Improvement of porous beta-TCP scaffolds with rhBMP-2 chitosan carrier film for bone tissue application

Ceramic materials are osteoconductive matrices extensively used in bone tissue engineering approaches. The performance of these types of biomaterials can be greatly enhanced by the incorporation of bioactive agents and materials. It is previously reported that chitosan is a biocompatible, biodegradable material that enhances bone formation. In the other hand, bone morphogenetic protein-2 (BMP-2) is a well-known osteoinductive factor. In this work we coated porous beta-tricalcium phosphate (beta-TCP) scaffolds with recombinant human BMP-2 (rhBMP-2) carrier chitosan films and studied how they could modify the ceramic physicochemical properties, cellular response, and in vivo bone generation. Initial beta-TCP disks with an average diameter of 5.78 mm, 2.9 mm thickness, and 53% porosity were coated with a chitosan film. These coating properties were studied by X-ray diffraction, Fourier transform-infrared analysis, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray analysis (EDX). Treatment modified the scaffold porous distribution and increased the average hardness. The biocompatibility did not seem to be altered. In addition, adhered C2C12 cells expressed alkaline phosphatase activity, related to cell differentiation toward osteogenic lineage, due to the incorporation of rhBMP-2. On the other hand, in vivo observations showed new bone formation 3 weeks after surgery, a much shorter time than control beta-TCP ceramics. These results suggest that developed coating improved porous beta-TCP scaffold for bone tissue applications and added osteoinductive properties.     

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。     

The performance of BMP-2 loaded TCP/HAP porous ceramics with a polyelectrolyte multilayer film coating

Delivering rhBMP-2 (recombinant Bone Morphogenic Protein-2) at low but therapeutically efficient dose is one of the current challenges for bone tissue repair. In this context, Polyelectrolyte Multilayer films (PEM) represent an attractive rhBMP-2 carrier due to their ability to protect proteins from denaturation and to coat a wide variety of materials with complex geometry. Herein, we coated macroporous TCP/HAP granules with a biopolymeric PEM film to deliver rhBMP-2 in a "matrix-bound" manner. In vitro release kinetics indicated that the PEM-coated granules sequestered significant amounts of rhBMP-2. The degree of film cross-linking influenced the quantity of rhBMP-2 trapped within the films. Bare (uncoated) TCP/HAP scaffolds were also able to retain rhBMP-2. Bioactivity of rhBMP-2 in the PEM-coated granules was confirmed on two cell markers: luciferase expression on BMP-responsive-element/Luc C2C12 cells and alkaline phosphatase activity induction on C2C12 cells. Promisingly, rhBMP-2 adsorbed onto PEM-coated and on bare granules in a lesser extent, could be stored and remained bioactive over at least 3 weeks. In vivo, both uncoated and PEM-coated TCP/HAP granules loaded with rhBMP-2 exhibited both osteoconductive and osteoinductive properties. This opens perspective for coating these bioactive PEM on other types of implantable materials, including metal alloy that do not exhibit any affinity for rhBMP-2.     

Bone morphogenetic protein 2 incorporated into biomimetic coatings retains its biological activity

We have previously shown that proteins can be incorporated into the latticework of calcium phosphate layers when biomimetically coprecipitated with the inorganic components, upon the surfaces of titanium-alloy implants. In the present study, we wished to ascertain whether recombinant human bone morphogenetic protein 2 (rhBMP-2) thus incorporated retained its bioactivity as an osteoinductive agent. Titanium alloy implants were coated biomimetically with a layer of calcium phosphate in the presence of different concentrations of rhBMP-2 (0.1-10 microg/mL). rhBMP-2 was successfully incorporated into the crystal latticework, as revealed by protein blot staining. rhBMP-2 was taken up by the calcium phosphate coatings in a dose-dependent manner, as determined by ELISA. Rat bone marrow stromal cells were grown directly on these coatings for 8 days. Their osteogenicity was then assessed quantitatively by monitoring alkaline phosphatase activity. This parameter increased as a function of rhBMP-2 concentrations within the coating medium. rhBMP-2 incorporated into calcium phosphate coatings was more potent in stimulating the alkaline phosphatase activity of the adhering cell layer than was the freely suspended drug in stimulating that of cell layers grown on a plastic substratum. This system may be of osteoinductive value in orthopedic and dental implant surgery.     

