Preclinical studies with autologous adipose-derived stem cells (ASCs) in regenerative medicine for orthopaedic applications

Treatment of severe bone defects remains one of the most important issues in orthopaedic surgery in human and veterinary fields. Surgical techniques should provide primary stability to reach osseous integration and secondary remodelling of bone grafts and substitute materials. None of the currently available substitute materials provides osteoconduction and osteogenesis comparable to human allografts and autografts. Nowadays, an innovative approach to solve this problem includes tissue engineering techniques in which the use of autologous cells and bio-degradable/ bio-absorbable scaffolds with adequate mechanical properties and interconnecting pores is required to allow cellular infiltration, graft integration and vascularisation. We know that adipose-derived stem cells (ASCs), may represent, alone or in combination with osteoconductive scaffolds, an efficient approach for bone regeneration. In this study, autologous ASCs, isolated from interscapular adipose tissue, were expanded in vitro and re-implanted in a full-thickness bone defect in the proximal epiphysis of rabbit’s tibia. Animals have been divided in 4 groups: lesions treated by ASCs seeded on HA-disk (ASCs-HA), ASCs alone, disk of hydroxyapatite (HA) and untreated (sham-just defect). Each ASCs population was tested in vitro: all of them show a homogenous high proliferation rate, a marked clonogenic ability, an osteogenic differentiation potential evaluated either by collagen type I production and extracellular calcified matrix deposition. Moreover, rabbit ASCs, cultured on HA granules for 14 days, produce an increased level of collagen type I respect to cells maintained on Petri dish. 8-weeks after surgery, bone features and scaffold readsorption were evaluate by macroscopic and microscopic analyses. The macroscopic analyses of the tibias show 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 osteointegration of HA was radiographically observed and similar amount of scaffold has been detectable in the lesion site by histomorphometry. Similar quantities of new formed bone were also present in HA and ASCs-HA groups, with a marked difference regarding its spatial distribution: indeed, in ASCs-HA treated tibia, new bone tissue was observed not only along the wall of the pores, as in HA group, but also in the inner part of the construct, as also confirmed by the presence of collagen type I and osteopontin expression. Furthermore, ASCs-treated defects have shown an improvement in bone mechanical properties suggesting that these constructs are able to bear mechanical loading with an increase of 19.8% in stiffness and 31.6% in hardness for ASCs-HA group respect to HA group. We conclude that autologous ASCs-HA construct may be a promising treatment for critical bone defects.