纳米晶重组类人胶原基/聚乳酸复合支架材料的表征及细胞相容性研究

目的　制备纳米羟基磷灰石/重组类人胶原基/聚乳酸复合支架材料 (nano-hydroxyapatite/ recombinant human- like collagen/polylactic acid,nHA/RHLC/PLA),观察材料的形貌特征,探讨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nHA/RHLC/PLA复合支架材料,应用X 光衍射分析（XRD）、红外光谱分析（FTIR）、ZWICK Z005 测试机对样品的化学成分、机械性能测试和压缩强度进行测试,通过扫描电镜检查等方法观察材料的表征;将犬骨髓基质细胞（BMSCs）接种在支架材料上培养,检测材料-细胞的黏附情况及材料对细胞生长增殖的影响。 结果　nHA/RHLC/PLA复合支架材料压缩强度均大于1MPa,达到了天然松质骨的最低强度。扫描电镜结果显示：支架材料呈三维多孔结构,孔为不规则多边形,孔的走向多样,纵向和横向孔隙互为交通,孔径在几十微米到300微米不等,孔隙率为75%~83%。nHA/RHLC/PLA复合支架材料表面BMSCs的黏附、生长良好;而BMSCs的增殖能力与对照组相比,差异无显著性意义(P>0.05)。 结论　nHA/RHLC/PLA复合支架材料符合组织工程骨支架的力学要求,具有良好的微观结构,无细胞毒性,细胞与支架生物相容性良好。利用重组类人胶原代替动物源性胶原制备纳米晶骨修复材料,规避了动物胶原交叉感染的风险,有望成为一种理想的骨组织工程支架材料。     

Hierarchically biomimetic bone scaffold materials: nano-HA/collagen/PLA composite

A bone scaffold material (nano-HA/ collagen/PLA composite) was developed by biomimetic synthesis. It shows some features of natural bone both in main composition and hierarchical microstructure. Nano-hydroxyapatite and collagen assembled into mineralized fibril. The three-dimensional porous scaffold materials mimic the microstructure of cancellous bone. Cell culture and animal model tests showed that the composite material is bioactive. The osteoblasts were separated from the neonatal rat calvaria. Osteoblasts adhered, spread, and proliferated throughout the pores of the scaffold material within a week. A 15-mm segmental defect model in the radius of the rabbit was used to evaluate the bone-remodeling ability of the composite. Combined with 0.5 mg rhBMP-2, the material block was implanted into the defect. The segmental defect was integrated 12 weeks after surgery, and the implanted composite was partially substituted by new bone tissue. This scaffold composite has promise for the clinical repair of large bony defects according to the principles of bone tissue engineering.     

Preparation of fibroin/recombinant human-like collagen scaffold to promote fibroblasts compatibility

Recombinant human-like collagen (RHLC) was added to fibroin solution to prepare a novel hybrid scaffold material for skin tissue engineering. The morphology of the scaffold had highly homogeneous and interconnected pores with pore sizes 136 +/- 32 mum measured by scanning electron microscopy (SEM). FTIR analysis indicated intermolecular crosslinkages between fibroin and RHLC formed. The viscosity of the blend solution increased because of the interaction between fibroin and RHLC, and then it restrained the unwanted fibroin aggregation in freezing process that generally appeared in fibroin scaffold preparation with freeze drying method. After methanol treatment the fibroin/RHLC scaffold became water-stable. The porosity of scaffolds was >90%, the compressive strength and modulus were up to 662 +/- 32 KPa and 7.8 +/- 0.64 MPa, respectively. Fibroblasts cultured within fibroin/RHLC scaffolds were investigated by SEM, laser scanning confocal microscopy (LSCM), and MTT assay, which showed that the adding of RHLC significantly enhanced the cells adhesion, proliferation, and viability compare with fibroin scaffolds. These results suggest that the hybrid scaffolds have favorable characteristics for skin tissue engineering.     

