Improving bone formation in a rat femur segmental defect by controlling bone morphogenetic protein-2 release

Nonunion is a common complication in open fractures and other severe bone injuries. Recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered on a collagen sponge enhances healing of fractures. However, the burst release of rhBMP-2 necessitates supra-physiological doses of rhBMP-2 to achieve a robust osteogenic effect, which introduces risk of ectopic bone formation and severe inflammation and increases the cost. Although the concept that the ideal pharmacokinetics for rhBMP-2 includes both a burst and sustained release is generally accepted, investigations into the effects of the release kinetics on new bone formation are limited. In the present study, biodegradable polyurethane (PUR) and PUR/microsphere [PUR/poly(lactic-co-glycolic acid)] composite scaffolds with varying rhBMP-2 release kinetics were compared to the collagen sponge delivery system in a critical-sized rat segmental defect model. Microcomputed tomography analysis indicated that a burst followed by a sustained release of rhBMP-2 from the PUR scaffolds regenerated 50% more new bone than the collagen sponge loaded with rhBMP-2, whereas a sustained release without the burst did not form significantly more bone than the scaffold without rhBMP-2. This study demonstrated that the putative optimal release profile (i.e., burst followed by sustained release) for rhBMP-2 can be achieved using PUR scaffolds, and that this enhanced pharmacokinetics regenerated more bone than the clinically available standard of care in a critical-sized defect in rat femora.     

Effect of autologous bone marrow stromal cell seeding and bone morphogenetic protein-2 delivery on ectopic bone formation in a microsphere/poly(propylene fumarate) composite

A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8 microg BMP-2 per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2-loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2-impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8 microg/mg BMP-2-loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2-impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.     

In vivo degradation of porous poly(propylene fumarate)/poly(DL-lactic-co-glycolic acid) composite scaffolds

This study investigated the in vivo degradation of poly(propylene fumarate) (PPF)/poly(DL-lactic-co-glycolic acid) (PLGA) composite scaffolds designed for controlled release of osteogenic factors. PPF/PLGA composites were implanted into 15.0mm segmental defects in the rabbit radius, harvested after 12 and 18 weeks, and analyzed using histological techniques to assess the extent of polymer degradation as well as the tissue response within the pores of the scaffolds. Polymer degradation was limited to micro-fragmentation of the scaffold at the ends and edges of the implant at both 12 and 18 weeks. The tissue within the pores of the scaffold consisted of fibrous tissue, blood vessels and some inflammatory cells. In areas where polymer breakdown was evident, an increased inflammatory response was observed. In contrast, areas of bone ingrowth into the polymer scaffold were characterized by minimal inflammatory response and polymer degradation. Our results show that minimal degradation of porous PPF occurs within 18 weeks of implantation in a rabbit model. Further, the in vivo degradation data of porous PPF/PLGA scaffolds are comparable with earlier obtained in vitro data.     

Effects of compatibility of deproteinized antler cancellous bone with various bioactive factors on their osteogenic potential

Combinations of calcium phosphate scaffolds and bioactive factors are promising niche-mimetic solutions for repairing large-sized bone defects. However, the importance of compatibility between scaffolds and bioactive factors on their osteogenic outcomes has been largely ignored. This study aimed to investigate the compatibility of calcinated antler cancellous bone (CACB) scaffolds with various bioactive factors including icariin (ICA), velvet antler polypeptides (VAP) or recombinant human bone morphogenetic protein-2 (rhBMP-2) as well as their combinational osteogenic potential in vitro and in vivo. Scanning electron microscopy and fourier transform infrared spectroscopy confirmed the uniform distribution and chemical stability of the reagents on CABC. In vitro release profiles showed relative steady release of ICA from ICA/CACB, burst VAP release from VAP/CACB, and minimal rhBMP-2 release from rhBMP-2/CACB composites. When compared with VAP and rhBMP-2, incorporation of ICA within CACB resulted in most increased cell attachment, proliferation, alkaline phosphatase activity, osteogenic gene expression, and mineralization of rat bone marrow mesenchymal stem cells. In rabbit mandible critical-sized defects, the most extensive osteogenesis and neovascularization were observed in the ICA/CACB group. Differences between the VAP/CACB and rhBMP-2/CACB groups were not apparent. Interestingly, low pro-inflammatory (TNF-α, IL-6) and high anti-inflammatory (IL-10) mRNA levels were observed at scaffold implantation sites which were in close association with amount of new bone formation. These findings highlight that the compatibility between scaffolds and bioactive factors should been taken into account when considering the formula of optimized bone defect repair.     

