类骨磷灰石涂覆改性聚(乙交酯/丙交酯)的细胞亲和性研究

目的 研究具有类骨磷灰石涂层的聚(乙交酯/丙交酯)[Poly(L-lactide-co-glycolide),PLGA]材料作为骨组织工程细胞支架材料的可行性.方法 采用氧等离子体预处理结合模拟体液1.5 SBF0孵育的方法对PLGA进行类骨磷灰石涂覆改性.用鼠OCT-1类成骨细胞作为种子细胞进行体外培养和扩增后分别种植于PLGA致密膜和多孔支架上,观察细胞在致密膜和多孔支架上的黏附率、黏附形态、增殖活力和生长形态.结果 经类骨磷灰石涂覆改性的PLGA,其表面OCT-1类成骨细胞的黏附率和增殖活力都得到了提高,细胞的黏附形态和生长形态良好;在多孔支架上培养的细胞能够迁移到支架的内部旺盛生长.结论 具有类骨磷灰石涂层的PLGA具有更高的细胞亲和性,可望成为一类新的骨组织工程细胞支架材料.     

新型羟磷灰石涂层的聚乳酸支架体外细胞相容性研究

目的研究新型的用固定化尿素酶的方法制备的羟基磷灰石／聚乳酸（HA／PLLA）复合材料的生物相容性和成骨活性。方法将成骨样MG63细胞种植在HA／PLLA和PLLA（对照材料）三维支架材料上。在培养2,4和6天后通过改良的MTI＂法检测细胞在材料上的增殖情况;用pNPP磷酸酶检测试剂盒测定ALP含量来评估成骨样细胞在材料上的分化情况;在培养4天后用扫描电镜观察细胞在材料上的形貌变化：用RT—PCR检测磷酸酶（AIJP）,骨钙素（0C）,1型胶原等基因变化情况。结果在培养2天和4天后,HA／PLLA材料上的细胞数量要明显多于PLLA材料组,说明相对于传统的PLLA材料,HA／PLLA材料更能促进成骨细胞的增殖。电镜结果提示,在培养4天后HA／PuJA材料上的MG63细胞铺展在材料孔壁表面,提示细胞具有良好的活力,而PLLA组的细胞表现为圆球形,未见明显的细胞伪足形成,其形态明显不及HA／PuA材料组。ALP检测结果提示,HA／PLLA组的细胞具有更高的ALP活性。RT—PCT结果也提示,HA／PLLA组具有更高的ALP和0c基因表达．提示HA／PLLA材料更能促进成骨细胞的分化。结论通过固定化尿素酶的方法制备的新型羟基磷灰石／聚乳酸材料相比传统的PLLA材料更能促进成骨细胞的增殖和分化,提示该材料是一种良好的骨组织工程支架材料。     

纳米支架与犬骨髓基质干细胞体外生物相容性的实验研究

目的研究具有纳米结构二嵌段共聚物左旋聚乳酸-聚己内酯（PLLA-b-PCL）与犬骨髓基质干细胞（BMSCs）的体外生物相容性，探讨其作为软骨组织工程支架的可行性。方法开环聚合制备PLLA-b—PCL，液-液相分离制备PLLA-b-PCL纳米支架，扫描电镜观察材料结构。分离培养犬BMSCs，取第3代BMSCs接种于PLLA-b—PCL膜进行复合二维培养，MTT法检测细胞毒性；通过倒置显微镜、Hoechst33342荧光法观察细胞的形态与黏附情况。另取第3代BMSCs与纳米PLLA—b-PCL支架材料（实验组）、PLLA—b—PCL支架材料（对照组）进行三维培养3周，扫描电镜观察BMSCs的形态、黏附、生长情况，Hoechst33258荧光法检测复合物中细胞DNA含量，BCA法测定蛋白质含量。结果PLLA-b-PCL无细胞毒性，BMSCs在纳米PLLA—b—PCL支架上黏附、增殖良好。随时间延长，BMSCs在支架材料上的DNA和蛋白质含量逐渐增加，DNA和蛋白质含量均明显高于对照组，差异有统计学意义（P〈0．05）。结论PLLA-b—PCL纳米支架能为BMSCs的生长分化提供较好的环境，具有良好的生物相容性，有望成为一种较好的软骨组织工程支架材料。     

