Luisa Nierhoff
Pronouns: she/her/hers
Research Mentor(s): Lisa Larkin
Research Mentor School/College/Department: University of Michigan / Medicine
Program: UROPF
Session: Session 4 (1:40pm – 2:30pm)
Authors: Luisa Nierhoff, Christopher Kennedy, Lisa Larkin, Eileen Su, Makayla Kelley, Ryan Ettner, Emmanuel Vega-Soto, Claudia Loebel
Abstract
Volumetric Muscle Loss (VML) can be defined as the loss of more than 30% of skeletal muscle tissue due to a traumatic injury or a surgical procedure that results in an impaired function of the remaining muscle. Current therapies for VML injuries include muscle transfers or autologous muscle grafts. However, these treatments are limited by certain constraints such as tissue availability and/or donor site morbidity. Therefore, hydrogels and tissue engineered skeletal muscle units that result in decreased fibrosis, as well as boost muscle regeneration at the VML site, provide promising alternatives for current treatments. This study assesses the biocompatibility of commercially available poly(ethylene glycol) (PEG), methacrylated gelatin (Gel-MA), and hyaluronic acid (HA) hydrogels, as well as the efficacy of combining HA with our fabricated skeletal muscle units (SMU) to repair muscle structure. To do so, the immune-response to hydrogels and the implanted SMU was analyzed, by performing an immunohistochemistry stain for M1 and M2 macrophages and neutrophils following a 6-week recovery period from a 30% VML in the peroneus tertius (PT) muscle in an ovine model. This study was conducted using 4.5-month-old male Polypay wether sheep with a weight of 19-32 kilograms. These sheep were divided into the following groups: VML+SMU(n=5) , VML+SMU+HA (n=5), VML only(n=5), VML+HA (n=5), VML+PEG (n=5), VML+Gel-MA (n=5). All procedures conducted with the large animal models were done in accordance with The Guide for Care and Use of Laboratory Animals (Public Health Service, 2011 NIH Publication No. 83-23). The procedure was performed by creating an incision in the skin above the PT muscle to expose the fascia as well as the peroneal nerve. While avoiding major vasculature and the nerve, a v-cut longitudinal portion of the PT constituting 30% total muscle volume was dissected. After closing the fascia, the HG was injected behind the fascia and into the site of the VML, and the skin was closed with sutures and staples. After a 6-week recovery period, the animal was euthanized and the surgical (left) and contralateral (right) PT muscles were dissected, weighed, and prepared for histology. The muscle was cryosectioned and stained with Masson’s Trichrome, Hematoxylin and Eosin, and Picrosirus Red. Additionally, the cryosectioned muscle was immunostained for myosin heavy chain (MF-20) and laminin. The tissue has been stained using CD68 and CD163 antibodies to mark M1 and M2 macrophages, respectively, and with anti-neutrophil antibodies to mark for neutrophils. The samples are currently being imaged with a Nikon Ti2 microscope. Using ImageJ/Fiji image analysis software, the images will be analyzed by looking for macrophage and neutrophil concentration around the VML site to assess the biocompatibility of the hydrogels. All of the animals presented with a normal weight gain over the 6-week recovery period. A histological analysis of the explanted muscles with Masson’s trichrome staining presented increased collagen content near the repair site in all surgical groups, when compared with the contralateral muscle. While more regenerated fibers as well as enhanced migration into the VML injury site were seen in the PEG and HA experimental groups, small fiber muscles indicating muscle regeneration was observed in all the repair sites. The analysis of the M1/M2 stained sections are currently in the process of being imaged with the Nikon Ti2 microscope, results have yet to be produced. Although we have not been able to collect data for the immune cell staining, we expect that lower concentrations of immune cells within the VML site indicate lower immune response to the hydrogel and/or SMU implant used to repair the VML site. On the other hand, higher concentrations of immune cells would indicate an increased immune response to the implanted therapies. The results of this study will emphasize the impact that hydrogels and tissue engineered constructs have on the ability to regenerate skeletal muscle tissue in VML sites.
