Regulatory effect of TGF-β3, BMP-2 and Noggin during the induction of endochondral bone formation

Liu, Heng (2022): Regulatory effect of TGF-β3, BMP-2 and Noggin during the induction of endochondral bone formation: an in vitro study using rectus abdominis muscle from rat. Dissertation, LMU München: Medizinische Fakultät

Abstract

Background: The regeneration of bone remains a worldwide dilemma for tissue engineering, with the biological mechanism regarding proper growth factor/s application in bone tissue engineering (BTE) still require more extensive elucidation. Muscle tissue is an appealing source for studying the effect of morphogenesis as it consists of multiple cell types and is one of the key tissues to assess the induction of bone formation in vivo. Transforming growth factor-beta 3 (TGF-β3) and bone morphogenetic protein-2 (BMP-2) are suggested to play vital roles in endochondral bone formation, while Noggin, acts as an antagonist of certain BMPs, usually plays a regulatory role for osteogenesis. Therefore, the present study applied these three growth factors to induce endochondral bone formation in muscle tissue, with the aim directed towards to confirm the potential of morphogen-induced muscle tissue for BTE and try to unravel the underlining molecular interaction mechanisms during the differentiation process. Methods: A series of recombinant rat (r) BMP-2, rTGF-β3, and rNoggin were applied continuously to the rat collected rectus abdominis muscle tissue over the designated culturing period. The bone induction effect was assessed at 7, 14, and 30 days by immunohistochemistry (IHC), histology, and RT-qPCR. Apart from ultimately aimed osteogenesis biomarkers (Alp, Runx2, Bmp-2, Ocn, Col1a1 genes and OCN protein), the chondrogenesis (Col2a1, Acan, Sox9 genes and ACAN protein), the articular chondrogenesis (Six1 and Abi3bp genes,) and the angiogenesis (Vegfa and Col4a1 genes) related molecules were also assessed. Results: All the detected chondrogenesis and osteogenesis related biomarkers underwent significant upregulation during the differentiation, both at protein and gene level, which happened in nearly all of the stimulated groups but excepted the rNoggin alone and rBMP-2 + rNoggin combination stimulated groups. Besides, the Vegfa and Col4a1 genes were also upregulated in most morphogen/s treated groups. At 7 days, the rBMP-2 single group displayed a peaked and significant gene expression, but rBMP-2 + rTGF-β3 group was non-significant. After that, the rBMP-2 + rTGF-β3 combination showed the highest gene expression on day 14 and kept stable until day 30. rTGF-β3 + rNoggin combination displayed relatively high gene expression on days 7 and 30, but rNoggin stopped the upward trend of rTGF-β3 on day 14. rTGF-β3 + rBMP-2 + rNoggin triple stimulation showed the highest gene expression on day 30. In addition, rBMP-2 application inhibited Bmp-2 gene expression, but rNoggin and rTGF-β3 + rNoggin upregulated Bmp-2 gene expression, and rTGF-β3 + rNoggin combination became the only one that showed significant Bmp-2 gene expression on day 7. Conclusions: Both rBMP-2 and rTGF-β3 showed their capability of inducing muscle tissue to-wards endochondral bone formation alone and stimulating the involved chondrogenesis as well. rNoggin inhibited rBMP-2 activities reliably and repressed rTGF-β3 function in the middle 14-day stage, but showed positive or at least no inhibitory function when used with rTGF-β3 in the initial and late stages of osteo-chondrogenesis and in combination with rTGF-β3 + rBMP-2 in the late stage of differentiation. This strongly suggests that TGF-b3 seems to regulate the Noggins molecule antagonistic effect possibly showing how bone formation is internally modulated by the expression of specific growth factors with time. Additionally, rTGF-β3 + rBMP-2 combination presented an early antagonistic action but later a synergistic interaction. The cellular microenvironment may be a key factor in determining the complex interaction mechanisms among these signaling molecules. Moreover, the negative feedback loops between protein/gene and protein/antag-onist were detected in this differentiation system. The data of this study provided some key evidence in how endochondral bone formation is regulated and shows that only through a temporal specific application of multiple growth factors and other modulatory factors can proper osteogenesis be achieved.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 19. September 2022
• 1. Bericht­erstatter:in: Müller, Peter