骨组织工程中种子细胞与材料黏附的有关蛋白及影响因素

骨组织工程近年的迅速发展为人们解决大段骨缺损的治疗难题提供了另一种途径。在骨组织工程研究中，细胞与支架材料的相互作用一直都是研究的重要领域，其核心是种子细胞在细胞外基质支架材料上的黏附。作为锚合依赖性细胞，成骨细胞在种植体表面的黏附、丛集是接触成骨的关键。只有当细胞与材料界面发生适当的黏附后，细胞才能进行迁移、增殖和分化。可以说，组织工程成功与否的关键之一就在于细胞在支架材料表面的黏附程度。     

Pluronic F-127表面修饰生物衍生骨的体外实验研究

目的：探讨成骨细胞复合PluronicF-127表面修饰生物衍生骨的三维培养，对成骨细胞活力及功能表达的影响。方法：将新鲜猪肋骨加工制成生物生骨，应用PluronicF-127对生物衍生骨进行表面修饰，然后体外复合第3代成骨细胞三维培养，实验为A、B和C组，A组为单纯生物衍生骨复合成骨细胞组，B组为PluronicF-127表面修饰生物衍生骨复合成骨细胞组，C组为单纯成骨细胞组，应用倒置相差显微镜和扫描电镜对各组成骨细胞生长及黏附进行观察，并对其细胞活力碱性磷酸酶（ALP）活性进行检测，结果：B组成骨细胞在支架材料上黏附，伸展及生长良好，成骨细胞活力和ALP活性表达与A组成骨细胞比较均无统计学意义（P＞0．05）；A、B组与C组比较，成骨细胞活性和ALP活性均明显降低，有统计学意义（P＜0．01）。结论：生物衍生骨经PluronicF-127表面修饰后具有良好的细胞相容性，可作为负载生物活性分子的载体修饰生物衍生骨。     

不同保存方法对生物衍生骨支架材料细胞相容性的影响

目的了解不同保存方法对生物衍生骨支架材料细胞相容性的影响.方法冻干生物衍生骨用两种不同保存液同等条件4℃冷藏保存3个月,以保存相同时间冻干生物衍生骨为对照.实验分为A组:成骨细胞复合 1号保存液处理材料培养;B组:成骨细胞复合 2号保存液处理材料培养;C组:成骨细胞复合冻干骨培养;D组:单纯成骨细胞培养.以2×106个/ml密度的成骨细胞悬液复合材料培养1、3、5和7天.用倒置相差显微镜和扫描电镜观察细胞的黏附和生长情况、检测细胞活力、碱性磷酸酶(alkaline phosphatase,ALP)活性及流式细胞仪分析细胞周期.结果成骨细胞与三种不同方法保存的材料均可黏附,并在材料孔隙内生长、分化和增殖.复合培养1和3天,各组间无显著差异;复合培养5和7天,黏附于材料的细胞活力大小依次为 A组>C组>B组 (P＜0.01,P＜0.05).复合培养7天,黏附于材料的细胞ALP活性大小依次为 A组>C组>B组(P＜0.01) .各组细胞周期未见明显变化,未见异倍体细胞.结论选择适宜的保存液对生物衍生骨支架材料的细胞相容性有一定优化作用.     

骨形态发生蛋白-2活性多肽修饰的重组胶原矿化骨对骨髓基质干细胞生物学行为的影响

目的 探讨骨形态发生蛋白-2(BMP-2)活件多肽修饰的重组胶原矿化骨复合材料对骨髓基质干细胞(BMSCs)增殖、黏附及分化等生物学行为的影响. 方法制备重组胶原矿化骨支架材料,将BMP-2活性多肽通过交联剂共价结合到材料上,扫描电镜观察支架材料表面微观形貌;取第3代BMSCs接种到材料上,以未结合多肽的重组胶原矿化骨作为对照,采用MTT法检测BMSCs在材料表面的增殖;沉淀法检测BMSCs在材料表面的黏附率;扣描电镜观察比较BMSCs在材料表面的生长形态;通过检测细胞中的碱性磷酸酶活性及钙含量,观察BMSCs在材料表面的分化情况. 结果扫描电镜结果显示:支架材料旱多孔状;X射线光电子能谱法证实BMP-2活性多肽成功共价结合到材料表面;BMP-2活性多肽修饰的重组胶原矿化骨复合材料表面BMSCs的黏附和向成骨细胞方向分化能力均高于对照组,差异有统计学意义(P＜0.05),而BMSCs的增殖能力与对照组相比,差异无统计学意义(P＞0.05). 结论BMP-2活性多肽可以显著改善重组胶原矿化骨复合材料的细胞相容性和生物活性,经BMP-2活性多肽修饰的重组胶原矿化骨复合材料是一种理想的骨组织工程支架材料.     

