程化组织如肾脏、输尿管、膀胱、尿道等的实验和临床研究报道层出不穷。 目的：总结近年组织工程支架材料在泌尿系统损伤修复中的应用情况。 方法：由作者应用计算机检索万方数据库(http://med.wanfangdata.com.cn/),检索时限1998-01/2010-12。检索关键词：输尿管;膀胱;尿道;支架材料;组织工程。纳入标准：①泌尿系组织工程所用生物支架材料制备方面的文章。②组织工程支架材料修复泌尿系统损伤方面的文章。排除标准：重复研究或较陈旧文献。根据纳入排除标准共保留相关文献41篇。 结果与结论：近年来,泌尿系组织工程支架材料的研究较为迅速,主要有天然细胞外基质,如胶原、蛋白多糖、糖蛋白等;细胞外基质衍生物,如膀胱细胞外基质、尿道细胞外基质、小肠黏膜下层等;合成聚合物,如聚羟基乙酸、聚乳酸等。这类材料具有最接近人体的网架结构、生物力学性能、部分活性因子,与上皮细胞和平滑肌细胞共同培养,有利于细胞黏附生长发挥生理功能。目前泌尿外科组织工程技术正处于从基础研究向临床应用转化的关键阶段。     

TGF-beta1 immobilized tri-co-polymer for articular cartilage tissue engineering

Tri-co-polymer with composition of gelatin, hyaluronic acid and chondroitin-6-sulfate has been used to mimic the cartilage extracellular matrix as scaffold for cartilage tissue engineering. In this study, we try to immobilize TGF-beta1 onto the surface of the tri-co-polymer sponge to suppress the undesired differentiation during the cartilage growth in vitro. The scaffold was synthesized with a pore size in a range of 300-500 microm. TGF-beta1 was immobilized on the surface of the tri-co-polymer scaffold with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Tri-co-polymer scaffolds with and without TGF-beta1 were seeded with porcine chondrocytes and cultured in a spinner flask for 2, 4, and 6 weeks. The chondrocytes were characterized by the methods of immunohistochemical staining with anti-type II collagen and anti-S-100 protein monoclonal antibody, and RT-PCR. After culturing for 4 weeks, chondrocytes showed positive in S-100 protein, Alcian blue, and type II collagen for the scaffold with TGF-beta1 immobilization. There is no observed type I and type X collagen expression in the scaffolds from the observation of RT-PCR. In addition, the scaffold without TGF-beta1 immobilization, type X collagen, can be detected after cultured for 2 weeks. Type I collagen was progressively expressed after 4 weeks. These results can conclude that TGF-beta1 immobilized scaffold can suppress chondrocytes toward prehypertrophic chondrocytes and osteolineage cells. The tri-co-polymer sponge with TGF-beta1 immobilization should have a great potential in cartilage tissue engineering in the future.     

聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架与兔软骨细胞的相容性

背景：传统的支架材料存在疏水性强,材料表面缺乏细胞表面受体特异结合的生物活性分子,材料的酸性降解产物易引发无菌性炎性反应等不足。根据仿生原理及软骨真实结构和构成来选择和制备组织工程软骨支架能够获得理想效果。 目的：制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架,评价其与兔膝关节软骨细胞的生物相容性,探讨其应用于关节软骨组织工程的可行性。 方法：采用二次相分离技术制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石复合支架,将第3代新西兰兔软骨细胞接种至复合支架材料上复合培养,倒置相差显微镜下观察细胞生长情况。细胞-支架复合物在24孔板中培养5 d以后,将其植入裸鼠皮下8周。 结果与结论：聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架材料经化学合成后,具有合适的三维多孔结构,孔隙率为90%,孔径300~450 μm;植入裸鼠皮下8周后Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色显示细胞-支架复合物中的软骨细胞可以像天然软骨一样分泌黏多糖和Ⅱ型胶原。提示生物材料聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石对于兔软骨细胞有良好的生物相容性,可作为生物组织工程支架。     

Macrochanneled bioactive ceramic scaffolds in combination with collagen hydrogel: a new tool for bone tissue engineering