Fabrication of nano-hydroxyapatite on electrospun silk fibroin nanofiber and their effects in osteoblastic behavior

In this study, a novel tissue engineering scaffold material of electrospun silk fibroin/nano-hydroxyapatite (nHA) biocomposite was prepared by means of an effective calcium and phosphate (Ca-P) alternate soaking method. nHA was successfully produced on regenerated silk fibroin nanofiber as a substrate within several minutes without any pretreatments. The morphologies of both nonmineralized and mineralized nanofibers were analyzed using a field-emission scanning electron microscopy (FESEM). The crystallographic phases of the nHA were analyzed using X-ray diffraction (XRD). Fourier transform infrared (FTIR) spectrophotometer and thermogravimetry analyses (TGA) were employed to determine the type of functional groups and the amount of nHA presenting in the silk/nHA biocomposite nanofibers, respectively. The osteoblastic activities of this novel nanofibrous biocomposite scaffold were also investigated by employing osteoblastic-like MC3T3-E1 cell line. The cell functionality such as alkaline phosphatase (ALP) activity was ameliorated on mineralized nanofibers. All these results indicated that this silk/nHA biocomposite scaffold material may be a promising biomaterial for bone tissue engineering.     

Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl(2). After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process.     

纳米羟基磷灰石/I型胶原复合人工骨组成结构及成骨活性的实验研究

按照仿生的方法合成纳米羟基磷灰石/I型胶原人工骨支架材料.采用扫描电镜、X线衍射分析、孔隙率测定的方法对人工骨支架材料进行分析.制作兔颅骨缺损模型,植入人工骨支架材料,组织切片观察支架材料在体内的反应.纳米羟基磷灰石/I型胶原人工骨支架材料呈疏松海绵状,具有100～300 μm的孔径和90%以上的孔隙率,具有类似天然骨的结构.植入兔颅骨缺损模型未出现急性或慢性的炎症反应,在4周左右人工骨支架内出现大量新生毛细血管,12周新生骨完全修复骨缺损,并形成成熟的骨小梁结构.纳米羟基磷灰石和I型胶原复合人工骨约3个月左右降解完全.     

骨髓间充质细胞复合胶原-壳聚糖材料联合骨搬移修复胫骨缺损：随机对照实验方案

背景：骨髓间充质干细胞发挥成骨作用需要支架材料的辅助,一方面支架材料不仅可将细胞运载至骨缺损区域,另一方面还可作为新骨生长的框架结构。胶原-壳聚糖复合材料是骨组织工程较为理想的支架材料之一,同时其具有骨诱导性,比常规支架材料更优越的成骨能力。骨搬移技术在临床上在修复长段骨缺损方面已得到广泛应用,但也存在成骨慢、外固定时间长、骨不连等缺憾。如何进一步加快骨形成速度,减少并发症发生,已成当前亟待解决的问题。实验假设：骨髓间充质干细胞复合胶原-壳聚糖支架移植能提高胫骨缺损骨搬移修复效果。 方法/设计：随机对照动物实验。分为体外和体内实验两部分。体外实验中取月龄一两个月的新西兰大白兔股骨骨髓,提取骨髓间充质干细胞,培养至第3代,将细胞悬液滴于胶原-壳聚糖支架材料,构建骨髓间充质干细胞复合胶原-壳聚糖支架。体内实验选用24只三四月龄新西兰大白兔,被随机分配接受如下干预：骨搬移、支架植入、骨搬移联合支架植入。研究的主要观察指标为植入材料与骨缺损界面的生长情况、X射线检测的缺损区骨修复情况、苏木精-伊红染色及扫描电镜观察缺损区成骨情况、免疫组织化学染色检测成骨区Ⅰ型胶原蛋白的表达情况、扫描电子显微镜观察移植材料与宿主骨的界面键合情况、超微结构及新骨的生成。 讨论：实验结果将有助于确定对骨缺损进行骨搬移治疗过程中,应用骨髓间充质干细胞复合胶原-壳聚糖支架移植促进骨缺损再生修复效果的可行性。 实验方案获基金支持情况：获辽宁省科学技术计划项目资助(2012225019)。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog

The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactide-co-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs' mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration.     

Repair of segmental bone defects in rabbit tibiae using a complex of beta-tricalcium phosphate, type I collagen, and fibroblast growth factor-2

The objective of this study was to evaluate the effects of a complex of beta-tricalcium phosphate (beta-TCP) granules, collagen, and fibroblast growth factor-2 (FGF-2) on cortical bone repair in rabbits. Segmental bone defects of 5 mm in length were created in the middle of the tibial shaft. The defect was stabilized with a plate and screws, and was filled with 0.3 ml of a complex of beta-TCP granules and 5% collagen, with or without 200 microg of recombinant human fibroblast growth factor-2 (rhFGF-2). Bone regeneration and beta-TCP resorption were assessed by X-ray and micro-CT scanner. A three-point bending test was also performed. The results showed that the segmental bone defect was not only radiologically, but also mechanically healed with cortical bone 12 weeks after implantation of the complex with rhFGF-2. In contrast, after implantation of the complex without rhFGF-2, most of the defect was filled with beta-TCP and only a small amount of bone formation was found. These results suggest that resorption of beta-TCP is important for bone formation and may be promoted by FGF-2 in the beta-TCP implantation site. In addition, the complex of beta-TCP granules and collagen combined with rhFGF-2 provides a paste-like material that is easy to handle. This material may be of considerable use in the treatment of cortical bone defects.     