Histological characterization of the early stages of bone morphogenetic protein-induced osteogenesis

On the basis of currently available knowledge, we hypothesize that the initial bone formation, as induced by bone morphogenetic protein (BMP), is influenced by the chemical composition and three-dimensional spatial configuration of the used carrier material. Therefore, in the current study, the osteoinductive properties of porous titanium (Ti) fiber mesh with a calcium phosphate (Ca-P) coating (Ti-CaP), insoluble bone matrix (IBM), fibrous glass membrane (FGM), and porous particles of hydroxy apatite (PPHAP) loaded with rhBMP-2 were compared in a rat ectopic assay model at short implantation periods. Twelve Ti-CaP, 12 IBM, 12 FGM, and 12 PPHAP implants, loaded with rhBMP-2, were subcutaneously placed in 16 Wistar King rats. The rats were sacrificed at 3, 5, 7, and 9 days post-operative, and the implants were retrieved. Histological analysis demonstrated that IBM and Ti-CaP had induced ectopic cartilage and bone formation by 5 and 7 days, respectively. However, in PPHAP, bone formation and cartilage formation were seen together at 7 days. At 9 days, in Ti-CaP, IBM, and PPHAP, cartilage was seen together with trabecular bone. At 9 days, in FGM, only cartilage was observed. Quantitative rating of the tissue response, using a scoring system, demonstrated that the observed differences were statistically significant (Wilcoxon rank sum test, p < 0.05). We conclude that IBM, CaP-coated Ti mesh, FGM, and PPHAP provided with rhBMP-2 can indeed induce ectopic bone formation with a cartilaginous phase in a rat model at short implantation periods. Considering the different chemical composition and three-dimensional spatial configuration of the carrier materials used, these findings even suggest that endochondral ossification is present in rhBMP-2-induced osteogenesis, even though the amount of cartilage may differ.     

Carrier dependent cell differentiation of bone morphogenetic protein-2 induced osteogenesis and chondrogenesis during the early implantation stage in rats

To evaluate the osteoinductive effects of recombinant human bone morphogenetic protein (rhBMP)-2 during the early stages of rat ectopic bone formation, we prepared two distinct carriers. Two carriers, insoluble bone matrix (IBM) and fibrous glass membrane (FGM) were combined with rhBMP-2 and implanted into the backs of rats to evaluate the osteoinductive effects of the two rhBMP-2 carrier systems. Insoluble bone matrix particle size was 320 to 620 microm. Fibrous glass membrane was constructed from unwoven glass fibers 1 microm in diameter. Alkaline phosphatase (ALP) activity and type II collagen were detected in IBM/rhBMP-2 at 5 days postimplantation. Calcium (Ca) was also detected in IBM/rhBMP-2 at 7 and 9 days postimplantation. In contrast, ALP and type II collagen were detected in FGM/rhBMP-2 at 7 days. Calcium was undetected, indicating that the bone formation in IBM/rhBMP-2 proceeded faster than in FGM/rhBMP-2 during the early stage of BMP-induced osteogenesis. In addition, mRNA expression level of KDR, a receptor for vascular endothelial growth factor, was also increased in IBM/rhBMP-2. To investigate the in vivo release profile of rhBMP-2, iodine 125 ((125)I)-labeled BMP-2-incorporating IBM and FGM implants were inserted into the back subcutis of mice. More than 60% of the rhBMP-2 was released from the IBM/rhBMP-2 carrier within 1 day after implantation, whereas 50% of the rhBMP-2 was released from the FGM/rhBMP-2 10 days postimplantation. These results indicated that osteo- and chondrogenesis depends highly upon the geometry of the carrier and the in situ retention of rhBMP-2 during the early stage of rhBMP-2 induced bone formation.     

