去抗原羊椎松质骨支架微观空间结构的分析

背景：实验证实不同去除免疫原性方法对去抗原异种骨材料微观空间结构有着不同的影响。 目的：比较不同方法处理条件下去抗原异种松质骨的微观空间结构。 方法：取成年绵羊新鲜椎骨松质部分制备成特定形状的圆柱体,其长轴与所取松质骨骨小梁排列方向一致,行振动冲洗、不同频率超声冲洗后随机分为3组：物理煅烧组采用甲醇/氯仿、过氧化氢等化学制剂序列脱脂脱细胞脱蛋白后,与焦磷酸钠溶液水浴并直接在煅烧炉内1 000 ℃煅烧3 h;化学处理组采用甲醇/氯仿、过氧化氢等化学制剂序列脱脂脱细胞脱蛋白;对照组直接置于室温自然晾干。通过孔隙率测定、扫描电镜观察、X射线衍射分析、X射线原子能谱元素分析比较各组样品的微观空间结构。 结果与结论：与对照组相比,物理煅烧组、化学处理组均不同程度保持了原骨组织的天然网状孔隙结构,大孔孔径为50-600 μm,微孔孔径为2 μm左右,孔隙率55%-70%;物理煅烧组的主要成分为羟基磷灰石和一定量的β-磷酸三钙,化学处理组与对照组的主要成分为羟基磷灰石;经化学或物理煅烧处理的松质骨立体空间结构并无很大破坏,保留了孔径不等、交通性良好、表面开放的孔隙和高度的孔隙间连接;并且经物理煅烧步骤可更彻底地去除异种松质骨的抗原成分,制成的去抗原异种骨支架材料基本符合组织工程骨支架材料的要求。     

骨髓基质干细胞复合煅烧骨的皮下成骨

背景：研究证明骨髓基质干细胞与煅烧骨支架材料结合后可形成组织工程化骨,但在动物体内的生物相容性及皮下诱导成骨的能力国内报道较少。 目的：观察骨髓基质细胞复合异种煅烧骨植入BALB/c裸鼠背部皮下的成骨性能及煅烧骨材料作为组织工程骨支架材料的可行性。 方法：选用经脱脂及脱蛋白处理后高温煅烧形成的骨支架材料与梯度密度离心法分离培养至第3代的羊骨髓基质干细胞构建细胞-煅烧骨复合物植入BALB/c裸鼠背部皮下,选同期对侧背部皮下植入单纯煅烧骨为对照组。 结果与结论：煅烧后的松质骨块为白垩色,表面呈蜂窝状多孔结构,保留了天然松质骨的多孔状空间结构。骨小梁结构完整,孔隙相互连通。骨髓基质干细胞接种到煅烧骨后24 h可见大量细胞黏附于支架上,7 d后细胞分泌大量细胞外基质,细胞与基质分界不清,细胞能在材料上良好地黏附、增殖与生长,细胞活性未受到支架材料的影响。植入4周后,两组均可见煅烧骨边缘出现少量残片,细胞-煅烧骨复合物组煅烧骨孔隙周边可发现骨细胞,对照组煅烧骨表面可见纤维结缔组织包绕。植入后8周,两组均可见到煅烧骨部分降解为片状类骨质,周围有成纤维细胞包绕,排列紧密,形态多样,细胞-煅烧骨复合物组煅烧骨孔隙内可见煅烧骨表面有排列成行的成骨细胞,孔隙间有散在淋巴细胞浸润。对照组标本可见孔隙内有大量结缔组织长入,未见明显成骨迹象。结果说明,经高温煅烧后的松质骨材料,具有良好的生物相容性和生物安全性,可作为骨髓基质干细胞的良好载体,复合后植入体内能够诱导新生骨组织形成,可作为骨缺损组织工程修复的支架材料。     

