缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导骨形成

背景：重组人骨形态发生蛋白2在体内半衰期短、易降解代谢,达不到理想的骨再生效果。 目的：制备缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料,并观察其缓释性能、骨诱导活性。 方法：将重组人骨形态发生蛋白2与壳聚糖混合制备壳聚糖膜,涂覆于生物骨修复材料表面,ELISA方法检测其体外释药性能。茜素红染色检测缓释型人骨形态发生蛋白2/壳聚糖生物骨材料、重组人骨形态发生蛋白2生物骨材料、单纯骨填充材料诱导C2C12细胞骨钙蛋白的形成,观察其诱导成骨细胞能力。同时将3种骨修复材料植入清洁级KM小鼠股部肌袋内,2周后检测新生骨Ca2+离子含量,评价其异位骨诱导能力。 结果与结论：材料表面的壳聚糖膜分布均匀,负载的重组人骨形态发生蛋白2呈团簇状。重组人骨形态发生蛋白2/壳聚糖生物骨修复材料体外释药存在突释,前4 d释放量达总药量的50%,持续至12 d,释药量达到90%,第18天时释放完全。与单纯骨填充材料、重组人骨形态发生蛋白2生物骨材料相比,缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导C2C12细胞向成骨晚期分化能力与异位骨形成能力显著增强(P < 0.05)。结果提示缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料缓释性能好,促进骨形成能力强。     

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.     

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.     

New scaffold for recombinant human bone morphogenetic protein-2

A bioactive and resorbable scaffold is necessary to exhibit the osteoinductive potency of recombinant human bone morphogenetic protein-2 (rhBMP-2). In a previous study, we found that synthetic octacalcium phosphate (OCP) enhances bone regeneration and is replaced by newly formed bone after it is resorbed. We hypothesized that OCP may be useful as an effective scaffold for rhBMP-2 to enhance bone regeneration. To test this hypothesis, the present study was designed to investigate whether an OCP/BMP composite implant could more effectively enhance bone regeneration. A critical-sized defect was made in a rat calvarium and 1. 15 mg of OCP combined with 10 microg of rhBMP-2 (OCP/BMP 10 microg), 2. 15 mg of OCP combined with 1 microg of rhBMP-2 (OCP/BMP 1 microg), or 3. OCP (OCP alone) was implanted into the defect and fixed at 4 or 8 weeks after implantation. The percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. A statistical analysis showed that n-Bone% with OCP/BMP was significantly higher than that with OCP at both time points, whereas the difference in n-Bone% between OCP/BMP 10 microg and OCP/BMP 1 microg was not significant. The present results suggest that OCP can be used as an effective scaffold for rhBMP-2 and this OCP delivery system may be able to reduce the standard effective dose of rhBMP-2, which would be beneficial because low doses (<100 microg/g OCP) of rhBMP-2 enhance bone regeneration.     

Orthotopic bone formation by implantation of apatite-coated poly(lactide-co-glycolide)/hydroxyapatite composite particulates and bone morphogenetic protein-2

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. In the present study, we tested the suitability of apatite-coated poly(D,L-lactide-co-glycolide)/nanohydroxyapatite (PLGA/HA) particulates as carriers for the controlled release of BMP-2. The release of BMP-2 from apatite-coated PLGA/HA particulates was sustained for at least 4 weeks in vitro. A delivery system of apatite-coated PLGA/HA particulates suspended in fibrin gel further slowed the BMP-2 release rate. In vivo implantation of either Fibrin gel + BMP-2 or Fibrin gel + apatite-coated PLGA/HA particulates showed enhanced new bone formation in critical-sized calvarial defects of rats 8 weeks after implantation, compared to implantation of fibrin gel only. Importantly, new bone formation was much higher in the defects treated with BMP-2 delivery using apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + PLGA/HA + BMP-2 group) than in the defects treated either with apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + BMP-2 group) or with BMP-2 delivery using fibrin gel alone (Fibrin gel + BMP-2 group). BMP-2 and osteoinductive HA had an additive effect on orthotopic bone formation. In conclusion, the apatite-coated PLGA/HA particulates showed good results as carriers for BMP-2. The BMP-2 delivery system showed high osteogenic capability in a rat calvarial bone defect model. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and would enhance bone regeneration efficacy for the treatment of large bone defects.     