A new bone-inducing biodegradable porous beta-tricalcium phosphate

A new type of degradable biomaterial with bone-inducing capacity was made by combining porous beta-tricalcium phosphate (beta-TCP) with a delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2). The BMP delivery system consisted of a block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG), a known biocompatible and biodegradable material. The efficacy of this biomaterial in terms of its bone-inducing capacity was examined by ectopic bone formation in the dorsal muscles of the mouse. In the beta-TCP implants coated with the PLA-DX-PEG polymer containing more than 0.0025% (w/w) of rhBMP-2, new ectopic bone tissues with marrow were consistently found on the surface of implants. The radiographic density of beta-TCP was diminished in a time-dependent manner. On histological examination, numerous multinucleated osteoclasts with positive tartrate-resistant acid-phosphatase (TRAP) staining were noted on the surface of the beta-TCP. These experimental results indicate that beta-TCP implants coated with synthetic rhBMP-2 delivery system might provide effective artificial bone-graft substitutes with osteoinductive capacity and biodegradable properties. In addition, this type of biomaterial may require less rhBMP-2 to induce significant new bone mass.     

重组人骨形态发生蛋白2-肝素-人工骨复合材料的骨诱导活性

背景：已有将重组人骨形态发生蛋白2应用于骨再生及修复的报道,但由于其在生物体内半衰期短而导致诱导骨形成的能力受到限制。 目的：制备具有较好缓释效果的重组人骨形态发生蛋白2-肝素-人工骨复合材料,并检测其缓释性能及骨诱导活性。 方法：通过高效液相色谱法检测重组人骨形态发生蛋白2-肝素复合物对于酶解的保护作用。将重组人骨形态发生蛋白2与肝素溶液混匀后复合于人工骨材料表面,ELISA方法检测其体外释药性质,茜素红染色法检测其诱导成骨细胞的能力,应用小鼠体内实验评价其异位骨诱导能力。 结果与结论：成功制备了具有良好缓释效果的重组人骨形态发生蛋白2-肝素-人工骨复合材料,具有较强的诱导骨钙蛋白及异位骨形成能力。     

The effect of polyelectrolyte multilayer coated titanium alloy surfaces on implant anchorage in rats

Advances have been achieved in the design and biomechanical performance of orthopedic implants in the last decades. These include anatomically shaped and angle-stable implants for fracture fixation or improved biomaterials (e.g. ultra-high-molecular-weight polyethylene) in total joint arthroplasty. Future modifications need to address the biological function of implant surfaces. Functionalized surfaces can promote or reduce osseointegration, avoid implant-related infections or reduce osteoporotic bone loss. To this end, polyelectrolyte multilayer structures have been developed as functional coatings and intensively tested in vitro previously. Nevertheless, only a few studies address the effect of polyelectrolyte multilayer coatings of biomaterials in vivo. The aim of the present work is to evaluate the effect of polyelectrolyte coatings of titanium alloy implants on implant anchorage in an animal model. We test the hypotheses that (1) polyelectrolyte multilayers have an effect on osseointegration in vivo; (2) multilayers of chitosan/hyaluronic acid decrease osteoblast proliferation compared to native titanium alloy, and hence reduce osseointegration; (3) multilayers of chitosan/gelatine increase osteoblast proliferation compared to native titanium alloy, hence enhance osseointegration. Polyelectrolyte multilayers on titanium alloy implants were fabricated by a layer-by-layer self-assembly process. Titanium alloy (Ti) implants were alternately dipped into gelatine (Gel), hyaluronic acid (HA) and chitosan (Chi) solutions, thus assembling a Chi/Gel and a Chi/HA coating with a terminating layer of Gel or HA, respectively. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bones’ response to polyelectrolyte surfaces in vivo. 48 rats were randomly assigned to three groups of implants: (1) native titanium alloy (control), (2) Chi/Gel and (3) Chi/HA coating. Mechanical fixation, peri-implant bone area and bone contact were evaluated by pull-out tests and histology at 3 and 8 weeks. Shear strength at 8 weeks was statistically significantly increased (p < 0.05) in both Chi/Gel and Chi/HA groups compared to the titanium alloy control. No statistically significant difference (p > 0.05) in bone contact or bone area was found between all groups. No decrease of osseointegration of Chi/HA-coated implants compared to non-coated implants was found. The results of polyelectrolyte coatings in a rat model showed that the Chi/Gel and Chi/HA coatings have a positive effect on mechanical implant anchorage in normal bone.     

