Tissue engineering in bone regeneration using autologous adipose-derived stem cells in small animals

The loss of bone due to trauma, infection, tumors and congenital deficiency is still one of the most important issues in orthopedic surgery. An innovative approach to solve this problem includes tissue engineering techniques in which the use of cells and bio-degradable or bio-absorbable scaffolds with adequate mechanical properties and interconnecting pores is required to allow cellular infiltration, graft integration and vascularisation. Since few years, we have been studying mesenchymal stem cells isolated from adipose tissue (ASCs), and we have shown their multi-differentiative potential towards osteogenic, chondrogenic and adipogenic lineages either in humans and animals. We believe that ASCs, with their great availability and strong osteogenic potential, may represent, alone or in combination with osteoconductive scaffolds, an efficient approach for bone regeneration; however some aspects need to be faced before moving to the clinic: 1. May the ability of ASCs to osteo-differentiate be affected by the donor’s physiological and pathological conditions? 2. May the known reduced immunogenicity of ASCs allow their future allogenic use? 3. Are preclinical models suitable in defining a successful cellular therapeutic approach? The influence of age and the body mass was studied comparing hASCs from subcutaneous adipose tissue of middle age females and young ones with or without pathological obesity. The clonogenic ability, the immunophenotype and the osteogenic potential may be affected by both physiological and pathological conditions suggesting that not all the hASC’s sources may be ideal for future clinical applications. For this reason, we have also observed that hASCs do not elicit any immune-response when cultured with activated peripheral blood leucocytes suggesting that they may be used in allogenic transplantation. Next, since we wanted to evaluate the ability of ASCs to regenerate osteochondral defects in pre-clinical models, we have characterized ASCs isolated from rabbit (rbASCs) and pig (pASCs) and we have shown their ability to maintain an undifferentiated state and to efficiently differentiate towards osteogenic lineage, alone or with biomaterials. For this reason, as a first preclinical model we have chosen to use autologous ASCs in a critical bone defect in rabbit. rbASCs were isolated from interscapular adipose tissue, expanded in vitro and implanted in a full-thickness bone defect in the tibial crest of rabbits. The 4 experimental animal groups were ASCs alone, disk of hydroxyapatite (HA), ASCs seeded on HA-disk, , and sham. Eight weeks after surgery, gross appearance, X-ray, and histological analyses were performed. The macroscopic analyses of the tibias show a satisfactory filling of the lesion without any significant differences in term of stiffness between groups treated with or without cells. In both scaffold-treated groups, a good osteo-integration was observed by X-ray. Even if HA was not completely reabsorbed, ASCs-loaded HA showed a more efficient scaffold reabsorption than the empty disks. Histology shows a marked osteogenic ability of the scaffold-treated defects compared to the scaffold cell-free samples, and the new formed bone appears more mature and similar to native bone in presence of scaffold filled with ASCs. Our data suggest that bioconstruct made by autologous ASCs loaded on HA disk may be a potential treatment for large bone defects.