Heparin-conjugated poly(lactic-co-glycolic acid) nanospheres enhance large-wound healing by delivering growth factors in platelet-rich plasma.

Platelet-rich plasma (PRP) contains many growth factors that are involved in tissue regeneration processes. For successful tissue regeneration, protein growth factors require a delivery vehicle for long-term and sustained release to a defect site in order to maintain their bioactivity. Previously, we showed that heparin-conjugated poly(lactic-co-glycolic acid) nanospheres (HCPNs) can provide long-term delivery of growth factors with affinity for heparin. In this study, we hypothesize that treatment of a skin wound with a mixture of PRP and HCPNs would provide long-term delivery of several growth factors contained in PRP to promote the skin wound healing process with preservation of bioactivity. The release of platelet-derived growth factor-BB (PDGF-BB), contained in PRP, from HCPN with fibrin gel (FG) showed a prolonged release period versus a PRP mixture with FG alone (FG-PRP). Also, growth factors released from PRP with HCPN and FG showed sustained human dermal fibroblast growth for 12 days. Full-thickness skin wound treatment in mice with FG-HCPN-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 9 compared with treatment with FG-HCPN or FG-PRP. The enhanced wound healing using FG-HCPN-PRP may be due to the prolonged release not only of PDGF-BB but also of other growth factors in the PRP. The delivered growth factors accelerated angiogenesis at the wound site.

Fibrinogen-based cell and spheroid sheets manipulating and delivery for mouse hindlimb ischemia.

In this research, we introduced a novel strategy for fabricating cell sheets (CSs) prepared by simply adding a fibrinogen solution to growth medium without using any synthetic polymers or chemical agents. We confirmed that the fibrinogen-based CS could be modified for target tissue regardless of size, shape, and cell types. Also, fibrinogen-based CSs were versatile and could be used to form three-dimensional (3D) CSs such as multi-layered CSs and those mimicking native blood vessels. We also prepared fibrinogen-based spheroid sheets for the treatment of ischemic disease. The fibrinogen-based spheroid sheets had much higherin vitrotubule formation and released more angiogenic factors compared to other types of platform in this research. We transplanted fibrinogen-based spheroid sheets into a mouse hindlimb ischemia model and found that fibrinogen-based spheroid sheets showed significantly improved physiological function and blood perfusion rates compared to the other types of platform in this research.

The efficacy of bone morphogenetic protein-2 depends on its mode of delivery.

Bone morphogenetic protein-2 (BMP-2) induces bone regeneration in a dose-dependent manner, with higher doses of BMP-2 inducing greater bone formation. Previously, we showed that long-term delivery of BMP-2 provides better ectopic bone formation than short-term delivery of an equivalent dose. In the present study, we investigated the efficacy of orthotopic bone formation over a range of BMP-2 doses, using different delivery modes. Heparin-conjugated poly(lactic-co-glycolic acid) nanospheres suspended in fibrin gel were used as a long-term delivery system, and fibrin gel was used as a short-term delivery system. Different doses of BMP-2 were delivered to mouse calvarial defects using either long-term or short-term delivery systems. Eight weeks after treatment, bone regeneration was evaluated by histomorphometry. For both delivery systems, bone regeneration increased as the BMP-2 dose increased up to 1 µg and did not increase beyond this dose. Importantly, at BMP-2 doses higher than 1 µg, long-term delivery resulted in much greater bone formation than short-term delivery. This study shows that long-term delivery of BMP-2 is more effective at enhancing orthotopic bone formation than short-term delivery over a range of doses.

Oxidative Epigallocatechin Gallate Coating on Polymeric Substrates for Bone Tissue Regeneration.

Plant derived flavonoids have not been well explored in tissue engineering applications due to difficulties in efficient formulations with biomaterials for controlled presentation. Here, the authors report that surface coating of epigallocatechin gallate (EGCG) on polymeric substrates including poly (L-lactic acid) (PLLA) nanofibers can be performed via oxidative polymerization of EGCG in the presence of cations, enabling regulation of biological functions of multiple cell types implicated in bone regeneration. EGCG coating on the PLLA nanofiber promotes osteogenic differentiation of adipose-derived stem cells (ADSCs) and is potent to suppress adipogenesis of ADSCs while significantly reduces osteoclastic maturation of murine macrophages. Moreover, EGCG coating serves as a protective layer for ADSCs against oxidative stress caused by hydrogen peroxide. Finally, the in vivo implantation of EGCG-coated nanofibers into a mouse calvarial defect model significantly promotes the bone regeneration (61.52 ± 28.10%) as compared to defect (17.48 ± 11.07%). Collectively, the results suggest that EGCG coating is a simple bioinspired surface modification of polymeric biomaterials and importantly can thus serve as a promising interface for tuning activities of multiple cell types associated with bone fracture healing.

