Mapping the Response of Human Osteocytes in Native Matrix to Mechanical Loading Using RNA Sequencing
JBMR PLUS(2023)
Abstract
ABSTRACTOsteocytes sense mechanical loads and transduce mechanical signals into a chemical response. They are the most abundant bone cells deeply embedded in mineralized bone matrix, which affects their regulatory activity in the mechanical adaptation of bone. The specific location in the calcified bone matrix hinders studies on osteocytes in the in vivo setting. Recently, we developed a three‐dimensional mechanical loading model of human osteocytes in their native matrix, allowing to study osteocyte mechanoresponsive target gene expression in vitro. Here we aimed to identify differentially expressed genes by mapping the response of human primary osteocytes in their native matrix to mechanical loading using RNA sequencing. Human fibular bone was retrieved from 10 donors (age: 32–82 years, 5 female, 5 male). Cortical bone explants (8.0 × 3.0 × 1.5 mm; length × width × height) were either not loaded or mechanically loaded by 2000 or 8000 μɛ for 5 minutes, followed by 0, 6, or 24 hours post‐culture without loading. High‐quality RNA was isolated, and differential gene expression analysis performed by R2 platform. Real‐time PCR was used to confirm differentially expressed genes. Twenty‐eight genes were differentially expressed between unloaded and loaded (2000 or 8000 μɛ) bone at 6 hours post‐culture, and 19 genes at 24 hours post‐culture. Eleven of these genes were related to bone metabolism, ie, EGR1, FAF1, H3F3B, PAN2, RNF213, SAMD4A, and TBC1D24 at 6 hours post‐culture, and EGFEM1P, HOXD4, SNORD91B, and SNX9 at 24 hours post‐culture. Mechanical loading significantly decreased RNF213 gene expression, which was confirmed by real‐time PCR. In conclusion, mechanically loaded osteocytes differentially expressed 47 genes, of which 11 genes were related to bone metabolism. RNF213 might play a role in mechanical adaptation of bone by regulating angiogenesis, which is a prerequisite for successful bone formation. The functional aspects of the differentially expressed genes in bone mechanical adaptation requires future investigation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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Key words
3D,HUMAN BONE,MECHANICAL LOADING,NATIVE MATRIX,OSTEOCYTES,RNA SEQUENCING
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