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 vitro growth factor release from injectable calcium phosphate cements containing gelatin microspheres

To improve the in vivo resorption of an injectable calcium phosphate cement (CPC) for bone tissue engineering purposes, in previous experiments macroporosity was introduced by the in situ degradation of incorporated gelatin microspheres. Gelatin microspheres are also suitable carriers for osteoinductive drugs/growth factors, where release occurs concomitantly with degradation of the hydrogel. Introduction of these microspheres into CPC can alter the release pattern of the cement, which usually shows a marginal release of incorporated drugs. The goal of this study was to determine the in vitro release characteristics of gelatin microsphere CPC. For this, recombinant human TGF-beta1, bFGF, and BMP-2 were labeled with (125)I and loaded onto gelatin type A (porcine, pI = 7.0-9.0)/type B (bovine, pI = 4.5-5.0) microspheres for a short (instant) and longer (prolonged) time before mixing them with the cement. Radioactivity of the resulting 5 or 10 wt % gelatin microsphere CPC composites was monitored for 6 weeks when subjected to proteolytic medium. Drug-loaded CPC was used as control. Results showed that release pattern/efficiency of gelatin microsphere CPCs and CPC controls was highly dependent on the type of growth factor but unaffected by the amount of growth factor. With gelatin microsphere CPC, release was also dependent on the type of gelatin, total volume of incorporated microspheres, and loading method.     

Bone regeneration using a microstereolithography-produced customized poly(propylene fumarate)/diethyl fumarate photopolymer 3D scaffold incorporating BMP-2 loaded PLGA microspheres

Bony defects have been three-dimensionally (3D) created in many clinical circumstances; however, many defects cannot be reconstructed because most of the current bony substitutes cannot provide the necessary exact 3D structure. Therefore, to overcome this limitation, a 3D scaffold with embedded growth factor-delivering microspheres was developed by solid free-form fabrication (SFF) technology using computer-aided design/manufacturing (CAD/CAM). In this study, BMP-2-loaded poly(DL-lactic-co-glycolic acid) (PLGA) microspheres were incorporated into a 3D scaffold that was fabricated using a microstereolithography (MSTL) system with a suspension of microspheres and a poly(propylene fumarate) (PPF)/diethyl fumarate (DEF) photopolymer. By measuring release profiles in vitro, we verified that the fabricated microsphere-containing 3D scaffold could gradually release growth factor. The effects of BMP-2 were also assessed in vitro by observing cell differentiation using MC3T3-E1 pre-osteoblasts. Finally, we confirmed that SFF scaffolds created by MSTL were superior to traditional scaffolds produced using a particulate leaching/gas foaming method. In addition, based on in vivo tests, the scaffolds that released BMP-2 promoted bone formation. Based on these results, we concluded that our 3D scaffold might be a useful tool for enhancing reconstruction quality in many complex bony defects that should be reconstructed using a customized 3D scaffold.     

Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague–Dawley rats (n = 9). Following implantation, the local release profiles were determined by measuring the activity of ¹²⁵I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, μCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p = 0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p = 0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.     

Controlled release by biodegradable hydrogels enhances the ectopic bone formation of bone morphogenetic protein

The objective of this study is to develop a carrier for the controlled release of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of the bone regeneration activity. Hydrogels with different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Following subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled BMP-2 into the back of mice, the in vivo retention period of BMP-2 prolonged with a decrease in the water content of hydrogels used, although every time period was much longer than that of BMP-2 solution injection. Ectopic bone formation studies demonstrated that the alkaline phosphatase (ALP) activity and osteocalcin content around the implanted site of BMP-2-incorporated gelatin hydrogels were significantly high compared with those around the injected site of BMP-2 solution. The values became maximum for the gelatin hydrogel incorporating BMP-2 with a middle period of BMP-2 retention, while bone formation was histologically observed around the hydrogel incorporating BMP-2. The ALP activity was significantly higher than that of the collagen sponge incorporating BMP-2. We concluded that the controlled release technology of BMP-2 for a certain time period was essential to induce the potential activity for bone formation.     

Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels

Transforming growth factor-beta1 (TGF-beta1) is of great relevance to cartilage development and regeneration. A delivery system for controlled release of growth factors such as TGF-beta1 may be therapeutic for cartilage repair. We have encapsulated TGF-beta1 into poly(DL-lactide-co-glycolide) (PLGA) microspheres, and subsequently incorporated the microspheres into biodegradable hydrogels. The hydrogels are poly(ethylene glycol) based, and the degradation rate of the hydrogels is controlled by the non-toxic cross-linking reagent, genipin. Release kinetics of TGF-beta1 were assessed using ELISA and the bioactivity of the released TGF-beta1 was evaluated using a mink lung cell growth inhibition assay. The controlled release of TGF-beta1 encapsulated within microspheres embedded in scaffolds is better controlled when compared to delivery from microspheres alone. ELISA results indicated that TGF-beta1 was released over 21 days from the delivery system, and the burst release was decreased when the microspheres were embedded in the hydrogels. The concentration of TGF-beta1 released from the gels can be controlled by both the mass of microspheres embedded in the gel, and by the concentration of genipin. Additionally, the scaffold permits containment and conformation of the spheres to the defect shape. Based on these in vitro observations, we predict that we can develop a microsphere-loaded hydrogel for controlled release of TGF-beta1 to a cartilage wound site.     

Skull bone regeneration in nonhuman primates by controlled release of bone morphogenetic protein-2 from a biodegradable hydrogel

The objective of this study was to investigate the feasibility of biodegradable gelatin hydrogels as the controlled-release carrier of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration at a skull defect of nonhuman primates. Hydrogels with 3 different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. A critical-sized defect (6 mm in diameter) was prepared at the skull bone of skeletally mature cynomolgus monkeys, and gelatin hydrogels incorporating various doses of BMP-2 were applied to the defects. When the bone regeneration was evaluated by soft radiography and bone mineral density (BMD) examinations, the gelatin hydrogel incorporating BMP-2 exhibited significantly higher osteoinduction activity than did an insoluble bone matrix that incorporated BMP-2 (one of the best osteoinduction systems), although the activity depended on the water content of hydrogels. BMD enhancement was highest for the gelatin hydrogel that had a water content of 97.8 wt% among all types of hydrogels. Moreover, the gelatin hydrogel enabled BMP-2 to induce the bone regeneration in nonhuman primates even at low doses. We conclude that the controlled release of BMP-2 for a certain time period was essential to inducing the osteoinductive potential of BMP-2.     

Controlled release of growth factors based on biodegradation of gelatin hydrogel

To develop a carrier for the controlled release of biologically-active growth factors, biodegradable hydrogels were prepared through glutaraldehyde cross-linking of gelatin with isoelectric points (IEP) of 5.0 and 9.0, i.e. 'acidic' and 'basic' gelatins, respectively. Radioiodinated growth factors were used to investigate their sorption and desorption from the hydrogel of both types of gelatin. Basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGF-beta1) were well sorbed with time to the acidic gelatin hydrogel, while less sorption was observed for the basic gelatin hydrogel. This could be explained in terms of the electrostatic interaction between the growth factors and the acidic gelatin. However, bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), though their IEPs are higher than 7.0, were sorbed to the acidic gelatin hydrogel to a smaller extent than the two other growth factors. Under in vitro non-degradation conditions, approximately 20% of the incorporated bFGF and TGF-beta1 was desorbed from the hydrogels within the initial 40 min, followed by no further substantial desorption, whereas large initial desorption was observed for BMP-2 and VEGF. When implanted in the back subcutis of mice, gelatin hydrogels were degraded over time. Each growth factor was retained in vivo being incorporated in the acidic gelatin hydrogel: the smaller the in vitro desorption amount from the hydrogel, the longer the in vivo retention time. The in vivo profile of bFGF and TGF-beta1 retention was in good accordance with that of the hydrogel. These findings indicate that the growth factor immobilized to the acidic gelatin hydrogel through ionic interaction was released in vivo as a result of hydrogel degradation.     