新型双相陶瓷材料复合成骨细胞构建人工骨及其体内成骨的观察

目的 观察骨髓来源的成骨细胞与新型双相陶瓷材料复合培养后细胞生长状况及其体内异位成骨能力。方法 将来源于兔骨髓的成骨细胞与新法制备的HA／TCP共培养2周，通过相差显微镜、扫描电镜观察体外细胞生长情况；然后将复合物自体异位植入体内，6周后观察体内成骨情况。结果 扫描电镜显示材料表面和孔隙内均有成骨细胞生长，有良好增殖活动性和稳定细胞表型，材料中心区未见细胞生长；植入体内6周后取材见内植物有新骨形成、多位于内植物表面，可见成骨细胞、骨细胞、髓腔样结构、板层样骨基质等正常骨组织结构。结论 新型双相陶瓷支架可用作组织工程骨的细胞外基质材料，骨髓来源的成骨细胞可用作骨组织工程的种子细胞。     

纳米PLLA膜复合内皮生长晕细胞的相容性研究

目的 通过观察内皮生长晕细胞(EOCs)与纳米聚-L-乳酸(PLLA)有序纤维膜体外复合培养的生物相容性及黏附、增殖情况,为构建组织修复材料提供理论依据.方法 通过静电纺丝技术制备的纳米PLLA纤维支架,行低温等离子体技术改性及 Ⅰ 型胶原表面涂覆,与EOCs复合培养,测定细胞黏附率及增殖率,观察细胞生长曲线,荧光显微镜及扫描电镜下观察支架材料与种子细胞EOCs形态特征和生物相容性.结果 制备的纳米PLLA膜孔径在300～400 nm之间,孔隙率>90%.各组(单纯细胞组、无序膜组、有序膜组及超级有序膜组)吸光度(A)值均随共培养时间的增加而逐步升高; 从第5天起,有序膜和超级有序膜组A值与无序膜和单纯细胞组比较,差异有统计学意义(P＜0.05); 单纯细胞组和无序膜组各检测时间点A值差异无统计学意义(P>0.05 ).复合培养12 h及24 h后有序膜组和超级有序膜组黏附率则明显高于无序膜组,差异有统计学意义(P＜0.01).复合培养1 d、3 d及7 d后,有序膜组和超级有序膜组的增殖率明显高于无序膜组(P＜0.05,P＜0.01).细胞在支架膜上生长良好,纳米无序膜的细胞生长较散在、杂乱; 有良好空间定向效果的有序膜及超级有序膜支架有利于细胞沿纤维定向附着、伸展、增殖,以超级有序膜更有利于保持其结构.结论 EOCs是理想的组织工程种子来源细胞; 纳米PLLA有序及超级有序膜支架能促进种子细胞在材料表面的黏附、增殖,并能较好地保持细胞的形态,是一种理想的组织修复材料.     

人脂肪来源干细胞与左旋聚乳酸/聚己内酯支架的体内外生物相容性研究

目的观察人脂肪来源干细胞(Human adipose-derived stem cells,hADSCs)在左旋聚乳酸/聚己内酯(Poly-Llactide/Polycaprolactone,PLLA/PCL)复合支架材料中的生长情况及其生物相容性。方法体外分离、培养和扩增hADSCs,制备PLLA/PCL复合支架。取PLLA/PCL浸提液培养hADSCs,CCK-8法检测细胞活力,评价支架的细胞毒性。hADSCs传代扩增后,接种到PLLA/PCL支架材料上。体外培养1周,裸鼠皮下分别培养1周、2周,HE染色观察细胞在支架上的生长情况。免疫荧光检测裸鼠体内复合支架材料内细胞平滑肌肌动蛋白(Smooth muscle actin,SMA)表达情况。结果hADSCs在PLLA/PCL浸提液中可保持较高的增殖率,表明PLLA/PCL浸提液无细胞毒性。hADSCs接种于PLLA/PCL支架上,经体外、体内培养后,均能长入PLLA/PCL支架的孔隙内,且体内培养比体外培养有更多的细胞长入支架内部。经体内培养后,复合细胞的支架比单纯支架有更多的细胞渗入支架内部。细胞材料复合物体内培养2周后,免疫荧光检测发现,支架材料内部分细胞SMA表达阳性。结论 PLLA/PCL复合支架材料,安全无毒,与hADSCs生物相容性好,可作为种子细胞的载体用于组织工程膀胱缺损修复的研究。     