In vitro osteogenic differentiation of human mesenchymal stem cells and in vivo bone formation in composite nanofiber meshes

Tissue engineering has become an alternative method to traditional surgical treatments for the repair of bone defects, and an appropriate scaffold supporting bone formation is a key element in this approach. In the present study, nanofibrous organic and inorganic composite scaffolds containing nano-sized demineralized bone powders (DBPs) with biodegradable poly(L-lactide) (PLA) were developed using an electrospinning process for engineering bone. To assess their biocompatibility, in vitro osteogenic differentiation of human mandible-derived mesenchymal stem cells (hMSCs) cultured on PLA or PLA/DBP composite nanofiber scaffolds were examined. The mineralization of hMSCs cultured with osteogenic supplements on the PLA/DBP nanofiber scaffolds was remarkably greater than on the PLA nanofiber scaffold during the first 14 days of culture but reached the same level after 21 days. The in vivo osteoconductive effect of PLA/DBP nanofibrous scaffolds was further investigated using rats with critical-sized skull defects. Micro-computerized tomography revealed that a greater amount of newly formed bone extended across the defect area in PLA/DBP scaffolds than in the nonimplant and PLA scaffolds 12 weeks after implantation and that the defect size was almost 90% smaller. Therefore, PLA/DBP composite nanofiber scaffolds may serve as a favorable matrix for the regeneration of bone tissue.     

柚皮苷-壳聚糖/羟基磷灰石复合支架修复大鼠颅骨缺损

背景:骨碎补的有效成分柚皮苷具有补肝肾强筋骨的传统功效,能增加骨痂厚度,提高骨折愈合质量。目的:探究载中药骨碎补有效成分柚皮苷壳聚糖/羟基磷灰石复合支架的骨传导和骨诱导性能。方法:将一定钙磷比的羟基磷灰石前体液与含柚皮苷的壳聚糖溶液在碱性条件下原位结晶、冷冻干燥,获得柚皮苷-壳聚糖/羟基磷灰石多孔支架。将15只成年SD大鼠随机分成空白组(n=5)、对照组(n=5)和实验组(n=5),建立直径5 mm颅骨骨缺损模型,空白组未填充生物材料,对照组填充壳聚糖/羟基磷灰石支架,实验组填充柚皮苷-壳聚糖/羟基磷灰石复合支架。术后4周取材,CT扫描观察颅骨修复情况,苏木精-伊红染色观察颅骨修复的形态学,骨形态发生蛋白2和血管内皮生长因子免疫组化染色后观察缺损区域局部成骨活性因子的表达。结果与结论:(1)CT扫描显示,空白组大鼠颅骨未见明显骨生成,仅在缺损边缘可见少量新生骨;对照组于缺损孔隙可见新生骨形成,新生骨较少;实验组骨缺损修复良好,新生骨组织与缺损孔隙周围颅骨密度相似,大面积新生骨广泛填充了缺损孔隙。(2)苏木精-伊红染色显示,空白组缺损区填充以稀薄的疏松网状纤维组织,可见大量炎性反应病灶,...     

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.     

