Gajadhar Bhakta

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.

The influence of collagen and hyaluronan matrices on the delivery and bioactivity of bone morphogenetic protein-2 and ectopic bone formation.

Bone morphogenetic protein-2 (BMP-2) is known to enhance fracture healing when delivered via a bovine collagen sponge. However, collagen rapidly releases BMP-2 with a high burst phase that is followed by a low sustained phase. As a result, supra-physiological doses of BMP-2 are often required to successfully treat bone defects. High BMP-2 dosing can introduce serious side effects that include edema, bone overgrowth, cyst-like bone formation and significant inflammation. As the release behavior of BMP-2 carriers significantly affects the efficacy of fracture healing, we sought to compare the influence of two BMP-2 delivery matrices with contrasting release profiles on BMP-2 bioactivity and ectopic bone formation. We compared a thiol-modified hyaluronan (Glycosil™) hydrogel that exhibits a low burst followed by a sustained release of BMP-2 to a collagen sponge for the delivery of three different doses of BMP-2, the bioactivities of released BMP-2 and ectopic bone formation. Analysis of bone formation by micro-computed tomography revealed that low burst followed by sustained release of BMP-2 from a hyaluronan hydrogel induced up to 456% more bone compared to a BMP-2 dose-matched collagen sponge that has a high burst and sustained release. This study demonstrates that BMP-2 released with a low burst followed by a sustained release of BMP-2 is more desirable for bone formation. This highlights the therapeutic potential of hydrogels, particularly hyaluronan-based, for the delivery of BMP-2 for the treatment of bone defects and may help abrogate the adverse clinical effects associated with high dose growth factor use.

Bone-marrow derived mesenchymal stem cells assembled with low dose BMP-2 in a three dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

BACKGROUND CONTEXT The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. PURPOSE We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. STUDY DESIGN/SETTING A prospective study using a rodent model of posterolateral spinal fusion was carried out. PATIENT SAMPLE Thirty-six syngeneic Fischer rats comprised the patient sample. METHODS Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. RESULTS Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). CONCLUSION In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.

Mode of heparin attachment to nanocrystalline hydroxyapatite affects its interaction with bone morphogenetic protein-2

Heparin has a high affinity for bone morphogenetic protein-2 (BMP-2), which is a key growth factor in bone regeneration. The aim of this study was to investigate how the rate of release of BMP-2 was affected when adsorbed to nanosized hydroxyapatite (HAP) particles functionalized with heparin by different methods. Heparin was attached to the surface of HAP, either via adsorption or covalent coupling, via a 3-aminopropyltriethoxysilane (APTES) layer. The chemical composition of the particles was evaluated using X-ray photoelectron spectroscopy and elemental microanalysis, revealing that the heparin grafting densities achieved were dependent on the curing temperature used in the fabrication of APTES-modified HAP. Comparable amounts of heparin were attached via both covalent coupling and adsorption to the APTES-modified particles, but characterization of the particle surfaces by zeta potential and Brunauer-Emmett-Teller measurements indicated that the conformation of the heparin on the surface was dependent on the method of attachment, which in turn affected the stability of heparin on the surface. The release of BMP-2 from the particles after 7 days in phosphate-buffered saline found that 31% of the loaded BMP-2 was released from the APTES-modified particles with heparin covalently attached, compared to 16% from the APTES-modified particles with the heparin adsorbed. Moreover, when heparin was adsorbed onto pure HAP, it was found that the BMP-2 released after 7 days was 5% (similar to that from unmodified HAP). This illustrates that by altering the mode of attachment of heparin to HAP the release profile and total release of BMP-2 can be manipulated. Importantly, the BMP-2 released from all the heparin particle types was found by the SMAD 1/5/8 phosphorylation assay to be biologically active.

Novel Protamine-Based Polyelectrolyte Carrier Enhances Low-Dose rhBMP-2 in Posterolateral Spinal Fusion.

Study Design. A rodent posterolateral spinal fusion model. Objective. This study evaluated a protamine-based polyelectrolyte complex (PEC) developed to use heparin in enhancing the biological activity of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) in spinal fusion. Summary of Background Data. rhBMP-2 is commonly regarded as the most potent bone-inducing molecule. However, poor pharmacokinetics and short in vivo half-life means that large amounts of the bioactive growth factor are required for consistent clinical outcomes. This has been associated with a number of adverse tissue reactions including seroma and heterotopic ossification. Glycosaminoglycans including heparin are known to stabilize rhBMP-2 bioactivity. Previous studies with poly-L-lysine (PLL) and heparin-based PEC carriers amplified the therapeutic efficacy of low-dose BMP-2. However, questions remained on the eventual clinical applicability of relatively cytotoxic PLL. In the present study, a protamine-based PEC carrier was designed to further enhance the safety and efficacy of BMP-2 by delivering lower dose within the therapeutic window. Methods. A polyelectrolyte shell was deposited on the surface of alginate microbead templates using the polycation (protamine)/polyanion (heparin) layer-by-layer polyelectrolyte self-assembly protocol. rhBMP-2 was loaded onto the outermost layer via heparin affinity binding. Loading and release of rhBMP-2 were evaluated in vitro. The bone-inductive ability of 20-fold reduction of rhBMP-2 with the different carrier vehicle was evaluated using a posterolateral spinal fusion model in rats. Results. In vitro uptake and release analysis, protamine-based PEC showed higher uptake and significantly enhanced control release than PLL-based PEC (P < 0.05). In vivo implantation with protamine-based and PLL-based PEC showed better fusion performances than absorbable collagen sponge–delivered same dose of rhBMP-2, and negative control group through manual palpation, micro–computed tomography, and histological analyses. Conclusion. Solid posterolateral spinal fusion was achieved with 20-fold reduction of rhBMP-2 when delivered using protamine-based PEC carrier in the rat posterolateral spinal fusion model. Level of Evidence: N/A

DNA-encapsulated magnesium phosphate nanoparticles elicit both humoral and cellular immune responses in mice

The efficacy of pEGFP (plasmid expressing enhanced green fluorescent protein)-encapsulated PEGylated (meaning polyethylene glycol coated) magnesium phosphate nanoparticles (referred to as MgPi-pEGFP nanoparticles) for the induction of immune responses was investigated in a mouse model. MgPi-pEGFP nanoparticles induced enhanced serum antibody and antigen-specific T-lymphocyte responses, as well as increased IFN-? and IL-12 levels compared to naked pEGFP when administered via intravenous, intraperitoneal or intramuscular routes. A significant macrophage response, both in size and activity, was also observed when mice were immunized with the nanoparticle formulation. The response was highly specific for the antigen, as the increase in interaction between macrophages and lymphocytes as well as lymphocyte proliferation took place only when they were re-stimulated with recombinant green fluorescence protein (rGFP). Thus the nanoparticle formulation elicited both humoral as well as cellular responses. Cytokine profiling revealed the induction of Th-1 type responses. The results suggest DNA-encapsulated magnesium phosphate (MgPi) nanoparticles may constitute a safer, more stable and cost-efficient DNA vaccine formulation.

Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications

BACKGROUND CONTEXT Interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site. PURPOSE We recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device. STUDY DESIGN/SETTING A combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds. MATERIALS AND METHODS Freshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120-150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology. RESULTS Progenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants. CONCLUSIONS The combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery.

Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications.