Abstract
Despite continuous expansion of tissue engineering research and development of promising technologies, the number of Tissue Engineered Medical Products (TEMPs) used as clinical therapies remains relatively low. Experts in the field attribute this, at least in part, to challenges regarding reproducibility and scalability of the biomanufacturing processes. Skeletal muscle regeneration research represents a large portion of tissue engineering research. Volumetric muscle loss injuries, by definition, exceed the endogenous capacity for skeletal muscle regeneration. These injuries result in significant scar formation and fibrosis, and a loss of muscle strength and function. These devastating injuries are prevalent in both military and civilian populations as a result from blast injuries, car accidents, table saw injuries, or other traumatic accidents. The current standard of care involves muscle flap transplant, which results in donor site morbidity and carries a risk of flap failure. As such, several groups are exploring tissue engineered solutions for VML injuries. Furthermore, a few of these groups are utilizing advanced biomanufacturing methods such as bioprinting. While the work described in this dissertation is not the first implementation of bioprinting for VML, it does represent a novel application of bioprinting: seeding a dense monolayer of cells onto both sides of a sheet-based scaffold. Importantly, this technology could be applied to improving reproducibility and scalability of any sheet-based therapy.
The focus of this dissertation and the development of these advanced biomanufacturing methods is the Tissue Engineered Muscle Repair (TEMR) construct – a pre-clinical therapy for VML, previously developed by the Christ Lab. TEMR has been tested in several rodent models of VML, and has been found to facilitate functional recovery of up to 90% in the rat latissimus dorsi. TEMR consists of a porcine bladder acellular matrix scaffold seeded with skeletal muscle progenitor cells (MPCs). During the TEMR maturation process, the seeded scaffolds are statically differentiated, and then exposed to uniaxial cyclic stretch bioreactor preconditioning. Developing bioprinting methods for automated seeding of the TEMR construct necessitated the development of a bioreactor system that would facilitate transfer between the steps of the maturation process: bioprinting, differentiation, and cyclic stretch preconditioning. Towards this end, the Fully Enclosed Bioreactor Environment (FEBE) was developed to reduce manual manipulation of the scaffold, improve reproducibility of the TEMR construct, and facilitate bioprinting on the sheet-based scaffold.
Prior to in vivo assessment, the bioprinting methods were evaluated for multiple tissue-relevant cell types including several primary muscle progenitor cell types and vascular and neuronal cell types. The bioprinting methods were found to be compatible with each of the assessed cell types, and in co-cultures of several cell types. The FEBE system was also evaluated. Several metrics of success of TEMR maturation, or critical quality attributes (CQAs) were determined and evaluated. The combination of bioprinting methods and the FEBE system allowed for consistently high cell viability (> 80%), cell coverage (> 80%), and cell alignment, as quantified by a coherency value (> 0.3).
Upon consistently successful implementation in primary human MPCs, these methods were translated to primary rat MPCs, and assessed in a rat tibialis anterior (TA) model of VML. Specifically, a 20% by weight defect was created in the rat TA and treated with either (1) no repair, (2) a manually seeded TEMR, matured in the FEBE, or (3) a bioprinted TEMR, also matured in the FEBE. CQAs of the implanted TEMRs were analyzed, the function of the TA muscle was assessed at 4-, 8-, and 12-weeks post injury, and histological analysis explored muscle fiber size and quantity, as well as collagen deposition within the muscle as an indication of fibrosis. This study yielded several key findings:
(1) bioprinting allowed for accelerated, more consistent, and more biomimetic functional recovery
(2) both cell coverage and alignment on the TEMR were directly correlated with force production at 12 weeks
(3) the FEBE system allowed for increased reproducibility and the elimination of the TEMR “non-responder” group previously reported in all TEMR publications in the TA model
Overall, the body of work presented in this dissertation demonstrates the utility of our automated bioprinting methods and an advanced biomanufacturing bioreactor system for increased reproducibility, applied to a therapy for volumetric muscle loss. Furthermore, this system could be applied to other sheet-based tissue engineered therapies. The results of these studies not only inform the TEMR maturation process, but offer important insights to the field of volumetric muscle loss research.
