Belgian Symposium on Tissue Engineering (BSTE) 2012 location:Leuven date:17-18 September 2012
Introduction: In vitro engineering of tissue intermediates that are highly recognised by in vivo systems may guide tissue regeneration more effectively than using fully-matured tissue engineering constructs. Essentially, these intermediates should contain engineered modalities that comprise biomimetic hierarchical structures and signaling motifs capable to initiate the full cascade of tissue/organ development upon implantation. In this study, we examined if the deposition of a biologically-derived extracellular mineralised matrix onto 3D open porous scaffolds via a novel in vitro cell-mediated biomineralisation process (CMBio), would support osteoinduction.
Methods: We first screened for the osteogenicity of synthetic calcium phosphate (CaP) 2D substrates [prepared through chemical precipitation by mixing equal volume of various concentrations of Ca2+ and PO43- -enriched media (ranges from 0, 2, 4, 6, 8 and 10 mM) on cell culture plastics] and studied the in vitro proliferation and osteogenic differentiation of a MSC-like osteoprogenitors derived from human periosteum (i.e. hPDCs) on these substrates. The optimum [Ca2+] and [PO43-] were then identified and translated into the formulations of biomineralisation medium (BM) to facilitate CMBio of 3D porous Ti6Al4V scaffolds after seeding with 150 x 103 hPDCs per scaffold and culturing for 21 days. The cell growth in BM was monitored by metabolic activity measurements. After 21 days, samples were harvested and decellularised via three freeze-thaw cycles in liquid nitrogen and 37 oC water bath. Two million freshly expanded hPDCs were seeded onto the decellularised carriers and implanted subcutaneously in nude mice. Implants were retrieved after 8 weeks for histological analysis.
Results: In vitro study showed that CaP 2D substrates induced significantly higher cell growth (p<0.001) (figure 1b) and alkaline phosphatase activity (data not shown) than the controls after 21 days of culture. This allowed us to select 6 and 4 mM of Ca2+ and PO43- to formulate three types of BM1, 2 and 3 (figure 1a), and successfully induce a range of 3D cell and mineralised matrix depositions on Ti6Al4V porous scaffolds (figure 1C). Ectopic bone formation assay showed that all decellularised carriers (in combination with freshly seeded hPDCs) induced new bone formation along the surface of scaffolds (figure 1d). Interestingly, under condition BM2 produced carriers resulted in bone formation that covered the scaffold struts with presented bone marrow compartments. In contrast, non-mineralised scaffold (OM-) seeded with fresh cells failed to induce bone formation.
Discussion & Conclusion: In this study, we showed that the proposed CMBio process has potential as novel surface functionalisation strategy to enhance the osteoinductivity of 3D porous biomaterials, through the introduction of a biologically-derived matrix that contain biomimetic and functional modalities.