TGF-beta 1 release from biodegradable polymer microparticles: Its effects on marrow stromal osteoblast function

Background: Controlled release of transforming growth factor-beta1 (TGF-bet a1) to a bone defect may be beneficial for the induction of a bone regenera tion cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF- beta1 delivery and the effects of released TGF-beta1 on the proliferation a nd differentiation of marrow stromal cells in vitro. Methods: Recombinant human TGF-beta1 was incorporated into microparticles o f blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glyco l) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kineti cs and the degradation of PLGA were determined in vitro for as long as 28 d ays. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of re leased TGF-beta1 was determined after 3 days in culture. The effects of TGF -beta1 released from PLGA/PEG microparticles on marrow stromal cell prolife ration and osteoblastic differentiation were assessed during a 21-day perio d. Results: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content r esulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG cont ent and significantly accelerated at acidic pH conditions. Rat marrow strom al cells cultured on PPF substrates showed a dose response to TGF-beta1 rel eased from the microparticles similar to that of added TGF-beta1, indicatin g that the activity of TGF-beta1 was retained during microparticle fabricat ion and after growth factor release. At an optimal TGF-beta1 dosage of 1.0 ng/ml after 3 days, the released TGF-beta1 enhanced the proliferation and o steoblastic differentiation of marrow stromal cells over 21 days of culture , with increased total cell number, alkaline phosphatase activity, and oste ocalcin production. Conclusions: PLGA/PEG blend microparticles can serve as delivery vehicles f or controlled release of TGF-beta1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitr o. Clinical Relevance: Controlled release of TGF-beta1 from PLGA/PEG micropart icles is representative of emerging tissue engineering technologies that ma y modulate cellular responses to encourage bone regeneration at a skeletal defect site.