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Abstract

Articular cartilage (AC) is a viscoelastic avascular tissue mainly composed of chondrocytes embedded in a rich extracellular matrix that covers the joints and supports load distribution of the joints. The absence of vessels restricts its regenerative capability. Hence, joint motion facilitates nutrient deposition and cell waste disposal. Mechanical stimulation contributes to the homeostasis of functional AC by supporting delivery of nutrients, cytokines and growth factors between the distant chondrocytes. Current techniques to treat AC defects still fail to entirely heal and to achieve a native-like AC. As the knee joint has neighboring niches of stem cells, we hypothesized that mechanical stimulation might enhance the mobilization of endogenous mesenchymal stem/stromal cells (MSCs) from nearby niches as the bone marrow (BM), when the subchondral bone is opened. To test this hypothesis, we developed a compression bioreactor system in vitro for simultaneous application of mechanical stimulation and cell cultivation. This study aimed to evaluate the role of dynamic of mechanical stimulation on mobilizing MSCs toward scaffolds in a bioreactor system. The novel mechanical system for evaluating mobilization of MSCs in a 3D context in vitro consisted of a) a compression bioreactor able to induce loading on scaffolds, b) custom-made software for settings for management and data recording, c) cell loading experiments, and d) 3D image-based biological evaluation. The mechanical stimulation acted on an acellular scaffold made of alginate, functionalized-alginate with laminin-521(alginate-Ln) or collagen-I (col-I), and a cell reservoir containing porcine or human BM-MSCs (pBM-MSCs and hBM-MSCs, respectively) below it. The mechanical loading program was set up as 10 % strain regarding the original height of the scaffold, 24 hours at 0.3 Hz, using dynamic continuous or intermittent loading regime, with breaks of 10 seconds each 180 cycles, when intermittent loading was used. Supporting our hypothesis, we found that intermittent mechanical stimulation induced the mobilization of hBM-MSCs in col-I scaffolds 10-fold compared to the unloaded control (245 ± 42 viable cells/mm3 vs. 22 ± 6 viable cells/mm3, respectively; p-value < 0.0001), as well as pBM-MSCs mobilized 4-fold in alginate-Ln scaffold when intermittently loaded (194 ± 39 cells/mm3 vs. 48 ± 21 cells/mm3 for the unloaded control. In addition, we found that the bioreactor was able to stimulate the scaffolds and the cells for 23.99 ± 0.94 hours in 137.72 ± 13.21 periods, exerting compression with vertical piston displacements of 230.08 ± 54.07 μm, force of 1.08 ± 0.13 N for hBM-MSCs and force-amplitude of 1.86 ± 1.46 N for pBM-MSCs. Remarkably, the viability of mobilized cells was not compromised by intermittent mechanical loading application as evaluated with an optimized and validated protocol for counting and viability cell detection in 3D. As a first step to induce cartilage regeneration in situ, this study shows enriched acellular scaffolds with viable MSCs after mechanical stimulation, and provides an useful tool to understand better the regeneration of AC in situ.