Vascularization and bone regeneration in a critical sized defect using 2-N,6-O-sulfated chitosan nanoparticles incorporating BMP-2

An ideal bone tissue engineering graft should have both excellent pro-osteogenesis and pro-angiogenesis to rapidly realize the bone regeneration in vivo. To meet this goal, 2-N,6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was successfully developed and showed a dose-dependent enhancement on angiogenesis in vitro. For the repair of a critical sized defect in rabbit radius, we developed BMP-2 loaded S-NP (BMP-2/S-NP) with protein loading efficiency of 1.4 ± 0.2% and fabricated a gelatin sponge (G) based implant loaded with BMP-2/S-NP (BMP-2/S-NP/G). This implant exerted a delivery of BMP-2 with an initial burst release of 15.3 ± 4.1% in first 24 h and a gradual release for 21 days to 77.8 ± 3.6%. The in vitro ALP assay revealed that the activity of released BMP-2 from BMP-2/S-NP/G was maintained after 3-d and 7-d delivery and further enhanced after 14-d delivery compared with the original BMP-2. Furthermore, the in vivo effects of BMP-2/S-NP/G on the bone regeneration and vessel formation in the critical sized defect (18 mm) of rabbit radius were investigated by synchrotron radiation-based micro-computed tomography (SRμCT) imaging, three dimensional micro-computed tomographic (μCT) imaging, histological analysis, immunohistochemistry and biomechanical measurement. Based on the results, both peripheral vessel and new vessel formation were significantly increased by the BMP-2/S-NP/G treatment, along with the bridged defects at as early as 2 weeks, the healed defects at 8 weeks and the reunion of bone marrow cavity at 12 weeks. The results indicated that both controlled release of active BMP-2 and favorable vascularization at the defect site contributed by BMP-2/S-NP/G played a crucial role in accelerating and promoting bone augmentation. This study suggests that BMP-2/S-NP/G demonstrates promise for vascularization and bone regeneration in clinical case of large defect.     

Modulation of osteogenic activity of BMP-2 by cellulose and chitosan derivatives

Polysaccharides with structure and potential bioactivity similar to heparin were synthesized based on cellulose which was regioselectively sulfated, carboxylated or carboxymethylated, and chitosan that was sulfated only. Osteogenic activity of these derivatives was studied in cooperation with BMP-2 using C2C12 myoblast cells as a model system measuring alkaline phosphatase (ALP) activity and the expression of the genes Osterix, Noggin and Runx-2. It was found that highly sulfated chitosan showed the strongest osteogenic activity of all polysaccharides, but only at lower concentrations, while higher concentrations were inhibitory. By contrast, cellulose with a low or intermediate degree of sulfation showed increasing ALP activity and expression of Osterix and Noggin with rising concentrations. Lower sulfated cellulose with a high degree of carboxylation was less osteogenic, but had a positive effect on cell viability, while carboxymethylated cellulose had almost no osteogenic activity. The results indicate that regioselectively sulfated as well as carboxylated cellulose and chitosan possess an osteogenic activity, which makes them interesting candidates for application in tissue engineering of bone.     

Heparin microparticle effects on presentation and bioactivity of bone morphogenetic protein-2

Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration.     

BMP-2 plasmid loaded PLGA/HAp composite scaffolds for treatment of bone defects in nude mice

We studied three different types of scaffolds, encapsulating bone morphogenetic protein-2 (BMP-2) plasmid, in terms of their performances in bone regeneration in nude mice. The plasmid was loaded into fibrous matrices in three different ways: coating of naked DNA (Group A) or DNA/chitosan nanoparticles (Group B) onto scaffolds after fiber fabrication by dripping, and encapsulation of DNA/chitosan nanoparticles into scaffold by mixing them with PLGA/DCM solution before fiber fabrication (Group C). Their individual performances were examined by soft X-ray observation, histological analysis and immunostaining of bone tissue. In addition, the BMP-2 protein concentration and alkaline phosphatase (ALP) activity in serum were monitored. The results revealed that the bioactivity of BMP-2 plasmid released from all three kinds of scaffolds was well maintained; this eventually helped improve the healing of segmental defects in vivo. Interestingly, the three kinds of scaffolds released DNA or DNA nanoparticles in different modes and their performances in bone healing were diverse. These observations demonstrate that the in vivo performance of these newly developed DNA delivery devices correlates well with their in vitro release profiles.     