物理联合化学或化学方法处理去抗原异种松质骨支架与骨髓间充质干细胞的细胞相容性**

背景：研究证明去抗原异种松质骨支架具有良好的理化性能和三维立体多孔结构,但应用于临床还需要考虑其安全性和生物相容性。 目的：比较物理联合化学及化学方法处理去抗原异种松质骨支架与羊骨髓间充质干细胞的细胞相容性。 方法：用梯度密度离心法分离培养羊骨髓间充质干细胞,四甲基偶氮唑盐比色法检测物理联合化学及化学方法处理去抗原异种松质骨支架材料对骨髓间充质干细胞增殖的影响。取第3代骨髓间充质干细胞,接种在两种支架上共同培养,倒置相差显微镜和扫描电镜观察细胞在两种支架材料上的形态、黏附、生长和增殖情况。 结果与结论：物理联合化学组细胞毒性为0或1级,细胞能在材料上良好地黏附、增殖、生长,细胞活性未受到支架材料的影响。化学组细胞毒性为3级,细胞在材料上生长受到抑制,支架孔隙内无细胞黏附。提示经过物理联合化学处理的去抗原异种松质骨支架材料与羊骨髓间充质干细胞具有良好的生物相容性;单纯经过化学处理的支架材料生物相容性较差,不符合生物材料安全性标准。     

三种去抗原异种松质骨材料生物相容性的比较CSCD

背景:经过一系列理化处理去抗原后的异种骨支架材料,其形态与结构类似于人体骨组织。目的:检测3种不同方法制备去抗原异种骨支架材料的生物相容性。方法:将经过物理、化学及物理联合化学方法制备的去抗原异种骨支架与羟基磷灰石生物陶瓷4组材料分别植入裸鼠背部皮下组织,术后4,8,12周行组织学观察。以经过物理、化学及物理联合化学方法制备的去抗原异种骨支架材料浸提液分别培养羊骨髓间充质干细胞7d,观察细胞黏附、生长、增殖及基质分泌情况。结果与结论:术后4周时,4组材料周围均有较强的炎症反应;术后12周时,物理及物理联合化学制备的去抗原异种骨支架和羟基磷灰石生物陶瓷材料孔隙内及周围组织炎症反应基本消失,极少量炎症细胞存在,材料降解较术后8周时多。化学制备的去抗原异种骨支架材料孔隙内及周围组织炎症反应仍存在;表明物理及物理联合化学制备的去抗原异种骨支架具有良好的组织相容性。羟基磷灰石生物陶瓷和物理联合化学法制备的去抗原异种骨支架材料周围有少量成骨细胞规则排列,并可见少量成骨,表明有异位成骨的趋势。经过物理或物理联合化学方法去除抗原的异种骨支架材料具有良好的细胞相容性;单纯经过化学方法处理的异种骨材料细胞相容性较差,不符合生物材料安全性标准。     

松质骨支架的制备及结构特征观察

目的研究猪松质骨支架结构特征。方法采用物理化学方法对猪松质骨进行处理得到猪松质骨支架,采用扫描电子显微镜(SEM)观察材料的结构、图像分析测量孔径,液体静力称重法测量材料的孔隙率,DDW-100型万能实验机进行材料抗压测试。结果猪松质骨支架外观呈乳白色、透光、无异味,维持原有形态并有一定的强度;骨块呈蜂窝状多孔结构,表面孔隙与深层孔隙相连通;扫描电镜观察材料表现为三维多孔网状结构,孔隙均匀且孔隙相互连通;孔径为(387.54±21.60)μm,孔隙率为78.26%±2.01%,最大抗压强度和抗压力分别为25MPa和12N。结论猪松质骨支架具有骨组织工程支架材料结构特征。     

异种生物衍生骨支架材料的制备及性能

背景：异种骨来源丰富,价格低廉,处理相对简单容易,处理后骨支架保留原有骨的微结构,具有良好的促成骨、骨传导及骨诱导活性。 目的：检测自制生物衍生骨支架材料的理化性质及体外细胞相容性。 方法：通过脱蛋白、脱脂、脱钙,深低温冻存制备猪源性松质骨支架材料。组织学检测松质骨处理前后的变化,扫描电镜观察材料结构及计算孔隙直径,采用液体置换法检测支架材料的孔隙率,体外降解速度,能谱分析及体外复合兔骨髓间充质干细胞的细胞相容性。 结果与结论：处理后的松质骨支架材料具有三维多孔结构,孔隙直径150.8-306.7 μm,孔隙率84.5%-89.7%。材料在前6周降解速度稍慢,6周后材料降解率曲线基本呈线性且降解速度明显加快,10周时材料接近完全降解,降解率达92.8%。松质骨支架材料孔隙大小适合骨髓间充质干细胞的黏附和增殖。表明生物衍生骨支架材料性能良好,细胞相容性良好,适用于构建组织工程骨。     

