Inkjet-based bio-patterning to spatially control osteo- and angiogenesis.
Michael R. Bykowski, M.S.1, James Cray, PhD1, Emily Lensie, BA1, Christopher Kinsella, MD1, Dai Fei Elmer Ker, BA2, Lee Weiss, PhD2, Phil Campbell, PhD2, Joseph Losee, MD1, Gregory Cooper, PhD1.
1University of Pittsburgh, Pittsburgh, PA, USA, 2Carnegie Mellon University, Pittsburgh, PA, USA.
Background: Three-dimensional control of tissue regeneration can enable substantial improvements in reconstruction of complex structures - e.g., repair of a comminuted mandible. Engineering specified and persistent spatial patterns of exogenous growth factors within biologically-relevant substrates is useful to control differentiation of host cells towards a desired tissue type with a precise shape. We hypothesize that low-dose bio-printing of bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) will enable spatial control of osteo- and angiogenesis within a mouse craniofacial defect.
Methods: A custom-built bio-printing system based on inkjet technology was used to precisely deposit and physically immobilize growth factors onto implantable, physiologically relevant substrates (e.g., acellular dermal matrix). To evaluate our ability to spatially control osteoblastic cell differentiation using bio-printed BMP-2 in vitro, mouse myoblast cells (C2C12 cell line; 30,000 cells) were seeded and cultured atop 1 mm circular BMP2-bio-printed DermaMatrix, which was adhered to the bottom of the well. One half of the DermaMatrix was printed with BMP-2, while the other half was untreated. Five days after seeding, ALP activity - an early marker for osteoblast differentiation - was analyzed.
To test the in vivo effect of bio-printed BMP-2 and VEGF, adult male mice were anesthetized, a midline cranial incision made, periosteum removed, and the skull exposed. After a 5 mm circular defect was created, a DermaMatrix scaffold was implanted in each defect. One half of the scaffold was untreated while the other was bio-printed with BMP-2 + VEGF (0, 5, 10, or 20 overprints; n=4-7/group).
Results: ALP activity was elevated in spatial register to bio-printed BMP-2 after 5 days in cell culture. Osteoblastic cell differentiation was localized to the bio-printed portion of the scaffold. Four weeks post-implantation, radiographic data show that implants treated with BMP-2 only had the greatest bone area localized to the treated side (Figure 1). The area of remaining defect of the treated side of the scaffold was subtracted from the untreated side, resulting in indirect measurements for efficacy of and spatial control of bone regeneration. Spatial control of bone regeneration was inversely proportional to dose of VEGF bio-printed (Table 1 and Figure 1). MicroCT and histologic data are being collected and analyzed.
Conclusion: Inkjet-based bio-printing enables spatial control of osteoblastic cell differentiation superior to liquid-phase application of BMP-2 in vitro. Bio-printing of BMP-2 represents a viable option to enhance spatial control of osteogenesis in vivo. However, with addition of VEGF, unexpectedly, BMP2-induced bone regeneration decreased. The angiogenic effects of BMP-2 may support sufficient blood supply to new bone regenerate, whereas VEGF inhibited regeneration. Intelligent delivery systems are integral for spatially controlled bone formation and economic application of BMP-2. Taken broadly, this technology holds promise for therapeutic approaches to tissue regeneration through persistent, localized delivery of molecules.
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