电纺左旋聚乳酸和左旋聚乳酸羟基磷灰石纳米纤维细胞相容性的比较研究

目的：用MTT法检测电纺左旋聚乳酸和左旋聚乳酸/羟基磷灰石PLLA和PLLA/HA纳米纤维材料,对人牙周膜细胞生长曲线的影响,评价两种材料的细胞相容性。方法：通过电纺技术制备PLLA、PLLA/HA纳米纤维材料,体外分离、培养并鉴定人牙周膜细胞,用MTT法检测人牙周膜细胞在两种材料上的生长曲线,评价两种材料的细胞相容性及其用于口腔组织工程的可能性。结果：成功分离、培养了人牙周膜细胞,通过免疫组织化学染色鉴定细胞来源于中胚层,为牙周膜细胞。电纺法制备的PLLA、PLLA/HA纳米纤维材料具有三维网状空间结构,与人牙周膜细胞共培养,MTT检测结果显示,细胞能够在材料上生长增殖,在第七天时PLLA/HA组细胞增殖好于PLLA组和对照组。结论：电纺PLLA和PLLA/HA纳米纤维材料生物相容性良好,PLLA/HA组较单纯PLLA组能够促进细胞的增殖,有望成为良好的新型口腔组织工程支架材料。     

PGS/PLLA共纺膜片的降解性能及生物相容性研究

目的:观察PGS/PLLA共纺膜片体外降解性及细胞相容性,探究其作为山羊颞下颌关节盘组织工程支架的可行性,为进一步动物实验奠定基础。方法:将PGS/PLLA共纺膜片浸泡在磷酸盐缓冲液中(pH=7.4),不同时间点取样,对材料微观形貌、pH、力学性能等进行检测;将关节盘细胞-PGS/PLLA支架复合培养,MTT法检测支架的细胞毒性及对关节盘细胞增殖影响;在14 d时,取出关节盘细胞-PGS/PLLA复合物,行HE、番红O、免疫组化染色。结果:PGS/PLLA支架降解性能适宜;MTT结果示,支架无毒性,细胞增殖良好。结论:颞下颌关节盘细胞在支架上粘附良好、增殖明显、代谢活跃,PGS/PLLA支架有望作为颞下颌关节盘组织工程支架。     

鹿角粉复合支架与纳米级羟基磷灰石复合支架的性能比较

目的探索不同胶体材料结合鹿角粉和纳米级羟基磷灰石(n-HA)支架的理化、生物性能。方法取鹿角粉和纳米级羟基磷灰石,分别与15%聚乙烯醇(PVA)、5%丝素蛋白液(SF)、15%PVA和5%丝素蛋白液(4∶1)混合,制备鹿角粉/PVA支架、鹿角粉/SF支架、鹿角粉/PVA/SF支架、n-HA/PVA支架、n-HA/SF支架、n-HA/PVA/SF支架,检测其抗溶解性能、抗压缩力、抗剪切力及支架细胞毒性。结果鹿角粉/SF支架、n-HA/SF支架溶解变形程度为Ⅲ级,其余4组为Ⅱ级。鹿角粉/SF支架、n-HA/SF支架抗剪切力、抗压缩力均较差,鹿角粉/PVA支架、鹿角粉/PVA/SF支架分别与n-HA/PVA支架、nHA/PVA/SF支架抗剪切性能相似,鹿角粉/PVA支架、鹿角粉/PVA/SF支架抗压缩性能分别优于n-HA/PVA支架、n-HA/PVA/SF支架。鹿角粉/PVA支架、鹿角粉/SF支架、鹿角粉/PVA/SF支架细胞相容性优于n-HA/PVA支架、n-HA/SF支架、n HA/PVA/SF支架。结论鹿角粉复合支架具有良好的机械性能及细胞相容性,可以作为异种骨支架材料研究的新方向。单纯丝素作为水凝胶复合支架抗溶解性不佳,机械性能差,无法满足骨组织工程支架要求。     

