RESUMEN
Juvenile chondrocytes are robust in regenerating articular cartilage, but their clinical application is hindered by donor scarcity. Stem cells offer an abundant autologous cell source but are limited by slow cartilage deposition with poor mechanical properties. Using 3D co-culture models, mixing stem cells and chondrocytes can induce synergistic cartilage regeneration. However, the resulting cartilage tissue still suffers from poor mechanical properties after prolonged culture. Here we report a microribbon/hydrogel composite scaffold that supports synergistic interactions using co-culture of adipose-derived stem cells (ADSCs) and neonatal chondrocytes (NChons). The composite scaffold is comprised of a macroporous, gelatin microribbon (µRB) scaffolds filled with degradable nanoporous chondroitin sulfate (CS) hydrogel. We identified an optimal CS concentration (6%) that best supported co-culture synergy in vitro. Furthermore, 7 days of TGF-ß3 exposure was sufficient to induce catalyzed cartilage formation. When implanted in vivo, µRB/CS composite scaffold supported over a 40-fold increase in compressive moduli of cartilage produced by mixed ADSCs/NChons to ~330â¯kPa, which surpassed even the quality of cartilage produced by 100% NChons. Together, these results validate µRB/CS composite as a promising scaffold for cartilage regeneration using mixed populations of stem cells and chondrocytes.
Asunto(s)
Cartílago Articular , Condrocitos , Condrogénesis , Humanos , Hidrogeles , Recién Nacido , Regeneración , Células Madre , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, most tissue engineering strategies for cartilage regeneration encapsulate MSCs as single cells, partially due to the technical challenge to homogeneously encapsulate cell pellets in three-dimensional (3D) hydrogels. It remains unclear whether encapsulating MSCs as single cell suspension or cell aggregates in 3D hydrogels would enhance MSC-based cartilage formation. In this study, we determined that the optimal size of MSC micropellets (µPellets) that can be homogeneously encapsulated in hydrogels with high cell viability is 100 cells/pellet. Using optimized µPellet size, MSCs were encapsulated either as single cell suspension or µPellets in four soft hydrogel formulations with stiffness ranging 3-6 kPa. Regardless of hydrogel formulations, single cell encapsulation resulted in more neocartilage deposition with improved mechanical functions over µPellet encapsulation. For single cell encapsulation, polyethylene glycol (PEG) hydrogels containing chondroitin sulfate led to the most cartilage matrix deposition, with compressive modulus reaching 211 kPa after only 21 days, a range approaching the stiffness of native cartilage. The findings from this study offer valuable insights on guiding optimal method design for MSCs and hydrogel-based cartilage regeneration. The optimized µPellet encapsulation method may be broadly applicable to encapsulate other stem cell types or cancer cells as aggregates in hydrogels. Impact Statement While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, it remains unclear whether encapsulating MSCs as cell pellets in three-dimensional hydrogels would enhance MSC-based cartilage formation. In this study, we determined the optimal size of MSC micropellet (µPellet) that can be homogeneously encapsulated in hydrogels with high cell viability. Unexpectedly, single cell encapsulation resulted in more robust new cartilage formation than µPellet encapsulation. Furthermore, tuning hydrogel formulation led to rapid cartilage regeneration with stiffness approaching that of native cartilage. The findings from this study would facilitate clinical translation of MSCs and hydrogel-based therapies for cartilage regeneration with optimized parameters.
Asunto(s)
Cartílago Articular/fisiología , Hidrogeles/farmacología , Células Madre Mesenquimatosas/citología , Regeneración/efectos de los fármacos , Cartílago Articular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , FenotipoRESUMEN
Stem cells hold great promise for treating cartilage degenerative diseases such as osteoarthritis (OA). The efficacy of stem cell-based therapy for cartilage repair is highly dependent on their interactions with local cells in the joint. This study aims at evaluating the interactions between osteoarthritic chondrocytes (OACs) and adipose-derived stem cells (ADSCs) using three dimensional (3D) biomimetic hydrogels. To examine the effects of cell distribution on such interactions, ADSCs and OACs were co-cultured in 3D using three co-culture models: conditioned medium (CM), bi-layered, and mixed co-culture with varying cell ratios. Furthermore, the effect of transforming growth factor (TGF)-ß3 supplementation on ADSC-OAC interactions and the resulting cartilage formation was examined. Outcomes were analyzed using quantitative gene expression, cell proliferation, cartilage matrix production, and histology. TGF-ß3 supplementation led to a substantial increase in cartilage matrix depositions in all groups, but had differential effects on OAC-ADSC interactions in different co-culture models. In the absence of TGF-ß3, CM or bi-layered co-culture had negligible effects on gene expression or cartilage formation. With TGF-ß3 supplementation, CM and bi-layered co-culture inhibited cartilage formation by both ADSCs and OACs. In contrast, a mixed co-culture with moderate OAC ratios (25% and 50%) resulted in synergistic interactions with enhanced cartilage matrix deposition and reduced catabolic marker expression. Our results suggested that the interaction between OACs and ADSCs is highly dependent on cell distribution in 3D and soluble factors, which should be taken into consideration when designing stem cell-based therapy for treating OA patients.