Background: Controlled release of transforming growth factor-&betabeta;1 (TGF-&betabeta;1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-&betabeta;1 delivery and the effects of released TGF-&betabeta;1 on the proliferation and differentiation of marrow stromal cells in vitro.
Methods: Recombinant human TGF-&betabeta;1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (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 kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-&betabeta;1 was determined after 3 days in culture. The effects of TGF-&betabeta;1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period.
Results: TGF-&betabeta;1 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 resulted 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 content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-&betabeta;1 released from the microparticles similar to that of added TGF-&betabeta;1, indicating that the activity of TGF-&betabeta;1 was retained during microparticle fabrication and after growth factor release. At an optimal TGF-&betabeta;1 dosage of 1.0 ng/ml after 3 days, the released TGF-&betabeta;1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells over 21 days of culture, with increased total cell number, alkaline phosphatase activity, and osteocalcin production.
Conclusions: PLGA/PEG blend microparticles can serve as delivery vehicles for controlled release of TGF-&betabeta;1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitro.
Clinical Relevance: Controlled release of TGF-&betabeta;1 from PLGA/PEG microparticles is representative of emerging tissue engineering technologies that may modulate cellular responses to encourage bone regeneration at a skeletal defect site.
Lichun Lu, PhD; Michael J. Yaszemski, MD, PhD; Departments of Orthopedic Surgery and Bioengineering, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, U.S.A.
Antonios G. Mikos, PhD; Department of Bioengineering, Rice University, 6100 Main Street, MS-142, Houston, TX 77005-1892, U.S.A. E-mail address: email@example.com