骨形态发生蛋白复合支架的体外缓释性能

背景：临床试验证明纳米晶胶原基骨支架材料具有较好的组织相容性、合适的孔隙率及降解性能,但缺乏缓释生长因子的作用,且无成骨诱导性。 目的：将聚乙稀吡咯啉酮与骨形态发生蛋白复合后形成复合颗粒,再修饰纳米晶胶原基骨制备复合支架,观察骨形态发生蛋白的体外缓释效果。 方法：实验分3组,实验组取冻存管2支,每支放入5 mm×5 mm×5 mm 纳米晶胶原基骨1块,滴加聚乙稀吡咯啉酮混匀,-4 ℃冻存过夜,再滴加骨形态发生蛋白溶液,真空抽干后冻存;对照组1取冻存管2支,每支放入5 mm×5 mm×5 mm 纳米晶胶原基骨1块,滴加骨形态发生蛋白溶液,真空抽干后冻存;对照组2取冻存管2支,每支放入5 mm×5 mm×5 mm未脱钙的大鼠松质骨1块,滴加聚乙稀吡咯啉酮混匀,-4 ℃冻存过夜,再滴加骨形态发生蛋白溶液,真空抽干后冻存。观察14 d,采用ELISA法检测3组复合物骨形态发生蛋白的体外释放活性。 结果与结论：观察到14 d时,两对照组骨形态发生蛋白A值趋近于0,而实验组上清液中骨形态发生蛋白仍保持较高的A值,组间比较差异有显著性意义(P < 0.05)。表明经聚乙烯吡咯烷酮修饰后纳米晶胶原基骨支架具有明显缓释骨形态发生蛋白的作用。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：     

冻干去抗原羊松质骨支架的生物相容性评价

背景:异种骨具有与人类骨类似的天然多孔结构,在治疗骨缺损时可引导骨组织再生,但植入过程中也会引起不同程度的免疫反应。目的:采用冻干法制备去抗原羊脊椎松质骨支架材料,并评价其生物相容性。方法:取羊脊椎松质骨,制备两组去抗原异种骨支架,化学组经H2O2、甲醇/氯仿混合液等化学试剂处理;冻干组将经化学处理的羊松质骨在-80℃冰箱中低温冷冻4周,真空干燥,60Co照射消毒。①细胞毒性实验:分别采用化学组材料浸提液、冻干组材料浸提液与DMEM/F12培养基培养羊骨髓间充质干细胞。②热源实验、急性毒性实验:从兔耳缘静脉分别注射化学组材料浸提液、冻干组材料浸提液与生理盐水。③皮内刺激实验:分别在兔脊柱背部皮下注射化学组材料浸提液、冻干组材料浸提液、生理盐水及乙醇。结果与结论:冻干组支架无细胞毒性、急性毒性及热源反应,皮内刺激实验阴性;化学组支架有细胞毒性及轻微急性毒性反应,有致热源作用及轻度皮肤刺激性。结果表明经过化学处理羊脊椎松质骨支架的生物相容性相对较差,而化学处理配合低温冷冻、真空干燥及60Co照射的羊脊椎松质骨生物相容性较好,基本能达到骨组织工程支架材料的要求。     

镀膜工艺结合3D打印制备Mg-F膜/淫羊藿素膜/β-磷酸三钙支架的表征及成骨能力

背景：修饰生长因子的骨组织工程支架在骨修复材料中具有广阔的应用前景,但生长因子释放过快导致复合支架仅能在早期促进骨修复,镀膜工艺为解决该问题提供了新思路。目的：制备Mg-F膜/淫羊藿素/β-磷酸三钙支架,表征该支架的生物学特性。方法：应用3D打印技术制备β-磷酸三钙支架,通过低能电子束沉积技术制备淫羊藿素膜/β-磷酸三钙支架,再通过脉冲激光沉积技术制备Mg-F膜/淫羊藿素膜/β-磷酸三钙支架,检测支架的微观结构、孔隙直径、孔隙率、丝径、抗压强度及元素组成,并分析淫羊藿素的结合力和缓释性能。将兔骨髓间充质干细胞分别与β-磷酸三钙支架、淫羊藿素/β-磷酸三钙支架与Mg-F膜/淫羊藿素膜/β-磷酸三钙支架浸提液共培养,利用CCK8法检测细胞增殖;将兔骨髓间充质干细胞分别与上述3种支架共培养,加入成骨诱导培养基,利用茜素红染色观察成骨分化能力。结果与结论：(1)扫描电镜下可见,β-磷酸三钙支架结构较为规则,孔隙连通率好;淫羊藿素膜/β-磷酸三钙支架表面有较为致密的淫羊藿素膜覆盖原微观孔隙;Mg-F膜在淫羊藿素膜表面沉积,留下较为粗糙、疏松的表面,存在微观孔隙;3种支架的孔隙直径、孔隙率、丝径、...     

