Table 2.
Application of in vitro techniques in signalling pathway studies.
| In vitro technique | Type of method | Principle | Samples Analysed | Key Findings | References |
|---|---|---|---|---|---|
| Genomics | Microarray analysis | Identification of gene expression profiles involved in osteogenic differentiation | Study of gene expression profiling microarray data during osteogenic differentiation of BM-MSCs, focusing on several signalling pathways based on KEGG pathway analysis: LPS-mediated, PI3K/AKT, and Wnt. | Microarray data from database repository revealed several upregulated differentially expressed genes (DEGs) related to the biological processes and pathways. | 99 |
| Real-time PCR (qRT-PCR) | Assessment of expression of bone-specific markers or gene expression of the signalling molecules in the osteogenic differentiated cells | Study of the effect of the PI3K inhibitor, LY3023414, on osteogenesis and osteoclastogenesis for bone remodelling using a preosteoblast cell line and bone marrow-derived macrophage cells (BMMs). | Treatment with a PI3K inhibitor led to reduced mRNA expression of osteoblast-specific genes in the preosteoblast cell line as well as expression of AKT in the preosteoblast cell during osteoclastogenesis in the BMMs. | 96 | |
| Proteomics | Specific inhibition assay | Assessment of the effect of pathway knockdown | Preosteoblast cell line MC3T3-E1 was subjected to a specific inhibitor targeting the PI3K pathway | Effect of inhibitor treatment decreased the mRNA expression of osteoblast-specific genes (Runx2, ALP, OCN), and expression of phosphorylated AKT related to the downstream process of the pathway. | 96,100 |
| Western blotting | Evaluation of protein expression and signalling molecules after cells were treated with pathway inhibitors. . |
MC3T3-E1 cells treated with C3G to enhance osteoblast regulation were subjected to ERK1/2 inhibition to study the involvement of the ERK/MAPK pathway in osteoblast proliferation. | Inhibition resulted in decreased phosphorylated ERK1/2 and OCN protein expression. Although Runx2 and ALP protein were not inhibited, it can be concluded that ERK was partially responsible for osteoblast differentiation due to the inhibitor effect on OCN. | 101 | |
| Immunostaining | Analysis of protein expression, distribution, and localisation of osteogenic differentiated cells | Immunofluorescence staining to examine the expression level of type I collagen in β-ecdysterone-treated MC3T3-E1 cells to study the osteogenic effect. | Expression of type I collagen in the treated group was significantly increased compared to the control group, which shows that β-ecdysterone can be implemented for bone regeneration due to its osteogenic properties. | 102 | |
| Mass spectrometry (MS) | Analysis of intercellular signalling proteins and the regulation of cellular posttranslational modifications | Comparative proteomics of gingival tissues and alveolar bone after tooth extraction to study the protein interaction and molecular mechanisms involved in periodontal bone tissue healing. | Several proteins and canonical pathways in both soft tissue and bone are interconnected for cellular organization and maintenance. This shows that both soft tissue and hard tissue are involved in bone repair and regeneration. | 103 |