ABSTRACT
OBJECTIVE: Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for 'de novo' regrowth or application as tissue 'patches' that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat gastrocnemius medialis muscle through perfusion of the main feeding arteries. RESULTS: Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) ex situ assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) in situ within the bioreactor environment assessment of the sample's passive mechanical elasticity. CONCLUSION: We find DNA and sarcomere-free constructs after 72 h of 0.1% SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs. SIGNIFICANCE: Our MyoBio represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.
Subject(s)
Tissue Engineering , Tissue Scaffolds , Animals , Bioreactors , DNA , Extracellular Matrix , Muscle, Skeletal , Perfusion , Rats , Tissue Engineering/methodsABSTRACT
OBJECTIVE: Muscle biomechanics is set by the spacing of repetitive striation patterns of individual sarcomeres within single muscle fibres of stacked myofibrils. Sarcomere lengths (SL) are rather unequally distributed than of equal distance. This non-uniformity may affect both, force production as well as passive-elastic deformation. However, online recording of SL during axially imposed strains is cumbersome due to a lack of compact technologies. METHODS: To fuse SL pattern recognition with restoration force assessments during quasi-static axial stretch, we implemented live tracking of SL distributions simultaneous to voice-coil actuated stretch and restoration force recordings in our MyoRobot 2.0 automated biomechatronics platform. Both were obtained online during stretch-relaxation cycles of murine single muscle fibres. RESULTS: Under quasi-static stretch conditions ( â¼ 1 µm/s fibre length changes), almost no apparent hysteresis was detected in single fibres. SL showed a non-uniform distribution. While mean SL varied between 2.6 µm and 3.4 µm upon 140% stretch, two populations of fibres were noticed: one showing a minor change in SL distribution with stretch, and one becoming more equally distributed upon stretch. CONCLUSION: A roughly 5% SL variability under rest either diminishes or remains almost unaltered upon elastic axial deformation. This may reflect differential impact of mostly extra-sarcomeric components to stretch in this stretch range. SIGNIFICANCE: The augmented functionality of the MyoRobot 2.0 towards online sarcomere analyses within single fibres shall provide a valuable tool for the muscle community to study the contribution of serial elastic and force producing elements in health and disease models.
