重组骨脱细胞细外基复合Wistar大鼠成骨细胞体外培养的形态学观察

目的新型重组骨脱细胞细胞外基质(REAECM)的制备及其细胞相容性的初步检测，为骨组织工程寻找一种新型的细胞外支架提供实验依据。方法应用体外细胞培养技术，对鼠成骨细胞和REAECM体外进行联合培养1-4周，通过相差显微镜、光镜、电镜观察细胞在材料中的生长情况。结果成骨细胞可以在REAECM上发生良好的黏附、增殖，并且可以长入REAECM的孔隙内。结论REAECM可作为构建组织工程骨的一种较好的支架材料，具有网状孔隙结构；在体外和成骨细胞复合培养时表现出良好的细胞相容性，可以作为一种天然的骨组织替代材料。     

生物衍生骨在骨组织工程研究中的应用

支架材料的选取是骨组织工程研究的关键 ,生物衍生骨具有较好的生物相容性和材料界面 ,三维立体孔隙 -网架合理 ,可塑性强 ,可降解 ,并具备一定的力学强度 ,兼备良好的骨传导及一定的骨诱导能力。可作为种子细胞的支架材料应用于骨组织工程研究。     

生物衍生骨在骨组织工程研究中的应用

支架材料的选取是骨组织工程研究的关键，生物衍生骨具有较好的生物相容性和材料界面，三维立体孔隙-网架合理，可塑性强，可降解，并具备一定的力学强度，兼备良好的骨传导及一定的骨诱导能力。可作为种子细胞的支架材料应用于骨组织工程研究。     

改良法制备异种脱蛋白骨体内植入修复骨缺损的免疫学分析

背景:在支架材料选择中,目前尚未完全解决异种骨的免疫原性问题。目的:观察改良法制备异种脱蛋白骨作为组织工程支架材料修复骨缺损后机体的免疫学改变。方法:SPF级青山羊18只,随机分为3组,正常骨组不截骨,自体骨组、异种脱蛋白骨组在羊右侧胫骨中下段造成胫骨总长度20%骨膜和骨缺损。自体骨组在缺损处植入羊自体骨;异种脱蛋白骨组培养收集羊自体骨髓间充质干细胞,调整细胞数为1×109L-1,将2mL细胞悬液接种于以猪股骨为原料改良制备的猪脱蛋白骨上,再加入重组人骨形成蛋白2,半环槽外固定。采用流式细胞仪进行CD4+及CD8+T淋巴细胞、血清抗体IgG免疫学检测,以双能X射线测量仪分析骨缺损修复效果。结果与结论:异种脱蛋白骨组植入后3,7,14,28d外周血CD4+和CD8+T淋巴细胞的含量基本正常(P0.05),血清抗体IgG水平略高于自体骨组(P0.05);植入后24周骨密度、骨矿物含量与自体骨组基本相似(P0.05),表现为骨缺损区两断端之间高密度钙化影。3组抗压缩压强及极限压强、抗弯曲载荷及极限载荷、抗扭转转矩及极限转矩均基本相似(P0.05)。植入后24周与自体骨组比较,异种脱蛋白骨组的成骨能力无明显差异(P0.05)。提示改良法制备的异种脱蛋白骨不引起明显的细胞和体液免疫反应,有良好的组织相容性,不影响自体骨髓间充质干细胞成骨,新骨生物力学性能与自体骨相当。     

股骨头缺损模型的建立及评估

目的:模拟临床上治疗Ⅱ期股骨头坏死的手术过程,制备兔股骨头缺损模型,并植入部分脱蛋白骨,评估股骨头缺损模型在Ⅱ期股骨头坏死治疗方面的应用价值。方法:成年健康新西兰白兔16只,切开关节囊,在头颈交界处开窗,建立股骨头缺损模型。32侧股骨头随机分为2组:(部分脱蛋白骨)PDPB组植入部分脱蛋白骨;另一组为空白对照。术后2,4,8周处死动物,标本行X射线及病理学观察。结果:X射线、病理学观察及图像分析结果表明:PDPB组在术后2,4,8周股骨头缺损区修复组织存在新生骨组织,而空白对照组至术后8周仍修复组织以纤维为主。结论:所建立的缺损模型在评价组织工程方法治疗Ⅱ期股骨头坏死的疗效方面具有可行性。     

