New strategies for tissue regeneration

My PhD project is divided in two parts, focusing on the development of new strategies for orthopedic tissue regeneration. In particular, the first part is about cartilage regeneration using human lipoaspirate as autologous injectable active scaffold for one-step repair of cartilage defects, the second part is about bone regeneration, through an injectable medicated graft substitute active on bone tissue regeneration. I. Cartilage regeneration Research on mesenchymal stem cells from adipose tissue (ASC) shows promising results for cell-based therapy in cartilage lesions: in these studies cells have been isolated, expanded, and differentiated in vitro, before transplantation into the damaged cartilage, or onto materials used as scaffolds to deliver cells to the impaired area. The present study employed in vitro assays to investigate the potential of intra-articular injection of micro-fragmented lipoaspirate, as a one-step repair strategy; it aimed to determine whether adipose tissue can act as a scaffold for cells naturally present at their anatomical site. Cultured clusters of lipoaspirate showed a spontaneous outgrowth of cells with mesenchymal phenotype and with multilineage differentiation potential. Transduction of lipoaspirate clusters by lentiviral vectors expressing GFP underlined the propensity of the outgrown cells to repopulate fragments of damaged cartilage. On the basis of the results, which showed an induction of proliferation and extracellular matrix (ECM) production of human primary chondrocytes, it was hypothesized that lipoaspirate may play a paracrine role. Moreover, the structure of a floating culture of lipoaspirate, treated for three weeks with chondrogenic growth factors, changed: tissue with a high fat component was replaced by a tissue with a lower fat component and connective tissue rich in glycosaminoglycan (GAG) and in collagen type I, increasing the mechanical strength of the tissue. From these promising in vitro results, it may be speculated that an injectable autologous biologically-active scaffold (lipoaspirate), employed intra-articularly, may: 1) become a fibrous tissue that provides mechanical support for the load on the damaged cartilage; 2) induce host chondrocytes to proliferate and produce ECM; 3) provide cells at the site of injury, which could regenerate or repair the damaged or missing cartilage. II. Bone regeneration With the aim to obtain an injectable medicated scaffold, which speeds bone formation in sinus lift augmentation, in bony void and in fracture repair, we have developed a three-dimensional (3D) jelly collagen containing Lysophosphatidic acid (LPA) and 1a,25-Dihydroxyvitamin D3 (1,25D3) using soluble native collagen prepared from rat tail tendons. We have demonstrated with an in vitro 3D culture model of bone fracture an osteoblasts’Rho-kinase mediated contraction of the collagen that causes an approach of human bone trabecular fragments with the formation of new union tissue within 3 weeks of organ culture. The contraction was faster in LPA medicated collagen while 1,25D3 enhanced the mineralization of the new formed tissue that showed also increased tensile strength. LPA was shown to modulate gel contraction rate not only mechanically, working in cytoskeleton reorganization, but also osteoconductively evidencing activity on proliferation, differentiation and migration of human primary osteoblasts (hOB). When LPA was used in combination with 1,25D3 a synergism on hOB’s activity in term of alkaline phosphatase and mineralization was seen. On the basis of these data, collagen can be considered as an injectable natural scaffold that allows the migration of cells from the side of bone defect and its enrichment with LPA and 1,25D3 could be used in vivo to accelerate bone growth and fracture healing.