The release profiles and bioactivity of parathyroid hormone from poly(lactic-co-glycolic acid) microspheres

Poly(lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) or human parathyroid hormone (PTH)(1-34) were prepared using a double emulsion method with high encapsulation efficiency and controlled particle sizes. The microspheres were characterized with regard to their surface morphology, size, protein loading, degradation and release kinetics, and in vitro and in vivo assessments of biological activity of released PTH. PLGA5050 microspheres degraded rapidly after a 3-week lag time and were degraded completely within 4 months. In vitro BSA release kinetics from PLGA5050 microspheres were characterized by a burst effect followed by a slow release phase within 1-7 weeks and a second burst release at 8 weeks, which was consistent with the degradation study. The PTH incorporated PLGA5050 microspheres released detectable PTH in the initial 24h, and the released PTH was biologically active as evidenced by the stimulated release of cAMP from ROS 17/2.8 osteosarcoma cells as well as increased serum calcium levels when injected subcutaneously into mice. Both in vitro and in vivo assays demonstrated that the bioactivity of PTH was maintained largely during the fabrication of PLGA microspheres and upon release. These studies illustrate the feasibility of achieving local delivery of PTH to induce a biologically active response in bone by a microsphere encapsulation technique.     

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.     

Neovascularization effect of biodegradable gelatin microspheres incorporating basic fibroblast growth factor

Biodegradable microspheres were prepared through glutaraldehyde cross-linking of gelatin without using any surfactants as a carrier matrix of basic fibroblast growth factor (bFGF). In the in vitro system, bFGF was sorbed to microspheres of acidic gelatin with an isoelectric point (IEP) of 5.0, but not to those of basic gelatin with an IEP of 9.0. The rate of bFGF sorption to the acidic gelatin microsphere in phosphate-buffered saline solution (pH 7.4) was smaller than that in water. Following incorporation of bFGF into the microspheres at 4 degrees C for 12 h, bFGF release from the bFGF-incorporating microspheres was studied. Approximately 30% of incorporated bFGF was released from the acidic gelatin microsphere within the initial 3 h, followed by no substantial release, whereas the basic gelatin microsphere released almost completely the incorporated bFGF within 1 day. It is likely that when basic bFGF molecules were immobilized to the acidic gelatin constituting microspheres through polyion complexation, they were not readily released under the in vitro nondegradation condition of gelatin. Incorporation of anionic carboxylmethyl cellulose (CMC) into the acidic gelatin microspheres reduced the amount of bFGF desorbed initially. This indicates that the initial burst is ascribed to free bFGF which is not ionically interacted with the acidic gelatin. CMC will function as a bFGF sorbent to suppress the initial leakage from the microspheres. When injected subcutaneously into the mouse back, bFGF-incorporating acidic gelatin microspheres were degraded over time and induced neovascularization around the injection site, in marked contrast to bFGF in the solution form. CMC incorporation slowed down the biodegradation and vascularization effect of bFGF-incorporating gelatin microspheres. It was concluded that the gelatin microsphere was a promising carrier matrix of bFGF to enhance the vascularization effect.     

Segmental bone regeneration using an rhBMP-2-loaded gelatin/nanohydroxyapatite/fibrin scaffold in a rabbit model

We aimed to develop a hybrid scaffold with a porous structure and similar composition as natural bone for the controlled release of bone morphogenetic protein-2 (BMP-2) to enhance bone regeneration. We fabricated a gelatin/nanohydroxypatite (nHAP) scaffold by glutaraldehyde chemical cross-linking a gelatin aqueous solution with nHAP granules at a 5:1 ratio (v/w). Then, fibrin glue (FG) mixed with recombinant human BMP-2 (rhBMP-2) was infused into the gelatin/nHAP scaffold and lyophilized to develop an rhBMP-2-loaded gelatin/nHAP/FG scaffold. On scanning electron microscopy, the composite had a 3-D porous structure. The rhBMP-2 release kinetics from the hybrid scaffold was sustained and slow, and release of rhBMP-2 was complete at 40 days. Immunohistochemistry, azo-coupling and alizarin S-red staining were used to study in vitro differentiation of human bone-marrow mesenchymal cells (hBMSCs). Strong positive staining results confirmed that rhBMP-2 released from the scaffold could improve osteocalcin (OCN) and alkaline phosphatase (ALP) expression and calcium deposition formation. RT-PCR results showed significantly high mRNA expression of ALP and OCN in hBM-MSCs cultured on the gelatin/nHAP/FG scaffold with rhBMP-2. DNA assay demonstrated that the scaffold was noncytotoxic and could promote hBMSC proliferation from the components of the hybrid scaffold, not released rhBMP-2. The hybrid scaffolds were then used to repair critical-size segmental bone defects of rabbit radius. Gross specimen, X-ray, bone histomorphology and bone mineral density assay demonstrated that the rhBMP-2-loaded gelatin/nHAP/FG scaffold had good osteogenic capability and could repair the segmental bone defect completely in 12 weeks.     

