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Ye L, Li H, Zhang W, Zhou Y, Lan X, Wang Y. Blue light-emitting diode promotes mineralization of stem cells from the apical papilla via cryptochrome 1/Wnt/β-catenin signaling. J Mol Histol 2025; 56:125. [PMID: 40167571 DOI: 10.1007/s10735-025-10400-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2024] [Accepted: 03/16/2025] [Indexed: 04/02/2025]
Abstract
This study aimed to determine whether low-intensity blue LED light (4 J/cm2) promotes mineralization of stem cells from the apical papilla (SCAPs) by modulating CRY1 expression and to elucidate the underlying molecular mechanisms. SCAPs identity was validated using flow cytometry. In a controlled experimental design, SCAPs were exposed to blue LED light, followed by quantitative assessment of CRY1 and osteogenic markers (Runx2, OSX, DSPP, DMP-1) via qRT-PCR, Western blotting, and osteogenic staining. To investigate the role of CRY1 in SCAPs osteogenic differentiation, CRY1 was overexpressed using lentiviral transfection. Additionally, the Wnt/β-catenin pathway was analyzed using specific inhibitors (XAV-939) to elucidate the underlying molecular mechanisms. Blue LED irradiation reduced CRY1 mRNA expression to 80% (day 7) and 45% (day 14) of control levels. CRY1 overexpression significantly increased CRY1 mRNA and protein levels (P < 0.001) but decreased ALP activity and ARS staining (P < 0.001). Blue LED treatment restored mineralization parameters to 80% of control levels. Key osteogenic genes (DMP-1, DSPP, Runx2, OSX) showed lower mRNA and protein levels in the CRY1 overexpression group compared to controls. Blue LED exposure increased these levels to 60-74% (mRNA) and 45-67% (protein) of control values. In the Wnt/β-catenin pathway, CRY1 overexpression elevated GSK-3β and reduced p-GSK-3β, β-catenin, and nuclear β-catenin levels. Blue LED treatment restored these levels to 33-54% of control values, indicating pathway activation. Inhibition of the Wnt/β-catenin pathway (using XAV-939) abolished differences in osteogenic gene expression and mineralization between CRY1 overexpression and blue LED-treated groups, confirming its critical role. Blue LED light at 4 J/cm2 enhances SCAPs mineralization by modulating CRY1 expression and activating the Wnt/β-catenin pathway. These findings provide mechanistic insights into photobiomodulation (PBM) in bone regeneration and highlight its potential for tissue engineering and regenerative medicine.
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Affiliation(s)
- Lin Ye
- Department of Preventive Dentistry, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou, 646000, China
- Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou, 646000, China
- Institute of Stomatology, Southwest Medical University, Luzhou, 646000, China
| | - Hao Li
- Institute of Stomatology, Southwest Medical University, Luzhou, 646000, China
- Pangang Group General Hospital, Panzhihua, 617023, China
| | - Wantong Zhang
- Department of Preventive Dentistry, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou, 646000, China
- Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou, 646000, China
- Institute of Stomatology, Southwest Medical University, Luzhou, 646000, China
| | - Yan Zhou
- Department of Preventive Dentistry, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou, 646000, China
- Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou, 646000, China
- Institute of Stomatology, Southwest Medical University, Luzhou, 646000, China
| | - Xiaorong Lan
- Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou, 646000, China
| | - Yao Wang
- Department of Preventive Dentistry, The Affiliated Stomatological Hospital of Southwest Medical University, Luzhou, 646000, China.
- Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou, 646000, China.
- Institute of Stomatology, Southwest Medical University, Luzhou, 646000, China.