Bone regeneration using photocrosslinked hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles

Although rhBMP-2 has excellent ability to accelerate the repair of normal bone defects, limitations of its application exist in the high cost and potential side effects. This study aimed to develop a composite photopolymerisable hydrogel incorporating rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (PH/rhBMP-2/NPs) as the bone substitute to realize segmental bone defect repair at a low growth factor dose. Firstly rhBMP-2 loaded 2-N, 6-O-sulfated chitosan nanoparticles (rhBMP-2/NPs) were prepared and characterized by DLS and TEM. Composite materials, PH/rhBMP-2/NPs were developed and investigated by SEM-EDS as well as a series of physical characterizations. Using hMSCs as an in vitro cell model, composite photopolymerisable hydrogels incorporating NPs (PH/NPs) showed good cell viability, cell adhesion and time dependent cell ingrowth. In vitro release kinetics of rhBMP-2 showed a significantly lower initial burst release from the composite system compared with the growth factor-loaded particles alone or encapsulated directly within the hydrogel, followed by a slow release over time. The bioactivity of released rhBMP-2 was validated by alkaline phosphatase (ALP) activity as well as a mineralization assay. In in vivo studies, the PH/rhBMP-2/NPs induced ectopic bone formation in the mouse thigh. In addition, we further investigated the in vivo effects of rhBMP-2-loaded scaffolds in a rabbit radius critical defect by three dimensional micro-computed tomographic (μCT) imaging, histological analysis, and biomechanical measurements. Animals implanted with the composite hydrogel containing rhBMP-2-loaded nanoparticles underwent gradual resorption with more pronounced replacement by new bone and induced reunion of the bone marrow cavity at 12 weeks, compared with animals implanted with hydrogel encapsulated growth factors alone. These data provided strong evidence that the composite PH/rhBMP-2/NPs are a promising substitute for bone tissue engineering.     

Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits

In this study, the osteoinductive properties of porous calcium phosphate (Ca-P) cement loaded with bone morphogenetic protein 2 (rhBMP-2) were evaluated and compared with rhBMP-2 loaded absorbable collagen sponge (ACS). Discs with a diameter of 8mm were loaded with a buffer solution with or without 10 microg rhBMP-2 and inserted in 8mm full thickness cranial defects in rabbits for 2 and 10 weeks of implantation. Histological analysis revealed excellent osteoconductive properties of the Ca-P material. It maintained its shape and stability during the implantation time better than the ACS but showed no degradation like the ACS. Quantification of the Ca-P cement implants showed that bone formation was increased significantly by administration of rhBMP-2 (10 weeks pore fill: 53.0+/-5.4%), and also reached a reasonable amount without rhBMP-2 (43.1+/-10.4%). Remarkably, callus-like bone formation outside the implant was observed frequently in the 2 weeks rhBMP-2 loaded Ca-P cement implants, suggesting a correlation with the presence of growth factor in the surrounding tissue. However, an additional in vitro assay revealed an accumulative release of no more than 9.7+/-0.9% after 4 weeks. We conclude that: (1). Porous Ca-P cement is an appropriate candidate scaffold material for bone engineering. (2). Bone formation can be enhanced by lyophilization of rhBMP-2 on the cement. (3). Degradation of porous Ca-P cement is species-, implantation site- and implant dimension-specific.     

