Self Contained Bioreactor For Bone Regeneration
Pratima Labroo, MS1, Ching-wen Li, PhD2, Himanshu Sant, PhD1, Bruce Gale, PhD1, Jill E. Shea, PhD1, Jayant Agarwal, MD1.
1University of Utah, Salt lake city, UT, USA, 2National Ching Hsing University, Taipei city, Taiwan.
Purpose: The current gold standard for the repair of segmental bone defects is autologous bone grafts or flaps, however these are limited by donor site morbidity, limited graft tissue availability, and potential for complications at the harvest site. ASCs (adipose derived stem cells) have potential to aid bone regeneration due to their ability to differentiate into osteoblasts. BMP-2 (bone morphogenetic protein) has been shown to induce osteogenic differentiation of ASCs and enhance bone growth. We present a bioreactor for bone regeneration consisting of three main components: (i) a biodegradable PLLA scaffold seeded with ASCs, (ii) a drug reservoir made of PLLA and (iii) a controlled drug diffusion system for sustained release of BMP-2 to repair segmental bone defects. The drug reservoir is attached to the porous polymer scaffold seeded with ASCs and is capable of delivering growth factors for prolonged periods.
Methods: The components for the drug delivering setup were made from poly-lactic-acid (Purac Biomaterials) using solvent casting method. The device consists of two concentric tubes and a reservoir in between the tubes that stores BMP-2. Six 180 µm diffusion holes were drilled into the inner tube by pulsing a laser cutter. In vitro release tests were performed over a 30-day period. The concentration of BMP-2 released from devices was determined with an enzyme linked immunosorbent assay (R&D systems). The PLLA scaffolds were manufactured by a salt leaching method. ASC seeded scaffolds were inserted in the inner tube. We first evaluated the dosage(s) of BMP-2 that allows for the differentiation of ASCs into osteogenic cells using RT-PCR. ASC seeded devices were then loaded with BMP-2 and in vitro osteogenic differentiation of the ASCs was evaluated by direct delivery of BMP-2 from the drug reservoir to the seeded scaffold.
Results: The RT-PCR data showed increased expression of osteogenic markers such as ALK and Runx-2 in ASCs treated with 100ng/ml BMP-2 compared to no BMP-2 (p<0.05). Alizarin Red staining results showed that we were able to achieve osteogenic differentiation of ASCs with 100ng/ml BMP-2/day and the percentage of mineralization was greater in the PLLA scaffold compared to ‘no scaffold’ (p<0.05). The results for drug release kinetics showed that the drug delivery setup test was effective in achieving controlled local release of BMP-2 for 30 days. The results of the sealed (no hole) devices (n=2) validated sealing techniques for the drug-release reservoirs. The diffusion tests (n=6) indicated that 6x180 µm holes allowed for a sustainable and controlled BMP-2 release in the range of 50-100 ng/ml/day for 30 days.
Conclusion: The bone bioreactor was able to release BMP-2 for 30 days in a controlled manner. The ASC seeded PLLA scaffold exposed to 50-100ng/m/day of BMP-2 produced significantly higher mineralization as compared to no scaffold. Further in vivo testing will be done to demonstrate that directly delivering BMP-2 from our reservoir to the ASC seeded scaffold enhances bone regeneration.
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