Study on Cav3.3-Mediated Endochondral Ossification in 3D Bioprinted GelMA Hydrogel

Research Background

Bone development is a complex process, in which the growth plate (GP) plays a crucial role in the longitudinal growth of long bones. The growth plate regulates bone maturation through the process of endochondral ossification (EO). However, GP dysfunction can lead to growth retardation and skeletal dysplasia. Although research on the growth plate is significant for understanding bone development and related diseases, in vivo studies are limited due to its complex spatiotemporal changes. In recent years, the development of three-dimensional (3D) bioprinting technology has provided new models for studying the physiological and pathological functions of the growth plate in vitro. However, simulating the endochondral ossification process remains a key challenge in bone organoid research.

This study aims to explore the role of the T-type voltage-dependent calcium channel (T-VDCC) subtype Cav3.3 in the hypertrophic differentiation of chondrocytes and to simulate the endochondral ossification process using a 3D bioprinted gelatin methacryloyl (GelMA) hydrogel model. This research not only provides new insights into understanding bone development but also offers potential technical support for the construction of bone organoids.

Source of the Paper

This study was conducted by researchers including Zhi Wang, Xin Wang, and Yang Huang from the Chongqing Key Laboratory of Precision Medicine in Joint Surgery, Center for Joint Surgery, Southwest Hospital, Army Medical University (Third Military Medical University). The paper was published online on November 26, 2024, in the journal Bio-design and Manufacturing, with the DOI 10.1007/s42242-024-00287-1.

Research Process and Results

1. Research Process

a) Animal Experiments and Chondrocyte Isolation

The study first used 12 C57BL/6 mice and 9 Cacna1i knockout mice (Cacna1i−/−) at different ages (1, 2, 4, and 6 months). The development of the growth plate was observed using hematoxylin-eosin (H&E) staining and safranin O/fast green staining. The results showed that the growth plates of Cacna1i−/− mice exhibited accelerated ossification at 4 months, similar to that of 6-month-old wild-type mice, indicating that Cav3.3 plays a negative regulatory role in the maturation of the growth plate.

b) Chondrocyte Culture and Cav3.3 Interference

Primary chondrocytes were isolated from the femoral condyles of mice, and the ATDC5 cell line was used for experiments. Cav3.3-specific knockdown ATDC5 cells (shCav3.3) were constructed using shRNA technology, and the downregulation of Cav3.3 expression was verified through immunofluorescence staining, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blot. The results showed that Cav3.3 expression was significantly reduced in shCav3.3 cells, and the intensity of intracellular calcium signaling was significantly decreased.

c) Preparation of GelMA Hydrogel and 3D Bioprinting

The study used 5% GelMA hydrogel as a bioink to construct a 5 mm × 5 mm × 1.5 mm hydrogel scaffold using 3D bioprinting technology. Rheological tests showed that the GelMA hydrogel had good printability at 15°C. The printed hydrogel scaffold was crosslinked by irradiation with 405 nm ultraviolet light for 10 seconds, and the cell survival rate exceeded 90%. Scanning electron microscopy (SEM) revealed that the hydrogel scaffold had a uniform pore structure suitable for cell adhesion and proliferation.

d) 3D Culture and Analysis of Hypertrophic Differentiation-Related Gene Expression

shCav3.3 cells were loaded into the GelMA hydrogel for 3D culture, and differentially expressed genes (DEGs) were analyzed through mRNA sequencing. The results showed that after Cav3.3 knockdown, extracellular matrix (ECM)-related genes and hypertrophic differentiation-related genes (such as Shh and TGF-β) were significantly upregulated. Western blot and immunofluorescence staining further confirmed that the expression of hypertrophic chondrocyte markers (such as Col-X, MMP-13, and Runx-2) was significantly increased in shCav3.3 cells, while the expression of hyaline cartilage markers (such as Col-II, Sox-9, and Acan) was significantly reduced.

e) Mineralization Process and Alkaline Phosphatase (ALP) Activity Detection

The deposition of ECM in the GelMA hydrogel was evaluated using alcian blue staining and alizarin red S staining. The results showed that calcium deposition was significantly increased in the shCav3.3 group, while the intensity of hyaline cartilage markers was significantly reduced. Additionally, ALP activity assays indicated that ALP activity in the shCav3.3 group increased significantly over time, further confirming the role of Cav3.3 in regulating chondrocyte hypertrophic differentiation and mineralization.

2. Main Results

• Expression and Function of Cav3.3 in Chondrocytes: Cav3.3 is the most highly expressed T-VDCC subtype in chondrocytes, and its knockdown significantly reduced intracellular calcium signaling intensity and accelerated chondrocyte hypertrophic differentiation.
• Printability and Biocompatibility of GelMA Hydrogel: The 5% GelMA hydrogel exhibited good printability and biocompatibility at 15°C, making it suitable for 3D bioprinting.
• Promotion of Hypertrophic Differentiation in 3D Culture: In the 3D culture environment, Cav3.3 knockdown cells showed a stronger tendency toward hypertrophic differentiation, with significant upregulation of ECM-related genes and hypertrophic differentiation-related genes.
• Simulation of the Mineralization Process: The GelMA hydrogel loaded with shCav3.3 successfully simulated the mineralization process during endochondral ossification, providing a functional bioink for the future construction of layered and orderly mineralized growth plate organoids.

3. Conclusions and Significance

This study is the first to confirm the negative regulatory role of Cav3.3 in chondrocyte hypertrophic differentiation and to successfully simulate the endochondral ossification process using a 3D bioprinted GelMA hydrogel model. This research not only provides new molecular mechanisms for understanding bone development but also offers potential technical support for the construction of bone organoids. Future studies can further explore the therapeutic potential of Cav3.3 in skeletal developmental diseases and optimize 3D bioprinting technology to achieve more complex bone organoid construction.

4. Research Highlights

• Novel Research Perspective: First revealed the negative regulatory role of Cav3.3 in chondrocyte hypertrophic differentiation.
• Innovative Experimental Methods: Combined 3D bioprinting technology with GelMA hydrogel to successfully simulate the endochondral ossification process.
• Potential Application Value: Provides a new technical platform for the construction of bone organoids and research on skeletal developmental diseases.

Summary

This study revealed the key role of Cav3.3 in chondrocyte hypertrophic differentiation by regulating its expression and successfully simulated the endochondral ossification process using 3D bioprinting technology. This research not only deepens our understanding of the mechanisms of bone development but also provides new ideas for the construction of bone organoids and the treatment of related diseases.