Topographically Defined, Biodegradable Nanopatterned Patches to Regulate Cell Fate and Acceleration of Bone Regeneration.

If only allowed to proceed naturally, the bone-healing process can take several weeks, months, or even years depending on the injury size. In terms of bone-healing speed, many studies have been conducted investigating the deliverance of various growth factors of implantable biomaterials to shorten the time for bone regeneration. However, there may be side effects such as nerve pain, infection, or ectopic bone formation. As an alternative method, we focused on biophysical guidance, which provided similar topographical cues to the cellular environment to recruit host cells for bone defect healing. In this study, we hypothesized that aligned nanotopographical features have enhanced osteoblast recruitment, migration, and differentiation without external stimuli. We designed and fabricated a biodegradable poly(lactic- co-glycolic acid) nanopatterned patch using simple solvent casting and capillary force lithography. We confirmed that a biodegradable nanopatterned patch (BNP) accelerated the migration of osteoblasts according to the orientation of the patterned direction. These highly aligned osteoblasts may contribute to in vitro osteogenic differentiation, such as alkaline phosphate activity, mineralization, and calcium deposition, compared to the biodegradable flat patch (BFP). To demonstrate bone defect healing by BNP guidance in vivo, we implanted either whole or bridge BNP on the critical size defect of mouse calvarial ( ø 4 mm) or tibia bone (3 × 7 mm2). Only the BNP-treated group showed faster new bone formation and compact bone regeneration at the calvarial or tibia bone defect area compared to BFP at 4 or 8 weeks. Bridge BNP guided, in particular, the regeneration of new bone formation along the parallel direction of nanopatterned substrates. Here, we show that a BNP with biophysical guidance should be suitable for use in bone tissue regeneration through accelerated migration of the intact host cell.

Biodegradable Nerve Guidance Conduit with Microporous and Micropatterned Poly(lactic-co-glycolic acid)-Accelerated Sciatic Nerve Regeneration.

An innovative technique combining capillary force lithography and phase separation method in one step is applied to fabricate artificial nerve guidance conduit (NGC) for peripheral nerve regeneration. Biodegradable porous, patterned NGC (PP-NGC) using poly(lactic-co-glycolic acid) is fabricated. It has micro-grooves and microporosity on the inner surface to promote axonal outgrowth and to enhance permeability for nutrient exchange. In this study, it is confirmed that the inner surface of micro-grooves can modulate neurite orientation and length of mouse neural stem cell compared to porous flat NGC (PF-NGC) in vitro. Coating with 3,4-dihydroxy-l-phenylalanine (DOPA) facilitates the hydrophilic inner surface of PF- and PP-NGCs via bioinspired catechol chemistry. For in vivo study, PF-NGC and PP-NGC coated with or without DOPA are implanted in the 10 mm sciatic nerve defect margins between proximal and distal nerves in rats. Especially, PP-NGC coated with DOPA shows higher sciatic function index score, onset-to-peak amplitude, and muscle fiber diameter compared to other groups. The proposed hybrid-structured NGC not only can serve as a design for functional NGC without growth factor but also can be used in clinical application for peripheral nerve regeneration.

Effects of culture conditions on osteogenic differentiation in human mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (hBMMSCs) must differentiate into osteogenic cells to allow for successful bone regeneration. In this study, we investigated the effects of different combinations of three soluble osteogenic differentiation-inducing factors [L-ascorbic acid (AC), beta-glycerophosphate (betaG), and bone morphogenic protein-2 (BMP-2)] and the presence of a hydroxyapatite (HA) substrate on hBMMSC osteogenic differentiation in vitro. hBMMSCs were cultured in medium containing various combinations of the soluble factors on culture plates with or without HA coating. After 7 days of culture, alkaline phosphatase (ALP) activity, calcium deposition, and osteoprotegerin (OPG) and osteopontin (OPN) expression were measured. The effects of individual and combined factors were evaluated using a factorial analysis method. BMP-2 predominantly affected expression of early markers of osteogenic differentiation (ALP and OPG). HA had the highest positive effect on OPN expression and calcium deposition. The interaction between AC, betaG, and HA had the second highest positive effect on ALP activity.