Nitric oxide and prostaglandin E2 production in response to ultra-high molecular weight polyethylene particles depends on osteoblast maturation state

BACKGROUND: Recent studies have shown that osteoblast-like cells respond directly to ultra-high molecular weight polyethylene particles in culture, suggesting that they may be involved in aseptic loosening of endoprostheses. We tested the hypothesis that the state of cell maturation plays a role in the response of osteogenic cells to ultra-high molecular weight polyethylene particles. METHODS: MG63 cells (immature osteoblast-like cells), OCT-1 cells (mature secretory osteoblast-like cells), and MLO-Y4 cells (osteocyte-like cells) were treated for twenty-four hours with commercial ultra-high molecular weight polyethylene particles with an average diameter of 1 mm. The effect of particle treatment on cell proliferation was assessed by measuring the number of cells, whereas the effects on differentiation and local factor production were assessed by measuring the production of osteocalcin, prostaglandin E2, and nitric oxide. The effect of particles on apoptosis was also evaluated. RESULTS: The addition of ultra-high molecular weight polyethylene particles increased the number of MG63 cells, did not affect the number of OCT-1 cells, and led to a decrease in the number of MLO-Y4 cells. The observed changes in cell number were not due to programmed cell death, as no more than 3% of the cells in cultures treated with the highest concentration of particles were undergoing apoptosis. Osteocalcin production was not affected by the addition of particles. Prostaglandin E2 production was increased in all three types of cultures, but the effect was greatest in OCT-1 cell cultures, as was the absolute amount of prostaglandin E2 produced. Nitric oxide production was unaffected in MG63 cell cultures, but it was stimulated in OCT-1 and MLO-Y4 cell cultures. CONCLUSIONS: The results of the present study support the hypothesis that osteoblast cell maturation state plays an important role in the response to ultra-high molecular weight polyethylene particles and that the terminally differentiated osteocyte may be involved in the bone response to wear debris in vivo.     

Repair of rat cranial bone defects with nHAC/PLLA and BMP‐2‐related peptide or rhBMP‐2

An ideal artificial substitute has good biocompatibility properties and is able to provide for rapid bone formation. Bone morphogenetic protein‐2 (BMP‐2) is considered as one of the most important growth factors for bone regeneration. In this study, a synthetic BMP‐2‐related peptide (designated P24) corresponding to residues of the knuckle epitope of BMP‐2 was introduced into a bioactive scaffold based on nano‐hydroxyapatite/collagen/poly(L ‐lactic acid) (nHAC/PLLA); its in vitro release kinetics was then measured. A 5 mm diameter cranial bone defect was created in the calvariae of 30 rats and randomly implanted with three groups of biomaterials: Group A (nHAC/PLLA alone); Group B (P24/nHAC/PLLA composite); and Group C (recombinant human BMP‐2 (rhBMP‐2)/nHAC/PLLA composite). The P24/nHAC/PLLA implants significantly stimulated bone growth similarly to the rhBMP‐2/nHAC/PLLA implants based on the radiographic and three‐dimensional CT evaluation and histological examination, thereby confirming the enhanced bone healing rate of these compounds compared with the stand‐alone nHAC/PLLA scaffold material. The osteoinductive ability of 3 mg P24 was similar to that of 1 µg rhBMP‐2. P24/nHAC/PLLA is a promising scaffold biomaterial for bone tissue regeneration. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1745–1752, 2011     

兔骨髓间充质干细胞复合纳米羟基磷灰石/左旋聚乳酸修复兔软骨缺损的实验研究

目的评价兔骨髓间充质干细胞复合纳米羟基磷灰石/左旋聚乳酸(Nano-HA/PLLA)修复兔膝关节全层软骨缺损的效果。方法体外分离培养兔骨髓间充质干细胞(rBMSCs),取第3代将其与Nano-HA/PLLA在体外构建组织工程软骨(tissue engineering cartilage,TEC)。将18只新西兰大白兔股骨内髁关节面造一直径4.5 mm、深度5 mm的软骨缺损模型,随机分成3组,即实验组、支架组和空白对照组,每组6只,分别予复合细胞的Nano-HA/PLLA、Nano-HA/PLLA及空白填充。术后12、24周取材行大体标本、组织学、免疫组织化学观察及Wakitani组织学评分。结果实验组显示缺损有骨软骨组织形成,软骨下骨基本达到生理整合,组织学及免疫组化显示有大量细胞外基质形成;支架组及对照组显示出有限的再生重建。以上三组12周Wakitani组织学评分分别为5.5±1.401,9.3±1.304和10.2±1.052,24周时为2.2±0.837,7.4±1.144和8.2±1.225,两个时间点实验组均优于支架组及空白组,差异有统计学意义(P<0.05);且实验组24周时优于12周(P<0.05)。结论复合骨髓间充质干细胞的多孔Nano-HA/PLLA能提高成年兔膝关节承重部的骨软骨缺损的修复。     

Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D3.