Blood permeability of a novel ceramic scaffold for bone morphogenetic protein-2

A functionally graded apatite (fg-HAp) with body fluid permeability was developed from bovine bone. The tissue reaction of fg-HAp and its efficacy as a scaffold for recombinant human bone morphogenetic protein-2 (BMP-2) were evaluated histomorphometrically, and a component of permeable fluid into the fg-HAp was analyzed by immunoblotting assay. The fg-HAp block (27 mm(3)) combined with and without BMP-2 (5 microg) was implanted subcutaneously in 4-week-old Wistar rats. Histological examination showed that the surface and bulk degradations of the fg-HAp proceeded extensively and giant cells appeared on the fg-HAp at 2 weeks. Body fluid permeation was found inside the fg-HAp, and the fluid component was immunopositive for albumin. In addition, albumin was detected as a main component among proteins collected from the in vivo implanted fg-HAp. The bioabsorption of the fg-HAp was accelerated as BMP-2-induced bone matured. Histomorphometrical analysis at 4 weeks in the BMP-2/fg-HAp implant showed 59.0% in the total volume of bone and marrow. These results indicate that fg-HAp is an innovative, bioabsorbable bioceramic with fluid permeability characteristic, and may become a biointegrated scaffold for bone engineering.     

Interleukin-11 acts synergistically with bone morphogenetic protein-2 to accelerate bone formation in a rat ectopic model.

We previously demonstrated that recombinant human interleukin-11 (rHuIL-11) induced osteoblast differentiation of C3H10T1/2 progenitor cells and also acted synergistically with recombinant human bone morphogenetic protein-2 (rHuBMP-2) in performing the same function. In this study, we investigated the effect of rHuIL-11 and rHuBMP-2 on bone formation in a rat ectopic model. When placed in rats, implants consisting of polymer-coated gelatin sponges containing various concentrations of rHuBMP-2 showed a dose-dependent increase in calcium content. This was confirmed by radiographic analysis of the implants. Although implants containing rHuIL-11 alone did not accumulate calcium, implants containing a combination of rHuBMP-2 and rHuIL-11 had significantly higher calcium levels than those containing rHuBMP-2 alone. This increase was rHuIL-11 dose dependent. The synergistic effect of 20 micrograms rHuIL-11 and 6 micrograms rHuBMP-2 on bone formation was estimated to be 1 week in advance of that of 6 micrograms rHuBMP-2 alone. Histologic examination revealed that the combination of rHuIL-11 and rHuBMP-2 caused spindle cells to accumulate around implants and induced cell infiltration into implants. Bone formation occurred faster in implants with the combination of rHuIL-11 and rHuBMP-2 compared with rHuBMP-2 alone. These results suggest that rHuIL-11 acts synergistically with rHuBMP-2 to more rapidly stimulate bone formation compared with rHuBMP-2 alone. This novel combined therapy may be of great clinical benefit in bone healing.     

Hybrid coating of hydroxyapatite and collagen within poly(D,L-lactic-co-glycolic acid) scaffold

To improve the cell-affinity of biodegradable polymer scaffold, coating hydroxyapatite (HA) or collagen on the surface of polymer materials seemed to be a strategy to combine both advantages of them. The objective of this study was to develop a novel method to introduce HA and collagen inside polymer scaffold uniformly. HA and collagen suspension was mixed with paraffin microspheres, and molded to form a composite sample. After the sample was dried, HA/collagen composite was left among and on the surface of paraffin microspheres. Poly(D,L-lactic-co-glycolic acid) (PLGA) (50/50) solution was cast into the inter-space of the paraffin microspheres and dried. Afterwards, the paraffin was dissolved and removed, HA/collagen was transferred to the surface and even inside of the pore wall of PLGA scaffolds. Collagen fibers and HA particles which were inlaid inside the PLGA pore wall could help to enhance the coating strength between HA/Col coating and the pore wall surface of the PLGA scaffold. The scaffolds with HA/Col coating were expected to exhibit desirable properties in bone tissue engineering.     