Osteoinduction of porous bioactive titanium metal

This is the first report of bone induction in a non-osseous site by titanium metal, which has long been recognized as a non-bioactive material. After undergoing specific chemical and thermal treatments, porous bioactive titanium induced bone formation without the need of additional osteogenic cells or osteoinductive agents. Four types of titanium implants were implanted in the dorsal muscles of mature beagle dogs, and were examined histologically after periods of 3 and 12 months. Chemically and thermally treated titanium, as well as pure titanium, was implanted either as porous blocks or as fibre mesh cylinders. Bone formation was found only in the chemically and thermally treated porous block implants removed after 12 months. The present study shows that even a non-soluble metal that contains no calcium or phosphorus can be an osteoinductive material when treated to form an appropriate macrostructure and microstructure. This finding may elucidate the nature of osteoinduction, and lead to the advent of epochal osteoinductive biomaterials for tissue regeneration.     

The biocompatibility of nanostructured calcium phosphate coated on micro-arc oxidized titanium

To achieve improved osseointegration, there have been many efforts to modify the surface composition and topography of dental implants. Recently, the anodic oxidation treatment of titanium (Ti) has attracted a great deal of attention. Meanwhile, calcium phosphate is commonly applied to metallic implants as a coating material for fast fixation and firm implant-bone attachment on the account of its demonstrated bioactive and osteoconductive properties. In the present study, anodized surface and calcium phosphate deposition by electron beam evaporation were combined. Nanostructured calcium phosphate film was deposited on the micro-arc oxidized Ti. New apatite layer formed easily on the coated film when incubating in DPBS solution at 37 degrees C. By adding basic fibroblast growth factor (bFGF) in the DPBS solution, the bFGF could be immobilized in the newly formed apatite layer. The coated film enhanced osseointegration of Ti implants in vivo.     

Potential of porous poly-D,L-lactide-co-glycolide particles as a carrier for recombinant human bone morphogenetic protein-2 during osteoinduction in vivo.

Several different biodegradable bone graft materials are in clinical or preclinical use for the repair of bone defects in orthopedics, maxillofacial surgery, and periodontics. This study tested the hypothesis that poly-D,L-lactide-co-glycolide copolymer (PLG) can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) and that the composite has osteoinductive ability. Porous PLG rods were shredded to a particle size ranging from 250 to 850 microm. Active and inactive demineralized freeze-dried bone allografts (DFDBA) with a comparable particle size were used as positive and negative controls, respectively. PLG particles were treated with vehicle or with 5 or 20 microg rhBMP-2. DFDBA and PLG particles were placed in gelatin capsules, mixed with vehicle or rhBMP-2, and implanted at intramuscular sites in male Nu/Nu (nude) mice. Each mouse underwent bilateral implantation with implants of the same formulation, resulting in five groups of four mice per group: active DFDBA, inactive DFDBA, PLG, PLG + 5 microg rhBMP-2, and PLG + 20 microg rhBMP-2. After 56 days, the implants were recovered and processed for histology. Bone induction was assessed by use of a semiquantitative scoring system based on the amount of new bone formed in representative histological sections. Histomorphometry was also used to measure the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induced the formation of ossicles containing new bone with bone marrowlike tissue, whereas inactive DFDBA or PLG particles alone did not induce new bone. The addition of rhBMP-2 to PLG particles resulted in new bone formation that had a greater bone induction score than active DFDBA. Moreover, the histomorphometric analysis showed that the addition of rhBMP-2 to PLG particles induced the formation of a greater area of new bone and bone marrowlike tissue than active DFDBA. The resorption of the PLG particles was markedly increased with the addition of rhBMP-2, suggesting that rhBMP-2 may attract and regulate resorptive cells at the implantation site. The results of the present study indicate that PLG copolymers are good carriers for BMP and promote the induction of new bone formation. Further, the PLG copolymers with rhBMP-2 had a greater effect in inducing new bone formation and resorbing the implanted material than active DFDBA alone.     

