The skeletal system forms the structural framework of the body, with bones providing support, protection, and mobility. Bone tissue is primarily composed of collagen, minerals such as calcium, and specialized non-collagenous proteins that together provide strength and flexibility. Bone health is maintained through the coordinated activity of osteoblasts, the cells responsible for building and mineralizing bone, osteoclasts, which break down bone tissue, and osteocytes, which help regulate bone remodeling. Among these, osteoblasts play a particularly important role in biomineralization, the process through which minerals are deposited within the collagen framework to form hardened bone tissue. However, the molecular mechanisms regulating osteoblast differentiation and mineralization remain incompletely understood.
Previous studies identified carbonic anhydrase III (CAR3) as a protein associated with osteoblast differentiation, with its expression increasing during osteoblast maturation. However, its precise role in skeletal development and bone formation had remained unclear.
To address this gap, a research team led by Dr. Fangfang Song and Professor Yufeng Zhang from Wuhan University investigated the function of CAR3 in skeletal development using genetically modified mouse models. Their findings were published on May 19, 2026, in the International Journal of Oral Science .
"As osteoblasts have critical roles in skeletal development, we examined the molecular mechanisms involved in osteoblast differentiation, which can potentially aid in developing novel therapeutic strategies for bone disorders," explained Dr. Song while discussing the motivation behind the study.
The researchers first analyzed publicly available single-cell RNA sequencing datasets from developing mouse cranial bone to examine the spatial and temporal expression of Car3, the gene encoding CAR3. They found that Car3 was highly activated in osteoblast-lineage cells during early embryonic cranial development, particularly between embryonic days 14.5 and 15.5, a period associated with active bone mineralization. In addition to craniofacial bones, Car3 expression was also observed in limb, rib, and spinal bones. Interestingly, while Car3 expression in young mice was closely associated with collagen-producing osteoblasts, its expression shifted toward adipocytes in aged mice.
Further experiments revealed that RUNX2, a master transcription factor involved in bone formation, directly regulated Car3 expression during osteoblast differentiation. The researchers also discovered that CAR3 formed a molecular complex with collagen type I alpha 1 (COL1A1) and subsequently recruited bone sialoprotein (BSP), a key structural component involved in mineral deposition. This ternary complex promoted collagen intrafibrillar mineralization, an essential process for generating strong and properly mineralized bone tissue.
To examine the functional importance of CAR3, the team selectively deleted Car3 in Prx1-lineage skeletal stem cells, which contribute to the development of skeletal and connective tissues. Although early femur development remained largely unaffected, adult mice lacking Car3 showed impaired osteoblast activity, defective collagen mineralization, reduced bone formation, and decreased bone density, highlighting the critical role of CAR3 in maintaining skeletal integrity.
The researchers then explored the therapeutic potential of CAR3 in bone regeneration. In mouse models with bone defects, implantation of recombinant CAR3-functionalized collagen scaffolds significantly enhanced new bone formation, increased bone volume, promoted osteoblast recruitment, and improved bone matrix mineralization after eight weeks.
Commenting on the potential implications of the findings, Prof. Zhang stated: "Our mouse experiments revealed that application of collagen functionalized with CAR3 promoted bone formation. Thus, the regulatory effects of Car3 on osteoblast differentiation can be harnessed to treat bone disorders."
Together, these findings identify CAR3 as a previously unrecognized regulator of osteoblast differentiation and collagen mineralization. By clarifying how CAR3 coordinates bone formation and regeneration, the study provides new insights into skeletal biology and highlights the potential of CAR3-based biomaterials and regenerative strategies for treating osteoporosis, fractures, and other bone disorders.