异种生物衍生骨材料与骨髓间充质干细胞的生物相容性

背景:异种生物衍生骨保存了原骨组织的天然网状孔隙结构,且具有免疫原性低、细胞相容性好的特点。目的:验证异种生物衍生骨材料与骨髓间充质干细胞的生物相容性。方法:取新鲜猪股骨制备生物衍生骨,扫描电镜观察材料结构。将第3代兔骨髓间充质干细胞以2×109 L-1的浓度接种于生物衍生骨材料的松质骨面,复合培养7 d内,采用扫描电镜观察细胞生长情况;复合培养8 d内,计数材料上附着的细胞数。结果与结论:生物衍生骨表面粗糙,孔隙不规则并相互通联,构成网状结构。复合培养3 d,细胞在衍生骨材料表面发生附着,且细胞形态不均一;复合培养培养5 d,细胞连接成片呈层状生长,细胞之间紧密接触;复合培养7 d,细胞呈现多层生长状态,成堆生长,部分区域存在细胞外基质分泌现象。复合培养后,前2 d为潜伏适应期,3-6 d细胞生长呈出线性曲线,处于细胞生长对数期;第6天后,细胞生长曲线逐渐变得平缓,细胞增殖速度下降,增殖进入平台期。表明异种生物衍生骨材料与骨髓间充质干细胞具有良好的生物相容性。     

天然骨衍生支架材料的应用

支架材料的研究在骨组织工程中十分重要.异体/异种骨等天然产物作为骨组织工程支架材料的研究取得一定进展,同时在研究和应用中也遇到一些问题.本文对目前天然骨衍生支架材料的骨组织工程中的应用情况作一概述和分析,并对遇到的问题进行了探讨.     

生物衍生骨支架材料的制备及其体内组织相容性的研究

目的：制备生物衍生骨支架材料，研究其生物安全性和生物相容性，从而为骨组织工程提供最佳的支架材料。方法：制备生物衍生骨支架材料，将BALB／c小鼠分为三组，分别为对照组、生物衍生骨支架植入组和异种骨植入组。植入21天后分别采用肌肉刺激实验，刀豆蛋白A（ConA）诱导的脾淋巴细胞转化实验和补体依赖性细胞毒实验分析生物衍生骨对动物机体局部组织的影响和免疫功能的影响。取植人物周围组织做HE染色进行组织学分析。结果：支架组材料周围未见明显的炎症反应，而异种骨组骨组织周围有大量的炎细胞浸润，并有坏死组织。支架组的脾淋巴细胞转化实验和补体依赖性细胞毒实验结果与对照组相比较均无明显差异；而异种骨组结果则均明显高于对照组，具有显著性差异。结论：生物衍生骨支架材料无细胞毒性，具有良好的组织相容性。     

猪源性骨支架材料的制备及性能研究

目的:制备猪源性骨支架材料,并与人源性骨支架材料对比检测其理化性能和组织相容性。方法:经低温深冻、超声清洗、H2O2、酒精浸泡、冻干、辐照制备猪源性骨支架材料和人源性骨支架材料。扫描电镜观察,测定材料孔隙率、蛋白质含量、钙磷含量及弹性模量。2种材料的浸提液与脂肪源间充质干细胞复合培养,观察细胞一般形态,流式细胞仪PI法检测细胞生命周期。皮下植入2种材料,在植入4、8、12、16周取材做病理切片观察和扫描电镜观察。结果:2种材料均具有骨本身的天然网状三维支架系统。猪源性骨支架材料的孔隙率高于人源性骨支架材料,蛋白含量低于人源性骨支架材料,弹性模量分别为无显著差异。材料浸提液组及空白对照组的细胞生长状态良好。流式细胞仪PI法检测细胞周期见G1期、G2期细胞百分率接近。皮下植入试验表明,随着植入时间的延长,炎症反应逐步减轻,材料降解增加,新生软骨样结构逐渐增多。结论:猪源性异种骨支架材料在理化性能和材料毒性等方面与同种异体骨支架材料接近,具有良好的应用前景。     

异种/异体骨衍生骨组织工程支架材料研究进展

支架材料是组织工程研究中的重要内容之一。本文就用于骨组织工程中的异种 /异体骨衍生骨支架材料的加工方法、材料的生物相容性和生物功能性进行了综述 ,并对需要进一步研究的内容进行了探讨     

Fabrication of porous ultra-short single-walled carbon nanotube nanocomposite scaffolds for bone tissue engineering