Collagen-infiltrated porous hydroxyapatite coating and its osteogenic properties: in vitro and in vivo study

Bone-implant interface is critical for the early fixation of orthopedic implants. In this study, porous hydroxyapatite (HA) coatings were prepared through a liquid precursor plasma spraying process and were infiltrated with the collagen, alone and with the additional incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) and RGD peptide (RGD). The results showed significantly improved mesenchymal stem cell (MSC) adhesion, proliferation, and differentiation on collagen-modified HA coatings, partially benefited from the formation of a fibrous network due to the self-reconstitution of collagen on the HA surface. Further enhancements on MSC proliferation and differentiation were generally observed through the additional incorporation of bone morphogenetic protein (BMP) and RGD. The osteoinductive and osteoconductive properties of the collagen/BMP-modified HA coatings were studied in vivo. Clear ectopic bone formation and significantly accelerated bone growth rate (29% increase, p < 0.05) have been observed after 1-month implantation of HA-collagen/rhBMP-2-coated Ti alloy samples into the rabbit muscle and dog femora, respectively. Overall, our results suggest that collagen-modified HA coating surface is a far superior substrate for cell attachment, proliferation, and differentiation, and collagen can be used an efficient carrier for BMP in vivo. Therefore, modification of HA coating with collagen is a simple but effective biomimetic approach to enhancing the osteointegration and early fixation of bone-implant interface.     

Evaluation of BMP-2 tethered polyelectrolyte coatings on hydroxyapatite scaffolds in vivo

The goal of this in vivo study was to evaluate the osteoinductive and angio-inductive properties of a porous hydroxyapatite (HAp) scaffold with immobilized recombinant bone morphogenetic protein-2 (rhBMP-2) on the surface. It was hypothesized in this study that the use of a rhBMP-2 incorporated polyelectrolyte coating on the HAp scaffold would allow for controlled exposure of rhBMP-2 into the tissue and would provide a sound platform for tissue growth. The scaffolds were characterized for porosity and interconnectivity using pycnometry, scanning electron microscopy and micro-ct. These scaffolds were then divided into the following four groups: (a) HAp scaffold (n-HAp group), (b) rhBMP-2 physically adsorbed on HAp scaffold (HAp-BMP-2 Group), (c) polyelectrolyte coating on HAp scaffold without rhBMP-2 (HAp-PEI Scaffold Group), and (d) polyelectrolyte coating tethered with rhBMP-2 on HAp scaffold (HAp-PEI-BMP-2 Scaffold Group). Using 18 skeletally matured New Zealand white rabbits, these scaffolds were evaluated in a nonload bearing femoral condyle plug model. The negative controls for this study have defects that were left untreated and the positive controls have defects that were filled with autologous bone graft harvested from epsilateral iliac crest. Bone induction, vessel growth, and scaffold-bone contact were analyzed after 8-week implantation using micro-CT and histomorphometry. It was concluded from this study that the use of scaffold with an attached rhBMP-2 increased the vascularization around the implant when compared with the uncoated n-HAp scaffold, a necessary step of bone regeneration. The open-pore HAp scaffold was also concluded to provide a platform for tissue growth, drug loading, and tissue interaction. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

Ectopic bone formation in mice associated with a lactic acid/dioxanone/ethylene glycol copolymer-tricalcium phosphate composite with added recombinant human bone morphogenetic protein-2