In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane) composite scaffold for bone tissue engineering

Scaffolds for bone tissue engineering should provide an osteoconductive surface to promote the ingrowth of new bone after implantation into bone defects. This may be achieved by hydroxyapatite loading of distinct scaffold biomaterials. Herein, we analyzed the in vitro and in vivo properties of a novel nanosize hydroxyapatite particles/poly(ester-urethane) (nHA/PU) composite scaffold which was prepared by a salt leaching–phase inverse process. Microtomography, scanning electron microscopy and X-ray spectroscopy analyses demonstrated the capability of the material processing to create a three-dimensional porous PU scaffold with nHA on the surface. Compared to nHA-free PU scaffolds (control), this modified scaffold type induced a significant increase in in vitro adsorption of model proteins. In vivo analysis of the inflammatory and angiogenic host tissue response to implanted nHA/PU scaffolds in the dorsal skinfold chamber model indicated that the incorporation of nHA particles into the scaffold material did not affect biocompatibility and vascularization when compared to control scaffolds. Thus, nHA/PU composite scaffolds represent a promising new type of scaffold for bone tissue engineering, combining the flexible material properties of PU with the advantage of an osteoconductive surface.     

In vivo study on hydroxyapatite scaffolds with trabecular architecture for bone repair

The objective of this research was to investigate the bone formation and angio-conductive potential of hydroxyapatite (HA) scaffolds closely matched to trabecular bone in a canine segmental defect after 3 and 12 weeks post implantation. Histomorphometric comparisons were made between naturally forming trabecular bone (control) and defects implanted with scaffolds fabricated with micro-size (M-HA) and nano-size HA (N-HA) ceramic surfaces. Scaffold architecture was similar to trabecular bone formed in control defects at 3 weeks. No significant differences were identified between the two HA scaffolds; however, significant bone in-growth was observed by 12 weeks with 43.9 +/- 4.1% and 50.4 +/- 8.8% of the cross-sectional area filled with mineralized bone in M-HA and N-HA scaffolds, respectively. Partially organized, lamellar collagen fibrils were identified by birefringence under cross-polarized light at both 3 and 12 weeks post implantation. Substantial blood vessel infiltration was identified in the scaffolds and compared with the distribution and diameter of vessels in the surrounding cortical bone. Vessels were less numerous but significantly larger than native cortical Haversian and Volkmann canals reflecting the scaffold architecture where open spaces allowed interconnected channels of bone to form. This study demonstrated the potential of trabecular bone modeled, highly porous and interconnected, HA scaffolds for regenerative orthopedics.     

Porous collagen-apatite nanocomposite foams as bone regeneration scaffolds

We have created a porous bioresorbable nanocomposite bone scaffold that chemically, structurally and mechanically matched natural bone so that it could be recognized and remodeled by natural bone. Containing collagen fibers and synthetic apatite nanocrystals, our scaffold has high strength for supporting the surrounding tissue. The foam-like scaffold has a similar microstructure as trabecular bone, with nanometer-sized and micron-sized pores. The apatitic phase of the scaffold exhibited similar chemical composition, crystalline phase and grain size as the trabecular bone apatite. The nanocomposite scaffold demonstrated excellent bioactivity for promoting cell attachment and proliferation. It was osteoconductive and successfully healed a non-union fracture in rat femur as well as a critical-sized defect in pig tibia.     