In this study we assessed whether osteogenic cells respond in a differential manner to changes in surface roughness depending on their maturation state. Previous studies using MG63 osteoblast-like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhibited and differentiation (osteocalcin production) was stimulated by culture on titanium (Ti) surfaces of increasing roughness. This effect was further enhanced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we examined the response of three additional cell lines at three different maturation states: fetal rat calvarial (FRC) cells (a mixture of multipotent mesenchymal cells, osteoprogenitor cells, and early committed osteoblasts), OCT-1 cells (well-differentiated secretory osteoblast-like cells isolated from calvaria), and MLO-Y4 cells (osteocyte-like cells). Both OCT-1 and MLO-Y4 cells were derived from transgenic mice transformed with the SV40 large T-antigen driven by the osteocalcin promoter. Cells were cultured on Ti disks with three different average surface roughnesses (Ra): PT, 0.5 microm; SLA, 4.1 microm; and TPS, 4.9 microm. When cultures reached confluence on plastic, vehicle or 10(-7) M or 10(-8) M 1,25(OH)2D3 was added for 24 h to all of the cultures. At harvest, cell number, alkaline phosphatase-specific activity, and production of osteocalcin, transforming growth factor beta1 (TGF-beta1) and prostaglandin E2 (PGE2) were measured. Cell behavior was sensitive to surface roughness and depended on the maturation state of the cell line. Fetal rat calvarial (FRC) cell number and alkaline phosphatase-specific activity were decreased, whereas production of osteocalcin, TGF-beta1, and PGE2 were increased with increasing surface roughness. Addition of 1,25(OH)2D3 to the cultures further augmented the effect of roughness for all parameters in a dose-dependent manner; only TGF-beta1 production on plastic and PT was unaffected by 1,25(OH)2D3. OCT-1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE2, osteocalcin, and TGF-beta1 were increased on SLA and TPS. Response to 1,25(OH)2D3 varied with the parameter being measured. Addition of the hormone to the cultures had no effect on cell number or TGF-beta1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE2 was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO-Y4 cultures, cell number was decreased on SLA and TPS; alkaline phosphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF-beta1, and PGE2 were increased on SLA and TPS. Although 1,25(OH)2D3 had no effect on cell number, alkaline phosphatase, or production of TGF-beta1 or PGE2 on any surface, the production of osteocalcin was stimulated by 1,25(OH)2D3 on SLA and TPS. These results indicate that surface roughness promotes osteogenic differentiation of less mature cells, enhancing their responsiveness to 1,25(OH)2D3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roughness.     

成骨细胞株与强韧化纳米人工骨支架联合培养实验研究

目的 :观察成骨细胞株 ( 3T3 E1)在新型纳米氧化锆强韧化高孔隙率人工骨支架 (下文简称人工骨支架 )材料上生长情况。方法 :成骨细胞株 ( 3T3 -E1)与人工骨支架联合培养 ,通过光镜观察和细胞计数的方法了解成骨细胞与支架结合能力 ,扫描电镜观察细胞生长的情况。结果 :新型强韧化纳米人工骨材料与建株成骨细胞有良好的结合能力 ,成骨细胞株 ( 3T3 E1)在人工骨材料上生长良好。结论 :纳米骨支架是较理想的骨组织工程支架材料 ,成骨细胞复合支架用于骨缺损的修复 ,具有广阔的临床应用前景。     