聚乳酸-纳米羟基磷灰石-丝素蛋白引导骨再生膜的制备及表面特征

目的制备一种新型的聚乳酸-纳米羟基磷灰石-丝素蛋白(polylactic acid/nano-hydroxyapatite/silk fi-broin,PLLA/n-HA/SF)纳米纤维引导骨组织再生膜,初步探讨其作为引导骨再生屏障膜的可行性。方法采用热致相分离法制备PLLA/n-HA/SF纳米纤维复合膜,通过扫描电镜对其形貌进行研究,利用傅里叶红外光谱仪分析纳米羟基磷灰石和丝素蛋白的加入对所制备的复合膜结构的影响,并计算其孔隙率。结果扫描电镜显示该复合膜具有纳米纤维状三维网络结构。纤维直径约为160～320nm,孔径大小为1～4μm,丝素蛋白和纳米羟基磷灰石在复合膜中分散均匀。红外光谱结果表明PLLA/n-HA/SF复合膜具备聚乳酸、纳米羟基磷灰石及丝素蛋白的特征峰表现,3种组分之间结合良好。该膜的孔隙率为92.600%。结论热效相分离法制备的PLLA/n-HA/SF复合膜具有良好的微观结构和较高的孔隙率。     

胶原凝胶复合快速成型材料异位成软骨的研究

目的:通过凝胶包埋软骨细胞复合于快速成型(RP)材料在动物体内异位成软骨情况,探讨其作为组织工程载体的可行性.方法:胶原凝胶包埋软骨细胞接种快速成型聚乳酸-聚羟基乙酸(PLGA)三维多孔支架.体外培养3 d,扫描电镜和倒置显微镜观察细胞在支架内的生长状况.复合物植入动物体内,大体观察,组织学染色检查软骨形成情况.结果:扫描电镜和倒置显微镜观察软骨细胞旱圆形均匀分布于支架材料中.组织学结果显示软骨细胞在体内生长增殖良好,12周时可见软骨组织形成.结论:胶原凝胶包埋快速成型支架材料可作为载体复合细胞进行软骨组织工程研究.     

新型聚乳酸复合支架材料在组织工程骨构建中的实验研究

目的:探索新型聚乳酸复合支架材料对兔骨髓基质细胞(bone marrow stromal cells,BMSC)生物学行为的影响及组织工程骨的构建方法。方法:实验组选择兔骨 MSC 诱导生成成骨细胞,接种于改良型聚乳酸复合新材料,包括新型聚乳酸(polylactic acid,PLA)、聚乳酸-乙醇酸共聚物(polylactide-co-glycolide,PLGA)、聚乳酸-乙二醇共聚物(polylactic acid-polyethylene glycol block copolymers,PLA-PEG)复合制作的三维支架中培养,用无机材料磷酸三钙(tricalcium phosphate,TCP)作为对照组,观察种子细胞在支架材料上的吸附迁移、生长增殖情况。结果:成骨细胞在新型三维支架材料表面分布较为均匀,生物学行为活跃。结论:可以通过 MSC 诱导获得成骨细胞。PLA、PLGA、PLA-PEG、TCP 具有良好的生物相容性,可作为支架材料与成骨细胞共同培养,获得具有成骨能力的三维立体结构组织工程骨。     

丝素蛋白-软骨脱细胞外基质复合取向支架的制备及评价

目的 制备丝素蛋白（SF）-软骨脱细胞外基质（CECM）仿生支架材料,检测其理化性能特性。方法 采用改良温度梯度热诱导相分离（TIPS）技术结合冷冻干燥法制备出CECM取向支架,然后将CECM浆料与制备好的SF溶液按照质量比1∶1混合后配制成质量分数6%的浆料,通过TIPS技术制备出SF-CECM复合取向支架。对SF-CECM复合取向支架进行扫描电子显微镜（SEM）观察和组织学染色,同时测定支架孔隙率、吸水性以及力学性能。结果 SEM观察可见,支架横断面呈多孔网状结构,纵剖面呈垂直的管状结构。组织学染色显示复合支架脱细胞彻底,有蛋白多糖、胶原成分,与天然软骨成分相似。支架孔隙率、吸水率和纵向压缩弹性模量分别为95.733%±1.010%、94.309%±1.302%和（65.40±4.09）kPa。结论 SF-CECM复合取向支架具有良好的理化特性和生物力学性能,有望成为较理想的组织工程软骨支架。     