Synergetic effect of 3,4-dihydroxy-l-phenylalanine-modified poly(lactic-co-glycolic acid) nanopatterned patch with fibroblast growth factor-2 for skin wound regeneration.

Nanoscale topography substrates have potential in guiding cell polarization and migration. Wound healing can be accelerated by nanotopographical substrates which provided appropriate direction to host cells around wound site. We fabricated biodegradable poly (lactic-co-glycolic acid) (PLGA) nanopatterned patch (nP-patch) using capillary force lithography method. Simple surface modification was applied using 3,4-dihydroxy-l-phenylalanine (LD), which has been reported as effective adhesion molecules with similar structure as mussel protein, to immobilize fibroblast growth factor-2 (FGF2) on PLGA patches. In present study, we hypothesized that nP-patch could be enhanced the cell migration in vitro by a guide of nanotopography and that nP-patch with soluble growth factor could accelerate skin wound healing in vivo. To investigate its nanotopographical effect on cell behavior, human dermal fibroblast (HDF) cells were cultured on nP-patch and flat PLGA patch (F-patch). The rate of surface coverage was measured on nP-patch with vertical or parallel orientation. The surface coverage rate was significantly enhanced by nP-patch with vertical orientation compared to the parallel orientation or the F-patch. We made a full thickness (Ø 18 mm) wound on the back of athymic mice and implanted PLGA patches with or without FGF2. FGF2-loaded nP-patch showed much faster wound closure in 21 days compared to others. Histological analysis showed that regenerated tissue had a similar structure as native skin. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 594-604, 2017.

In Vitro and In Vivo Dentinogenic Efficacy of Human Dental Pulp-Derived Cells Induced by Demineralized Dentin Matrix and HA-TCP.

Human dental pulp cells have been known to have the stem cell features such as self-renewal and multipotency. These cells are differentiated into hard tissue by addition of proper cytokines and biomaterials. Hydroxyapatite-tricalcium phosphates (HA-TCPs) are essential components of hard tissue and generally used as a biocompatible material in tissue engineering of bone. Demineralized dentin matrix (DDM) has been reported to increase efficiency of bone induction. We compared the efficiencies of osteogenic differentiation and in vivo bone formation of HA-TCP and DDM on human dental pulp stem cells (hDPSCs). DDM contains inorganic components as with HA-TCP, and organic components such as collagen type-1. Due to these components, osteoinduction potential of DDM on hDPSCs was remarkably higher than that of HA-TCP. However, the efficiencies of in vivo bone formation are similar in HA-TCP and DDM. Although osteogenic gene expression and bone formation in immunocompromised nude mice were similar levels in both cases, dentinogenic gene expression level was slightly higher in DDM transplantation than in HA-TCP. All these results suggested that in vivo osteogenic potentials in hDPSCs are induced with both HA-TCP and DDM by osteoconduction and osteoinduction, respectively. In addition, transplantation of hDPSCs/DDM might be more effective for differentiation into dentin.

Dual delivery of growth factors with coacervate-coated poly(lactic-co-glycolic acid) nanofiber improves neovascularization in a mouse skin flap model.

Random skin flaps are commonly used in plastic and reconstructive surgery for patients suffering from severe or large scale wounds or in facial reconstruction. However, skin flaps are sometimes susceptible to partial or complete necrosis at the distal parts of the flaps due to insufficient blood perfusion in the defected area. In order to improve neovascularization in skin flaps, we developed an exogenous growth factor (GF) delivery platform comprised of coacervate-coated poly(lactic-co-glycolic acid) (PLGA) nanofibers. We used a coacervate that is a self-assembled complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., vascular endothelial growth factor (VEGF) and/or transforming growth factor beta 3 (TGF-ß3)). The coacervate was coated onto a nanofibrous PLGA membrane for co-administration of dual GFs. In vitro proliferation of human dermal fibroblasts and endothelial tube formation using human umbilical vein endothelial cells indicated an enhanced bioactivity of released GFs when both VEGF and TGF-ß3 were incorporated into coacervate-coated PLGA nanofibers (Coa-Dual NFs). Moreover, an in vivo study using a mouse skin flap model demonstrated that implantation of Coa-Dual NF reduced necrosis and enhanced blood perfusion in skin flap areas after 10 days, as compared to any single GF-loaded coacervate/PLGA fiber (Coa-Single NF) along with direct administration of the other GF onto the defect site. Moreover, Coa-Dual NFs exhibited a well-composed skin appendage and a significantly higher number of blood vessels. Based upon these results, we conclude that Coa-Dual NFs may stimulate cellular activity by enhancing the bioactivity of the released GF, leading to a synergetic effect of dual GFs for reducing necrosis in the random skin flaps. Therefore, Coa-Dual NFs could be a valuable drug delivery platform for a variety of potential clinical applications for skin tissue regeneration applications.