Efficient Bone Regeneration Induced by Bone Morphogenetic Protein-2 Released from Apatite-Coated Collagen Scaffolds

Abstract

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. Clinically utilized BMP-2 uses a type-I collagen scaffold as a carrier. Here we hypothesized that an apatite coating on a type-I collagen scaffold would prolong the BMP-2 release period and enhance bone regeneration in calvarial defects in mice. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluid. BMP-2 release kinetics and bioactivity were evaluated by enzyme-linked immunosorbent assay and alkaline phosphatase activity measurement of cultured osteoblasts. Computed tomography and histomorphometry were performed eight weeks after various doses of BMP-2 were delivered to mouse calvarial defects using either non-modified or apatite-coated collagen scaffolds. Apatite-coated collagen scaffolds released 91.8 ± 11.5% of the loaded BMP-2 over 13 days in vitro, whereas non-modified collagen scaffolds released 98.3 ± 2.2% over the initial one day. The in vivo study showed that BMP-2 delivery with apatite-coated collagen scaffolds resulted in a significantly greater bone formation area and higher bone density than that with non-modified collagen scaffolds. This study suggests that simple apatite coating on collagen scaffolds can enhance the bone regeneration efficacy of BMP-2 released from collagen scaffolds.     

Development of biodegradable poly(propylene fumarate)/poly(lactic-co-glycolic acid) blend microspheres. II. Controlled drug release and microsphere degradation

This article describes the effects of six processing parameters on the release kinetics of a model drug Texas red dextran (TRD) from poly(propylene fumarate)/poly(lactic-co-glycolic acid) (PPF/PLGA) blend microspheres as well as the degradation of these microspheres. The microspheres were fabricated using a double emulsion-solvent extraction technique in which the following six parameters were varied: PPF/PLGA ratio, polymer viscosity, vortex speed during emulsification, amount of internal aqueous phase, use of poly(vinyl alcohol) in the internal aqueous phase, and poly(vinyl alcohol) concentration in the external aqueous phase. We have previously characterized these microspheres in terms of microsphere morphology, size distribution, and TRD entrapment efficiency. In this work, the TRD release profiles in phosphate-buffered saline were determined and all formulations showed an initial burst release in the first 2 days followed by a decreased sustained release over a 38-day period. The initial burst release varied from 5.1 (+/-1.1) to 67.7 (+/-3.4)% of the entrapped TRD, and was affected most by the viscosity of the polymer solution used for microsphere fabrication. The sustained release between day 2 and day 38 ranged from 7.9 (+/-0.8) to 27.2 (+/-3.1)% of the entrapped TRD. During 11 weeks of in vitro degradation, the mass of the microspheres remained relatively constant for the first 3 weeks after which it decreased dramatically, whereas the molecular weight of the polymers decreased immediately upon placement in phosphate-buffered saline. Increasing the PPF content in the PPF/PLGA blend resulted in slower microsphere degradation. Overall, this study provides further understanding of the effects of various processing parameters on the release kinetics from PPF/PLGA blend microspheres thus allowing modulation of drug release to achieve a wide spectrum of release profiles.     

PLGA microsphere/calcium phosphate cement composites for tissue engineering: in vitro release and degradation characteristics