新短肽P17-骨形态发生蛋白2/胶原基质矿化磷灰石材料生物活性的评价

文题释义：胶原基质矿化磷灰石：具有良好的生物相容性,不产生排斥反应,降解速度与成骨的速度相适应,其降解不会影响周围环境的pH值。该材料在微米尺度上具有互联孔洞结构,孔隙尺寸为100-500 µm,孔隙率为70%-90%,结构和成分与自体骨相似,能够更好的诱导自体骨生长,具有良好的骨修复作用,其机械耐受性、可塑性、强度接近松质骨。 新短肽P17-骨形态发生蛋白2：通过FMOC/tBu固相多肽合成法合成的具有17个氨基酸的新型活性短肽中包含磷酸化的丝氨酸及天冬氨酸,能够极好地模拟天然骨基质的促发及指导矿化的功能,在局部形成偏酸环境,促进局部的钙磷沉积、成核和生物自组装矿化。短链多肽活性位点能充分暴露并与细胞表面受体结合,生物活性更强。 背景：胶原基质矿化磷灰石材料具有仿生的化学组成及良好的生物学性能,已被用于某些骨缺损修复;新短肽P17-骨形态发生蛋白2具有良好的生物相容性和成骨诱导生物活性,因此将新短肽P17-骨形态发生蛋白2与胶原基质矿化磷灰石材料制备成复合支架材料可望提升骨修复效率和效果。 目的：探讨新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料的生物活性。 方法：将兔骨髓间充质干细胞分别接种于新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料与胶原基质矿化磷灰石材料上,培养3,7 d后,利用RT-PCR检测细胞碱性磷酸酶 mRNA相对表达。将新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料(实验组)与胶原基质矿化磷灰石材料(对照组)分别埋置于SD大鼠皮下,植入12,35 d后进行Masson染色后组织学分析。将新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合材料(实验组)与胶原基质矿化磷灰石材料(对照组)分别植入日本大耳白兔下颌骨箱状缺损处,植入5,15周后进行大体与X射线检查。实验经中国医科大学附属口腔医院伦理委员会批准。 结果与结论：①复合材料组培养7 d的碱性磷酸酶mRNA表达高于胶原基质矿化磷灰石组(P < 0.05);②皮下埋植实验显示两组材料和组织界面均未引起明显的急性炎症反应,植入后35 d实验组可见更多的纤维细胞与材料嵌合;③骨缺损修复实验中,大体观察显示两种材料均具有良好的骨修复能力,植入5周时缺损区已有缩小趋势,植入15周缺损表面比较平整;X射线检查显示与对照组相比,实验组缺损区缩小趋势更明显;④结果表明,新型P17-骨形态发生蛋白2/胶原基质矿化磷灰石复合支架材料具有比胶原基质矿化磷灰石更为优良的生物活性与骨缺损修复能力。 ORCID: 0000-0002-1196-5954(张雪) 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     

Reconstruction of extensive long bone defects in sheep using resorbable bioceramics based on silicon stabilized tricalcium phosphate

In this study we evaluated the performance of Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (Si-TCP), in promoting the repair of a large-sized, experimentally induced defect in a weight-bearing long bone sheep model. Eighteen 2-year-old ewes were used in this study. Animals were sacrificed at 3, 6, and 12 months. One animal entered a very prolonged followup and was sacrificed 2 years after surgery. Bone formation and scaffold resorption were evaluated by sequential x-ray studies, CT scans, histology, immunohistology, microradiography, and quantitative analysis of x-ray studies (optical density) and microradiographs (percentage of bone and scaffold area). Our data show an excellent implant integration and significant bone regeneration within the bone substitute over the course of the experiment. Progressive osteoclastic resorption of the biomaterial was also evident. At 1 year from surgery, the remaining scaffold was approximately 10-20% of the scaffold initially implanted, while after 2 years it was essentially completely resorbed. At the end of the observation period, the segmental defect was filled with newly formed, highly mineralized, lamellar bone.     

骨组织工程三维复合支架修复兔桡骨骨缺损

背景：前期实验构建的丝素/壳聚糖/纳米羟基磷灰石复合支架具有良好的理化性质。 目的：观察丝素/壳聚糖/纳米羟基磷灰石三维复合支架修复兔桡骨大段骨缺损的效果。 方法：取新西兰大白兔36只,建立右侧桡骨长段骨缺损模型,随机均分为3组,实验组于骨缺损处植入丝素/壳聚糖/纳米羟基磷灰石复合支架,对照组于骨缺损处植入丝素/壳聚糖复合支架,空白对照组造模后不作任何处理。术后4,8,12,16周进行X射线摄片、标本大体观察、组织病理学观察。 结果与结论：术后16周,实验组缺损区X射线影像与正常骨组织无区别,骨髓腔完全再通,有明显的骨组织生成,苏木精-伊红染色可见骨小梁和较多核深染的长梭形骨细胞;对照组X射线骨密度影略低于正常骨组织,部分骨髓腔再通,苏木精-伊红染色可见骨细胞周围有不少软骨细胞,未见明显的骨小梁或骨板结构,排列较紊乱;空白对照组断端骨钙化影同正常骨组织一致,断端各自封闭形成骨不连,苏木精-伊红染色可见较多的纤维组织和少量的类骨组织。表明丝素/壳聚糖/纳米羟基磷灰石三维复合支架可较好地修复兔桡骨大段骨缺损。     