Controlled Retention of BMP-2-Derived Peptide on Nanofibers Based on Mussel-Inspired Adhesion for Bone Formation.

Although bone morphogenetic protein-2 (BMP-2) has been frequently used to stimulate bone formation, it has several side effects to be addressed, including the difficulty in optimization of clinically relevant doses and unwanted induction of cancerous signaling processes. In this study, an osteogenic peptide (OP) derived from BMP-2 was investigated as a substitute for BMP-2. In vitro studies showed that OP was able to enhance the osteogenic differentiation and mineralization of human mesenchymal stem cells (hMSCs). The peptides were then conjugated onto biocompatible poly-ι-lactide electrospun nanofibers through polydopamine chemistry. Surface chemical analysis proved that more than 80% of the peptides were stably retained on the nanofiber surface after 8 h of polydopamine coating during at least 28 days, and the amount of peptides that was retained increased depending on the polydopamine coating time. For instance, about 65% of the peptides were retained on nanofibers after 4 h of polydopamine coating. Also, a relatively small dose of peptides could effectively induce bone formation in in vivo critical-sized defects on the calvarial bones of mice. More than 50.4% ± 16.9% of newly formed bone was filled within the defect after treatment with only 10.5 ± 0.6 µg of peptides. Moreover, these groups had similar elastic moduli and contact hardnesses with host bone. Taken together, our results suggest that polydopamine-mediated OP immobilized on nanofibers can modulate the retention of relatively short lengths of peptides, which might make this an effective therapeutic remedy to guide bone regeneration using a relatively small amount of peptides.

Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues To Regulate Skeletal Muscle Maturation and Vascularization.

Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here, we have developed biomimetic nanopatterned poly(lactic-co-glycolic acid) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles.

In Situ Bone Tissue Engineering With an Endogenous Stem Cell Mobilizer and Osteoinductive Nanofibrous Polymeric Scaffolds.

Classical bone tissue engineering involves the use of culture-expanded cells and scaffolds to produce tissue constructs for transplantation. Despite promising results, clinical adoption of these constructs has been limited due to various drawbacks, including extensive cell expansion steps, low cell survival rate upon transplantation, and the possibility of immuno-rejection. To bypass the ex vivo cell culture and transplantation process, the regenerative capacity of the host is exploited by mobilizing endogenous stem cells to the site of injury. Systemic injection of substance P (SP) induce mobilization of CD29+ CD105+ CD45- cells from bone marrow and enhance bone tissue regeneration in a critical-sized calvarial bone defect model. To provide an appropriate environment for endogenous stem cells to survive and differentiate into osteogenic lineage cells, electrospun nanofibrous polycaprolactone (PCL) scaffolds are functionalized with hydroxyapatite (HA) particles via a polydopamine (PDA) coating to create highly osteoinductive PCL-PDA-HA scaffolds that are implanted in defects. The combination of the PCL-PDA-HA scaffold and SP treatment enhance in situ bone tissue formation in defects. Thus, this in situ bone regeneration strategy, which combines recruitment of endogenous stem cells from the bone marrow to defective sites and implantation of a highly biocompatible and osteoinductive cell-free scaffold system, has potential as an effective therapeutic in regenerative medicine.

Electroconductive Nanopatterned Substrates for Enhanced Myogenic Differentiation and Maturation.