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Jia Y, Duan M, Yang Y, Li D, Wang D, Tang Z. The local pulsatile parathyroid hormone delivery system induces the osteogenic differentiation of dental pulp mesenchymal stem cells to reconstruct mandibular defects. Stem Cell Res Ther 2025; 16:119. [PMID: 40050973 PMCID: PMC11887249 DOI: 10.1186/s13287-025-04258-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2025] [Accepted: 02/27/2025] [Indexed: 03/09/2025] Open
Abstract
BACKGROUND Tumors and injuries often lead to large mandibular defects. Accelerating the osteogenesis of large bone defect areas is a major concern in current research. In this study, dental pulp mesenchymal stem cells (DPSCs) were used as seed cells, and the local pulsatile parathyroid hormone (PTH) delivery system was used as an osteogenic-inducing active ingredient to act on DPSCs and osteoblasts, which were applied to the jaw defect area to evaluate its therapeutic effect on bone regeneration. METHODS Pulsatile delivery systems, both with and without PTH, were developed following the protocols outlined in our previous study. In vitro, the biocompatibility of the pulsatile delivery system with DPSCs was assessed using the Cell Counting Kit-8 (CCK8) assay and live/dead cell staining. Osteogenic differentiation was evaluated through alkaline phosphatase staining and alizarin red staining. In vivo, critical bone defects with a diameter of 10 mm were created in the mandibles of white rabbits. The osteogenic effect was further assessed through gross observation, X-ray imaging, and histological examination. RESULTS In vitro experiments using CCK8 assays and live/dead cell staining demonstrated that DPSCs successfully adhered to the surface of the PTH pulsatile delivery system, showing no significant difference compared to the control group. Furthermore, alkaline phosphatase staining and Alizarin Red staining confirmed that the localized pulsatile parathyroid hormone delivery system effectively induced the differentiation of DPSCs into osteoblasts, leading to the secretion of abundant calcium nodules. Animal studies further revealed that the PTH pulsatile delivery system promoted the osteogenic differentiation of DPSCs, facilitating the repair of critical mandibular bone defects. CONCLUSION The rhythmic release of PTH from the pulsatile delivery system effectively induces the osteogenic differentiation of DPSCs. By leveraging the synergistic interaction between PTH and DPSCs, this approach facilitates the repair of extensive mandibular bone defects.
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Affiliation(s)
- Yuanyuan Jia
- Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Guizhou Medical University, Guiyang, 550000, China
| | - Mianmian Duan
- Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Guizhou Medical University, Guiyang, 550000, China
| | - Yan Yang
- Center for Tissue Engineering and Stem Cell Research, Guizhou Medical University, Guiyang, 550000, China
| | - Duchenhui Li
- Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Guizhou Medical University, Guiyang, 550000, China
| | - Dongxiang Wang
- Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Guizhou Medical University, Guiyang, 550000, China
| | - Zhenglong Tang
- Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Guizhou Medical University, Guiyang, 550000, China.
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Liu C, Yu J, Liu B, Liu M, Song G, Zhu L, Peng B. BACH1 regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells. BMC Oral Health 2022; 22:536. [DOI: 10.1186/s12903-022-02588-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Accepted: 11/14/2022] [Indexed: 11/25/2022] Open
Abstract
Abstract
Background
The preservation of biological and physiological vitality as well as the formation of dentin are among the main tasks of human dental pulp for a life time. Odontoblastic differentiation of human dental pulp stem cells (hDPSCs) exhibits the capacity of dental pulp regeneration and dentin complex rebuilding. Exploration of the mechanisms regulating differentiation and proliferation of hDPSCs may help to investigate potential clinical applications. BTB and CNC homology 1 (BACH1) is a transcription repressor engaged in the regulation of multiple cellular functions. This study aimed to investigate the effects of BACH1 on the proliferation and odontoblastic differentiation of hDPSCs in vitro.
Methods
hDPSCs and pulpal tissues were obtained from extracted human premolars or third molars. The distribution of BACH1 was detected by immunohistochemistry. The mRNA and protein expression of BACH1 were examined by qRT-PCR and Western blot analysis. BACH1 expression was regulated by stable lentivirus-mediated transfection. Cell proliferation and cell cycle were assessed by cell counting kit-8 assay, 5-Ethynyl-2'-deoxyuridine assay and flow cytometry. The expression of mineralization markers, alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability.
Results
BACH1 expression was stronger in the odontoblast layer than in the cell rich zone. The total and nuclear protein level of BACH1 during odontoblastic differentiation was downregulated initially and then upregulated gradually. Knockdown of BACH1 greatly inhibited cell proliferation, arrested cell cycle, upregulated the heme oxygenase-1 (HO-1) expression and attenuated ALP activity, decreased calcium deposits and downregulated the expression of mineralization markers. Treatment of Tin-protoporphyrin IX, an HO-1 inhibitor, failed to rescue the impaired odonto/osteogenic differentiation capacity. Overexpression of BACH1 increased cell proliferation, ALP activity and the expression of mineralization markers.
