Introduction: Composite musculoskeletal injuries are very common in civilian and military populations and remain a significant clinical challenge due to their extensive damage to multiple tissue types. Typically, these injuries consist of segmental bone defects and volumetric muscle loss (VML)[1]. Existing clinical treatment of composite bone-muscle injuries involve multiple surgeries such as skeletal and soft tissue stabilization and free functional muscle transfer[2]. One of the major setbacks of these treatments is the inability to regenerate functionalized muscle tissue due to donor site morbidity, long operative time, and prolonged denervation of the transferred muscle tissue. As VML persists, bone regeneration will be negatively impacted due to the lack of essential cytokines and stem cells, continous inflammation, and fibrotic tissue formation[1]. In order to overcome these issues, a tissue engineered scaffold derived from muscle decellularized extracellular matrix (mdECM) can be generated to provide an environment that supports muscle and bone tissue regeneration using the biochemical cues naturally found in decellularized muscle tissue. It is hypothesized that the mdECM will recruit relevant cell populations at a level similar to known chemoattractant agents. In this study we will determine the chemoattractant ability of mdECM in vitro. The migration of relevant musculoskeletal cells, human bone marrow stem cells (hBMSCs) and C2C12 myoblasts, in response to mdECM will be analyzed using a 3D timelapse chemotaxis assay. The goal of this work is to provide the foundation for a mdECM-based scaffold that can be used to regenerate tissue in bone-muscle composite injuries.
Materials and
Methods: Fresh skeletal muscle from the tibialis anterior of New Zealand white rabbits (Envigo) was harvested for decellularization[3]. Post decellularization, the samples were lyophilized and then solubilized[4]. The resulting solution had a mdECM concentration of 20 mg/mL and kept at physiological pH. Cell migration studies were conducted with either isolated hBMSCs (Lonza; donor 24-year-old male) or C2C12 myoblasts (ATCC; CRL-1772) using μ-slide chambers from Ibidi [5]. The outside reservoirs of the chemotaxis slides were loaded with either basal media without FBS, basal medium containing the known chemoattractant growth factor (50 ng/mL SDF1- α for hBMSCs, 50 ng/mL HGF for myoblasts), or mdECM (10 mg/mL)[5]. The mdECM is used at a concentration of 10 mg/mL due to the chemoattractant loading mechanism for the assay, where the chemoattract is diluted by half with basal media. Cells are embedded into a 3D ECM-like matrix made from rat tail collagen and then loaded into the middle observational channel with a concentration of either 3x106 hBMSCs/mL or 1.5x106 myoblasts/mL. For live cell imaging, a stage-top incubator and phase-contrast microscopy was used to image the observational channel every 10 minutes over the course of 30 hours. 40 Cells within the observational area during the entire duration were randomly selected for cell tracking. Data was then analyzed using Chemotaxis and Migration Tool (version 2.0, Ibidi) to obtain cell trajectory plots and chemotactic parameters, such as forward migration index, center of mass, and directness.
Results, Conclusions, and Discussions: We performed studies with primary hBMSCs, which participate in the regeneration of both bone and muscle tissue. The migration tracks for hBMSCs show random movement when there is no chemoattractant present, but a more direct downwards movement was seen when SDF1- α or mdECM is present (Figure 1A). These observations are further clarified by analyzing the forward migration index in the y-axis (FMIy), center of mass in the y-axis (COMy), directness of cell path, and the percent of cells moving towards the chemoattractant. FMIy indicates the efficiency of the forward migration of cells, COMy provides insight on the direction the cells primarily travelled, and directness measures the straightness of the cell path towards the chemoattractant [5]. For all of these parameters, the mdECM group showed similarity to the SDF1- α(Figure 1B-E). This showcases that mdECM can effectively recruit hBMSCs to the same level as a known chemoattractant. The following study utilized HGF as it can activate and recruit both muscle satellite cells and myoblasts. The migration tracks of the myoblasts display a similar trend to the hBMSCs, where there is random movement when no chemoattractant is applied and a distinct downwards movement towards the HGF and mdECM reservoirs (Figure 2A). There were minor differences noticed in FMIy and directness of cell path between HGF and mdECM groups, but similar behavior is seen for COMy and percent of cells moving towards the chemoattractant (Figure 2B-E). These results establish that the mdECM can recruit myoblasts to a similar level to HGF. Overall, these results demonstrate the chemotactic ability of mdECM. This study showcased that mdECM can recruit relevant cell types found in bone and muscle tissue. This is an essential characteristic for a scaffold used for bone-muscle compact injuries as it can encourage muscle regeneration in response to VML, which in turn will promote bone regeneration. Future work will be conducted on fabricating a scaffold derived from mdECM for this purpose. Overall, this study is the foundation for an in-situ tissue regeneration approach using mdECM as a therapy to treat composite bone-muscle injuries.
Acknowledgements (Optional):
References: (1) Subbian et al., Acta Biomaterialia 2021; 127: 180-192. (2) McKinley et al., Journal of Orthopaedic Research 2023; 41(9): 1890-1901. (3)Fishman et al., Annals of Otology, Rhinology & Laryngology 2012; 121(2):129-138. (4) Saldin et al, Acta Biomaterialia 2017; 49: 1-15. (5) Golebiowska et al., Annals of Biomedical Engineering 2023.
Acknowledgement: National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (#R01EB030060 & R01EB020640)