材料孔隙结构对骨长入的影响

材料的孔隙结构对骨长入有关键性的影响.高孔隙率和大孔径有利于骨长入,但有损于力学性能.因此,在保证材料力学性能的基础上,探索最佳的孔隙结构足很有必要的.随着材料科学技术的发展,人们可以相对自由地设计特定的孔隙结构,为骨组织工程的进一步探索提供条件.就材料孔隙结构对骨长入的影响,从离体细胞学研究以及材料孔隙率.孔径大小等方面进行综述.     

Preparation of macroporous polymer scaffolds using calcined cancellous bone as a template

A technique for preparing porous polymeric scaffolds using calcined cancellous bone as template has been developed. The scaffolds prepared by this method were morphologically similar to the structure of the demineralized bone matrix (DBM), i.e. scaffolds prepared by this novel method possessed high porosity, interconnected pores and uniform pore distribution. In addition, the mechanical properties of the scaffolds prepared by this novel method were significantly improved as compared to that of the scaffolds prepared by conventional solvent cast-particle leaching (SC-PL) method. The effects of concentration of the polymer solution on the structure, porosity and mechanical properties of the scaffolds were evaluated and the results showed that the mechanical properties of the scaffolds could be controlled by adjusting the concentration of the polymer solution. The advantages mentioned above suggest that this novel method might be a useful technique to prepare polymer scaffolds for tissue engineering.     

Preparation of macroporous polymer scaffolds using calcined cancellous bone as a template

A technique for preparing porous polymeric scaffolds using calcined cancellous bone as template has been developed. The scaffolds prepared by this method were morphologically similar to the structure of the demineralized bone matrix (DBM), i.e. scaffolds prepared by this novel method possessed high porosity, interconnected pores and uniform pore distribution. In addition, the mechanical properties of the scaffolds prepared by this novel method were significantly improved as compared to that of the scaffolds prepared by conventional solvent cast–particle leaching (SC-PL) method. The effects of concentration of the polymer solution on the structure, porosity and mechanical properties of the scaffolds were evaluated and the results showed that the mechanical properties of the scaffolds could be controlled by adjusting the concentration of the polymer solution. The advantages mentioned above suggest that this novel method might be a useful technique to prepare polymer scaffolds for tissue engineering.     

Three-dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at -20 degrees C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140 degrees C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 microm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.     

Three-dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at ?20°C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140°C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 μm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.     

Preparation of porous hydroxyapatite scaffolds by combination of the gel-casting and polymer sponge methods

A new technique of combining the gel-casting and polymer sponge methods is introduced in this study to prepare macroporous hydroxyapatite scaffolds, which provides a better control over the microstructures of scaffolds and enhances their mechanical properties. With this technique, we were able to produce scaffolds with mechanical and structural properties that cannot be attained by either the polymer sponge or gel-casting method. The scaffolds prepared have an open, uniform and interconnected porous structure with a pore size of 200-400 microm. A compressive yield strength of approximately 5 MPa equivalent to that of cancellous bone and a compressive modulus of approximately 8 GPa similar to that of cortical bone were achieved. The pore morphology, size, and distribution of the scaffolds were characterized using a scanning electron microscope. X-ray diffraction and Fourier transform infrared spectroscopy were used to determine the crystal structure and chemical composition of scaffolds, respectively. Scaffolds with desired porosity, pore size, and geometry can be prepared by using polymer sponges of appropriate structures.     

Novel porous gelatin scaffolds by overrun/particle leaching process for tissue engineering applications

Porous gelatin scaffolds were prepared using a modified overrun process, which is a novel method for preparing a porous matrix by injecting air and mixing polymer solution at low temperature. The pores in the scaffolds formed by the overrun process exhibited a dual-pore structure due to the injection of air bubbles and ice recrystallization. However, the morphology of the overrun-processed gelatin scaffolds had closed pore structures. The closed pore structure was reformed into a uniformly distributed and interconnected open structure by the combination of the overrun process and a particle-leaching technique (NaCl and sucrose). The mechanical strength and biodegradation rate of gelatin scaffolds were controlled by the matrix porosity and concentration of gelatin solution. Despite higher porosity, overrun processed gelatin scaffolds showed similar mechanical strength to freeze-dried scaffolds. After 1 week of in vitro culturing, the fibroblasts in overrun-processed scaffolds were widely distributed on the surface of the scaffold pores, whereas cells seeded in freeze-dried scaffolds were mainly placed on the top and bottom of the scaffolds. Therefore, the overrun process combined with a particle-leaching technique can be applied to fabricate porous scaffolds with a desirable cellular structure for tissue engineering applications.     