Enhanced healing of rat calvarial defects with sulfated chitosan-coated calcium-deficient hydroxyapatite/bone morphogenetic protein 2 scaffolds

Calcium phosphate cements (CPCs), which are widely used in bone regeneration, possess good biocompatibility and osteoconductivity and have been demonstrated to be candidate carriers for bone growth factors. However, limited release of growth factors from CPCs and slow degradation of the materials are not desirable for certain clinical applications. Previous studies have shown that calcium-deficient hydroxyapatite (CDHA) from CPCs presents more rapid degradation rate than CPCs. In this study, a hybrid growth factor delivery system was prepared by using bone morphogenetic protein 2 (BMP-2) loaded CDHA porous scaffold with sulfated chitosan (SCS) coating for improved release profile. We tested the BMP-2 release characteristic of CDHA/BMP-2/SCS composite in vitro and its ability to repair rat calvarial bone defects. A higher percentage of BMP-2 was released when sulfated chitosan coating was present compared with CDHA/BMP-2 group. Eight weeks postoperation, the repaired crania were evaluated by microcomputed tomography, sequential fluorescent labeling, histological analysis, and immunohistochemistry. CDHA/BMP-2/SCS group promoted the most extensive new bone formation than CDHA/BMP-2 and CDHA groups. Our observations suggest that sulfated chitosan coating could enhance the release profile of CDHA/BMP-2 composite in vitro and promote new bone formation in vivo. The hybrid CDHA/BMP-2/SCS system is a promising growth factor delivery strategy for bone regeneration.     

Enhanced osteogenic activity of bone morphogenetic protein-2 by 2-O-desulfated heparin

The objective of this study was to investigate the effect of heparin sulfate groups on the osteogenic activity of bone morphogenetic protein-2 (BMP-2) in vitro and in vivo. Three types of desulfated (DS) derivatives of heparin (2-O-DS, 6-O-DS, and N-DS) were prepared and their bioactivity in rat bone marrow derived mesenchymal stem cells (MSC) in the absence or presence of BMP-2 was evaluated. When cultured with the 2-O-DS derivative and BMP-2 MSC showed enhanced proliferation, alkaline phosphatase activity, and Runx2 mRNA expression, compared with heparin and other derivatives. A similar tendency was observed for MSC cultured on two-dimensional substrates coated with heparin or the derivatives and in three-dimensional hydrogels containing heparin or the derivatives. A binding experiment demonstrated a greater binding affinity of 2-O-DS for BMP-2 than that of heparin and the other derivatives. Following implantation into the back subcutis of mice significantly greater ectopic bone formation in terms of bone weight, amount of calcium, and histology were observed for the gelatin hydrogels incorporating 2-O-DS and containing BMP-2. In addition, the gelatin hydrogels incorporating 2-O-DS showed controlled release of BMP-2 in vitro and in vivo. These findings demonstrated that the 2-O-DS derivative of heparin has a synergistic effect on the in vitro and in vivo osteogenic activity of BMP-2.     

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.     

Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4–PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.     

BMP Stimulation of Alkaline Phosphatase Activity in Pluripotent Mouse C2C12 Cells is Inhibited by Dermatopontin,One of the Most Abundant Low Molecular Weight Proteins in Demineralized Bone Matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl₂, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of γ-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.     

BMP stimulation of alkaline phosphatase activity in pluripotent mouse C2C12 cells is inhibited by dermatopontin, one of the most abundant low molecular weight proteins in demineralized bone matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.     

