RESUMO
Chemonucleolysis has become an established method of producing whole organ culture models of intervertebral disc (IVD) degeneration. However, the field needs more side-by-side comparisons of the degenerative effects of the major enzymes used in chemonucleolysis towards gaining a greater understanding of how these organ culture models mimic the wide spectrum of characteristics observed in human degeneration. In the current work we induced chemonucleolysis in bovine coccygeal IVDs with 100 µL of papain (65 U/mL), chondroitinase ABC (chABC, 5 U/mL), or collagenase II (col'ase, 0.5 U/mL). Each enzyme was applied in a concentration projected to produce moderate levels of degeneration. After 7 days of culture with daily dynamic physiological loading (0.02-0.2 MPa, 0.2 Hz, 2 h), the cellular, biochemical and histological properties of the IVDs were evaluated in comparison to a PBS-injected control. Papain and collagenase, but not chABC, produced macroscopic voids in the tissues. Compared to day 0 intact IVDs, papain induced the greatest magnitude glycosaminoglycan (GAG) loss compared to chABC and col'ase. Papain also induced the greatest height loss (3%), compared to 0.7%, 1.2% and 0.4% for chABC, col'ase, and PBS, respectively. Cell viability in the region adjacent to papain and PBS-injection remained at nearly 100% over the 7-day culture period, whereas it was reduced to 60%-70% by chABC and col'ase. Generally, enzyme treatment tended to downregulate gene expression for major ECM markers, type I collagen (COL1), type II collagen (COL2), and aggrecan (ACAN) in the tissue adjacent to injection. However, chABC treatment induced an increase in COL2 gene expression, which was significant compared to the papain treated group. In general, papain and col'ase treatment tended to recapitulate aspects of advanced IVD degeneration, whereas chABC treatment captured aspects of early-stage degeneration. Chemonucleolysis of whole bovine IVDs is a useful tool providing researchers with a robust spectrum of degenerative changes and can be utilized for examination of therapeutic interventions.
RESUMO
Tendons and ligaments have a poor innate healing capacity, yet account for 50% of musculoskeletal injuries in the United States. Full structure and function restoration postinjury remains an unmet clinical need. This study aimed to assess the application of novel three dimensional (3D) printed scaffolds and induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) overexpressing the transcription factor Scleraxis (SCX, iMSCSCX+ ) as a new strategy for tendon defect repair. The polycaprolactone (PCL) scaffolds were fabricated by extrusion through a patterned nozzle or conventional round nozzle. Scaffolds were seeded with iMSCSCX+ and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSCSCX+ -seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence. iMSCSCX+ -seeded on microgrooved scaffolds showed upregulation of tendon markers and increased organization and linearity of cells compared to non-patterned scaffolds in vitro. In vivo gait analysis showed improvement in the Scaffold + iMSCSCX+ -treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold + iMSCSCX+ group compared with the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold + iMSCSCX+ group. This study demonstrates the potential of 3D-printed scaffolds with cell-instructive surface topography seeded with iMSCSCX+ as an approach to tendon defect repair. Further studies of cell-scaffold constructs can potentially revolutionize tendon reconstruction by advancing the application of 3D printing-based technologies toward patient-specific therapies that improve healing and functional outcomes at both the cellular and tissue level.
