Delivery System For Bone Morphogenetic Protein-2 Research Articles

A silk hydrogel-based delivery system of bone morphogenetic protein for the treatment of large bone defects

The use of tissue grafting for the repair of large bone defects has numerous limitations including donor site morbidity and the risk of disease transmission. These limitations have prompted research efforts to investigate the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. The goal of this study was to use a critically-sized rat femoral segmental defect model to investigate the efficacy of a delivery system consisting of an electrospun polycaprolactone (PCL) nanofiber mesh tube with a silk fibroin hydrogel for local recombinant bone morphogenetic protein 2 (BMP-2) delivery. Bilateral 8 mm segmental femoral defects were formed in 13-week-old Sprague Dawley rats. Perforated electrospun PCL nanofiber mesh tubes were fitted into the adjacent native bone such that the lumen of the tubes contained the defect (Kolambkar et al., 2011b). Silk hydrogels with or without BMP-2 were injected into the defect. Bone regeneration was longitudinally assessed using 2D X-ray radiography and 3D microcomputed topography (μCT). Following sacrifice at 12 weeks after surgery, the extracted femurs were either subjected to biomechanical testing or assigned for histology. The results demonstrated that silk was an effective carrier for BMP-2. Compared to the delivery system without BMP-2, the delivery system that contained BMP-2 resulted in more bone formation (p<0.05) at 4, 8, 12 weeks after surgery. Biomechanical properties were also significantly improved in the presence of BMP-2 (p<0.05) and were comparable to age-matched intact femurs. Histological evaluation of the defect region indicated that the silk hydrogel has been completely degraded by the end of the study. Based on these results, we conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects.

Material-related effects of BMP-2 delivery systems on bone regeneration

Material-related effects of a brushite and a PLGA controlled release system loaded with two distinct doses of bone morphogenetic protein-2 (BMP-2) (3.5 and 17.5 μg), pre-encapsulated in poly(lactic-co-glycolic acid) (PLGA), were investigated in an intramedullary femur defect model in rabbits. The systems were characterized in vitro and in vivo over 12 weeks in terms of morphology, release kinetics, porosity, molecular weight, and composition using scanning electron microscopy, mercury porosimetry, radioactivity counting, X-ray diffractometry, differential scanning calorimetry, and gel permeation chromatography. During the experimental period the investigated systems underwent significant changes in vitro as well as in vivo. It should be stressed that the two in vitro release patterns were similar, however in vivo parallel profiles were observed with a higher burst effect for BMP-2 in the PLGA system. The PLGA system degraded and disintegrated significantly faster than the brushite system, which suffered slowly progressing external erosion and, additionally, material resorption by osteoclasts in vivo. The consequences of this were reflected in the degree of bone regeneration. Although a sustained delivery of BMP-2 was achieved with both systems, the brushite construct, independent of the loaded growth factor dose, failed to consistently induce defect repair, a result attributed to its slow resorption rate. In contrast, the PLGA system resulted in complete regeneration with mature trabecular bone formation 8 weeks after implantation.

Perlecan domain I-conjugated, hyaluronic acid-based hydrogel particles for enhanced chondrogenic differentiation via BMP-2 release

We have developed a biomimetic growth factor delivery system that effectively stimulates the chondrogenic differentiation of the cultured mesenchymal stem cells via the controlled presentation of bone morphogenetic protein-2 (BMP-2). Hyaluronic acid (HA)-based, microscopic hydrogel particles (HGPs) with inherent nanopores and defined functional groups were synthesized by an inverse emulsion polymerization technique. Recombinantly produced, heparan sulfate (HS)-bearing perlecan domain I (PlnDI) was covalently immobilized to HA HGPs (HGP–P 1) via a flexible poly(ethylene glycol) (PEG) linker through the lysine amines in the core protein of PlnDI employing reductive amination. Compared to HGP without PlnDI, HGP–P 1 exhibited significantly ( p < 0.05) higher BMP-2 binding capacity and distinctly different BMP-2 release kinetics. Heparitinase treatment increased the amount of BMP-2 released from HGP–P 1, confirming the HS-dependent BMP-2 binding. While BMP-2 was released from HGPs with a distinct burst release followed by a minimal cumulative release, its release from HGP–P 1 exhibited a minimal burst release followed by linear release kinetics over 15 days. The bioactivity of the hydrogel particles was evaluated using micromass culture of multipotent mesenchymal stem cells (MSCs), and the chondrogenic differentiation was assessed by the production of glycosaminoglycan, aggrecan and collagen type II. Our results revealed that BMP-2 loaded HGP–P 1 stimulates more robust cartilage specific ECM production as compared to BMP-2 loaded HGP, due to the ability of HGP–P 1 to potentiate BMP-2 and modulate its release with a near zero-order release kinetics. The PlnDI-conjugated, HA HGPs provide an improved BMP-2 delivery system for stimulating chondrogenic differentiation in vitro, with potential therapeutic application for cartilage repair and regeneration.

In Vivo Bone Formation Following Transplantation of Human Adipose–Derived Stromal Cells That Are Not Differentiated Osteogenically

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

Electrospun silk-BMP-2 scaffolds for bone tissue engineering

Silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and/or nanoparticles of hydroxyapatite (nHAP) prepared via electrospinning were used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs). BMP-2 survived the aqueous-based electrospinnig process in bioactive form. hMSCs were cultured for up to 31 days under static conditions in osteogenic media on the scaffolds (silk/PEO/BMP-2, silk/PEO/nHAP, silk/PEO/nHAP/BMP-2) and controls (silk/PEO, silk/PEO extracted). Electrospun silk fibroin-based scaffolds supported hMSC growth and differentiation toward osteogenic outcomes. The scaffolds with the co-processed BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers than in the controls, indicating that these nanofibrous electrospun silk scaffolds were an efficient delivery system for BMP-2. X-ray diffraction (XRD) analysis revealed that the apatite formed on the silk fibroin/BMP-2 scaffolds had higher crystallinity than on the silk fibroin scaffold controls. In addition, nHAP particles were incorporated into the electrospun fibrous scaffolds during processing and improved bone formation. The coexistence of BMP-2 and nHAP in the electrospun silk fibroin fibers resulted in the highest calcium deposition and upregulation of BMP-2 transcript levels when compared with the other systems. The results suggest that electrospun silk-fibroin-based scaffolds are potential candidates for bone tissue engineering. Furthermore, the mild aqueous process required to spin the fibers offers an important option for delivery of labile cytokines and other components into the system.