Abstract
The studies describe a biomimetic mode of juxtacrine insoluble signaling stimulation for target delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) to prolong its retention for use in bone tissue engineering that would provide localized release in a manner that is triggered by cellular activity. A porous three-dimensional scaffold of poly-(lactide-co-glycolide) was fabricated by means of gel molding and particulate leaching. Collagen immobilization onto the scaffold surface was produced by performing photo-induced graft polymerization of acrylic acid, and rhBMP-2 was tethered to the collagenous surface by covalent conjugation. On pharmacokinetic analysis, in vitro enzyme-linked immunosorbent and alkaline phosphatase assays revealed sustained, slow release of rhBMP-2 over 28 days, with a cumulative release of one third of the initial load diffusing out of the scaffold. Conjugation of rhBMP-2 inhibited the free lateral diffusion and internalization of the activated complex of rhBMP-2 and the bone morphogenetic protein receptor. Osteoprogenitor cells were used as bone precursors to determine the expression of biosignaling growth factor in regulating cell proliferation and differentiation. To identify the phenotype of cells seeded on the rhBMP-2–conjugated scaffold, cellular activity was evaluated with scanning electron microscopy and with viability, histological, and immunohistochemical testing. The rhBMP-2–conjugated scaffold prolonged stimulation of intracellular signal proteins in cells. Enhancement of cell growth and differentiation was considered a consequence of juxtacrine signaling transduction. Animal studies of rhBMP-2–containing filling implants showed evidence of resorption and de novo bone formation. A novel localization process utilizes acrylate-N-hydroxysuccinimide poly (ethylene glycol) (PEG) as a spacer arm to tether rhBMP-2 on a scaffold surface. Bone marrow–derived mesenchymal stem cells were used as osteogenic precursors to evaluate cellular morphology and phenotypic expression of the scaffolds, which were seeded with the cells in a rotating bioreactor. Bilateral, full-thickness cranial defects in rabbits were selected to investigate the osteogenic effect of in vitro cultured mesenchymal stem cells for in vivo bone tissue engineering. Three-dimensional computed tomography and histology demonstrated that de novo bone formation was enhanced after surgery when PEG-tethered rhBMP-2 conjugate was introduced. Our work revealed the potential for biomimetic surface engineering by entrapping signaling growth factor–stimulated osteogenesis. It would create a new platform for bone-engineered stem cell therapies. Photopolymerized hydrogel based on poly (ethylene glycol) diacrylate (PEGDA) was applied to periosteum□derived osteoprogenitor cells encapsulation and orthopaedic tissue engineering. A need was recognized to incorporate rhBMP-2 in binding form into a PEG-tethering network to prevent its rapid, uncontrolled diffusion out of the matrix vehicle. Cell growth and differentiation are controlled by matrix-bound, insoluble signals. In vitro studies demonstrated that covalent conjugation of PEG-tethered rhBMP-2 can be utilized to mimic the extracellular matrix composition when generating engineered bone tissues. Fixation and incorporation of a tendon graft within the bone tunnel is a primary concern when employing tendon graft for ligament reconstruction. This study presents a novel technique for fabricating injectable PEGDA hydrogel photoencapsulated osteoprogenitor cells. A total of 28 adult New Zealand white rabbits were used. The long digitorum extensor tendon was transplanted into a bone tunnel of the proximal tibia. The tendon was pulled through a drill-hole in the proximal tibia and attached to the medial aspect of the tibia. Hydrogel suspension containing osteoprogenitor cells at a concentration of 20 million/ml was injected in the bone tunnel. Histological examination of the tendon-bone interface and biomechanical test for maximal pull-out load were evaluated at postoperative weeks 3 and 6. Histological analysis of tendon-bone interface showed an interface fibrous layer formed by photoencapsulation of periosteal cells between the tendon and the bone. This layer became progressive integrated with tendon and bone surface during the healing process. Biomechanical testing revealed higher maximal pull-out strength in the rhBMP-2 tethering group at all time points with a statistically significant difference at 3 and 6 weeks. The rhBMP-2 tethering group had a higher interface strength-to-length ratio and significant increase at 3 weeks and 6 weeks.