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The Damage Associated Molecular Pattern HMGB1 Limits The Formation Of Heterotopic Ossification In A Mouse Model
Fady P. Marji, MD, Kyle Parks, PhD, Gregory Cooper, PhD, Joseph Losee, Lucas Dvoracek.
University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Purpose
Heterotopic ossification (HO) is the pathological extra-skeletal or ectopic formation of lamellar bone in non-osseous tissues. The treatment of HO often requires large soft tissue resections with reconstruction via local tissue rearrangement or free flaps. Additionally, burn wounds have been found to develop HO- a severely debilitating complication. In this study, we generated a model of HO in mice by injection of rhBMP-2 subcutaneously, with an attempt to alter the process of HO development via modulation of inflammation using local treatment with HMGB1, a damage associated molecular pattern (DAMP). As the nonsurgical mitigation or reversal of HO has appreciable clinical implications, we hoped to discover a novel local strategy for potential treatment of this disease process using HMGB1.
Methods
Twenty mice were used in the study. All animals were injected subcutaneously in the abdomen with 1 g of rhBMP2 impregnated in 300 L of growth factor-reduced Matrigel as a model for the induction of heterotopic bone formation. All animals were maintained without additional treatment for four weeks for the development of heterotopic bone. At the end of four weeks, all animals underwent whole-body micro computed tomography (micro-CT) imaging for the evaluation of heterotopic bone formation at the injection site. Sixteen of the initial twenty mice had radiological evidence of heterotopic bone formation. Eight mice were randomly allocated respectively to the experimental and control arms of the study. In the treatment group, mice were treated with 10 g of HMGB1 in 250 L of phosphate buffered saline (PBS), injected subcutaneously directly at the site of palpable heterotopic bone, every other day, for a total of fourteen days. Control mice were injected with 250 L of PBS using the same treatment regimen. Imaging also occurred on treatment days seven and fourteen. Heterotopic tissues were recovered for histochemical analysis, including tartrate-resistant acid phosphatase (TRAP) staining, at the end of imaging. Heterotopic bone volumes were computed using 3D reconstruction of micro-CT imaging using 3D Slicer software.
Results
Though both groups continued growing bone over the course of the study, treatment with HMGB1 led to a significant lower mean volume of growth at time seven and fourteen-day timepoints. Seven days after the initiation of treatment, mean percent volume increase for the HMGB1 treated group and for the PBS treated group was 121.33 percent (16.02) and 145.98% (22.37), respectively, demonstrating a statistically significant decrease in heterotopic bone growth secondary to treatment with HMGB1 (p = 0.032). Fourteen days after the initiation of treatment, mean percent volume increase for the HMGB1 treated group and for the PBS treated group was 201.88 percent (56.76) and 253.4484 percent (53.23), respectively, again demonstrating a therapeutic effect in the HMGB1 treated group (p = 0.025). TRAP staining did not detect a difference in the number of osteoclasts between the control and treatment group.
Conclusions
The use of HMGB1 may have implications in the treatment of heterotopic ossification. Further investigations are needed to decipher the mechanism for which this DAMP limits the progression of heterotopic bone formation.


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