Bone cements with biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres have already been proven to provide a macroporous calcium phosphate cement (CPC) during in situ microsphere degradation. Furthermore, in vitro/in vivo release studies with these PLGA microsphere/CPC composites (PLGA/CPCs) showed a sustained release of osteo-inductive growth factor when drug was distributed inside/onto the microspheres. The goal of this study was to elucidate the mechanism behind drug release from PLGA/CPC. For this, in vitro release and degradation characteristics of a low-molecular-weight PLGA/CPC (M(w) = 5 kg/mol) were determined using bovine serum albumin (BSA) as a model protein. Two loading mechanisms were applied; BSA was either adsorbed onto the microspheres or incorporated inside the microspheres during double-emulsion. BSA release from PLGA microspheres and CPC was also measured and used as reference. Results show fast degrading polymer microspheres which produced a macroporous scaffold within 4 weeks, but also showed a concomitant release of acidic degradation products. BSA release from the PLGA/CPC was similar to the CPC samples and showed a pattern consisting of a small initial release, followed by a period of almost no sustained release. Separate PLGA microspheres exhibited a high burst release and release efficiency that was higher with the adsorbed samples. Combining degradation and release data we can conclude that for the PLGA/CPC samples BSA re-adsorbed to the cement surface after being released from the microspheres, which was mediated by the pH decrease during microsphere degradation.     

Introduction of gelatin microspheres into an injectable calcium phosphate cement

For tissue engineered bone constructs, calcium phosphate cement (CPC) has a high potential as scaffold material because of its biocompatibility and osteoconductivity. However, in vivo resorption and tissue ingrowth is slow. To address these issues, microspheres can be incorporated into the cement, which will create macroporosity after in situ degradation. The goal of this study was to investigate the handling properties and degradation characteristics of CPC containing gelatin microspheres. Setting time and injectability were determined and an in vitro degradation study was performed. Samples were assayed on mass, compression strength, E-modulus, and morphology. A supplementary degradation test with gelatin microspheres was performed to investigate the influence of physical conditions inside the cement on microsphere stability. The gelatin microsphere CPCs were easy to inject and showed initial setting times of less than 3 min. After 12-weeks in vitro degradation no increase in macroporosity was observed, which was supported by the small mass loss and stabilizing mechanical strength. Even a clear densification of the composite was observed. Explanations for the lack of macroporosity were recrystallization of the cement onto or inside the gelatin spheres and a delayed degradation of gelatin microspheres inside the scaffold. The supplementary degradation test showed that the pH is a factor in the delayed gelatin microsphere degradation. Also differences in degradation rate between types of gelatin were observed. Overall, the introduction of gelatin microspheres into CPC renders composites with good handling properties, though the degradation characteristics should be further investigated to generate a macroporous scaffold.     

Apatite-coated collagen scaffold for bone morphogenetic protein-2 delivery

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. BMP-2 is clinically used for spine fusion and bone fracture healing. Commercially available BMP-2 uses a type I collagen scaffold as a carrier, but it only releases BMP-2 for a short period of time, which may release the bone formation efficacy. In the present study, we hypothesize that apatite coating of a collagen scaffold increases the release period as well as the osteogenic efficacy of BMP-2. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluids (SBFs). Apatite coating on collagen scaffolds was confirmed by X-ray diffraction, electron spectroscopy for chemical analysis, attenuated total reflectance-Fourier transform infrared spectroscopy, and scanning electron microscopy. The rate and period of BMP-2 release from apatite-coated collagen scaffolds varied depending on the concentration of SBFs used. The 5× and 10× SBF apatite-coated collagen scaffolds released 91.8%±11.5% and 82.2%±13.1% of their loaded BMP-2 over 13 days in vitro, respectively, whereas noncoated collagen scaffold released 98.3%±2.2% over the initial one day. BMP-2 released from apatite-coated collagen scaffold significantly increased the alkaline phosphatase activity of cultured osteoblasts, compared with BMP-2 released from noncoated collagen scaffold. Computed tomography and histomorphometry showed that BMP-2 delivery using apatite-coated collagen scaffolds resulted in 2.5-fold higher bone formation volume and 4.0-fold higher bone formation area than BMP-2 delivery using noncoated collagen scaffolds. This study shows that simple apatite coating of a collagen scaffold results in a BMP-2 carrier that renders long-term release of BMP-2 and dramatically enhances osteogenic efficacy.     