We investigated the fabrication of highly porous scaffolds made of three different materials [poly(propylene fumarate) (PPF) polymer, an ultra-short single-walled carbon nanotube (US-tube) nanocomposite, and a dodecylated US-tube (F-US-tube) nanocomposite] in order to evaluate the effects of material composition and porosity on scaffold pore structure, mechanical properties, and marrow stromal cell culture. All scaffolds were produced by a thermal-crosslinking particulate-leaching technique at specific porogen contents of 75, 80, 85, and 90 vol%. Scanning electron microcopy, microcomputed tomography, and mercury intrusion porosimetry were used to analyze the pore structures of scaffolds. The porogen content was found to dictate the porosity of scaffolds. There was no significant difference in porosity, pore size, and interconnectivity among the different materials for the same porogen fraction. Nearly 100% of the pore volume was interconnected through 20microm or larger connections for all scaffolds. While interconnectivity through larger connections improved with higher porosity, compressive mechanical properties of scaffolds declined at the same time. However, the compressive modulus, offset yield strength, and compressive strength of F-US-tube nanocomposites were higher than or similar to the corresponding properties for the PPF polymer and US-tube nanocomposites for all the porosities examined. As for in vitro osteoconductivity, marrow stromal cells demonstrated equally good cell attachment and proliferation on all scaffolds made of different materials at each porosity. These results indicate that functionalized ultra-short single-walled carbon nanotube nanocomposite scaffolds with tunable porosity and mechanical properties hold great promise for bone tissue engineering applications.     

Bone growth in rapid prototyped porous titanium implants

Two porous titanium implants with a pore size diameter of 800 and 1200 microm (Ti800 and Ti1200) and an interconnected network were manufactured using rapid prototyping. Their dimensions and structure matched those of the computer assisted design. The porosity of the implants was around 60%. Their compressive strength and Young's modulus were around 80 MPa and 2.7 GPa, respectively. These values are comparable to those of cortical bone. The implants were implanted bilaterally in the femoral epiphysis of 15 New Zealand White rabbits. After 3 and 8 weeks, abundant bone formation was found inside the rapid prototyped porous titanium implants. For the Ti1200 implants, bone ingrowth was (23.9 +/- 3.5)% and (10.3 +/- 2.8)%, respectively. A significant statistical difference (p < 0.05) was found for bone ingrowth in the Ti1200 between the two delays. The percentage of bone directly apposited on titanium was (35.8 +/- 5.4)% and (30.5 +/- 5.0)%. No significant difference was found for bone-implant contact between the different time periods and pore sizes. This work demonstrates that manufacturing macroporous titanium implants with controlled shape and porosity using a rapid prototyping method is possible and that this technique is a good candidate for orthopedic and maxillofacial applications.     

Use of natural coralline biomaterials as reinforcing and gas-forming agent for developing novel hybrid biomatrices: microarchitectural and mechanical studies

This paper describes the first attempt in fabrication of three-dimensional macroporous composites of chitosan and natural coralline material with pore sizes of 300-400 microm, exceeding the upper pore size limit of 250 microm obtained with freeze-dried chitosan-based scaffolds. Natural coral particulates of less than 20 microm, which is mainly composed of calcium carbonate (CaCO3), was simultaneously used as reinforcing phase and gas-forming agent to obtain a structure with large pores and improved mechanical and biological properties. The reaction between the coralline material and the acidic chitosan polymer solvent, which produced carbon dioxide, was rapidly stopped by the subsequent thermally induced phase separation technique, leaving coralline particulates in the polymeric structure. Scaffolds containing five different proportions of coralline material (0, 25, 50, 75, and 100 wt%) were investigated. The coralline-chitosan weight ratio was studied for its effects on the physical properties of the scaffolds. The relation between scaffold microarchitecture and mechanical properties was assessed with scanning electron microscope (SEM), along with micro-CT imaging and compression testing. The scaffolds were used in bone marrow cell culturing experiments to assess the effect of composition on cell behavior through cell-material interaction and morphological observation by SEM. Higher coralline concentration increased the pore wall thickness and favored large pore formation. Varying the coralline particulate to chitosan polymer ratio from 0 to 75 wt% increased the average pore size from 80 microm to 400 microm while the porosity decreased from 91% to 78%. The compressive modulus was improved proportionally with the coralline content, and the 75 wt% composites had a significantly higher modulus than other chitosan-based scaffold groups. More cells were observed on scaffolds with higher coralline content. The cell culture experiments indicated that the scaffolds containing coralline material might have a high cell affinity, since it allowed fast cell attachment and spreading.     