人类涎腺细胞在几种生物材料上的生长

目的观察人类涎腺细胞在几种生物材料上的生物情况，筛选适合人类涎腺细胞生长的生物材料，材料和方法，将人类颌下腺上皮细胞系（HSG）细胞分别接种于聚乳酸，聚醚酯，丝素蛋白等生物材料上，噻唑盐比色试验（MTT）检测细胞生长。扫描电镜观察细胞的形态，结果：丝素蛋白膜上生长的HSG细胞数量量多，贴壁良好，形态为我边形，细胞突起多而长，聚醚酯膜上生长的HSG细胞数量少，贴壁较差。结论：丝素蛋白更适合HSG细胞生长。可作为组织工程人造涎腺支架的表面涂层材料。     

PLGA/PCL/nHA生物支架复合兔BMSCs体外培养的实验研究

目的:研究新型复合支架聚乳酸-聚乙醇酸/聚己内酯/纳米羟基磷灰石(PLGA/PCL/nHA)的生物相容性,探讨其作为细胞培养材料和骨组织工程支架的可行性.方法:将兔骨髓基质细胞(bone marrow stromal cells,BMSCs)接种于PLGA/PCL/nHA复合支架上,体外共同培养后,MTT法检测BMSC...     

蚕丝蛋白液与聚乙烯醇作为骨缺损修复材料的性能比较

目的探索不同材料与丝素蛋白液(SP)及聚乙烯醇(PVA)水凝胶混合后制成材料的理化、生物性能。方法取羊椎骨粉和纳米级羟基磷灰石,分别与丝素蛋白溶液、PVA水凝胶以及丝素蛋白复合PVA溶液混合,通过抗溶解性、圧缩力、剪切力及材料细胞毒性的检测,分析比较各组材料的机械生物性能。结果 (1)抗溶解性检测:丝素蛋白溶液混合材料72 h后均达到Ⅲ级溶解,丝素蛋白复合PVA溶液混合材料力学性能优于单纯丝素蛋白组;(2)力学性能检测:丝素蛋白溶液的力学性能较弱,PVA力学性能优良,丝素蛋白溶液混合PVA后力学性能可增强。各组间差异有统计学意义,抗压缩力F=333.667,P<0.05,抗剪切力F=59.997,P<0.05;(3)材料体外细胞毒性检测:各组毒性均为0级或1级,丝素蛋白溶液/骨粉组、PVA/骨粉组、丝素蛋白复合PVA溶液/骨粉组、丝素蛋白溶液/NHA组、PVA/NHA组、丝素蛋白复合PVA溶液/NHA组均无细胞毒性,单纯丝素蛋白溶液混合粉剂材料细胞相容性更好。结论丝素蛋白具有良好的生物相容性,但力学性能欠佳,可以将蚕丝蛋白混合其他材料作为组织工程骨研究的新方向。     

3D打印聚乙烯醇/纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的性能比较

目的　采用3D打印技术制备3D打印聚乙烯醇 /纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架,并对其进行表征。方法　采用3D打印技术制作聚乙烯醇/纳米羟基磷灰石支架以及丝素蛋白/聚乙烯醇/纳米羟基磷灰石复合支架。进行孔隙率、扫描电镜、压缩力学性能及细胞毒性检测。 结果　①扫描电镜观察：丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架结构规则,网状结构清晰,交通支连续,层层之间搭接良好,支架空隙均一。相同倍数下,聚乙烯醇/纳米羟基磷灰石支架网状结构连续性较差。②压缩力学性能：相同应力情况下（10 MPa）,3D打印丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的应变大于3D打印聚乙烯醇/纳米羟基磷灰石支架。③孔隙率：3D打印丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的孔隙率大于3D打印聚乙烯醇/纳米羟基磷灰石支架。④细胞毒性检测：不同时间点两组支架的细胞增殖率无明显差别。结论　结果表明：3D打印聚乙烯醇 /纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架具有良好的理化性能和细胞相容性。     