Conclusions
Our findings suggest that BACH1 is an important regulator of the proliferation and odontoblastic differentiation of hDPSCs in vitro. Manipulation of BACH1 expression may provide an opportunity to promote the regenerative capacity of hDPSCs.
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Novoa Díaz MB, Carriere P, Gigola G, Zwenger AO, Calvo N, Gentili C. Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide. World J Gastroenterol 2022; 28:3177-3200. [PMID: 36051345 PMCID: PMC9331538 DOI: 10.3748/wjg.v28.i26.3177] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Revised: 03/21/2022] [Accepted: 05/28/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Parathyroid hormone-related peptide (PTHrP) plays a key role in the development and progression of many tumors. We found that in colorectal cancer (CRC) HCT116 cells, the binding of PTHrP to its receptor PTHR type 1 (PTHR1) activates events associated with an aggressive phenotype. In HCT116 cell xenografts, PTHrP modulates the expression of molecular markers linked to tumor progression. Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC. Based on these data, we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.
AIM To elucidate the relationship among PTHR1, PTHrP, and Met in CRC models.
METHODS For in vitro assays, HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP (1-34) (10-8 M). Where indicated, cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide, the vehicle of the inhibitors. The protein levels were evaluated by Western blot technique. Real-time polymerase chain reaction (RT-qPCR) was carried out to determine the changes in gene expression. Wound healing assay and morphological monitoring were performed to evaluate cell migration and changes related to the epithelial-mesenchymal transition (EMT), respectively. The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan (CPT-11), oxaliplatin (OXA), or doxorubicin (DOXO) with or without PTHrP. For in vivo tests, HCT116 cell xenografts on 6-wk-old male N:NIH (S)_nu mice received daily intratumoral injections of PTHrP (40 μg/kg) in 100 μL phosphate-buffered saline (PBS) or the vehicle (PBS) as a control during 20 d. Humanitarian slaughter was carried out and the tumors were removed, weighed, and fixed in a 4% formaldehyde solution for subsequent treatment by immunoassays. To evaluate the expression of molecular markers in human tumor samples, we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr. José Penna (Bahía Blanca, Buenos Aires, Argentina) and the Hospital Provincial de Neuquén (Neuquén, Neuquén, Argentina) from January 1990 to December 2007. Seven cases with normal colorectal tissues were assigned to the control group. Tumor tissue samples and clinical histories of patients were analyzed. Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique; subsequently, representative histological samples were selected from each patient. From each paraffin block, tumor sections were stained for immunohistochemical detection. The statistical significance of differences was analyzed using proper statistical analysis. The results were considered statistically significant at P < 0.05.
RESULTS By Western blot analysis and using total Met antibody, we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells. In HCT116 cells, Met protein levels increased at 30 min (P < 0.01) and at 20 h (P < 0.01) whereas the levels diminished at 3 min (P < 0.05), 10 min (P < 0.01), and 1 h to 5 h (P < 0.01) of PTHrP treatment. Using an active Met antibody, we found that where the protein levels of total Met decreased (3 min, 10 min, and 60 min of PTHrP exposure), the status of phosphorylated/activated Met increased (P < 0.01) at the same time, suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP. The increment of its protein level after these decreases (at 30 min and 20 h) suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis (P < 0.05). We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/ activation of Met induced by PTHrP in HCT116 cells. By Western blot technique, we observed that PP1, a specific inhibitor of the activation of the proto-oncogene protein tyrosine kinase Src, blocked the effect of PTHrP on Met phosphorylation (P < 0.05). Furthermore, the selective inhibition of the ERK 1/2 mitogen-activated protein kinase (ERK 1/2 MAPK) using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation (P < 0.05). Using SU11274, the specific inhibitor of Met activation, and trypan blue dye exclusion test, Western blot, wound healing assay, and morphological analysis with a microscope, we observed the reversal of cell events induced by PTHrP such as cell proliferation (P < 0.05), migration (P < 0.05), and the EMT program (P < 0.01) in HCT116 cells. Also, PTHrP favored the chemoresistance to CPT-11 (P < 0.001), OXA (P < 0.01), and DOXO (P < 0.01) through the Met pathway. Taken together, these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells. By immunohistochemical analysis, we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met (0.190 ± 0.014) compared to tumors from control mice (0.110 ± 0.012; P < 0.05) and of its own receptor (2.27 ± 0.20) compared to tumors from control mice (1.98 ± 0.14; P < 0.01). Finally, assuming that the changes in the expression of PTHrP and its receptor are directly correlated, we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis. Comparing histologically differentiated tumors with respect to those less differentiated, we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner, respectively (P < 0.05).
CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model. More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.
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Affiliation(s)
- María Belén Novoa Díaz
- Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)- INBIOSUR (CONICET-UNS), Bahía Blanca 8000, Buenos Aires, Argentina
| | - Pedro Carriere
- Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)- INBIOSUR (CONICET-UNS), Bahía Blanca 8000, Buenos Aires, Argentina
| | - Graciela Gigola
- Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)- INBIOSUR (CONICET-UNS), Bahía Blanca 8000, Buenos Aires, Argentina
| | | | - Natalia Calvo
- Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)- INBIOSUR (CONICET-UNS), Bahía Blanca 8000, Buenos Aires, Argentina
| | - Claudia Gentili
- Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur (UNS)- INBIOSUR (CONICET-UNS), Bahía Blanca 8000, Buenos Aires, Argentina
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Lyu P, Li B, Li P, Bi R, Cui C, Zhao Z, Zhou X, Fan Y. Parathyroid Hormone 1 Receptor Signaling in Dental Mesenchymal Stem Cells: Basic and Clinical Implications. Front Cell Dev Biol 2021; 9:654715. [PMID: 34760881 PMCID: PMC8573197 DOI: 10.3389/fcell.2021.654715] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2021] [Accepted: 09/28/2021] [Indexed: 02/05/2023] Open
Abstract
Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) are two peptides that regulate mineral ion homeostasis, skeletal development, and bone turnover by activating parathyroid hormone 1 receptor (PTH1R). PTH1R signaling is of profound clinical interest for its potential to stimulate bone formation and regeneration. Recent pre-clinical animal studies and clinical trials have investigated the effects of PTH and PTHrP analogs in the orofacial region. Dental mesenchymal stem cells (MSCs) are targets of PTH1R signaling and have long been known as major factors in tissue repair and regeneration. Previous studies have begun to reveal important roles for PTH1R signaling in modulating the proliferation and differentiation of MSCs in the orofacial region. A better understanding of the molecular networks and underlying mechanisms for modulating MSCs in dental diseases will pave the way for the therapeutic applications of PTH and PTHrP in the future. Here we review recent studies involving dental MSCs, focusing on relationships with PTH1R. We also summarize recent basic and clinical observations of PTH and PTHrP treatment to help understand their use in MSCs-based dental and bone regeneration.
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Affiliation(s)
- Ping Lyu
- State Key Laboratory of Oral Diseases, Department of Cariology and Endodontics, West China Hospital of Stomatology, National Clinical Research Center for Oral Diseases, Sichuan University, Chengdu, China
| | - Bo Li
- State Key Laboratory of Oral Diseases, Department of Orthodontics, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Peiran Li
- State Key Laboratory of Oral Diseases, Department of Oral and Maxillofacial Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Ruiye Bi
- State Key Laboratory of Oral Diseases, Department of Oral and Maxillofacial Surgery, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Chen Cui
- Guangdong Province Key Laboratory of Stomatology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong, China
| | - Zhihe Zhao
- State Key Laboratory of Oral Diseases, Department of Orthodontics, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Xuedong Zhou
- State Key Laboratory of Oral Diseases, Department of Cariology and Endodontics, West China Hospital of Stomatology, National Clinical Research Center for Oral Diseases, Sichuan University, Chengdu, China
| | - Yi Fan
- State Key Laboratory of Oral Diseases, Department of Cariology and Endodontics, West China Hospital of Stomatology, National Clinical Research Center for Oral Diseases, Sichuan University, Chengdu, China
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