Controlled drug release from a novel injectable biodegradable microsphere/scaffold composite based on poly(propylene fumarate)

The ideal biomaterial for the repair of bone defects is expected to have good mechanical properties, be fabricated easily into a desired shape, support cell attachment, allow controlled release of bioactive factors to induce bone formation, and biodegrade into nontoxic products to permit natural bone formation and remodeling. The synthetic polymer poly(propylene fumarate) (PPF) holds great promise as such a biomaterial. In previous work we developed poly(DL-lactic-co-glycolic acid) (PLGA) and PPF microspheres for the controlled delivery of bioactive molecules. This study presents an approach to incorporate these microspheres into an injectable, porous PPF scaffold. Model drug Texas red dextran (TRD) was encapsulated into biodegradable PLGA and PPF microspheres at 2 microg/mg microsphere. Five porous composite formulations were fabricated via a gas foaming technique by combining the injectable PPF paste with the PLGA or PPF microspheres at 100 or 250 mg microsphere per composite formulation, or a control aqueous TRD solution (200 microg per composite). All scaffolds had an interconnected pore network with an average porosity of 64.8 +/- 3.6%. The presence of microspheres in the composite scaffolds was confirmed by scanning electron microscopy and confocal microscopy. The composite scaffolds exhibited a sustained release of the model drug for at least 28 days and had minimal burst release during the initial phase of release, as compared to drug release from microspheres alone. The compressive moduli of the scaffolds were between 2.4 and 26.2 MPa after fabrication, and between 14.9 and 62.8 MPa after 28 days in PBS. The scaffolds containing PPF microspheres exhibited a significantly higher initial compressive modulus than those containing PLGA microspheres. Increasing the amount of microspheres in the composites was found to significantly decrease the initial compressive modulus. The novel injectable PPF-based microsphere/scaffold composites developed in this study are promising to serve as vehicles for controlled drug delivery for bone tissue engineering.     

In vivo bone formation from human embryonic stem cell-derived osteogenic cells in poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffolds

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation (n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 microg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

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     

Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2

This work evaluated gelatin microparticles and biodegradable composite scaffolds for the controlled release of bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. Gelatin crosslinking (10 and 40mM glutaraldehyde), BMP-2 dose (6 and 60ng BMP-2 per mg dry microparticles), buffer type (phosphate buffered saline (PBS) and collagenase-containing PBS), and gelatin type (acidic and basic) were investigated for their effects on BMP-2 release. Release profiles were also observed using poly(lactic-co-glycolic acid) (PLGA) microparticles with varying molecular weights (8300 and 57,500). In vitro and in vivo studies were conducted using radiolabeled BMP-2; the chloramine-T method was preferred over Bolton-Hunter reagent for radioiodination with this system. BMP-2 release from PLGA microparticles resulted in a moderate burst release followed by minimal cumulative release, while BMP-2 release from gelatin microparticles exhibited minimal burst release followed by linear release kinetics in vitro. Growth factor dose had a small effect on its normalized release kinetics probably because of an equilibrium between gelatin-bound and unbound BMP-2. Differences in release from acidic and basic gelatin microparticles may result from the different pretreatment conditions used for gelatin synthesis. The in vitro release kinetics for both gelatin microparticles alone and within composite scaffolds were dependent largely on the extent of gelatin crosslinking; varying buffer type served to confirm that controlled release relies on enzymatic degradation of the gelatin for controlled release. Finally, in vivo studies with composite scaffolds exhibited minimal burst and linear release up to 28 days. In summary, dose effects on BMP-2 release were found to be minimal while varying gelatin type and release medium can alter release kinetics. These results demonstrate that a systematic control of BMP-2 delivery from gelatin microparticles can be achieved by altering the extent of basic gelatin crosslinking.     