New tissue-engineering tool for bone regeneration is described to facilitate homogeneous cell seeding and effective osteogenic development. Calcium phosphate (CaP) scaffolds with macrochanneled and well-defined pore structure was developed, however, a large portion of the cells seeded directly within the scaffold easily penetrates without good adhesion to the scaffold surface. To overcome this, a method was exploited to dispense cells evenly throughout the CaP scaffold using collagen hydrogel. Rat bone marrow-derived mesenchymal stem cells (MSCs) were mixed within a neutralized collagen solution, which was then infiltrated into the macrochanneled pore space and gelled to result in macrochanneled bioceramic scaffold combined with MSCs-hydrogel. MSCs contained within the hydrogel-CaP scaffolds were highly viable, with similar growth pattern to those in the collagen hydrogel. Cells seeded by this approach were initially almost double in number compared with those seeded directly onto the CaP scaffold and had an active proliferation more than 14 days. Assessments of the MSCs showed significantly higher alkaline phosphatase levels in the combined scaffold, which was accompanied by enhanced osteogenesis including the expression of genes [collagen type I, bone sialoprotein, and osteopontin (OPN)] and proteins (OPN and osteocalcin). Extracellular calcium was also elevated significantly in the combined scaffold compared to the CaP scaffold. In addition, mechanical strength of the constructs was improved significantly in the combined scaffold compared to the CaP scaffold. Based on these, the cell culturing and tissue engineering strategy within the macrochanneled bioactive ceramic scaffolds could be improved greatly by the combinatory approach of using collagen hydrogel.     

Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering

Hyaluronic acid (hyaluronan, HA) was immobilized onto the surface of macroporous biodegradable poly(D,L-lactic acid-co-glycolic acid) [PLGA] scaffolds to enhance the attachment, proliferation, and differentiation of chondrocytes for cartilage tissue engineering. The PLGA scaffolds were prepared by blending PLGA with varying amounts of amine-terminated PLGA-PEG di-block copolymer. They were fabricated by a gas foaming/salt leaching method. HA was chemically conjugated to the surface-exposed amine groups on the pre-fabricated scaffolds. The amount of surface exposed free amine groups was quantitatively determined by conjugating an amine-reactive fluorescent dye to the PLGA blend films. The extent of HA immobilization was also confirmed by measuring water contact angles. When chondrocytes were seeded within HA modified PLGA scaffolds, enhanced cellular attachment was observed compared to unmodified PLGA scaffolds. Furthermore, glycosaminoglycan and total collagen synthesis increased substantially for HA modified PLGA scaffolds. RT-PCR result and histological examination of the resultant cartilage tissue revealed that HA modified scaffolds excelled in inducing cartilage tissue formation in terms of collagen type II expression and tissue morphological characteristics.     

Mechanical properties and cellular proliferation of electrospun collagen type II

A suitable technique for articular cartilage repair and replacement is necessitated by inadequacies of current methods. Electrospinning has potential in cartilage repair by producing scaffolds with fiber diameters in the range of native extracellular matrix. Chondrocytes seeded onto such scaffolds may prefer this environment for differentiation and proliferation, thus approaching functional cartilage replacement tissue. Scaffolds of collagen type II were created by an electrospinning technique. Individual scaffold specimens were prepared and evaluated as uncross-linked, cross-linked, or crosslinked/seeded. Uncross-linked scaffolds contained a minimum and average fiber diameter of 70 and 496 nm, respectively, whereas cross-linked scaffolds possessed diameters of 140 nm and 1.46 microm. The average thickness for uncross-linked scaffolds was 0.20 +/- 0.02 mm and 0.52 +/- 0.07 mm for cross-linked scaffolds. Uniaxial tensile tests of uncross-linked scaffolds revealed an average tangent modulus, ultimate tensile strength, and ultimate strain of 172.5 +/- 36.1 MPa, 3.3 +/- 0.3 MPa, and 0.026 +/- 0.005 mm/mm, respectively. Scanning electron microscopy of cross-linked scaffolds cultured with chondrocytes demonstrated the ability of the cells to infiltrate the scaffold surface and interior. Electrospun collagen type II scaffolds produce a suitable environment for chondrocyte growth, which potentially establishes the foundation for the development of articular cartilage repair.