Electrically conductive materials provide a suitable platform for the in vitro study of excitable cells, such as skeletal muscle cells, due to their inherent conductivity and electroactivity. Here it is demonstrated that bioinspired electroconductive nanopatterned substrates enhance myogenic differentiation and maturation. The topographical cues from the highly aligned collagen bundles that form the extracellular matrix of skeletal muscle tissue are mimicked using nanopatterns created with capillary force lithography. Electron beam deposition is then utilized to conformally coat nanopatterned substrates with a thin layer of either gold or titanium to create electroconductive substrates with well-defined, large-area nanotopographical features. C2C12 cells, a myoblast cell line, are cultured for 7 d on substrates and the effects of topography and electrical conductivity on cellular morphology and myogenic differentiation are assessed. It is found that biomimetic nanotopography enhances the formation of aligned myotubes and the addition of an electroconductive coating promotes myogenic differentiation and maturation, as indicated by the upregulation of myogenic regulatory factors Myf5, MyoD, and myogenin (MyoG). These results suggest the suitability of electroconductive nanopatterned substrates as a biomimetic platform for the in vitro engineering of skeletal muscle tissue.

Biodegradable Nanotopography Combined with Neurotrophic Signals Enhances Contact Guidance and Neuronal Differentiation of Human Neural Stem Cells.

Biophysical cues provided by nanotopographical surfaces have been used as stimuli to guide neurite extension and regulate neural stem cell (NSC) differentiation. Here, we fabricated biodegradable polymer substrates with nanoscale topography for enhancing human NSC (hNSC) differentiation and guided neurite outgrowth. The substrate was constructed from biodegradable poly(lactic-co-glycolic acid) (PLGA) using solvent-assisted capillary force lithography. We found that precoating with 3,4-dihydroxy-l-phenylalanine (DOPA) facilitated the immobilization of poly-l-lysine and fibronectin on PLGA substrates via bio-inspired catechol chemistry. The DOPA-coated nanopatterned substrates directed cellular alignment along the patterned grooves by contact guidance, leading to enhanced focal adhesion, skeletal protein reorganization, and neuronal differentiation of hNSCs as indicated by highly extended neurites from cell bodies and increased expression of neuronal markers (Tuj1 and MAP2). The addition of nerve growth factor further enhanced neuronal differentiation of hNSCs, indicating a synergistic effect of biophysical and biochemical cues on NSC differentiation. These bio-inspired PLGA nanopatterned substrates could potentially be used as implantable biomaterials for improving the efficacy of hNSCs in treating neurodegenerative diseases.

Thermoresponsive nanofabricated substratum for the engineering of three-dimensional tissues with layer-by-layer architectural control.

Current tissue engineering methods lack the ability to properly recreate scaffold-free, cell-dense tissues with physiological structures. Recent studies have shown that the use of nanoscale cues allows for precise control over large-area 2D tissue structures without restricting cell growth or cell density. In this study, we developed a simple and versatile platform combining a thermoresponsive nanofabricated substratum (TNFS) incorporating nanotopographical cues and the gel casting method for the fabrication of scaffold-free 3D tissues. Our TNFS allows for the structural control of aligned cell monolayers which can be spontaneously detached via a change in culture temperature. Utilizing our gel casting method, viable, aligned cell sheets can be transferred without loss of anisotropy or stacked with control over individual layer orientations. Transferred cell sheets and individual cell layers within multilayered tissues robustly retain structural anisotropy, allowing for the fabrication of scaffold-free, 3D tissues with hierarchical control of overall tissue structure.

Nanopatterned muscle cell patches for enhanced myogenesis and dystrophin expression in a mouse model of muscular dystrophy.

Skeletal muscle is a highly organized tissue in which the extracellular matrix (ECM) is composed of highly-aligned cables of collagen with nanoscale feature sizes, and provides structural and functional support to muscle fibers. As such, the transplantation of disorganized tissues or the direct injection of cells into muscles for regenerative therapy often results in suboptimal functional improvement due to a failure to integrate with native tissue properly. Here, we present a simple method in which biodegradable, biomimetic substrates with precisely controlled nanotopography were fabricated using solvent-assisted capillary force lithography (CFL) and were able to induce the proper development and differentiation of primary mononucleated cells to form mature muscle patches. Cells cultured on these nanopatterned substrates were highly-aligned and elongated, and formed more mature myotubes as evidenced by up-regulated expression of the myogenic regulatory factors Myf5, MyoD and myogenin (MyoG). When transplanted into mdx mice models for Duchenne muscular dystrophy (DMD), the proposed muscle patches led to the formation of a significantly greater number of dystrophin-positive muscle fibers, indicating that dystrophin replacement and myogenesis is achievable in vivo with this approach. These results demonstrate the feasibility of utilizing biomimetic substrates not only as platforms for studying the influences of the ECM on skeletal muscle function and maturation, but also to create transplantable muscle cell patches for the treatment of chronic and acute muscle diseases or injuries.