Confocal laser scanning microscopy as a tool for imaging cancellous bone

Understanding the bimodal structure of cancellous bone is important for tissue engineering in order to more accurately fabricate scaffolds to promote bone ingrowth and vascularization in newly forming bone. In this study, confocal laser scanning microscopy (CLSM) was used to create detailed images of the bimodally porous intertrabecular space of defatted and deproteinized cancellous canine bone taken from the epiphysis of the humerus. The bimodal pore structure was imaged using both reflective and fluorescent modes in CLSM, resulting in four different, but complementary image types: (1) a Z-stack overlay, (2) a phi-Z scan, (3) a topographical map, and (4) a contour map. Submerging the bone in rhodamine B dye prior to fluorescent imaging enhanced the pore surface details, giving a more accurate pore size measurement. The average macropore diameter was found to be 260 +/- 97 microm while the average micropore diameter was 13 +/- 10 microm. When compared with common techniques, including microcomputed tomography, magnetic resonance imaging, scanning electron microscopy, and environmental scanning electron microscopy, for imaging cancellous bone, CLSM was found to be an effective tool, given its ability to nondestructively image the surface and near-surface pore structure.     

Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration

To meet the challenge of regenerating bone lost to disease or trauma, biodegradable scaffolds are being investigated as a way to regenerate bone without the need for an auto- or allograft. Here, we have developed a novel microsphere-based chitosan/nanocrystalline calcium phosphate (CaP) composite scaffold and investigated its potential compared to plain chitosan scaffolds to be used as a bone graft substitute. Composite and chitosan scaffolds were prepared by fusing microspheres of 500-900 microm in diameter, and porosity, degradation, compressive strength, and cell growth were examined. Both scaffolds had porosities of 33-35% and pore sizes between 100 and 800 . However, composite scaffolds were much rougher and, as a result, had 20 times more surface area/unit mass than chitosan scaffolds. The compressive modulus of hydrated composite scaffolds was significantly higher than chitosan scaffolds (9.29 +/- 0.8 MPa vs. 3.26 +/- 2.5 MPa), and composite scaffolds were tougher and more flexible than what has been reported for other chitosan-CaP composites or CaP scaffolds alone. Using X-ray diffraction, scaffolds were shown to contain partially crystalline hydroxyapatite with a crystallinity of 16.7% +/- 6.8% and crystallite size of 128 +/- 55 nm. Fibronection adsorption was increased on composite scaffolds, and cell attachment was higher on composite scaffolds after 30 min, although attachment rates were similar after 1 h. Osteoblast proliferation (based on dsDNA measurements) was significantly increased after 1 week of culture. These studies have demonstrated that composite scaffolds have mechanical properties and porosity sufficient to support ingrowth of new bone tissue, and cell attachment and proliferation data indicate composite scaffolds are promising for bone regeneration.     

Assessment of the suitability of chitosan/polybutylene succinate scaffolds seeded with mouse mesenchymal progenitor cells for a cartilage tissue engineering approach

In this work, scaffolds derived from a new biomaterial originated from the combination of a natural material and a synthetic material were tested for assessing their suitability for cartilage tissue engineering applications. In order to obtain a better outcome result in terms of scaffolds' overall properties, different blends of natural and synthetic materials were created. Chitosan and polybutylene succinate (C-PBS) 50/50 (wt%) were melt blended using a twin-screw extruder and processed into 5 x 5 x 5 mm scaffolds by compression moulding with salt leaching. Micro-computed tomography analysis calculated an average of 66.29% porosity and 92.78% interconnectivity degree for the presented scaffolds. The salt particles used ranged in size between 63 and 125 mum, retrieving an average pore size of 251.28 mum. Regarding the mechanical properties, the compressive modulus was of 1.73 +/- 0.4 MPa (E(sec) 1%). Cytotoxicity evaluation revealed that the leachables released by the developed porous structures were not harmful to the cells and hence were noncytotoxic. Direct contact assays were carried out using a mouse bone marrow-derived mesenchymal progenitor cell line (BMC9). Cells were seeded at a density of 5 x 10(5) cells/scaffold and allowed to grow for periods up to 3 weeks under chondrogenic differentiating conditions. Scanning electron microscopy analysis revealed that the cells were able to proliferate and colonize the scaffold structure, and MTS test demonstrated cell viability during the time of the experiment. Finally, Western blot performed for collagen type II, a natural cartilage extracellular matrix component, showed that this protein was being expressed by the end of 3 weeks, which seems to indicate that the BMC9 cells were being differentiated toward the chondrogenic pathway. These results indicate the adequacy of these newly developed C-PBS scaffolds for supporting cell growth and differentiation toward the chondrogenic pathway, suggesting that they should be considered for further studies in the cartilage tissue engineering field.     