Optimally porous and biomechanically compatible scaffolds for large-area bone regeneration

Large-area or critical-sized bone defects pose a serious challenge in orthopedic surgery, as all current treatment options present with shortcomings. Bone tissue engineering offers a more promising alternative treatment strategy. However, this approach requires mechanically stable scaffolds that support homogenous bone formation throughout the scaffold thickness. Despite advances in scaffold fabrication, current scaffold-based techniques are unable to support uniform, three-dimensional bone regeneration, and are limited to only the scaffold surface in vitro and in vivo. This is mainly because of inadequate scaffold pore sizes (<200?μm) and accessible pore volume, and the associated limited oxygen diffusion and vascular invasion. In this study, we have adopted a method combining microsphere-sintering and porogen-leaching techniques to fabricate scaffolds with an increased accessible pore volume. Of the scaffolds developed, moderately porous poly(85 lactide-co-15 glycolide) (PLGA) microsphere scaffolds were selected as most advantageous, since they retain mechanical strength in the range of human cancellous bone and display a significantly higher accessible pore volume, which is attributed to an increased percentage of larger pores (i.e., size range 200-600?μm). Unlike control scaffolds with a limited pore size and an accessible pore volume, moderately porous scaffolds displayed increased oxygen diffusion, pre-osteoblast cell infiltration, proliferation, and survival throughout the entire scaffold. Furthermore, moderately porous PLGA microsphere scaffolds displayed enhanced and homogenous mineralization in vitro. Since these newly designed moderately porous scaffolds are weight bearing, are fully osteoconductive, and have the ability to support vascularization, they may serve as effective scaffolds for large-area bone defect repair/regeneration. In addition, this study demonstrates the ability to modulate scaffold porosity and, in turn, to develop oxygen tension-controlled matrices that are effective for large-area bone regeneration.     

Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(L-lactic-co-glycolic acid) scaffold

In this study, a heparin-conjugated poly(l-lactic-co-glycolic acid) (HP-PLGA) scaffold was developed for the sustained delivery of bone morphogenetic protein-2 (BMP-2), and then used to address the hypothesis that BMP-2 delivered from this scaffold could enhance ectopic bone formation. We found the amount of heparin conjugated to the PLGA scaffolds could be increased up to 3.2-fold by using scaffolds made from star-shaped PLGA, as compared to scaffolds made from linear PLGA, and that the release of BMP-2 from the HP-PLGA scaffold was sustained for at least 14 days in vitro. The BMP-2 released from the HP-PLGA scaffold stimulated an increase in alkaline phosphatase (ALP) activity of osteoblasts for 14 days in vitro, suggesting that the HP-PLGA scaffold delivery system releases BMP-2 in a bioactive form for a prolonged period. By contrast, BMP-2 release from unmodified (no heparin) PLGA scaffolds induced a transient increase in ALP activity for the first 3 days and a decrease thereafter. In vivo bone formation studies showed the BMP-2-loaded HP-PLGA scaffolds induced bone formation to a much greater extent than did either BMP-2-loaded unmodified PLGA scaffolds or unloaded (no BMP-2) HP-PLGA scaffolds, with 9-fold greater bone formation area and 4-fold greater calcium content in the BMP-2-loaded HP-PLGA scaffold group compared to the BMP-2-loaded unmodified PLGA scaffold group. Collectively, these results demonstrate that the HP-PLGA delivery system is capable of potentiating the osteogenic efficacy of BMP-2, and underscore its importance as a possible bone regeneration strategy.     

Sustained release of vascular endothelial growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering

Strategies to engineer bone tissue have focused on either: (1) the use of scaffolds for osteogenic cell transplantation or as conductive substrates for guided bone regeneration; or (2) release of inductive bioactive factors from these scaffold materials. This study describes an approach to add an inductive component to an osteoconductive scaffold for bone tissue engineering. We report the release of bioactive vascular endothelial growth factor (VEGF) from a mineralized, porous, degradable polymer scaffold. Three dimensional, porous scaffolds of the copolymer 85 : 15 poly(lactide-co-glycolide) were fabricated by including the growth factor into a gas foaming/particulate leaching process. The scaffold was then mineralized via incubation in a simulated body fluid. Growth of a bone-like mineral film on the inner pore surfaces of the porous scaffold is confirmed by mass increase measurements and quantification of phosphate content within scaffolds. Release of ¹²⁵I-labeled VEGF was tracked over a 15 day period to determine release kinetics from the mineralized scaffolds. Sustained release from the mineralized scaffolds was achieved, and growth of the mineral film had only a minor effect on the release kinetics from the scaffolds. The VEGF released from the mineralized and non-mineralized scaffolds was over 70% active for up to 12 days following mineralization treatment, and the growth of mineral had little effect on total scaffold porosity.     

