Poster V13 - Construction of an Optimization System for Effective Mechanical Stimulation by in situ Predicted Cartilage Matrix Gene Expression

Introduction: Mechanical Stimulation is recognized to enhance the production of extracellular matrix (ECM) proteins in cultured cartilage. Traditional approaches to defining mechanical stimulation conditions rely on physical cues present in the natural environment, such as 1-12% tensile strain, 0.1-25% compressive strain and 0.4-20% shear strain induced in walking, running, and daily exercise, and these conditions are maintained consistently throughout the culture period. However, a heterogeneous mechanical field is generated in cartilage tissue by changes in the orientation of collagen fibers and protein density, which allows chondrocytes to receive various mechanical stimuli depending on their location within the tissue. Furthermore, this mechanical field gradually changes with the development of chondrogenesis and tissue maturation. Therefore, it is crucial to consider the mechanical field and temporal cellular responses to apply effective mechanical stimulation for efficient ECM production. In this study, the function of a gene-specific promoter was used to identify both the expression level and location of the target gene during cell culture. This approach introduces a novel system that adapts stimuli based on the mechanical field surrounding chondrocytes and the temporal cellular responses, utilizing in-situ estimation of Col2a1 mRNA expression, with the aiming of achieving more effective production of ECM.

Materials and

Methods: Plasmid vector: The previously identified Co2a1 promoter and enhancer regions for mouse chondrogenesis were inserted into the multi-cloning site of the pZsGreen1-1 vector (Col2a1-ZsGreen1). The cytomegalovirus promoter-driven DsRed-Express2 was used to correct the transfection efficiency and chondrogenesis level.

Mechanical stimulation: ATDC5 cells were seeded at 2.4 × 10^4 cells/cm^2 on a transparent silicone sheet coated with type I collagen. After 18 hours, Col2a1-ZsGreen1 and CMV-DsRed-Express2 plasmid vectors were transfected and incubated for the next 24 hours. The tensile displacement of the sheet was controlled to generate 1-11% of tensile strain (εxx) and 1-5% of compressive strain (εyy), and the combination of strain distributions varied depending on the tensile displacement. The tensile displacement was adjusted every 24 hours to maximize the predicted fluorescence intensity that expresses Col2a1 gene expression in an entire sheet. The predicted fluorescence values were calculated by using the historical information from day 1 to the day of observation at each strain field. These predicted fluorescence values were estimated using a Response Surface Method (RSM) composed of εxx, εyy, and the historical gene expression at each strain. The initial displacement was set at 0.96 mm and cyclic tensile stimulation (0.5 Hz, 5 hours/day) was applied. The control group without mechanical stimulation was prepared to evaluate the effect of variable mechanical stimulation.

Results, Conclusions, and Discussions: Results & Discussions: The fluorescence values of each strain filed, including the estimated values using RSM on day 1, are presented in Fig.1 (Bold: observation values, Normal: estimated values). It was observed that the fluorescence values on day 1 were equal to or higher than those of the control group (εxx, εyy=0,0). Figure 2 shows the changes in total fluorescence intensity on each culture day in both the control and stimulation groups, respectively. The total fluorescence intensity within the sheet was calculated by multiplying each fluorescence value by the area of strain expression in the stimulation group. Fluorescence levels in both groups increased throughout the culture period, and the stimulation group consistently exhibited higher levels than the control group. In addition, the transition of changes in tensile displacement applied to the silicone sheet is also plotted in Fig.2. The control group, which was not subjected to mechanical stimulation, exhibited an increase of fluorescence intensity, indicating that ATDC5 cells naturally progressed chondrocytic differentiation during culture. In the variable mechanical stimulation group, the sheet displacement initially generated tensile strain (εxx) of 2-7% and was subsequently adjusted to increase to 5-11%, leading to an increase in the proportion of high-strain regions in the sheet. However, the proportion of high-strain regions decreased on days 5 and 6. It has been reported that Col2a1 expression in chondrocytes increases at approximately 3-10% tensile strain. However, excessive tensile strain on chondrocytes can lead to cartilage degradation. Furthermore, the total fluorescence value calculated by the function of the response surface includes the effects of compressive strain (εyy) as well as those of tensile strain (εxx) on chondrocytes. Since the function of the response surface was modulated by predicted gene expression based on the expression history at each strain field, the effects of mechanical stress would be effectively reflected in the later period.

Conclusions: This study established a novel system capable of variably controlling optimal mechanical stimulation according to cellular response. This system might facilitate the comprehensive evaluation of target genes in response to mechanical fields and the generation of effective mechanical stimulation patterns for cartilage matrix production.

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