A scaffold-free graft for large critical size bone defect: preclinical evidence to clinical proof of concept

Abstract

Background & Aim Large critical-sized bone defect (CBD) remains a challenging pathology in orthopaedics. The direct application of adipose stem cells (ASCs) remains limited by a low homing efficiency and a low survival rate. This study aims to show the osteogenic role of ASCs in a scaffold-free approach. Methods, Results & Conclusion 3D scaffold-free grafts were characterized by q-RT-PCR (for osteogenesis/angiogenesis). The bioactivity of the graft was studied in 2 nude rat models: (i) the comparison of fresh and decellularized grafts in term of angiogenesis promotion up to 1 month in a fibrotic tissue (cauterized muscular pocket,n=20); (ii) the osteogenicity of the graft (compared to HA/bTCP bone substitute) at 1/2/3 months post-implantation, in an irreversible femoral CBD (n=28). Angiogenesis was assessed by histomorphometry, cellular engraftment by HLA-I staining, mineralization by micro-CTscan and osteogenic genes expression by q-RT-PCR. A 5-year-old boy with congenital pseudarthrosis of the tibia (previously treated by nailing and grafting without success) was proposed for the autologous scaffold-free graft approach in combination with the induced membrane technique. The pseudarthrosis area (fibula and tibia) was firstly resected and filled by a cement spacer. Then, ASCs were isolated from adipose tissue (AT) to produce the graft. After 3 months, the cement was removed, and the graft was placed into the defect to be followed clinically and radiologically. After intra-muscular transplantation in nude rats, cellular survival (with major osteogenic genes expression) and the promotion of angiogenesis (in a fibrotic/hypoxic site) was found. A complete integration and bone fusion were found for the graft in comparison to the bone substitute which revealed a lack of tissue remodelling and osteogenesis. Specific osteogenic genes were overexpressed in the CBD treated with the grafts (at 4 weeks). A large volume (>15cm3) of the 3D-graft was manufactured in GMP conditions and then implanted without any modification of the surgical procedure. The graft was easily handled and implanted. The graft demonstrated a continuous remodelling (with bone formation) during the first 2 years post-implantation to obtain a bone fusion (allowing walk without pain) and no recurrence of the disease. In conclusion, the scaffold-free 3D-graft (made of ASCs) plays a major role to promote ASCs engraftment and consequence to induce osteogenesis in a fibrotic environment and to recover a bone fusion in a critical-sized bone defect.