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Effect Of Burn Injury On Mesenchymal Stem Cells: Mechanism And Improved Imaging And Treatment Of Heterotopic Ossification
Benjamin Levi, MD, Jonathan Peterson, BS, Sara DeLaRosa, BS, Jeffrey Lisiecki, BS, Jacob Rinkinen, BA, Alexis Donneys, MD, Jill Bayliss, PhD, Grace Su, MD, Stewart Wang, MD, PhD, Steven R. Buchman, MD, PhD, Paul S. Cederna, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE: Heterotopic ossification (HO), or the abnormal development of bone in soft tissue locations, is a clinically devastating sequela of burn injury and trauma that leads to joint contractures and pain. An accepted mechanism for HO proposes that stem cell-like progenitors become aberrantly activated to form bone. We hypothesize that the inflammatory response to burn injury enhances the osteogenic capacity of human and mouse mesenchymal stem cells (MSCs) in vitro and HO formation in vivo. Furthermore, we hypothesize that this osteogenic potential and HO formation can be mitigated through Adenosine triphosphate (ATP) hydrolysis at the burn site.
METHODS:Human MSCs cells were harvested from adipose tissue of burn patients and age and sex matched controls. Mouse MSCs (adipose derived) were harvested 2, 6, and 24 hours after burn injury, burn injury + ATP inhibitor application (apyrase), or non-burn control (sham) (n=6 per group). Subsequently, osteogenic capacity was assessed by gene expression (qRT PCR), protein expression (western blot analysis) and standard in vitro osteogenic differentiation assays. In vivo heterotopic bone formation was assessed using an Achilles tenotomy model. Osteogenic signaling was assessed in vivo by immunohistochemistry and HO formation was analyzed by Raman spectroscopy, microCT, near infra-red imaging and histology.
RESULTS:
Human ASCs demonstrated increased expression in osteogenic gene expression of the early osteogenic marker Runt-related transcription factor-2 (RUNX2), and the late osteogenic marker osteocalcin (OCN), as well as BMP-2 ligand after burn injury (p<0.05). Similarly, burn injury resulted in a striking increase in osteogenic differentiation and osteogenic gene expression among mouse MSCs at all time points which was mitigated by ATP hydrolysis at the burn site. In both human and mouse cell lines, increased osteogenic differentiation correlated with increased BMP-2 signaling as demonstrated by Western Blot analysis. Burn injury stimulated increased HO formation and enhanced bone mineral density by histology, x-ray and microCT (Fig.1). Furthermore, near infrared imaging and Raman Spectroscopy (Fig. 2) allowed for detection of HO prior to CT evidence. This HO formation was blunted in mice after ATP hydrolysis at the burn site as demonstrated by all imaging modalities.
CONCLUSION:We demonstrate that MSCs exhibit enhanced osteogenic capacity after burn injury. The mechanism appears to be due to up-regulation of BMP-2 and this can be mitigated by ATP hydrolysis at the burn site both in vitro and in vivo. We establish a potential role for burn injury in modulating HO formation and demonstrate that direct manipulation of ATP levels at the burn site may have therapeutic utility in treatment regimens designed to prevent and remediate HO. We also validate transcutaneous Raman imaging that provide surgeons the ability to examine the extent of HO for surgical planning and the opportunity for early diagnosis which is currently unavailable.


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