Er:YAG Laser-Roughened Enamel Promotes Osteoblastic Differentiation

Abstract Objective: The aim of this study was to test whether Er:YAG laser-etched enamel of human teeth could act as a biologically active scaffold for tissue regeneration. Background data: Hydroxylapatite (HA) with rough surface created by acid etching treatment has been used as a scaffold for tissue engineering. However, whether tooth HA can be a scaffold for osteoblastic cell seeding is still unclear. Materials and methods: Enamel samples from human teeth were pretreated with an Er:YAG laser to create a rough surface. Then the surface of the laser-treated enamel was examined using a surface roughness profilometer and a scanning electron microscope. In addition, static water contact angles of the Er:YAG laser-treated enamel samples were measured using goniometry. To observe the effects of cell behavior on an Er:YAG laser-roughened enamel surface, we cultured MG63 osteoblast-like cells on the surface-modified enamel samples. Alkaline phosphatase activity, a marker of cell proliferation and differentiation, was monitored and compared with that in untreated control and acid-etched enamel samples. Results: Er:YAG laser treatment significantly improved the surface roughness of the enamel samples. Furthermore, MG63 osteoblast-like cells cultured on the Er:YAG laser-roughened enamel surface expressed more alkaline phosphatase activity and exhibited greater degrees of cellular differentiation than did cells that had been cultured on untreated enamel samples. Conclusions: These results demonstrate that Er:YAG laser-roughened enamel promotes osteoblastic differentiation. This finding suggests that Er:YAG laser-roughened enamel surfaces can potentially serve as a scaffold for tissue engineering.     

Nanobioengineered electrospun composite nanofibers and osteoblasts for bone regeneration

Abstract:  Bone defects represent a medical and socioeconomic challenge. Engineering bioartificial bone tissues may help to solve problems related to donor site morbidity and size limitations. Nanofibrous scaffolds were electrospun into a blend of synthetic biodegradable polycaprolactone (PCL) with hydroxyapatite (HA) and natural polymer gelatin (Gel) at a ratio of 1:1:2 (PCL/HA/Gel) compared to PCL (9%), PCL/HA (1:1), and PCL/Gel (1:2) nanofibers. These fiber diameters were around 411 ± 158 to 856 ± 157 nm, and the pore size and porosity around 5–35 µm and 76–93%, respectively. The interconnecting porous structure of the nanofibrous scaffolds provides large surface area for cell attachment and sufficient space for nutrient transportation. The tensile property of composite nanofibrous scaffold (PCL/HA/Gel) was highly flexible and allows penetrating osteoblasts inside the scaffolds for bone tissue regeneration. Fourier transform infrared analysis showed that the composite nanofiber contains an amino group, a phosphate group, and carboxyl groups for inducing proliferation and mineralization of osteoblasts for in vitro bone formation. The cell proliferation (88%), alkaline phosphatase activity (77%), and mineralization (66%) of osteoblasts were significantly ( P  < 0.001) increased in composite nanofibrous scaffold compared to PCL nanofibrous scaffolds. Field emission scanning electron microscopic images showed that the composite nanofibers supported the proliferation and mineralization of osteoblast cells. These results show that the fabrication of electrospun PCL/HA/Gel composite nanofibrous scaffolds has potential for the proliferation and mineralization of osteoblasts for bone regeneration.     

Repair of bone defect in femoral condyle using microencapsulated chitosan, nanohydroxyapatite/collagen and poly(L-lactide)-based microsphere-scaffold delivery system

Bone repair ability of microencapsulated chitosan, nanohydroxyapatite/collagen (nHAC), and poly(L-lactide) (PLLA)-based microsphere-scaffold delivery system was investigated in present research, with nHAC/PLLA composite scaffold as a control. Chitosan microspheres (CMs) encapsulated with bone morphogenetic protein-2-derived synthetic peptide were incorporated into nHAC and PLLA-based matrix via a thermally induced phase separation method, in which dioxane was used as the solvent for PLLA. Compared with the rapid release from CMs, the synthetic peptide was delivered from CMs/nHAC/PLLA microsphere-scaffold composite in a temporally controlled manner, depending on the degradation of both incorporated CMs and PLLA matrix. MC3T3-E1 osteoblastic cells were seeded into nHAC/PLLA and CMs/nHAC/PLLA scaffolds, respectively, and in vitro cytocompatibility was tested by scanning electron microscopy and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results indicated that, with the appearance of CMs in microsphere-scaffold composite, the osteoblasts exhibit better morphology and proliferation ability. In vivo tissue compatibility was evaluated by transplanting the scaffolds into rabbit femoral condyles with a defect 6 mm in diameter. After implanting for 4, 8, and 12 weeks, respectively, radiographic and histological observation revealed that the CMs/nHAC/PLLA composite can accelerate the regeneration of cancellous bone defect as compared with the nHAC/PLLA scaffold. The results demonstrated that the CMs/nHAC/PLLA possesses better biocompatibility, which should be attributed to both the incorporated chitosan component and the encapsulated bioactive synthetic peptide. The promising CMs/nHAC/PLLA microsphere-scaffold composite can be used as delivery system for multiple bioactive factors or as inductive implant scaffold for bone regeneration.