3,4-dihydroxyphenylalanine-assisted hydroxyapatite nanoparticle coating on polymer scaffolds for efficient osteoconduction.

For bone regeneration applications, scaffolds made from a composite of a biodegradable polymer and ceramic have advantages over scaffolds made from only one component (biodegradable polymer or ceramic alone). In this study, a simple and rapid method was developed to induce hydroxyapatite (HA) nanoparticle adsorption on polyglycolic acid (PGA) scaffold surfaces. PGA meshes were coated with HA nanoparticles by immersing the scaffolds in a buffer solution containing 3,4-dihydroxyphenylalanine (DOPA), a critical, functional element in mussel adhesive protein known to strongly bind to various materials. Substantial HA coating on PGA scaffolds was achieved within 24 hours of immersion, as determined according to selective staining of ceramic particles, scanning electron microscopy, X-ray photoelectron spectroscopy, and energy-dispersive spectroscopy. To evaluate the osteoconduction efficacy of the scaffolds in vivo, PGA scaffolds, DOPA-coated PGA scaffolds, PGA scaffolds immersed in HA solution, and HA- and DOPA-coated PGA (HA-DOPA-PGA) scaffolds were implanted in critical-sized defects in mouse skulls for 8 weeks. Micro-computed tomography and histological analyses showed that bone regeneration in vivo was far more extensive on HA-DOPA-PGA scaffolds than on the other scaffolds. DOPA offers an efficient and simple method of HA coating on polymer scaffolds. HA-polymer composite scaffolds fabricated using this method could be useful as bone graft.

Efficient bone regeneration induced by bone morphogenetic protein-2 released from apatite-coated collagen scaffolds.

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. Clinically utilized BMP-2 uses a type-I collagen scaffold as a carrier. Here we hypothesized that an apatite coating on a type-I collagen scaffold would prolong the BMP-2 release period and enhance bone regeneration in calvarial defects in mice. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluid. BMP-2 release kinetics and bioactivity were evaluated by enzyme-linked immunosorbent assay and alkaline phosphatase activity measurement of cultured osteoblasts. Computed tomography and histomorphometry were performed eight weeks after various doses of BMP-2 were delivered to mouse calvarial defects using either non-modified or apatite-coated collagen scaffolds. Apatite-coated collagen scaffolds released 91.8 ± 11.5% of the loaded BMP-2 over 13 days in vitro, whereas non-modified collagen scaffolds released 98.3 ± 2.2% over the initial one day. The in vivo study showed that BMP-2 delivery with apatite-coated collagen scaffolds resulted in a significantly greater bone formation area and higher bone density than that with non-modified collagen scaffolds. This study suggests that simple apatite coating on collagen scaffolds can enhance the bone regeneration efficacy of BMP-2 released from collagen scaffolds.

Heparin-conjugated fibrin as an injectable system for sustained delivery of bone morphogenetic protein-2.

Long-term release of bone morphogenetic protein-2 (BMP-2) can promote bone regeneration. We developed an injectable system for long-term delivery of BMP-2 by covalently conjugating heparin to fibrinogen. The heparin-conjugated fibrinogen formed an injectable, heparin-conjugated fibrin (HCF) gel when mixed with thrombin. HCF released 89.4 +/- 3.8% of the loaded BMP-2 for 13 days, whereas normal fibrin released 83.7 +/- 7.6% for the initial 3 days. BMP-2 released from HCF significantly increased alkaline phosphatase activity of cultured osteoblasts, whereas BMP-2 released from normal fibrin did not do so, indicating that BMP-2 released from HCF is bioactive and suggesting that long-term delivery of BMP-2 is advantageous over short-term delivery for bone regeneration. HCF, BMP-2-loaded HCF, and BMP-2-loaded normal fibrin containing free heparin were contained in polyester cylindrical tubes and implanted into the hind limb muscle pockets of rats for 8 weeks. Soft X-ray radiography, computed tomography, histomorphometry, calcium assay, and western blot analysis showed that BMP-2-loaded HCF yielded the most extensive bone formation among the groups. Since HCF can deliver BMP-2 over a long term, is an injectable system, and is made of clinically benign materials, this system would have advantages for clinical applications to regenerate bone.