Bone Tissue Engineering: Regulation of the BMP Pathway for Enhance Bone Tissue Regeneration

Bone is a dynamic tissue pivotal for providing structural support and enabling movement in the human body. Growth factors are essential for bone tissue remodeling because of their regulatory functions, such as cell adhesion, proliferation, and differentiation. Key growth factors involved in this process include vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). BMPs, which belong to the Transforming Growth Factor-Beta (TGF-β) signaling pathway, especially BMP-2, are increasingly utilized in bone tissue engineering due to their osteoinductive effects, which have the potential to enhance and facilitate bone tissue repair [1].

Regulation of biological pathways using molecular techniques has great potential in various fields including basic sciences, pharmaceutical industry, biotechnology, and tissue engineering. In the past 10 years, the knockdown of target genes by siRNAs has been the most important development in molecular biology studies. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)associated protein 9 (CRISPR-Cas9) has been the most popular genome-editing system [2].

Noggin, Chordin, Gremlin (Grem1), and twisted gastrulation-1 (Twsg1) are pivotal antagonists secreted to regulate BMP activity. These antagonists bind to BMP and prevent the formation of the BMP/receptor complex on the plasma membrane. In particular, Noggin serves as a primary extracellular antagonist of BMP-2. Targeting Noggin, by siRNA-mediated knockdown, shows promise for reducing the necessary BMP-2 dosage, thereby enhancing osteogenic differentiation. Various molecular biology and tissue engineering methods, including gene delivery by 3D scaffolds and gene editing approaches like CRISPR/Cas9, CRISPRa, and CRISPRi, are being explored for their potential roles in bone tissue regeneration [2].

In my master's studies, we were suppressed the synthesis of Noggin protein, an antagonist of the BMP signaling pathway, using Noggin-targeted siRNA (siNoggin) on the MC3T3-E1 pre-osteoblasts Afterward, the osteogenic differentiation of cells transfected with siNoggin was investigated on silk scaffolds. As a result, the osteogenic effect of BMP-2 and long-term Nog gene suppression was observed in siNoggin transfected cells with a 5-fold increase in Ocn gene expression. Also, we found that the siNoggin transfected cells formed dense fiber structures with mineralized regions, and dense calcium and phosphorus elements were accumulated on siNoggin transfected cells. Based on the results of this study, it has been concluded that using siNoggin transfected pre-osteoblastic cells together with silk scaffolds can be beneficial in bone tissue engineering by inducing differentiation.

Also, during my master's education, I participated in the Tübitak project. In this project, we hypothesized that the generation of Noggin knockout cells by using the CRISPR/Cas9 mechanism would show an enhanced osteogenic differentiation in 3D scaffolds and could be used as a functional cell source for bone tissue engineering. Therefore, in this study, expression of the BMP-2 antagonist Noggin in C2C12 cells was knocked out using CRISPR/Cas9, and genetically modified cells were seeded on controlled BMP-2-loaded silk scaffolds. Our findings demonstrated that: (i) a new cell source for bone tissue regeneration was obtained, (ii) BMP signaling was internally controlled by editing Noggin expression to reduce the required dose of BMP- 2, (iii) Noggin knockout and BMP-2 synergistically induced osteogenic differentiation of cells, and (iv) the CRISPR/Cas9 system has been proposed as an alternative endogenous approach to the use of high-dose exogenous growth factor.

In conclusion, the combined use of Noggin suppression and controlled release of BMP-2 has great potential for osteogenic differentiation of cells for bone tissue regeneration.

References:
[1] Fuerkaiti, S. N.; Çakmak, A. S.; Karaaslan, Ç.; Gümüşderelioğlu, M. Enhanced Osteogenic Effect in Reduced BMP-2 Doses with siNoggin Transfected Pre-Osteoblasts in 3D Silk Scaffolds. Int. J. Pharm. 2022, 612, No. 121352.
[2] Çakmak, A.S.; Fuerkaiti, S.; Karagüzel, D.; Karaaslan, Ç.; Gümüşderelioğlu, M. Enhanced Osteogenic Potential of Noggin Knockout C2C12 Cells on BMP-2 Releasing Silk Scaffolds. ACS Biomater. Sci. Eng. 2023, 9, 6175–6185.