Closing The Gap On Clinical Translation Of Cell-Based Therapies: A Novel Technique For Intraoperative Isolation Of The Stromal Vascular Fraction For Bone Tissue Engineering
Alexandra O. Luby, MD, MS, Jessie Hoxie, BS, Gina N. Sacks, MD, Lauren Buchman, BS, Lauren Patrick, BS, Kevin M. Urlaub, BS, Jeremy V. Lynn, BS, Noah S. Nelson, BS, MPH, Alexis Donneys, MD, Steven R. Buchman, MD.
University of Michigan, Ann Arbor, MI, USA.
Purpose: Adipose-derived stems cells (ASC) have demonstrated promise across many areas of regenerative medicine, including bone tissue engineering. One major barrier to clinical translation and broad application of these therapeutics is current protocols which require laboratory cell culture and enzymatic digestion to produce stem cell rich product. More optimal methodologies would circumvent the current protocols, and instead, employ mechanically-based techniques to produce ASC rich stromal vascular fraction (SVF) intraoperatively for immediate clinical application. Therefore, the purpose of this study was to develop a clinically translatable technique for intra-operative harvest, isolation, and implantation of SVF with the primary aim of enhancing bone healing at irradiated fracture sites.
Methods: Male Lewis rats (n=29) were divided into groups: Fracture (Fx), Radiation with Fracture (XRT), and Radiation with Fracture and SVF implantation (SVF). Experimental groups received 35Gy of radiation. All groups underwent mandibular osteotomy and external fixation. Inguinal fat pads were minced and serially processed using Tulip Sizing Transfers (2.4mm, 1.4mm, 1.2mm). Serial filtration (800 micron, 400 micron) and centrifugation was performed. The resultant oil and aqueous layers were discarded and the cell pellet was collected for immediate implantation at the osteotomy site. Animals were sacrificed on post-operative day 40. Gross pathology and MicroCT analysis were utilized to determine union rates and the quality of the bone formed at the osteotomy site. Biomechanical strength testing was performed until failure to evaluate yield and ultimate load of new bone at the osteotomy site.
Results: Immediate implantation of SVF increased union rates compared to XRT alone (79% vs. 20%). Additionally, MicroCT analysis demonstrated high quality new bone formation in irradiated fractures treated with SVF compared to the control based on bone mineral density (666.2 ± 32.0 vs. 312.2 ± 51.7; p=0.000) and bone volume fraction (0.744 ± 0.072 vs. 0.350 ± 0.041; p=0.000). In fact, implantation of SVF into irradiated fracture sites resulted in bone quality similar to the bone formed at non-irradiated fracture sites, as there was no significant difference found between groups (BMD: 666.2 ± 32.0 vs. 710.3 ± 38.0; p=0.390, BVF: 0.744 ± 0.072 vs. 0.803 ± 0.04; p=0.300). Radiation significantly diminished the biomechanical properties of bone, including yield (23.6 ± 28.2 vs. 81.9 ± 31.3; p=0.002) and ultimate load (33.7 ± 30.9 vs. 87.3 ± 26.7; p=0.005). SVF implantation improved yield (52.0 ± 28.6 vs. 81.9 ± 31.3; p=0.161) and ultimate load (33.7 ± 30.9 vs. 87.3 ± 26.7; p=0.145) of the irradiated bone to the level of the non-irradiated control, as there was no significant difference between groups.
Conclusions: Use of the stromal vascular fraction for bone tissue engineering demonstrates great potential, including applications in irradiated fracture healing. In this study, we developed a novel approach that eliminates laboratory dependent techniques and instead, utilizes mechanical methods that would enable intraoperative SVF harvest, isolation, and immediate implantation. While further studies are required to optimize this approach, the results of this study are incredibly promising for the long-awaited translation of cell-based therapeutics into the clinical arena.
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