The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2

Bacterial cellulose (BC) is a nanofibrous biological material with attractive physicochemical properties and biocompatibility. Its fiber is similar to the collagenous fiber of bone. To explore if BC could be utilized as a localized delivery system to increase the local concentration of cytokines for tissue engineering, we prepared the BC scaffold from Acetobacter xylinum X-2 (A.?xylinum X-2) and investigated the osteogenic potential of the BC scaffold coated with bone morphogenetic protein-2 (BMP-2). The data showed that BC had a good biocompatibility and induced differentiation of mouse fibroblast-like C2C12 cells into osteoblasts in the presence of BMP-2 in?vitro, as demonstrated by alkaline phosphatase (ALP) activity assays. Within a certain range (0?～?3?μg/scaffold), the osteogenic activity of induced osteoblasts was positively correlated to the concentrations of BMP-2. In in?vivo subcutaneous implantation studies, BC scaffolds carrying BMP-2 showed more bone formation and higher calcium concentration than the BC scaffolds alone at 2 and 4 weeks, respectively. The ALP activity assay and the measurement of calcium concentration of BC scaffolds also showed that more new bone was developed in the BC scaffolds carrying BMP-2 than in the BC scaffolds alone. Our studies suggest that BC is a good localized delivery system for BMPs and would be a potential candidate in bone tissue engineering.     

3D生物打印构建聚乳酸羟基乙酸/纳米羟基磷灰石支架骨形态发生蛋白2缓释复合体的实验研究

BACKGROUND: Tissue-engineered bone scaffold fabricated by 3D-bioprinting technique has good controllability in morphology and structure. However, construction of tissue-engineered bone/cell growth factor complex and time-dose effect of sustained-release factors are needed to be further researched.  OBJECTIVE: To fabricate a sustained-release composite of polylactic-co-glycolic acid (PLGA)/nano-hydroxyapatite (n-HA) scaffold carrying bone morphogenetic protein-2 (BMP-2) using 3D-bioprinting technique, and test the biological properties of the PLGA/n-HA scaffold carrying BMP-2 and the sustained-release properties, thereby to discuss its feasibility as the tissue-engineered bone scaffold composite.  METHODS: Temperature-sensitive chitosan hydrogel was prepared using chitosan and β-glycerophosphate to construct a sustained-release composite, chitosan nanoparticles carrying BMP-2 . 3D-bioprinting technique was utilized to fabricate the PLGA/n-HA scaffold carrying BMP-2. Biological features of the scaffold composite were tested, and time-dose effect of BMP-2 sustained-release was observed.  RESULTS AND CONCLUSION: The average pore size of the scaffold-cytokine composite was (431.31±18.40) μm, and the porosity was (73.64±1.82)%. The cumulative release rate of BMP-2 from the scaffold-cytokine composite that effectively controlled the burst release during 48 hours and 30 days were suitable for the physiological needs. In conclusion, the porosity, pore size, release property, degradation rate, and mechanical strength of the scaffold-cytokine composite all meet the biological requirements of tissue-engineered bone construction. 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     

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.     