新型梯度复合HA/ZrO2组织工程骨支架在猕猴颈椎融合中的应用

目的 构建新型梯度复合羟基磷灰石-二氧化锆(HA/ZrO₂)组织工程骨支架,并评估其在猕猴颈椎椎间融合中的效果。方法 通过离子交联法制备壳聚糖水凝胶作为骨形态发生蛋白2(BMP-2)的缓释载体,扫描电镜下观察其微观形态,检测其载药量、包封率及缓释速率;然后分离、培养猕猴源性骨髓间充质干细胞(BMSCs),并进行碱性磷酸酶、冯库萨染色等对其进行鉴定;最后将前期制备好的BMP-2明胶/壳聚糖凝水胶缓释系统和第3代猕猴BMSCs加载于HA/ZrO₂泡沫陶瓷,以构建新型组织工程骨,并观察其形态特征。将24只雄性猕猴按照随机数字表法分成4组,其中组织工程骨组(n=8)为植入梯度复合HA/ZrO₂泡沫陶瓷负载BMP-2明胶/壳聚糖水凝胶缓释系统和第3代猕猴BMSCs,泡沫陶瓷组(n=8)为植入梯度复合HA/ZrO₂泡沫陶瓷,自体髂骨组(n=4)为植入自体髂骨,假手术组(n=4)为只对相应部位的软组织进行切开、缝合,未破坏其椎间盘;观察术后颈椎X线(术后即刻、8周、16周)、组织形态学表现(术后8周、16周)及测试相应节段的生物力学(术后16周)。结果 扫描电镜下BMP-2明胶/壳聚糖水凝胶呈3D 网状结构,内见均匀分布的壳聚糖微球,其负载BMP-2后的包封率和载药率随时间逐渐降低:第1天分别为87.4%±0.9%和58.2%±0.5%、第15天分别为45.2%±0.6%和30.1%±0.4%,累积释放率第1天为12.6%±0.11%、第15天为55%±0.16%。显微镜下观察见BMSCs的形态呈多样性,以梭形及短棒形等较常见;第3代猕猴BMSCs成骨诱导后的碱性磷酸酶、冯库萨染色以及表面特异性抗原的检测结果显示,符合BMSCs的生物学特性。不同时点的X线及组织形态学观察显示材料内部新生骨量组织工程骨组较泡沫陶瓷组明显增多;生物力学测试结果显示,在最大载荷、抗压强度和能量吸收方面假手术组均低于其他3组(P值均＜0.05),在抗压强度上组织工程骨组与自体髂骨组差异无统计学意义(P＞0.05)。结论BMP-2明胶/壳聚糖水凝胶缓释系统复合猕猴第3代BMSCs种植于HA/ZrO₂泡沫陶瓷构建的新型组织工程骨支架,能有效促进猕猴颈椎椎间融合,在影像学及组织学表现和生物力学测试方面,可达到与自体骨相似的骨代替作用。     

Dual growth factor delivery from bilayered,biodegradable hydrogel composites for spatially-guided osteochondral tissue repair

The present work investigated the use of biodegradable hydrogel composite scaffolds, based on the macromer oligo(poly(ethylene glycol) fumarate) (OPF), to deliver growth factors for the repair of osteochondral tissue in a rabbit model. In particular, bilayered OPF composites were used to mimic the structural layers of the osteochondral unit, and insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) were loaded into gelatin microparticles and embedded within the OPF hydrogel matrix in a spatially controlled manner. Three different scaffold formulations were implanted in a medial femoral condyle osteochondral defect: 1) IGF-1 in the chondral layer, 2) BMP-2 in the subchondral layer, and 3) IGF-1 and BMP-2 in their respective separate layers. The quantity and quality of osteochondral repair was evaluated at 6 and 12 weeks with histological scoring and micro-computed tomography (micro-CT). While histological scoring results at 6 weeks showed no differences between experimental groups, micro-CT analysis revealed that the delivery of BMP-2 alone increased the number of bony trabecular islets formed, an indication of early bone formation, over that of IGF-1 delivery alone. At 12 weeks post-implantation, minimal differences were detected between the three groups for cartilage repair. However, the dual delivery of IGF-1 and BMP-2 had a higher proportion of subchondral bone repair, greater bone growth at the defect margins, and lower bone specific surface than the single delivery of IGF-1. These results suggest that the delivery of BMP-2 enhances subchondral bone formation and that, while the dual delivery of IGF-1 and BMP-2 in separate layers does not improve cartilage repair under the conditions studied, they may synergistically enhance the degree of subchondral bone formation. Overall, bilayered OPF hydrogel composites demonstrate potential as spatially-guided, multiple growth factor release vehicles for osteochondral tissue repair.     

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.