Bone marrow is one current source of adult stem cells for therapeutic purposes; however, the magnitude and accessibility of subcutaneous adipose tissue in humans make it an attractive alternative. Numerous in vitro studies have been conducted to determine how these cells act in vitro, but it is imperative to determine the vast abilities of these cells in vivo. The objective of this study was to evaluate in vivo migration and bone healing ability after transplanting adipose-derived stem cells (ADSC) in a swine model. Adipose-derived stem cells were isolated from subcutaneous adipose tissue of adult Yorkshire pigs and cultured in vitro. At 80 to 90% confluence/passage 3, the cells were trypsinized and labeled in suspension with carboxyfluorescein succinimidyl ester (CFDA-SE). This project included 20 pigs weighing between 63.5 and 81.7 kg. Bilateral mandibular osteoectomies with 10-mm defects were performed on each pig. Of the 20 pigs, half received a treatment of 2.5 million CFDA-SE labeled stem cells administered directly into each defect (DI), and the remaining half received a treatment of approximately 5 million CFDA-SE labeled stem cells through an ear vein injection via catheter (EVI). The time points were 1 h and 2 and 4 wk, with 2 pigs per time with the DI and EVI treatments. Pigs were slaughtered at each time, and spleen, liver, lung, kidney, ear vein, blood, and mandible tissues were collected. Blood samples were collected from the jugular vein with EDTA and processed via flow cytometry after collection. Tissues were fixed in 10% buffered formalin for histology. Fluorescent microscopy (CFDA-SE excitation/emission is 492/517 nm) has confirmed that transplanted ADSC do indeed migrate to a site of injury or trauma. Labeled cells were also present in blood collected from the 1-h time point group. Currently, we have not seen the presence of labeled ADSC in the other tissues (spleen, liver, lung, and kidney) after the 1-h time point. We did observe that ADSC administered by DI and EVI were able to significantly heal and regenerate bone defects within 4 wk post-surgery (P < 0.05, ANOVA with F-test), in contrast to bone defects in pigs that did not receive cell injections (control). Evidence of ADSC-related healing and bone regeneration was evident by gross visualization, dual-energy x-ray absorptiometry (DXA) and micro computer tomography (microCT) analysis. The clinical implications of these results are significant for treating many diseases in which inflammation or defects exist, such as cardiac disease, neurological disease, or traumatic injuries to both soft and hard tissue. If the adult stem cells can be harvested from fat, encouraged to produce bone or cartilage, and then reinserted into defects, treatment protocols for trauma victims could be developed that would reduce the need for alternate harvesting techniques for bone.
This work was support by a grant from the Illinois Regenerative Medicine Institute (IDPH # 63080017).
Porcine adipose-derived stem cells from buccal fat pad and subcutaneous adipose tissue for future preclinical studies in oral surgery
