Multiaxial filament winding of biopolymer microfibers with a collagen resin binder for orthobiologic medical device biomanufacturing.

Multiaxial filament winding is an additive manufacturing technique used extensively in large industrial and military manufacturing yet unexplored for biomedical uses. This study adapts filament winding to biomanufacture scalable, strong, three-dimensional microfiber (3DMF) medical device implants for potential orthopedic applications. Polylactide microfiber filaments were wound through a collagen 'resin' bath to create organized, stable orthobiologic implants, which are sized for common ligament (e.g. anterior cruciate ligament) and tendon (e.g. rotator cuff) injuries and can be manufactured at industrial scale using a small footprint, economical, high-output benchtop system. Ethylene oxide or electron beam sterilized 3DMF samples were analyzed by scanning electron microscopy (SEM), underwent ASTM1635-based degradation testing, tensile testing, ISO 10993-based cytocompatibility, and biocompatibility testing, quantified for human platelet-rich plasma (PRP) absorption kinetics, and examined for adhesion of bioceramics and lyophilized collagen after coating. 3DMF implants had consistent fiber size and high alignment by SEM. Negligible mass and strength loss were noted over 4 months in culture. 3DMF implants initially exceeded 1000 N hydrated tensile strength and retained over 70% strength through 4 months in culture, significantly stronger than conventionally produced implants made by fused fiber deposition 3D printing. 3DMF implants absorbed over 3xtheir weight in PRP within 5 min, were cytocompatible and biocompatible in vivo in rabbits, and could readily bind tricalcium phosphate and calcium carbonate coatings discretely on implant ends for further orthobiologic material functionalization. The additive manufacturing process further enabled engineering implants with suture-shuttling passages for facile arthroscopic surgical delivery. This accessible, facile, economical, and rapid microfiber manufacturing platform presents a new method to engineer high-strength, flexible, low-cost, bio-based implants for orthopedic and extended medical device applications.

Sensor technologies for quality control in engineered tissue manufacturing.

The use of engineered cells, tissues, and organs has the opportunity to change the way injuries and diseases are treated. Commercialization of these groundbreaking technologies has been limited in part by the complex and costly nature of their manufacture. Process-related variability and even small changes in the manufacturing process of a living product will impact its quality. Without real-time integrated detection, the magnitude and mechanism of that impact are largely unknown. Real-time and non-destructive sensor technologies are key for in-process insight and ensuring a consistent product throughout commercial scale-up and/or scale-out. The application of a measurement technology into a manufacturing process requires cell and tissue developers to understand the best way to apply a sensor to their process, and for sensor manufacturers to understand the design requirements and end-user needs. Furthermore, sensors to monitor component cells' health and phenotype need to be compatible with novel integrated and automated manufacturing equipment. This review summarizes commercially relevant sensor technologies that can detect meaningful quality attributes during the manufacturing of regenerative medicine products, the gaps within each technology, and sensor considerations for manufacturing.

Biopreservation of living tissue engineered nerve grafts.

Tissue engineered nerve grafts (TENGs) built from living neurons and aligned axon tracts offer a revolutionary new approach as "living scaffolds" to bridge major peripheral nerve defects. Clinical application, however, necessitates sufficient shelf-life to allow for shipping from manufacturing facility to clinic as well as storage until use. Here, hypothermic storage in commercially available hibernation media is explored as a potential biopreservation strategy for TENGs. After up to 28 days of refrigeration at 4℃, TENGs maintain viability and structure in vitro. Following transplantation into 1 cm rat sciatic defects, biopreserved TENGs routinely survive and persist for at least 2 weeks and recapitulate pro-regenerative mechanisms of fresh TENGs, including the ability to recruit regenerating host tissue into the graft and extend neurites beyond the margins of the graft. The protocols and timelines established here serve as important foundational work for the manufacturing, storage, and translation of other neuron-based tissue engineered therapeutics.

Biomanufacturing of Axon-Based Tissue Engineered Nerve Grafts Using Porcine GalSafe Neurons.

Existing strategies for repair of major peripheral nerve injury (PNI) are inefficient at promoting axon regeneration and functional recovery and are generally ineffective for nerve lesions >5 cm. To address this need, we have previously developed tissue engineered nerve grafts (TENGs) through the process of axon stretch growth. TENGs consist of living, centimeter-scale, aligned axon tracts that accelerate axon regeneration at rates equivalent to the gold standard autograft in small and large animal models of PNI, by providing a newfound mechanism-of-action referred to as axon-facilitated axon regeneration (AFAR). To enable clinical-grade biomanufacturing of TENGs, a suitable cell source that is hypoimmunogenic, exhibits low batch-to-batch variability, and able to tolerate axon stretch growth must be utilized. To fulfill these requirements, a genetically engineered, FDA-approved, xenogeneic cell source, GalSafe® neurons, produced by Revivicor, Inc., have been selected to advance TENG biofabrication for eventual clinical use. To this end, sensory and motor neurons were harvested from genetically engineered GalSafe day 40 swine embryos, cultured in custom mechanobioreactors, and axon tracts were successfully stretch-grown to 5 cm within 25 days. Importantly, both sensory and motor GalSafe neurons were observed to tolerate established axon stretch growth regimes of ≥1 mm/day to produce continuous, healthy axon tracts spanning 1, 3, or 5 cm. Once stretch-grown, 1 cm GalSafe TENGs were transplanted into a 1 cm lesion in the sciatic nerve of athymic rats. Regeneration was assessed through histological measures at the terminal time point of 2 and 8 weeks. Neurons from GalSafe TENGs survived and elicited AFAR as observed when using wild-type TENGs. At 8 weeks postrepair, myelinated regenerated axons were observed in the nerve section distal to the injury site, confirming axon regeneration across the lesion. These experiments are the first to demonstrate successful harvest and axon stretch growth of GalSafe neurons for use as starting biomass for bioengineered nerve grafts as well as initial safety and efficacy in an established preclinical model-important steps for the advancement of clinical-grade TENGs for future regulatory testing and eventual clinical trials. Impact statement Biofabrication of tissue engineered medical products requires several steps, one of which is choosing a suitable starting biomass. To this end, we have shown that the clinical-grade, genetically engineered biomass-GalSafe® neurons-is a viable option for biomanufacturing of our tissue engineered nerve grafts (TENGs) to promote regeneration following major peripheral nerve injury. Importantly, this is a first step in clinical-grade TENG biofabrication, proving that GalSafe TENGs recapitulate the mechanism of axon-facilitated axon regeneration seen previously with research-grade TENGs.