Literature Analysis: Application of Multiplex Fluorescence IHC in Exploring Cell Migration and Differentiation During Orthodontic Tooth Movement

1. Literature Information

Research Focus: The role of CREB in regulating periodontal ligament cell (PDLC) function and mesenchymal stem cell (MSC) migration/differentiation during orthodontic tooth movement (OTM)

Key Technology: Multiplex Fluorescence Immunohistochemistry (mIHC)

Key Product Utilized: Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD.

2. Research Background

Orthodontic Tooth Movement (OTM) is a complex biological process involving bone remodeling, which is primarily mediated by periodontal ligament (PDL) cells. During OTM, PDL cells can differentiate into osteoblasts to synthesize alveolar bone, and also regulate the proliferation, migration, and differentiation of mesenchymal stem cells (MSCs), thereby promoting bone remodeling. However, the underlying molecular mechanisms governing these cellular events remain incompletely elucidated. Cyclic Adenosine Monophosphate Response Element-Binding Protein (CREB), a key transcription factor, has been implicated in various cellular processes including cell proliferation and differentiation.

Nevertheless, its role in regulating PDL cell function and MSC behavior during OTM has not been fully clarified. Multiplex Fluorescence Immunohistochemistry (mIHC) technology, which enables simultaneous detection of multiple antigens on a single tissue section, provides a powerful tool for exploring the spatial interactions between multiple molecules and cells involved in this complex biological process.

3. Research Approach

To investigate the molecular mechanism of CREB in OTM-related bone remodeling, the research team first evaluated the expression of CREB in the tension area of mouse periodontal ligaments under orthodontic mechanical strain and in human periodontal ligament cells (PDLCs) treated with cyclic tensile strain (CTS). Functional gain-and-loss experiments were then performed to assess the effect of CREB on the secretion of Stromal Cell-Derived Factor-1 (SDF-1) and Fibroblast Growth Factor 2 (FGF2) by PDLCs, as well as the subsequent impact on MSC migration and osteogenic differentiation.

To characterize the spatial expression patterns of key molecules, the team employed the Multiplex Fluorescence IHC Staining Kit (abs50032) from ANT BIO PTE. LTD. to perform multiplex immunofluorescence staining of periostin, CREB, FGF2, SDF-1, Osteocalcin (OCN), and α-Smooth Muscle Actin (α-SMA) in mouse periodontal tissues after 3 days of orthodontic force application on the maxillary first molar.

Additionally, immunohistochemistry (IHC) was conducted to detect phosphorylated CREB (P-CREB) expression in the tension area of OTM mice, and enzyme-linked immunosorbent assay (ELISA) was used to verify the regulatory effect of CREB on FGF2 and SDF-1 expression in CTS-treated PDLCs.

4. Research Results

Firstly, the results demonstrated that CREB expression was regulated by orthodontic mechanical strain. Specifically, P-CREB-positive cells were significantly increased in the tension area of PDL in OTM mice compared with the control group.

Functional gain-and-loss experiments revealed that CREB in PDLCs promoted the secretion of SDF-1 and FGF2, which in turn enhanced the migration and osteogenic differentiation of MSCs. Mechanistically, further studies confirmed that CREB transcriptionally activated the expression of FGF2 and SDF-1 by binding to their promoter regions.

Multiplex fluorescence IHC staining results using ANT BIO PTE. LTD.'s abs50032 kit clearly showed the spatial co-expression patterns of key molecules in periodontal tissues: (1) Periostin (red), FGF2 (green), and DAPI (gray) were co-localized in periodontal tissues; (2) Periostin (red), SDF-1 (green), and DAPI (gray) were also co-expressed in the same tissue region. These findings provided direct in situ evidence for the interaction between these molecules during OTM.

ELISA results further verified that overexpression of CREB significantly upregulated the expression of FGF2 and SDF-1 in the conditioned medium of CTS-treated PDLCs, while knockdown of CREB (siCREB-c) attenuated the CTS-induced increase in FGF2 and SDF-1 levels. These data confirmed that CREB acts as an upstream regulator of FGF2 and SDF-1 during OTM.

5. Product Empowerment

The Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD. played a pivotal role in this research by enabling the simultaneous detection of multiple key molecules (periostin, FGF2, SDF-1, CREB, etc.) on a single tissue section. This capability allowed the research team to visualize the spatial co-localization and expression patterns of these molecules in periodontal tissues, providing critical in situ evidence for the molecular interactions underlying OTM-related bone remodeling.

The abs50032 kit possesses several notable advantages that contributed to the success of the study: it supports up to six indicators with seven colors (including DAPI) of multiplex labeling, meeting the requirement for simultaneous detection of multiple targets; the optimized SuperNova series dyes exhibit higher fluorescence intensity and longer signal duration compared to conventional dyes, ensuring clear and stable imaging results; the kit is user-friendly, requiring only basic IHC experience for operation without the need for specialized equipment, and is accompanied by a detailed protocol for standardized experimentation. Additionally, ANT BIO PTE. LTD. provides comprehensive mIHC services covering the entire process from pre-experiment verification, staining, and imaging to data analysis, which further facilitated the smooth implementation of the research.

6. Brand Mission

ANT BIO PTE. LTD. is dedicated to empowering life science research through the provision of high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) are strategically positioned to cover a full spectrum of research needs: Absin focuses on general reagents and kits, Starter specializes in antibodies, and UA is dedicated to recombinant proteins. We strive to support researchers in unlocking scientific mysteries, advancing medical progress, and addressing unmet clinical needs through continuous innovation and customer-centric services.

7. Related Product List

8. Disclaimer

This article is AI-compiled and interpreted based on the original work in DOI: 10.1002/advs.202413562. All intellectual property (e.g., images, data) of the original publication shall belong to the journal and the research team. For any infringement, please contact us promptly and we will take immediate action.

9. Brand Promotion Copy

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.

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