No abstract

Integrated image-based design and solid free-form fabrication can create scaffolds that attain desired elasticity and permeability while fitting any 3D craniofacial defect. The scaffolds could be manufactured from degradable polymers, calcium phosphate ceramics and titanium. The designed scaffolds supported significant bone regeneration for all pore sizes ranging from 300 to 1200 microns. These results suggest that designed scaffolds are clinically applicable for complex craniofacial reconstruction.

IntroductionNotochordal cells (NCs) are influential in development of the intervertebral disc (IVD) and species that retain NCs do not degenerate. IVD repair using bone marrow derived mesenchymal stem cells (MSCs) is an attractive approach and the harsh microenvironment of the IVD suggests pre-differentiation is a necessary first step. The goal of this study was to use soluble factors from NCs in alginate and NCs in their native tissue to differentiate human MSCs to a young nucleus pulposus (NP) phenotype.MethodsHuman MSCs (cultured under micromass conditions for 21 days in hypoxia) were differentiated with conditioned medium derived from porcine notochordal cells in native tissue (NCT) or in alginate beads (NCA), and compared with chondrogenic (TGFβ-3) or basal medium. A PCR array of 42 genes was utilized to screen a large number of genes known to be associated with the healthy NP phenotype and pellet cultures were also evaluated for glycosaminoglycan content, histology and viability. Proteomic analysis was used to assess candidate soluble factors in NCA and NCT.ResultsNotochordal cell conditioned media had diverse effects on MSC phenotype. NCT resulted in the highest levels of glycosaminoglycan (GAG), as well as up-regulation of SOX9 and Collagen II gene expression. NCA demonstrated effects that were catabolic yet also anti-fibrotic and minimally hypertrophic with down-regulation of Collagens I and III and low levels of Collagen X, respectively. Micromass culture and hypoxic conditions were sufficient to promote chondrogenesis demonstrating that both basal and chondrogenic media produced similar phenotypes. Candidate matricellular proteins, clusterin and tenascin were identified by proteomics in the NCA group.ConclusionsNCs secreted important soluble factors capable of differentiating MSCs to a NP phenotype synthesizing high levels of proteoglycan while also resisting collagen fiber expression and hypertrophy, yet results were sensitive to the conditions in which media was generated (cells in alginate versus cells in their native tissue) so that further mechanistic studies optimizing culture conditions and defining important NC secreted factors are required. Matricellular proteins, such as clusterin and tenascin, are likely to be important to optimize differentiation of MSCs for maximum GAG production and reduced collagen fiber expression.

Treatment of damaged intervertebral discs is a signifi cant clinical problem and, despite advances in the repair and replacement of the nucleus pulposus, there are few effective strategies to restore defects in the annulus fi brosus. An annular repair material should meet three specifi cations: have a modulus similar to the native annulus tissue, support the growth of disc cells, and maintain adhesion to tissue under physiological strain levels. We hypothesized that a genipin crosslinked fi brin gel could meet these requirements. Our mechanical results showed that genipin crosslinked fi brin gels could be created with a modulus in the range of native annular tissue. We also demonstrated that this material is compatible with the in vitro growth of human disc cells, when genipin:fi brin ratios were 0.25:1 or less, although cell proliferation was slower and cell morphology more rounded than for fi brin alone. Finally, lap tests were performed to evaluate adhesion between fi brin gels and pieces of annular tissue. Specimens created without genipin had poor handling properties and readily delaminated, while genipin crosslinked fi brin gels remained adhered to the tissue pieces at strains exceeding physiological levels and failed at 15-30%. This study demonstrated that genipin crosslinked fi brin gels show promise as a gap-fi lling adhesive biomaterial with tunable material properties, yet the slow cell proliferation suggests this biomaterial may be best suited as a sealant for small annulus fi brosus defects or as an adhesive to augment large annulus repairs. Future studies will evaluate degradation rate, fatigue behaviors, and long-term biocompatibility.