同种异体松质骨支架材料的制备及理化特性

背景：同种异体脱脂、脱蛋白松质骨具有与受体相同的三维立体结构,力学性能稳定,排异反应弱,细胞相容性好等独特的生物学性能。 目的：通过理化方法制备同种异体脱脂、脱蛋白松质骨支架材料,分析其理化特性。 方法：剥离兔髂骨10对,制作成约1.0 cm×0.8 cm×0.1 cm的骨条,经脱脂、脱蛋白、深低温冷冻处理制备骨支架材料,检测其生物化学性能。测定支架材料与骨髓间充质干细胞的黏附率;将支架植入同种属动物体内,观察其组织相容性、免疫反应。 结果与结论：同种异体脱脂、脱蛋白松质骨支架材料保留了天然骨组织的网状孔隙结构,孔隙率为(80.23±5.65)%,孔径最大为(318.11±17.51) μm,最小为(209.37±11.33) μm。骨髓间充质干细胞不仅能与支架黏附,而且能在支架上分裂、增殖。兔体内植入6周后支架周围界面未引起明显的炎症和排斥反应,并形成少量骨样组织。说明脱脂、脱蛋白松质骨支架具有适宜的三维多孔结构,与种子细胞黏附率高,有良好的生物相容性和细胞-材料界面作用;同时有一定的成骨作用。     

Preparation of a biphasic porous bioceramic by heating bovine cancellous bone with Na4P2O7.10H2O addition

Sintered bovine cancellous bone exhibited excellent biocompatiblity, high porosity and have an interconnecting porous structure allowing for bone ingrowth. However, the main mineral constitution of sintered bovine bone-hydroxyapatite (Ca10(PO4)6(OH)2, HAP) seems to be too stable in vivo. For improving its bioactivity, the calcined bovine bone removing the organic substance by burning process-with different quantities of sodium pyrophosphate (Na4P2O7.10H2O, NP) addition was heated to a high temperature to transform its crystalline phase constitution from HAP into TCP/HAP biphasic or other multiphasic structures. Results revealed that the calcined bovine bone without NP addition, exhibited a pure form of HAP characterized pattern during heating. Its thermal behavior was similar to stoichiometric HAP, it gradually lost its OH- ions and transformed into oxyhydroxyapatite at high temperature. After being doped into calcined bovine bone, NP would react with HAP to form betaBTCP and NaCaPO4 around 600 degrees C. At 900 degrees C, doped NP would completely react with HAP and the NaCaPO4 would further react with HAP to form more betaBTCP in the system. With NP increasing in the calcined bovine bone, HAP would gradually convert into different crystalline phase compositions of TCP/HAP, TCP/HAP/NaCaPO4 or TCP/NaCaPO4 at high temperature. By heating calcined bovine cancellouse bone with different quantities of NP we could obtain different crystalline phase compositions of natural porous bioceramic in this study.     

多孔纳米羟基磷灰石/聚酰胺复合骨修复材料的制备

BACKGROUND: Compared with dense bone repair materials, porous materials has lower intensity, but its three-dimensional porous network structure can ensure a larger surface area that is more conducive to cell adhesion, growth and division as well as nutrient transmission. OBJECTIVE: To investigate the preparation and performance of porous nano-hydroxyapatite/polyamide composites. METHODS: Porous nano-hydroxyapatite/polyamide composites were prepared using chemical foaming method, and different composite bone repair materials were made by regulating foaming agent amount and nano-hydroxyapatite content. Mechanical properties, porosity and composition of the composite bone repair materials were detected. RESULTS AND CONCLUSION: Porous nano-hydroxyapatite/polyamide composites were substantially tubular  channels, and had relatively good connectivity and uniform distribution, with a pore size of 260-400 μm, porosity of 35%-57%. Foaming agent amount, nano-hydroxyapatite content and density of composite materials all are influential factors of the total porosity. Porous nano-hydroxyapatite/polyamide composites have better mechanical properties and compressive strength than the cancellous bone, and the foaming agent has no influence on its shape, composition and diffraction peaks.