丝素蛋白/纳米羟磷灰石支架的实验研究

目的设计和构建三维丝素蛋白/羟磷灰石骨组织工程支架材料。方法联合运用丝素蛋白非编织方法和仿生矿化技术,制备并表征三维多孔丝素蛋白/纳米羟磷灰石的有机/无机组织工程支架。结果仿生矿化在非编织支架上形成的针状羟磷灰石晶体,直径20～60 nm,长100～300 nm。复合支架孔隙度为70%～78%,孔径为（163.4±42.6）μm。结论采用非编织丝素蛋白和仿生矿化的方法可制备孔隙度和孔径可控的组织工程支架。     

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目的探讨丝素胶原支架用作牙周组织工程支架材料的可行性。方法本研究于2008年9月在辽宁医学院实验中心进行。通过冻干法制备丝素胶原支架,用扫描电镜(SEM)观察、噻唑蓝(MTT)法及碱性磷酸酶(ALP)活性检测,了解人牙周膜成纤维细胞(PDLF)附着于丝素胶原支架的生长情况,评价其生物相容性。结果制备的复合膜孔隙率为80.7%;PDLF在材料浸提液中的生长、增殖状况与对照组相比,差异无统计学意义(P>0.05);ALP活性与对照组相比差异亦无统计学意义(P>0.05)。SEM观察可见复合材料具有良好的多孔网状结构,细胞在各复合膜上生长旺盛,伸展充分,形成良好。结论通过冻干法自制的丝素胶原复合物的孔隙率符合牙周组织工程的要求。其良好的三维空间结构和细胞相容性促进了PDLF的生长、黏附及增殖,且无细胞毒性,有望成为牙周组织工程的支架材料。     

Nano-hydroxyapatite mineralized silk fibroin porous scaffold for tooth extraction site preservation

ObjectiveTo fabricate a novel nano-hydroxyapatite mineralized silk fibroin (MSF) scaffold in order to diminish the resorption of alveolar ridge and accelerate new bone formation within tooth sockets. Also, to investigate the biocompatibility and osteogenic ability of the MSF in vitro, and the effect of site preservation of the MSF graft in post-extractive sockets in vivo.MethodsSEM, EDX, FTIR and XRD were used to analyze the mineral crystals deposited on the silk fibroin (SF) surface. Pre-osteoblasts (MC3T3-E1) were seeded on SF and MSF scaffolds. Cell viability, distribution and differentiation were examined using a live-dead assay, histological analysis and Alizarin Red S staining. Furthermore, prepared grafts (SF or MSF scaffold) were implanted into the maxillary right first molar sockets of Sprague Dawley rats for 6 weeks and newly formed bone tissue was analyzed by micro-CT and histological examination.ResultsThe SEM, EDX, FTIR and XRD analysis demonstrated that granulate nano-hydroxyapatite (nHA) crystals were uniformly distributed on the SF scaffold. In addition, the MSF hydrophilicity measured by water contact angle and swelling ratio was superior to plain SF scaffold. The effect of nHA inorganic crystals on osteogenic differentiation of MC3T3-E1 cells indicated the MSF scaffolds improved osteogenesis. Furthermore, MSF grafts induced more bone formation and reduced the height of alveolar bone resorption after tooth extraction.SignificanceThe MSF scaffold partially simulated the structure and composition of natural bone matrix. It induced osteogenic differentiation of MC3T3-E1 cells in vitro, and also promoted new bone regeneration in tooth extraction sockets in vivo, indicating it is a biomaterial with great potential for tooth extraction site preservation.     

