Wan Geun La

Delivery of a Therapeutic Protein for Bone Regeneration from a Substrate Coated with Graphene Oxide

The therapeutic efficacy of drugs often depends on the drug delivery carrier. For efficient delivery of therapeutic proteins, delivery carriers should enable the loading of large doses, sustained release, and retention of the bioactivity of the therapeutic proteins. Here, it is demonstrated that graphene oxide (GO) is an efficient carrier for delivery of therapeutic proteins. Titanium (Ti) substrates are coated with GO through layer-by-layer assembly of positively (GO-NH₃⁺) and negatively (GO-COO⁻) charged GO sheets. Subsequently, a therapeutic protein (bone morphogenetic protein-2, BMP-2) is loaded on the GO-coated Ti substrate with the outermost coating layer of GO-COO⁻ (Ti/GO⁻). The GO coating on Ti substrate enables loading of large doses and the sustained release of BMP-2 with preservation of the structure and bioactivity of the drug. The extent of in vitro osteogenic differentiation of human bone marrow-derived mesenchymal stem cells is higher when they are cultured on Ti/GO- carrying BMP-2 than when they are cultured on Ti with BMP-2. Eight weeks after implantation in mouse models of calvarial defects, the Ti/GO-/BMP-2 implants show more robust new bone formation compared with Ti, Ti/GO-, or Ti/BMP-2 implants. Therefore, GO is an effective carrier for the controlled delivery of therapeutic proteins, such as BMP-2, which promotes osteointegration of orthopedic or dental Ti implants.

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.

Dual roles of hyaluronic acids in multilayer films capturing nanocarriers for drug-eluting coatings

We developed hyaluronic acid (HA)-based multilayer films capturing polymeric nanocarriers (NCs) for drug delivery. The electrostatic interactions between positively charged linear polyethylene imines (LPEI) and negatively charged HAs are the main driving forces to form multilayers based on the layer-by-layer (LbL) deposition. NCs were easily incorporated within the multilayer film due to intra- and/or inter-hydrogen bonding among HA chains. The amount of NCs captured by the HA chains was varied by the ratio between HAs and NCs as well as the length (i.e., molecular weight) and absolute number density of HAs in solution. Biocompatibility of the NC-capturing HA multilayer films was tested with the human dermal fibroblast (HDF) culture. In addition, the controlled release of paclitaxel (PTX) from the HA multilayer films successfully led to the apoptosis of human aortic smooth muscle cells (hSMC) in vitro, implying that the NC-capturing HA multilayer films would be quite useful as drug-eluting stent systems to prevent the restenosis after surgery.

Bone morphogenetic protein-2 enhances bone regeneration mediated by transplantation of osteogenically undifferentiated bone marrow-derived mesenchymal stem cells.

We have hypothesized that human bone marrow-derived mesenchymal stem cells (BMMSCs), that are not osteogenically differentiated prior to implantation, would regenerate bone extensively inxa0vivo once exogenous bone morphogenetic protein-2 (BMP-2) was delivered to the implantation site. BMP-2 released from heparin-conjugated poly(lactic-co-glycolic acid) (HCPLGA) scaffolds stimulates osteogenic differentiation of cultured BMMSCs. Upon implantation, undifferentiated BMMSCs on BMP-2-loaded HCPLGA scaffolds induce far more extensive bone formation than either undifferentiated BMMSCs or osteogenically differentiated BMMSCs on HCPLGA scaffolds. These BMP-2-loaded HCPLGA scaffolds could prove invaluable for inxa0vivo regeneration of bone from undifferentiated human BMMSCs.

Controlled Delivery of Low-Dose Bone Morphogenetic Protein-2 Using Heparin-Conjugated Fibrin in the Posterolateral Lumbar Fusion of Rabbits

The heparin-conjugated fibrin (HCF) system has been developed to deliver bone morphogenetic proteins (BMPs) for a long-term period and thus enhance bone regeneration. In the present study, we tested the effectiveness of the delivery system for spinal fusion with a very low dose of BMP-2. A total of 15 rabbits underwent posterolateral lumbar spine, divided into three groups. The control group received only collagen sponges without BMP-2, another group (BMP-only group) received collagen sponges loaded with BMP-2 (10 μg each side), and the last group (BMP/HCF group) received collagen sponges filled with HCF loaded with BMP-2 (10 μg each side). All animals were euthanized 8 weeks after surgery, and the fusion was assessed by radiographs, manual palpation, computed tomography scan, and mechanical testing. No case in the BMP/HCF group or in the control group achieved solid fusion, while all cases in BMP-only group showed evidence of solid fusion. BMP/HCF group had significantly lower fusion rate and tensile strength than BMP-only group at the dose of 10 μg of BMP-2. The HCF long-term delivery system with the low dose of BMP-2 (10 μg) is ineffective for the induction of lumbar posterolateral fusion in the rabbits.

Efficient Bone Regeneration Induced by Bone Morphogenetic Protein-2 Released from Apatite-Coated Collagen Scaffolds

Abstract 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.