Regenerative Repair of Volumetric Muscle Loss Injury is Sensitive to Age.

In this study, the influence of age on effectiveness of regenerative repair for the treatment of volumetric muscle loss (VML) injury was explored. Tibialis anterior (TA) VML injuries were repaired in both 3- and 18-month-old animal models (Fischer 344 rat) using allogeneic decellularized skeletal muscle (DSM) scaffolds supplemented with autologous minced muscle (MM) paste. Within the 3-month animal group, TA peak contractile force was significantly improved (79% of normal) in response to DSM+MM repair. However, within the 18-month animal group, muscle force following repair (57% of normal) was not significantly different from unrepaired VML controls (59% of normal). Within the 3-month animal group, repair with DSM+MM generally reduced scarring at the site of VML repair, whereas scarring and a loss of contractile tissue was notable at the site of repair within the 18-month group. Within 3-month animals, expression of myogenic genes (MyoD, MyoG), extracellular matrix genes (Col I, Col III, TGF-ß), and key wound healing genes (TNF-α and IL-1ß) were increased. Alternatively, expression was unchanged across all genes examined within the 18-month animal group. The findings suggest that a decline in regenerative capacity and increased fibrosis with age may present an obstacle to regenerative medicine strategies targeting VML injury. Impact Statement This study compared the recovery following volumetric muscle loss (VML) injury repair using a combination of minced muscle paste and decellularized muscle extracellular matrix carrier in both a younger (3 months) and older (18 months) rat population. Currently, VML repair research is being conducted with the young patient population in mind, but our group is the first to look at the effects of age on the efficacy of VML repair. Our findings highlight the importance of considering age-related changes in response to VML when developing repair strategies targeting an elderly patient population.

Codelivery of Infusion Decellularized Skeletal Muscle with Minced Muscle Autografts Improved Recovery from Volumetric Muscle Loss Injury in a Rat Model.

Skeletal muscle is capable of robust self-repair following mild trauma, yet in cases of traumatic volumetric muscle loss (VML), where more than 20% of a muscle's mass is lost, this capacity is overwhelmed. Current autogenic whole muscle transfer techniques are imperfect, which has motivated the exploration of implantable scaffolding strategies. In this study, the use of an allogeneic decellularized skeletal muscle (DSM) scaffold with and without the addition of minced muscle (MM) autograft tissue was explored as a repair strategy using a lower-limb VML injury model (n = 8/sample group). We found that the repair of VML injuries using DSM + MM scaffolds significantly increased recovery of peak contractile force (81 ± 3% of normal contralateral muscle) compared to unrepaired VML controls (62 ± 4%). Similar significant improvements were measured for restoration of muscle mass (88 ± 3%) in response to DSM + MM repair compared to unrepaired VML controls (79 ± 3%). Histological findings revealed a marked decrease in collagen dense repair tissue formation both at and away from the implant site for DSM + MM repaired muscles. The addition of MM to DSM significantly increased MyoD expression, compared to isolated DSM treatment (21-fold increase) and unrepaired VML (37-fold) controls. These findings support the further exploration of both DSM and MM as promising strategies for the repair of VML injury.

Recovery from volumetric muscle loss injury: A comparison between young and aged rats.

Termed volumetric muscle loss (VML), the bulk loss of skeletal muscle tissue either through trauma or surgery overwhelms the capacity for repair, leading to the formation of non-contractile scar tissue. The myogenic potential, along with other factors that influence wound repair are known to decline with age. In order to develop effective treatment strategies for VML injuries that are effective across a broad range of patient populations, it is necessary to understand how the response to VML injury is affected by aging. Towards this end, this study was conducted to compare the response of young and aged animal groups to a lower extremity VML injury. Young (3months, n=12) and aged (18months, n=8) male Fischer 344 rats underwent surgical VML injury of the tibialis anterior muscle. Three months after VML injury it was found that young TA muscle was on average 16% heavier than aged muscle when no VML injury was performed and 25% heavier when comparing VML treated young and aged animals (p<0.0001, p<0.0001). Peak contractile force for both the young and aged groups was found to decrease significantly following VML injury, producing 65% and 59% of the contralateral limbs' peak force, respectively (p<0.0001). However, there were no differences found for peak contractile force based on age, suggesting that VML affects muscle's ability to repair, regardless of age. In this study, we used the ratio of collagen I to MyoD expression as a metric for fibrosis vs. myogenesis. Decreasing fiber cross-sectional area with advancing age (p<0.005) coupled with the ratio of collagen I to MyoD expression, which increased with age, supports the thought that regeneration is impaired in the aged population in favor of fibrosis (p=0.0241). This impairment is also exacerbated by the contribution of VML injury, where a 77-fold increase in the ratio of collagen I to MyoD was observed in the aged group (p<0.0002). The aged animal model described in this study provides a tool for investigators exploring not only the development of VML injury strategies but also the effect of aging on muscle regeneration.

Development of an infusion bioreactor for the accelerated preparation of decellularized skeletal muscle scaffolds.

The implantation of decellularized tissue has shown effectiveness as a strategy for the treatment of volumetric muscle loss (VML) injuries. The preparation of decellularized tissue typically relies on the diffusion driven removal of cellular debris. For bulky tissues like muscle, the process can be lengthy, which introduces opportunities for both tissue contamination and degradation of key ECM molecules. In this study we report on the accelerated preparation of decellularized skeletal muscle (DSM) scaffolds using a infusion system and examine scaffold performance for the repair of VML injuries. The preparation of DSM scaffolds using infusion was dramatically accelerated. As the infusion rate (1% SDS) was increased from 0.1 to 1 and 10ml/hr, the time needed to remove intracellular myoglobin and actin decreased from a maximum of 140 ± 3hrs to 45 ± 3hrs and 10 ± 2hrs respectively. Although infusion appeared to remove cellular debris more aggressively, it did not significantly decrease the collagen or glycosaminoglycan composition of DSM samples when compared to un-infused controls. Infusion prepared DSM samples retained the aligned network structure and mechanical integrity of control samples. Infusion prepared DSM samples supported the attachment and in-vitro proliferation of myoblast cells and was well tolerated by the host when examined in-vivo. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:745-755, 2016.