The combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) with poly(d,l lactide-co-glycolide) (PLGA) porous microspheres provided for "sustained release" of the protein from the microspheres. Soaking 50:50 PLGA microspheres in a buffered rhBMP-2 solution for a sufficient period of time to permit equilibrium binding enabled quantification of "free" and "bound" protein. "Free" protein is defined as protein present within the porous matrix of the microspheres, whereas "bound" refers to protein adsorbed to PLGA surfaces. Kinetics of the rhBMP-2-microsphere association revealed that equilibrium was attained within 8 hr for two buffer systems (arginine/histidine, pH 6.50; and glutamic acid/sodium glutamate, pH 4.50). Increasing the concentration of the rhBMP-2 stock solution used for the interaction studies from 0.025 to 1.0 mg/ml increased the amount of rhBMP-2 adsorbed and the concentration of free rhBMP-2. Beyond a 1.0 mg/mL concentration, only free rhBMP-2 levels increased. Linearized Langmuir treatment of the adsorption data yielded values corresponding to monolayer coverage of the microspheres (Cm) and the equilibrium adsorption constant (K) of 0.17 microgram/cm2 and 7.57 ml/mg, respectively. Studies performed to determine the effect of ionic strength revealed that increasing NaCl and buffer concentration decreased the amount of protein adsorbed. rhBMP-2 release studies, conducted in an isotonic phosphate buffered saline, pH 7.4 vehicle, revealed that free rhBMP-2 was released during an initial period of 72-96 hr. Following this period, there was no discernible release of rhBMP-2 from the microspheres for up to 7 days, suggesting that the bound protein would remain at a defect site and release slowly upon erosion of the polymer. Mass balances performed by using an extraction buffer of high ionic strength confirmed this prediction.
Novel unitary devices, prepared by lyophilization of viscous solutions of sodium carboxymethylcellulose (CMC) and methylcellulose (MC), were evaluated as sustained-release delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). In vitro characterization of the unitary devices, which contained rhBMP-2-loaded poly (d,l lactide-co-glycolide) (PLGA) bioerodible particles (BEPs), was conducted over a 2-month period. Determinations included buffer uptake, mass and molecular weight loss and rhBMP-2 release from the unitary devices. CMC devices imbibed approximately 16 times their weight of buffer, while with MC, equilibrium uptake was approximately 6 times the dry weight of the devices. Overall mass loss percentages were approximately 55 and 35%, respectively, for CMC and MC devices. rhBMP-2 release from the devices was essentially a triphasic process: an initial phase during which "free" protein (rhBMP-2 present on the surface and within the pores of the PLGA BEPs) was released, a lag period during which no release was discerned, and then release of "bound" rhBMP-2 (protein adsorbed to the BEPs). The release of bound protein correlated with the mass loss of the polymer which began after 3 weeks. Release from the unitary devices was lower than that from the BEPs alone, due to a retardation effect of the gelled CMC/MC polymers. In rabbits in which full-thickness cranial bone defects were created, the implants were well tolerated and induced significant new bone growth during an 8-week evaluation period. The CMC devices appear to have induced bone earlier (at 2 weeks), but this did not affect eventual 8-week results. CMC devices without rhBMP-2 appeared to provide some bone conduction, in contrast to the blank MC devices.
Porous 50:50 poly(d,l lactide-co-glycolide) microspheres containing varying amounts of "free" recombinant human bone morphogenetic protein-2 (rhBMP-2) were evaluated for their ability to induce/enhance expression of osteoblastic characteristics by pluripotent mesenchymal cells in vitro. "Free" protein (Fp) is defined as protein present on the surface and within the porous matrix of the microspheres. Four preparations of bioerodible particles (BEP) were used: blank--without rhBMP-2; low Fp--24 microg of free rhBMP-2 per g of particles; medium Fp--403 microg/g; and high Fp--884 microg/g. C3H10T1/2 cells (C3H) and bone marrow stromal cells (BMC) were cultured with 1 mg of BEP for up to 4 weeks, and cell growth and expression of osteogenic responses were determined weekly. For both cell types, control cultures (neither BEP nor rhBMP-2) and cultures with blank BEP exhibited no or minimal osteoblastic characteristics. Compared to control and blank BEP cultures, C3H cells responded to particles having medium and high amounts of free rhBMP-2 with increased cell growth and alkaline phosphatase activity, but osteocalcin secretion and mineralization were not markedly influenced. Low Fp BEP enhanced only the alkaline phosphatase activity of C3H cells. In contrast, although growth was not affected, rhBMP-2-loaded BEP increased alkaline phosphatase activity, osteocalcin secretion, and mineralization in BMC cultures in a dose-dependent manner (i.e., blank < low < medium < high Fp).
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