The evaluation of ectopic bone formation induced by delivery systems for bone morphogenetic protein-9 or its derived peptide

We have earlier shown that a peptide derived from the bone morphogenetic protein-9 (pBMP-9) stimulates mouse preosteoblasts MC3T3-E1 differentiation in vitro. Here, we evaluated the effects of two delivery systems (DSs) for pBMP-9, one based on collagen and the other on chitosan. The release kinetics of BMP-9 (used as control) and pBMP-9 from these DSs were first determined in vitro by using enzyme-linked immunosorbent assay and high performance liquid chromatography assays, respectively. Micro-computerized tomography and histological analysis were then performed to study in vivo the ectopic ossification induced by both DSs containing these molecules in C57BL/6 mouse quadriceps. We found that collagen DS released in vitro about 35% of its BMP-9 within 1?h, whereas chitosan DS released 80%. The pBMP-9 was released from both DSs more slowly for up to 10 days. These release kinetics seemed to fit the Korsmeyer-Peppas model. Only chitosan DS containing BMP-9 induced strong bone formation in all mice quadriceps within 24 days. All mice quadriceps treated by pBMP-9 trapped in this DS also favored bone structures that started to mineralize. However, pBMP-9 in collagen DS failed to promote ectopic ossification within 24 days in vivo. This study highlights the importance to optimize carrier, thus improving the efficiency of pBMP-9 in vivo.     

Stromal cell-derived factor-1 potentiates bone morphogenetic protein-2 induced bone formation

The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1α (SDF-1α) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients.     

Experimental studies on bone induction using low-molecular-weight poly (DL-lactide-co-glycolide) as a carrier for recombinant human bone morphogenetic protein-2

An appropriate carrier acting as a slow delivery vehicle for the BMPs is required for maximal clinical effectiveness of these bone-inductive proteins. The purpose of this study was to evaluate a low-molecular-weight PLGA copolymer as a synthetic, biodegradable carrier for rhBMP-2 implantation in vivo. Two, 10, or 50 microg of recombinant human BMP-2 were mixed with 10 mg of a poly (DL-lactide-co-glycolide) (PLGA) 50:50 copolymer and implanted into the calf muscles of Wistar rats. Soft X-ray analysis and histologic examination indicated that new bone formation occurred at all rhBMP-2-implanted sites within 3 weeks after implantation. Correlation of rhBMP-2 concentration with the amount of bone induction was confirmed by specific alkaline phosphatase activity and calcium content assay. In vitro analysis indicated that 78.5% of the PLGA copolymer was degraded to smaller molecular weight material after 14 days in PBS solution. It is suggested that rhBMP-2 was released in an active form at the implant site during the degradation of the copolymer, resulting in the induction of new bone formation. Thus this low-molecular-weight PLGA copolymer material represents a promising delivery vehicle for BMPs, and possibly other growth factors, around dental and orthopedic implants.     

The influence of collagen and hyaluronan matrices on the delivery and bioactivity of bone morphogenetic protein-2 and ectopic bone formation

Bone morphogenetic protein-2 (BMP-2) is known to enhance fracture healing when delivered via a bovine collagen sponge. However, collagen rapidly releases BMP-2 with a high burst phase that is followed by a low sustained phase. As a result, supra-physiological doses of BMP-2 are often required to successfully treat bone defects. High BMP-2 dosing can introduce serious side effects that include edema, bone overgrowth, cyst-like bone formation and significant inflammation. As the release behavior of BMP-2 carriers significantly affects the efficacy of fracture healing, we sought to compare the influence of two BMP-2 delivery matrices with contrasting release profiles on BMP-2 bioactivity and ectopic bone formation. We compared a thiol-modified hyaluronan (Glycosil?) hydrogel that exhibits a low burst followed by a sustained release of BMP-2 to a collagen sponge for the delivery of three different doses of BMP-2, the bioactivities of released BMP-2 and ectopic bone formation. Analysis of bone formation by micro-computed tomography revealed that low burst followed by sustained release of BMP-2 from a hyaluronan hydrogel induced up to 456% more bone compared to a BMP-2 dose-matched collagen sponge that has a high burst and sustained release. This study demonstrates that BMP-2 released with a low burst followed by a sustained release of BMP-2 is more desirable for bone formation. This highlights the therapeutic potential of hydrogels, particularly hyaluronan-based, for the delivery of BMP-2 for the treatment of bone defects and may help abrogate the adverse clinical effects associated with high dose growth factor use.     

Heterobifunctional poly(ethylene glycol)-tethered bone morphogenetic protein-2-stimulated bone marrow mesenchymal stromal cell differentiation and osteogenesis

We describe a biomimetic mode of insoluble signaling stimulation to provide target delivery of bone morphogenetic protein-2 (BMP-2), with the aim of prolonging the retention of BMP-2 use in bone tissue engineering and to enable its localized release in response to cellular activity. In our novel localization process, we used heterobifunctional acrylate-N-hydroxysuccinimide poly(ethylene glycol) (PEG) as a spacer to tether BMP-2 onto a poly(lactide-co-glycolide) scaffold. Use of PEG-tethered BMP-2 was feasible because BMP-2 retained its activity after covalent conjugation. The PEG-tethered BMP-2 conjugate sustained stimulation and retained its mitogenic activity, notably affecting pluripotent stem cell proliferation and differentiation. We seeded the scaffolds with bone marrow-derived mesenchymal stromal cells as progenitor cells to evaluate their morphology and phenotypic expression. We also created bilateral, full-thickness cranial defects in rabbits to investigate the osteogenic effect of cultured mesenchymal stromal cells on bone regeneration in vivo. Histomorphometry and histology demonstrated that the PEG-tethered BMP-2 conjugate enhanced de novo bone formation after surgery. Our work revealed the potential for biomimetic surface engineering by entrapping signaling growth factor to stimulate osteogenesis. Our technique may provide a new platform for bone-engineered stem cell therapies.     

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.     

Enhanced bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein-2 from a biodegradable hydrogel

The objective of this study is to investigate the feasibility of a biodegradable hydrogel of gelatin as the controlled release carrier of bone morphogenetic protein-2 (BMP-2) suitable for enhancement of bone regeneration at a segmental bone defect. Hydrogels with three different water contents were prepared through glutaraldehyde crosslinking of gelatin with an isoelectric point of 9.0 under varied reaction conditions. Segmental critical-sized defects (20 mm) were created at the ulnar bone of skeletally mature New Zealand white rabbits, and gelatin hydrogels incorporating BMP-2 (17 microg/hydrogel) were implanted into the defects. When bone regeneration was evaluated by soft x-ray observation and bone mineral density (BMD) measurement, the gelatin hydrogels incorporating BMP- 2 exhibited significantly high osteoinduction activity compared with that of free BMP-2, although the activity depended on the water content of the hydrogels. Significantly higher BMD enhancement was observed in the gelatin hydrogel with a water content of 97.8 wt% than that with the lower or higher water content. We concluded that the biodegradable gelatin hydrogel is a promising controlled release carrier of BMP-2 for bone regeneration at the segmental bone defect.     

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.     

Spatial immobilization of bone morphogenetic protein-4 in a collagen-PLGA hybrid scaffold for enhanced osteoinductivity

The introduction of bioactive molecules into three-dimensional porous scaffolds to mimic the in vivo microenvironment is a promising strategy for tissue engineering and stem cell research. In this study, bone morphogenetic protein-4 (BMP4) was spatially immobilized in a collagen-PLGA hybrid scaffold with a fusion BMP4 composed of an additional collagen-binding domain derived from fibronectin (CBD-BMP4). CBD-BMP4 bound to the collagen-PLGA hybrid scaffold and the BMP4-immobilized hybrid scaffold supported cell adhesion and proliferation. The osteogenic induction effect of the immobilized CBD-BMP4 was investigated with three-dimensional culture of human bone marrow-derived mesenchymal stem cells in the BMP4-immobilized collagen-PLGA hybrid scaffold. The in vivo implantation experiment demonstrated that the immobilized CBD-BMP4 maintained its osteoinductive activity, being capable of up-regulating osteogenic gene expression and biomineralization. The strong osteoinductivity of the BMP4-immobilized scaffold suggests it should be useful for bone tissue engineering, stem cell function manipulation and bone substitutes.