FRESH 3D Bioprinting of Decellularized Extracellular Matrix Scaffolds for Volumetric Muscle Loss

Introduction: Volumetric muscle loss (VML) due to trauma or disease is a severe condition characterized by the loss of >20% of muscle mass and impaired contractile function. VML overwhelms the innate regenerative capacity of skeletal muscles, leading to chronic injuries with permanent deficits. The current gold-standard treatment is autologous muscle grafts, but these suffer from limited graft efficacy, and challenges with donor site availability and morbidity. Regenerative medicine approaches using decellularized extracellular matrix (dECM) scaffolds hold promise for in vivo muscle regeneration by providing myogenic biochemical cues and have progressed to human clinical trials. However, these dECM scaffolds are commonly implanted as powders or thin sheets that are unable to effectively fill the VML space and provide structural cues. Given the crucial role of myofiber alignment in the functional contraction of skeletal muscle tissue, an improved solution would provide the necessary biochemical cues present in dECM together with physical guidance cues to recreate the 3D muscle tissue architecture. Here, we employ freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting to fabricate biomimetic scaffolds from dECM hydrogels. These scaffolds are designed to restore native skeletal muscle 3D architecture by modulating the immune response and guiding myofiber alignment.

Materials and

Methods: For the VML in vivo model, a surgical resection of ~20% of the tibialis anterior (TA) muscle in Sprague-Dawley rats was created, under approval from Carnegie Mellon’s IACUC. Both female and male rats were used, with weights ranging from 250 to 450 grams. Defect size was calculated from regression analysis that relates the weight of the rat to weight of TA muscle from pilot studies. For the short term studies, the calculation was 0.00148x + 0.07286 (n=16 , r2=0.975). For the long-term studies, the equation was 0.00145x + 0.10614 (n=35, r2=0.952). Scaffolds designed to fill the wound were aseptically FRESH 3D bioprinted and lightly crosslinked. Bioprinted dECM scaffolds were then implanted into the defect area and secured in place using fibrin glue. To address the intrinsically limited printability of the dECM, collagen type 1 (Col1) was incorporated into the bioink to enhance the handleability of the resulting implantable scaffolds. Rodents received implants of three different bioink compositions, with variations in the combination of dECM and Col1. A dECM only sheet and defect only groups were used as the positive and negative controls, respectively. Short-term studies at day five evaluated the impact of bioink composition and scaffold architecture on macrophage polarization and satellite cell activation through flow cytometry and histological analysis.

Results, Conclusions, and Discussions: Results
We have successfully built, implanted, and immobilized 3D bioprinted dECM scaffolds that emulate muscle architecture within the TA muscle of rodents (Fig 1A). While scaffolds with dECM alone encounter challenges in maintaining their shape post-implantation, the incorporation of collagen into the bioinks enables the scaffold to retain its structure (Fig 1B). At day 5 post-implantation, scaffolds with added Col1 are present with distinct fiber orientation still evident (Fig 1C). Preliminary flow data suggests that the addition of the ECM scaffolds increases the recruitment of macrophages to the wound site and delays the macrophage shift from M1 to M2 (Fig 1D and 1E). This, in turn, facilitates enhanced activation of satellite cells at the wound site (Fig 1F). Preliminary histological data verifies that M2 macrophages are present around the scaffold area with M1 macrophages surrounding the outside of the defect (Fig 1G and 1H).

Conclusions
This dECM scaffold has been implanted in more than 50 rodent models, with ongoing investigations. In vivo data acquisition is currently underway to comprehensively evaluate the scaffold's efficacy in directing skeletal muscle regeneration following severe, irreversible injuries. The successful outcome of these studies holds the promise of significantly mitigating patient morbidity associated with profound musculoskeletal trauma, thereby offering a transformative leap in biomedical engineering for enhanced patient care and recovery.

Discussion
Long-term investigations spanning up to 42 days are currently underway, employing a comprehensive approach that integrates both functional testing and histological analysis. The functional recovery of the TA muscle will be evaluated by eliciting peroneal nerve stimulation via needle electrodes positioned superficially through the skin, while the foot remains affixed to a custom-designed footplate attached to a force transducer. Dorsiflexion strength will be quantified by multiplying the force of the kick (in Newtons) by the foot length (in millimeters), and subsequently normalized to the rat's body weight (in kilograms). Histological examination will be utilized to assess various factors including de novo skeletal muscle generation, endogenous tissue remodeling, reinnervation, and revascularization. These analyses collectively aim to provide a comprehensive understanding of the scaffold's impact on skeletal muscle regeneration and functional restoration post-severe injury.

Acknowledgements (Optional): This work is funded by the NSF GRFP and DARPA.