聚乳酸/矿化胶原复合人工骨修复材料在兔股骨髁松质骨缺损中的应用研究*

目的 研究聚乳酸/矿化胶原人工骨修复材料（NBG）在兔股骨髁松质骨缺损中的疗效,以及该材料与自体骨、骨髓混合使用的效果,考察材料的安全性及有效性。方法 在兔双侧股骨髁造成松质骨缺损,将60只雄性大白兔进行不同骨材料植入。试验分为5组：空白（Blank）组、人工骨（NBG）组、人工骨混骨髓（NBG+ABM）组、人工骨混自体骨（NBG+AGB）组、自体骨（AGB）组,通过影像学观察、组织学评价、生物力学分析等方法考察5 组材料的骨修复性能。结果 观察新生骨和周围骨质结合情况,拍摄X线片并进行灰度值分析以及进行CT三维重建分析,不同时间点的组织切片观察,均表明NBG组、NBG+ABM组、NBG+AGB组、AGB组骨修复效果良好,生物力学测试也表明各实验组相对于空白组可以承载较高的压力载荷。AGB组、NBG+AGB组、NBG+ABM组、NBG组修复效果依次降低,但均接近完全修复。结论 聚乳酸/矿化胶原基人工骨修复材料有良好的生物相容性及骨诱导性,是良好的骨植入材料。单独使用即可以获得良好的骨修复效果,也可以和自体骨或骨髓混合使用,修复效果更加明显。     

Enhancement of osteogenesis by poly(lactide-co-glycolide) sponges loaded with surface-embedded hydroxyapatite particles and rhBMP-2

The bone mesenchymal stem cells (BMSCs) were seeded on [poly(lactide-co-glycolide) scaffolds with hydroxyapatite (HA) coating, and "s" stands for surface] (PLGA/HA-S), PLGA/HA-M (containing the same HA amount in the matrix as that of the PLGA/HA-S and "m" stands for matrix), and PLGA scaffolds, which were then cultured in a medium-containing Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2). In vitro culture of rat BMSCs found no different cell morphology in all the scaffolds, but the alkaline phosphatase activity and osteogenic gene expression of type I collagen (COL I) and osteocalcin (OCN) in the PLGA/HA-S scaffolds were always highest and were significantly improved in comparison with those in the PLGA scaffolds. In a rat calvarial defect model, new bone formation was enhanced in the PLGA/HA-S/ErhBMP-2 implants at 4 and 8 weeks after implantation too. Therefore, the PLGA/HA-S scaffold can better enhance the ErhBMP-2-induced osteogenic differentiation of BMSCs in vitro and osteogenesis in vivo.     