Hyaluronic acid-based hydrogels functionalized with heparin that support controlled release of bioactive BMP-2

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive factor, yet its clinical use is limited by a short biological half-life, rapid local clearance and propensity for side effects. Heparin (HP), a highly sulfated glycosaminoglycan (GAG) that avidly binds BMP-2, has inherent biological properties that may circumvent these limitations. Here, we compared hyaluronan-based hydrogels formulated to include heparin (Heprasil?) with similar gels without heparin (Glycosil?) for their ability to deliver bioactive BMP-2 in vitro and in vivo. The osteogenic activity of BMP-2 released from the hydrogels was evaluated by monitoring alkaline phosphatase (ALP) activity and SMAD 1/5/8 phosphorylation in mesenchymal precursor cells. The osteoinductive ability of these hydrogels was determined in a rat ectopic bone model by 2D radiography, 3D μ-CT and histological analyses at 8 weeks post-implantation. Both hydrogels sustain the release of BMP-2. Importantly, the inclusion of a small amount of heparin (0.3% w/w) attenuated release of BMP-2 and sustained its osteogenic activity for up to 28 days. In contrast, hydrogels lacking heparin released more BMP-2 initially but were unable to maintain BMP-2 activity at later time points. Ectopic bone-forming assays using transplanted hydrogels emphasized the therapeutic importance of the initial burst of BMP-2 rather than its long-term osteogenic activity. Thus, tuning the burst release phase of BMP-2 from hydrogels may be advantageous for optimal bone formation.     

Controlled dual delivery of BMP-2 and dexamethasone by nanoparticle-embedded electrospun nanofibers for the efficient repair of critical-sized rat calvarial defect

There is an urgent need to develop biomimetic bone tissue engineering scaffolds for the repair of critical-sized calvarial defect. In this study, we developed a new nanoparticle-embedded electrospun nanofiber scaffold for the controlled dual delivery of BMP-2 and dexamethasone (DEX). The scaffold was achieved by (1) the encapsulation of BMP-2 into bovine serum albumin (BSA) nanoparticles to maintain the bioactivity of BMP-2 and (2) the co-electrospinning of the blending solution composed of the BSA nanoparticles, DEX and the poly(ε-caprolactone)-co-poly(ethylene glycol) (PCE) copolymer. The in vitro studies showed that the bioactivity of DEX and BMP-2 was preserved in the dual-drug-loaded nanofiber scaffold, and a sequential release pattern in which most of the DEX was released in the original eight days and the BMP-2 release lasted up to 35 days was achieved. The in vitro osteogenesis study demonstrated that the drug-loaded groups exhibited a strong ability to induce differentiation toward osteoblasts. In vivo osteogenesis studies also revealed that the degrees of repair of rat calvarial defect achieved with the drug-loaded nanofiber scaffolds were significantly better than those obtained with the blank materials; in particular, the dual-drug-loaded nanofiber scaffold manifested the best repair efficacy due to a synergistic effect of BMP-2 and DEX. Therefore, the dual-drug-loaded nanofiber scaffold is deemed a strong potential candidate for the repair of bone defects in bone tissue engineering.     

Local suppression of pro-inflammatory cytokines and the effects in BMP-2-induced bone regeneration

The objective of this study is to investigate the effect of local inflammation suppression on the bone regeneration. Gelatin hydrogels incorporating mixed immunosuppressive triptolide-micelles and bone morphogenic protein-2 (BMP-2) were prepared. The controlled release of both the triptolide and BMP-2 from the hydrogels was observed under in vitro and in vivo conditions. When either J774.1 macrophage-like or MC3T3-E1 osteoblastic cells were cultured in the hydrogels incorporating mixed 2.5, 5 or 10 mg of triptolide-micelles and BMP-2, the expression level of pro- and anti-inflammatory cytokines including interleukin (IL)-6 and IL-10 was down-regulated, but the alkaline phosphatase (ALP) activity was promoted compared with those of hydrogels incorporating BMP-2 without triptolide-micelles. When implanted into a critical-sized bone defect of rats, the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 showed significantly lower number of neutrophils, lymphocytes, macrophages or dendritic and mast cells infiltrated into the defect, and lower expression level of IL-6, TNF-α, and IL-10 than those incorporating BMP-2 without triptolide-micelles. The reduced local inflammation responses at the defects implanted with the hydrogels incorporating mixed 2.5 or 5 mg of triptolide-micelles and BMP-2 subsequently enhanced the bone regeneration thereat. It is concluded that the proper local modulation of inflammation responses is a promising way to achieve the enhanced bone regeneration.     

Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing

Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications.     

Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle

Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.     

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

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

The effect of crosslinking heparin to demineralized bone matrix on mechanical strength and specific binding to human bone morphogenetic protein-2

Demineralized bone matrix (DBM) is a collagen-based scaffold, but its low mechanical strength and limited BMP-2 binding ability restrict its application in bone repair. It is known that heparin could be immobilized onto scaffolds to enhance their binding of growth factors with the heparin-binding domain. Here, we crosslinked heparin to DBM to increase its BMP-2 binding ability. To our surprise, the mechanical strength of DBM was also dramatically increased. The compression modulus of heparin crosslinked DBM (HC-DBM) have improved (seven-fold increased) under wet condition, which would allow the scaffolds to keep specific shapes in vivo. As expected, HC-DBM showed specific binding ability to BMP-2. Additional studies showed the bound BMP-2 exerted its function to induce cell differentiation on the scaffold. Subcutaneous implantation of HC-DBM carrying BMP-2 showed higher alkaline phosphatase (ALP) activity (2 weeks), more calcium deposition (4 and 8 weeks) and more bone formation than that of control groups. It is concluded that HC-DBM has increased mechanical intensity as well as specific BMP-2 binding ability; HC-DBM/BMP-2 enhances the osteogenesis and therefore could be an effective medical device for bone repair.     

A comparison of osteocyte bioactivity in fine particulate bone powder grafts vs larger bone grafts in a rat bone repair model

The osteogenic potential for bone grafts is based on numbers and activities of cells that survive transplantation. In this study, we compared the bioactivity of osteocytes in 300–500 μm fine particulate bone powder grafts to 2 mm larger bone grafts in a rat radial defect model. Expression levels of bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-β1), alkaline phosphatase (ALP), and collagen I were semi-quantified by both immunohistochemistry and RT-PCR at days 1 and 4, as well as weeks 1, 2, 4, 6 and 10 post-transplantation. Within two weeks post-transplantation, more cells stained positively for BMP-2, TGF-β1, ALP, and collagen I within the bone grafts and in the surrounding tissues in the group transplanted with the fine particulate bone powder grafts than in those with larger bone grafts (P < 0.05). The mRNA levels of all four markers in the group transplanted with fine particulate bone powder graft peaked earlier and were expressed more highly than in the larger bone graft group, suggesting that fine particulate bone powder grafts provide more viable and active osteocytes to accelerate bone defect healing than larger bone grafts.     

BMP-2/PLGA Delayed-Release Microspheres Composite Graft,Selection of Bone Particulate Diameters,and Prevention of Aseptic Inflammation for Bone Tissue Engineering

Autogenous bone grafts are widely used in the repair of bone defects. Growth factors such as bone morphogenetic protein 2 (BMP-2) can induce bone regeneration and enhance bone growth. The combination of an autogenous bone graft and BMP-2 may provide a better osteogenic effect than either treatment alone, but BMP-2 is easily inactivated in body fluid. The objective of this study was to develop a technique that can better preserve the in vivo activity of BMP-2 incorporated in bone grafts. In this study, we first prepared BMP-2/poly(lactic-co-glycolic acid) (PLGA) delayed-release microspheres, and then combined collagen, the delayed-release microspheres, and rat autologous bone particulates to form four groups of composite grafts with different combinations: collagen in group A; collagen combined with bone particulates in group B; collagen combined with BMP-2/PLGA delayed-release microspheres in group C; and collagen combined with both bone particulates and BMP-2/PLGA delayed-release microspheres in group D. The four groups of composite grafts were implanted into the gluteus maximus pockets in rats. The ectopic osteogenesis and ALP level in group D (experimental group) were compared with those in groups A, B, and C (control groups) to study whether it had higher osteogenic capability. Results showed that the composite graft design increased the utility of BMP-2 and reduced the required dose of BMP-2 and volume of autologous bone. The selection of bone particulate diameter had an impact on the osteogenetic potential of bone grafts. Collagen prevented the occurrence of aseptic inflammation and improved the osteoinductivity of BMP-2. These results showed that this composite graft design is effective and feasible for use in bone repair.