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.     

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.     

RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration

Calcium phosphate cement scaffold (CPC) has been widely used as bone graft substitutes, but undesirable osteoinductivity and slow degradability greatly hamper their clinic application. To address these problems, a recombinant human bone morphogenetic protein-2 (rhBMP-2)-loaded calcium silicate/calcium phosphate cement scaffold (CSPC) with hierarchical pores was developed in this study. The CSPC scaffold with both interconnected macropores on the order of 200–500 μm and micropores of 2–5 μm was synthesized from CPC and calcium silicate (CS) by a NaCl particulate-leaching method. In vitro cell culture with C2C12 model cells, in vivo ectopic bone formation and rabbit femur cavity defect repair were performed to evaluate the osteogeneic capacity of the CSPC/rhBMP-2 scaffold. CPC, CSPC and CPC/rhBMP-2 scaffolds were parallelly investigated for comparison. The results demonstrated that the hierarchical macro/microporous structure, whether in presence of CS or rhBMP-2, highly favored the adhesion of C2C12 cells and bone in-growth into the CPC-based scaffolds. But, in comparison to the CPC-based scaffolds with CS or rhBMP-2 alone, the CSPC/rhBMP-2 scaffold strongly promoted osteogenic differentiation in vitro and osteogenetic efficacy in vivo. Further studies demonstrated that Si ions derived from CSPC contributed mainly to maintain the conformation of rhBMP-2 and thus stimulate the synergistic action of CS and rhBMP-2 in osteogenic differentiation and osteoinductivity. Additionally, the incorporation of CS was also beneficial for the dissolution of the scaffold. Those results suggest that the CSPC has superior properties for incorporation of rhBMP-2 and our developed CSPC/rhBMP-2 scaffold have great potential for future use in bone tissue regeneration.     

Biocompatibility and osteogenesis of biomimetic Bioglass-Collagen-Phosphatidylserine composite scaffolds for bone tissue engineering

A novel biomimetic composite scaffold Bioglass-Collagen-Phosphatidylserine (BG-COL-PS) was fabricated with a freeze-drying technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the BG-COL-PS scaffolds exhibited interconnected porous structures with pore sizes of several microns up to about 300 μm. The scaffolds have a porosity of 75.40% and the corresponding compressive strength of 1.5469 Mpa. Rat mesenchymal stem cells (rMSCs) were seeded on BG-COL-PS or BG-COL scaffolds and cultured for 21 days in vitro. Based on the results of SEM, dsDNA content, alkaline phosphatase (ALP) activity, osteogenic gene expression analysis and alizarin red staining, the responses of MSCs to the scaffold exhibited a higher degree of attachment, growth as well as osteogenic differentiation than those on BG-COL scaffolds in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure BG-COL-PS scaffolds and MSC/scaffold constructs were implanted in rat femurs defects for 6 weeks and studied histologically and radiographically. The in vivo results showed that BG-COL-PS composite scaffolds exhibited good biocompatibility and extensive osteoconductivity with host bone. Moreover, the BG-COL-PS/MSC constructs dramatically enhanced the efficiency of new bone formation than pure BG-COL-PS scaffolds or BG-COL/MSC constructs. All these results demonstrate the usefulness of PS composited BG-COL-PS scaffolds for inducing enhanced bone formation. The BG-COL-PS scaffolds fulfill the basic requirements of bone tissue engineering scaffold and have the potential to be applied in orthopedic and reconstructive surgery.