3D打印羊椎骨粉/聚乙烯醇支架、纳米级羟基磷灰石/聚乙烯醇支架、羊椎骨粉/聚乙烯醇无孔骨板的性能比较

BACKGROUND: With the promotion of 3D printing technology, 3D printing scaffolds for bone tissue engineering have become the new ideas for jaw bone repair. OBJECTIVE: To compare the physical and biological properties of sheep vertebral bone meal/polyvinyl alcohol (PVA) scaffold, nano-hydroxyapatite (nHA)/PVA scaffold, and sheep vertebral bone meal/PVA nonporous bone plate. METHODS: 3D printing technology was used to print sheep vertebral bone meal/PVA scaffold, nHA/PVA scaffold, and sheep vertebral bone meal/PVA nonporous bone plate. Porosity, morphology, water absorption rate and mechanical properties of different scaffolds were detected. Three kinds of scaffolds were all used to culture bone marrow mesenchymal stem cells, and cell proliferation ability was detected using cell counting kit-8 at 1, 4, 7 days of culture. RESULTS AND CONCLUSION: Under scanning electron microscope, the sheep vertebral bone meal/PVA scaffold and nHA/PVA scaffold exhibited regular and interconnected pores with good continuity and clear network structure; the sheep vertebral bone meal/PVA nonporous bone plate had no obvious pores; however, it had dense and evenly distributed micropores with different sizes on its surface. The porosity of nHA/PVA scaffold was lower than that of the sheep vertebral bone meal/PVA scaffold (P < 0.05). The water absorption rate was highest for the nHA/PVA scaffold followed by the sheep vertebral bone meal/PVA scaffold and the sheep vertebral bone meal/PVA nonporous bone plate (P < 0.05). In contrast, the scaffold toughness was highest for the sheep vertebral bone meal/PVA nonporous bone plate, followed by the sheep vertebral bone meal/PVA scaffold and nHA/PVA scaffold. In addition, the cell proliferation activity of cells cultured on the sheep vertebral bone meal/PVA scaffold was significantly higher than that cultured on the other two kinds of scaffolds. Taken together, the 3D printing sheep vertebral bone/PVA scaffold has good physical and chemical performance.     

Repair of diaphyseal bone defects with calcitriol-loaded PLGA scaffolds and marrow stromal cells

Calcitriol (1,25(OH)2D3)-loaded porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds prepared by solvent casting/salt leaching method were used to repair a 1.5 cm diaphyseal segmental bone defect as a fully absorbable osteogenic biomaterial. The in vitro release of sulforhodamine B (SRB) from PLGA scaffold was measured using spectrophotometer, considering SRB as a model drug. The SRB released from SRB-incorporated PLGA scaffold during 3 months was with relatively low initial burst. The calcitriol-loaded PLGA scaffolds with or without marrow stromal cells (MSCs) were implanted in a critical-sized intercalated bone defect in rabbit femur. Defects were assessed by radiographs until 9 weeks. The bony union of the defect was observed only in the calcitriol-loaded groups. RT-PCR results indicated that MSCs, which were seeded into calcitriol-loaded scaffold, expressed an increased level of alkaline phosphatase, osteonectin, and type I collagen mRNA at day 10. After 2 and 4 weeks, the implanted scaffolds were evaluated by histology. New osteoid matrix and direct calcium deposits were more evident in calcitriol/PLGA/MSC group. Three-dimensional computed tomography and frontal tomographic images of repaired femur showed that normal femur anatomy had been restored with cortical bone with no implanted PLGA remnants at 20 weeks. It can be concluded that the porous calcitriol-loaded PLGA scaffold combined with MSCs may be a novel method for repairing the large loaded bone defect.     

Recent advances in gene delivery for structural bone allografts

In this paper, we review the progress toward developing strategies to engineer improved structural grafting of bone. Three strategies are typically used to augment massive bone defect repair. The first is to engraft mesenchymal stem cells (MSCs) onto a graft or a biosynthetic matrix to provide a viable osteoinductive scaffold material for segmental defect repair. The second strategy is to introduce critical factor(s), for example, bone morphogenetic proteins (BMPs), in the form of bone-derived or recombinant proteins onto the graft or matrix directly. The third strategy uses targeted delivery of therapeutic genes (using viral and nonviral vectors) that either transduce host cells in vivo or stably transduce cells in vitro for subsequent implantation in vivo. We developed a murine femoral model in which allografts can be revitalized via recombinant adeno-associated virus (rAAV) gene transfer. Specifically, allografts coated with rAAV expressing either the constitutively active BMP type I receptor Alk2 (caAlk2), or the angiogenic factor vascular endothelial growth factor (VEGF) combined with the osteoclastogenic factor receptor activator of NF-kappa B ligand (RANKL) have remarkable osteogenic, angiogenic, and remodeling effects that have not been previously documented in healing allografts. Using histomorphometric and micro computed tomography (muCT) imaging we show that rAAV-mediated delivery of caAlk2 induces significant osteoinduction manifested by a mineralized callus on the surface of the allograft, which resembles the healing response of an autograft. We also demonstrate that the rAAV-mediated gene transfer of the combination of VEGF and RANKL can induce significant vascularization and remodeling of processed structural allografts. By contrast, rAAV-LacZ coated allograft controls appeared similar to necrotic allografts and lacked significant mineralized callus, neovascularization, and remodeling. Therefore, innovations in gene delivery offer promising therapeutic approaches for tissue engineering of structural bone substitutes that can potentially have clinical applications in challenging indications.