Nano-mechanics of bone and bioactive bone cement interfaces in a load-bearing model

Many bioactive bone cements were developed for total hip replacement and found to bond with bone directly. However, the mechanical properties at the bone/bone cement interface under load bearing are not fully understood. In this study, a bioactive bone cement, which consists of strontium-containing hydroxyapatite (Sr-HA) powder and bisphenol-alpha-glycidyl dimethacrylate (Bis-GMA)-based resin, was evaluated in rabbit hip replacement for 6 months, and the mechanical properties of interfaces of cancellous bone/Sr-HA cement and cortical bone/Sr-HA cement were investigated by nanoindentation. The results showed that Young's modulus (17.6+/-4.2 GPa) and hardness (987.6+/-329.2 MPa) at interface between cancellous bone and Sr-HA cement were significantly higher than those at the cancellous bone (12.7+/-1.7 GPa; 632.7+/-108.4 MPa) and Sr-HA cement (5.2+/-0.5 GPa; 265.5+/-39.2 MPa); whereas Young's modulus (6.3+/-2.8 GPa) and hardness (417.4+/-164.5 MPa) at interface between cortical bone and Sr-HA cement were significantly lower than those at cortical bone (12.9+/-2.2 GPa; 887.9+/-162.0 MPa), but significantly higher than Sr-HA cement (3.6+/-0.3 GPa; 239.1+/-30.4 MPa). The results of the mechanical properties of the interfaces were supported by the histological observation and chemical composition. Osseointegration of Sr-HA cement with cancellous bone was observed. An apatite layer with high content of calcium and phosphorus was found between cancellous bone and Sr-HA cement. However, no such apatite layer was observed at the interface between cortical bone and Sr-HA cement. And the contents of calcium and phosphorus of the interface were lower than those of cortical bone. The mechanical properties indicated that these two interfaces were diffused interfaces, and cancellous bone or cortical bone was grown into Sr-HA cement 6 months after the implantation.     

Compressive mechanical properties of demineralized and deproteinized cancellous bone

A method to completely demineralize and deproteinize bone was used to investigate the mechanical properties of either the mineral or protein phase in cancellous bone and compared to an untreated one. Compression tests on cancellous bovine femur and elk antler (Cervus elaphus canadensis) were performed on demineralized, deproteinized, and untreated samples in an air-dry condition. Results showed that the elastic modulus and compressive strength of the demineralized (protein only) and deproteinized (mineral only) samples were far lower than that of the untreated ones, indicating a strong synergetic effect between the two phases. Experimental data showed that the demineralized, deproteinized, and untreated samples can be modeled as cellular solids, with the strong dependence of mechanical properties on the relative density. Deformed samples were examined under SEM and different failure mechanisms were observed. Plastic buckling was observed in demineralized samples while brittle crushing was found in deproteinized ones.     

脱蛋白联合冷冻干燥法制备异种骨的实验研究

目的通过脱蛋白联合冷冻干燥的方法制备异种骨,探讨其能否在保持其生物力学特性的同时良好地消除异种骨的抗原性,以满足骨移植的需求。
　　方法取牛股骨上端去除表面筋膜、结缔组织和皮质骨部分,制备成骨粒和圆柱形骨棒。将十二烷基苯磺酸钠（ABS）和脂肪醇聚氧乙烯醚硫酸钠（AES）以及蒸馏水以重量比13：7：80的比例配制复合表面活性剂。将牛骨粒或者骨棒与复合表面活性剂以重量比1：10的比例置于烧瓶中,超声振荡清洗,去除其抗原性,电镜下观察其结构。骨粒和骨棒经乙醇和乙醚脱水、脱脂,冷冻干燥。骨粒用作溶血试验和细胞毒性检测,骨棒进行新西兰大白兔的长期骨植入实验（4、12、26、52周）检测其生物相容性。乙醇抽吸法检测脱蛋白-冻干骨与未脱蛋白的单纯冻干骨的孔隙率。并且比较牛松质骨骨粒与脱蛋白-冻干松质骨骨粒的生物力学特性差异。
　　结果电镜下观察,制备的异种骨材料为天然多孔结构,保留了骨组织的三维结构,骨小梁间有200~650μm 骨髓腔。溶血率检测未超过5%,判定溶血率合格。细胞毒性检测为1级,极低细胞毒性,材料合格。长期骨植入实验表明,植入4周即有植入的骨棒与兔自身骨出现融合,未见严重炎症反应;52周时,植入骨已完全吸收。孔隙率检测表明,脱蛋白-冻干骨与未脱蛋白的单纯冻干骨相比无显著性差异,均可达到60%以上,符合骨移植材料的要求。脱蛋白-冻干骨以及未脱蛋白的单纯冻干骨的力学特性与新鲜松质骨块相比均有显著降低,但两者之间无显著性差异。结论通过脱蛋白联合冷冻干燥的方法制备的异种骨,能够在保持其生物力学特性的同时良好地消除抗原性;满足骨移植的需求。