A new putty-like material with bone-inducing capacity was made by combining a block copolymer of poly d,l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) and beta-tricalcium phosphate (beta-TCP) powder with added recombinant human bone morphogenetic protein-2 (rhBMP-2). To optimize the material's efficacy for bone formation, we formulated the optimal composition ratio of the respective constituent that gives the greatest osteoinductive efficacy in a mouse model of ectopic bone formation. In this series of studies, we investigated the size of ectopic bone mass induced 3 and 6 weeks after implantation of the materials composed of 30 mg of PLA-DX-PEG with 2 microg of rhBMP-2 and 0, 15, 30, or 60 mg of beta-TCP powder. An additional experiment was designed to investigate how content ratios of beta-TCP powder in 30 mg-putty implants (0%, 16.7%, 33.3%, 50%, 66.7%, 83.3%, or 100%) for a fixed dose (5 microg) of the rhBMP-2 altered the size of the induced ossicle. The results from the first experiment indicated that the bone yields were linearly dependent on the amount of additional beta-TCP powder. In the second experiment, the largest ossicles induced by 5 microg of rhBMP-2 were obtained when the polymer/beta-TCP ratio was 1/2 in mice. The data provide important insights into the fabrication of implants that provide efficacious delivery of rhBMP-2. The new putty-like material may be valuable for repairing or regenerating bone in a clinical setting.     

Biological performance of uncoated and octacalcium phosphate-coated Ti6Al4V

The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose. The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential. In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant. Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction. This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process.     

不同植骨材料在腰椎融合过程中的应用及转化生长因子β的表达

背景：骨形态发生蛋白单独应用于骨移植效果不佳。而转化生长因子β能明显增强其在体内的诱导成骨作用。 目的：观察自体骨及重组人骨形态发生蛋白2复合骨用于兔腰椎融合过程中转化生长因子β的基因表达。 方法：将新西兰大白兔60只随机分为自体骨组、复合骨组和异体骨组,分别于双侧L5-6横突间植入自体骨、重组人骨形态发生蛋白2复合骨及异体骨。植入后7,14,21,28,35 d取3组节段骨痂,应用实时荧光定量RT-PCR检测转化生长因子β的基因表达水平。 结果与结论：植入后28 d,复合骨组转化生长因子β达峰值,随后有所下降,但均高于自体骨组和异体骨组(P < 0.05)。结果证实,重组人骨形态发生蛋白2复合骨缓释重组人骨形态发生蛋白2,有效地促进了转化生长因子β的表达,明显增强了体内的诱导成骨效应。     

Mechanical property, degradation rate, and bone cell growth of chitosan coated titanium influenced by degree of deacetylation of chitosan

Chitosan has shown promise as a coating for dental/craniofacial and orthopaedic implants. However, the effects of degree of deacetylation (DDA) of chitosan on coating bond strength, degradation, and biological performance is not known. The aim of this project was to evaluate bonding, degradation, and bone cell growth on titanium coated with chitosans of different DDA and from different manufacturers. Three different chitosans, 80.6%, 81.7%, and 92.3% DDA were covalently bonded to titanium coupons via silane-glutaraldehyde molecules. Bond strengths were evaluated in mechanical tensile tests, and degradation, over 5 weeks, was conducted in cell culture medium with and without 100 microg/mL lysozyme. Cytocompatibility was evaluated for 10 days using UMR 106 osteoblastic cells. Results showed that mean chitosan coating bond strengths ranged from 2.2-3.8 MPa, and that there was minimal affect of DDA on coating bond strengths. The coatings exhibited little dissolution over 5 weeks in medium with or without lysozyme. However, the molecular weight (MW) of the chitosan coatings remaining on the titanium samples after 5 weeks decreased by 69-85% with the higher DDA chitosan coatings exhibiting less percent change in MW than the lower DDA materials. The growth of the UMR 106 osteoblast cells on the 81.7% DDA chitosan coating was lower on days 3 and 5, as compared with the other two coatings, but by day 10, there were no differences in growth among three coatings or to the uncoated titanium controls. Differences in growth were attributed to differences in manufacturer source material, though all coatings were judged to be osteocompatible in vitro.