Osteochondral regeneration of a critical size defect in a minipig model using adipose-derived stem cells in association with an hydrogel of oligo(polyethylene glycol) fumarate

Summary. Autologous and heterologous adipose derived stem cells associated to an oligo(polyethylene glycol) fumarate hydrogel scaffold are able to promote a complete regeneration of the subchondral bone and a cartilage-like tissue in a critical osteochondral defect in a minipig model Introduction. Osteochondral tissue loss caused by high-energy trauma, disease, and tumor resection have always been a very troublesome problem for orthopedic surgeons. During normal bone defect healing, undifferentiated mesenchymal stem cells, in the presence of suitable stimuli, proliferate and differentiate into osteoblasts and chondrocytes forming bone tissue and cartilage, and repairing the injury. However, in some cases, defects fail to healing and surgical intervention is often required. Tissue engineering is an innovative approach that overcome the standard surgical procedures, to achieve the regeneration of bone and cartilage tissues. Adipose-derived Stem Cells (ASCs), isolated from the stromal vascular fraction derived from subcutaneous fat tissue, show a multi-differentiative ability which may be exploited, alone or in association with suitable scaffolds, as novel and efficient tools for bone and cartilage regeneration. Methods. ASCs, isolated from seven adult male minipigs, expanded in culture, were used to repair a critical femoral osteochondral defect in association with an hydrogel scaffold of oligo(polyethylene glycol) fumarate (OPF). In the peripheral part of the trochlea of each animals four defects (diameter 9mm, depth 8mm) were created, and then treated with constructs made of OPF scaffold pre-seeded with either autologous or heterologous ASCs. Untreated defects and defects filled by just scaffold were included as controls. Results. ASCs were rapidly isolated with an average of 6.45x104±4.34x104 per ml of processed adipose tissue. All the swine ASC populations were analyzed in vitro: their doubling times were of 59.2±16.6 hours and their clonogenic ability was stable and greater than human ASCs (15.7±8.2%). Their osteogenic differentiation ability was shown by collagen production and extracellular calcified matrix deposition (+136% and +70% of osteo- vs undifferentiated ASCs, respectively). 4 weeks after subcutaneous fat withdrawal, constructs made with 3x106 of either autologous or heterologous undifferentiated ASCs were implanted in the bone defects. No side-effects have been observed during the follow up and 6 months later, animals were sacrificed and knees explanted. Gross appearance analyses showed quite satisfactory filling of the lesions in all the samples, with the exception of one animal, whose joint appeared infected and not healed. Good osteointegration was observed by MRI evaluation. Histological and immunohistochemical analyses revealed an increased expression of collagen type II in the ASCs+OPF treated groups compared to the groups treated with the OPF hydrogel alone. In addition, biomechanical analyses confirmed the histological results showing an improved elastic module of the new formed cartilage tissue. Conclusion. ASCs were able to promote a quite complete regeneration of the subchondral bone and a cartilage-like tissue and might be considered a treatment option for the regeneration of osteochondral defects. Moreover, since no differences were observed between autologous and heterologous constructs, the possibility of an allogeneic use of these cells is also supported and encouraging for a future clinical practice.