In vivo model of critical bone defects regeneration by autologous adipose-derived stem cells (ASCs) loaded onto hydroxyapatite scaffold

Bone defect can be provoked by several pathological conditions, like bone tumors, infections, major trauma with bone stock loss. Surgical techniques currently used for treating bone defects may count on different alternatives, including autologous or homologous vascularized bone grafts or prostheses, each one of them characterized by both specific complications and drawbacks, including donor site morbidity and limited available amount of transplantable tissue, possible immune response and viral transmission. Nowadays, an ideal alternative is the use of osteoconductive synthetic bone substitutes either alone or in combination with autologous cells able to enhance the regeneration process and thus to provide better results. Adipose-derived stem cells (ASCs), with their great availability and osteogenic potential may represent, in association with specific scaffolds, a novel and efficient approach for bone regeneration. Here we present the results of an experimental study using ASC alone or in association with custom-made hydroxyapatite scaffold (HA) to promote the repair of bone critical-size defect in a rabbit model. METHODS Twelve adult New Zealand White rabbits were divided into two experimental groups: - control group (n=6)  lesion without treatment (right tibia) / lesion + HA (left tibia) - experimental group (n=6)  lesion + rbASCs (right tibia) / lesion + rbASCs + HA (left tibia) rbASCs were isolated from interscapolar adipose tissue of each rabbit belonging to the experimental group; cells were then purified by plastic adherence and expanded in vitro. Two weeks after isolation, autologous rbASCs were reimplanted alone or in association with HA scaffold in the lesion site to evaluate their osteo-regenerative potential. The bone regeneration process was monitored during time performing X-ray of the lesions sites. 8 weeks after surgical interventions, gross appearance, X-ray, BMD and histological analyses were performed on each samples. At the same time we in vitro characterized rbASCs: cellular yield (number of cells per ml of raw adipose tissue), proliferation rate, cellular viability, clonogenic ability and osteogenic potential were assayed for each population of cells. RESULTS Regarding the in vitro study, the number of rbASCs per ml of raw adipose tissue was 2.8x105±1.9x105 cells. One week after isolation, rbASCs started to proliferate (doubling time: 65±20 hours), showing the typical fibroblast-like morphology of mesenchymal stem cells. All the tested cell populations were able to express specific osteogenic markers, confirming their ability to participate in the bone regeneration process. No fractures, infections or other complications have been observed in rabbits during the experiment. At final follow up, the gross appearance analyses and radiographs showed a good lesion filling in all the samples, but with relevant differences between lesions treated with rbASCs+HA or rbASCs alone compared to not treated lesions or just treated with HA. This differences is strongly confirmed by histological analyses, which showed the presence of a regenerated tissue similar to native mature bone just in lesions treated with rbASCs. CONCLUSIONS ASCs seem to be a valid alternative to treat severe bone defects. Indeed they could be easily harvested, expanded in vitro and then reimplanted in the lesion The in vitro data show their higher osteogenic potential and thus their ability in actively participating to the bone regeneration process.