Autologous injectable tissue-engineered cartilage by using platelet-rich plasma: experimental study in a rabbit model.

PURPOSE: Platelet-rich plasma (PRP) has been widely applied to promote tissue healing and used as a novel injectable scaffold in bone tissue engineering. However, there is no report about its feasibility to support chondrogenesis. This study aimed to investigate the feasibility of a PRP carrier to deliver chondrocytes and regenerate cartilage tissues in a rabbit model via injection. MATERIALS AND METHODS: Eight New Zealand rabbits were divided into a chondrocytes/PRP group (n = 4) and a PRP-alone group (n = 4). Chondrocytes harvested from the auricular root of New Zealand rabbits were cultured and harvested. The chondrocytes were then mixed with PRP solution to generate chondrocytes/PRP composites with final cellular density of 5.0 x 10(7)/mL. Bovine thrombin was used as a cross-linking agent to gel chondrocytes/PRP composites, then, the composites were injected subcutaneously into the dorsal tissue of cell donor animals. As controls, PRP alone was injected into another 4 rabbits. At the second month after injection, rabbits were prepared for magnetic resonance imaging. The samples were then harvested for macroscopical examination, histological analysis, and glycosaminoglycan quantification. RESULTS: Two months after injection, the hard knobbles were easily palpated under the dorsal skin of the animals in the chondrocytes/PRP group, and magnetic resonance images showed the presence of cartilage-like tissues. In histological analysis, formation of new cartilage was observed in the chondrocytes/PRP composites. Safranin-O staining and Masson's trichrome staining showed proteoglycan and collagen were produced in matrices. In contrast, no tissue formed in the PRP-alone group. CONCLUSIONS: This study suggests the feasibility of using PRP as injectable scaffold seeded with chondrocytes to regenerate cartilage and showed the potential of using this method for the reconstruction of cartilage defects.     

Engineering of human tracheal tissue with collagen-enforced poly-lactic-glycolic acid non-woven mesh: a preliminary study in nude mice

The purpose of the current study is to fabricate tissue engineered trachea with poly-lactic-glycolic acid (PLGA) non-woven mesh enforced by collagen type I. PLGA fibres coated with collagen solution were put together and fabricated into the shape of a human trachea, after drying and cross-linking treatment, a non-woven mesh with "C" shape formed. Chondrocytes from sheep nasal septum cartilage were expanded in vitro and seeded into PLGA/collagen non-woven mesh in the density of 5.0 x 10(7)mL(-1). After 5 days of in vitro incubation, six Cell-PLGA/collagen composites were implanted subcutaneously into the back of 6 nude mice to prefabricate a tissue engineering trachea. Eight weeks later, the cartilage formation was observed by gross inspection and histological examination. Cartilage-like tissue in the shape of the initial PLGA/collagen scaffold had been regenerated successfully without obvious inflammatory response. The tissue engineered trachea cartilage consisted of evenly spaced lacunae embedded in matrix stained red with safranin-O staining. The amount of GAGs in tissue engineered trachea cartilage reached 71.42% of normal value in native cartilage. This study demonstrated that collagen-enforced PLGA non-woven mesh facilitated the adhesion and proliferation of chondrocytes, it also owned adequate mechanical strength to serve as an ideal scaffold for trachea tissue engineering without internal support.     

软骨细胞移植修复髁突软骨损伤的实验研究

目的 研究软骨细胞移植修复髁突软骨损伤后的效果及修复组织的性质。方法 选用成年雄性白免５３只,分成５组。第１组：细胞移植组;第２组：单纯胶原膜植入组;第３组：在实验动物有髁突前斜面形成直径２ｍｍ的全层损伤后,未植入任何物品;第４组：空白手术对照组;第５组：正常对照组。结果 细胞移植组动物的髁突软骨损伤区形成软骨组织修复。结论 异体兔髁突软骨细胞移植能够形成类似正常的关节软骨修复髁突软骨缺损。