Dental pulp stem cells (DPSCs) are ideal seed cells for the regeneration of dental tissues. However, DPSC senescence restricts its clinical applications. Metformin (Met), a common prescription drug for type 2 diabetes, is thought to influence the aging process. This study is aimed at determining the effects of metformin on DPSC senescence. Young and aging DPSCs were isolated from freshly extracted human teeth. Flow cytometry confirmed that DPSCs expressed characteristic surface antigen markers of mesenchymal stem cells (MSCs). Cell Counting Kit-8 (CCK-8) assay showed that a concentration of 100μM metformin produced the highest increase in the proliferation of DPSCs. Metformin inhibited senescence in DPSCs as evidenced by senescence-associated β-galactosidase (SA-β-gal) staining and the expression levels of senescence-associated proteins. Additionally, metformin significantly suppressed microRNA-34a-3p (miR-34a-3p) expression, elevated calcium-binding protein 39 (CAB39) expression, and activated the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Dual-luciferase reporter assay confirmed that CAB39 is a direct target for miR-34a-3p. Furthermore, transfection of miR-34a-3p mimics promoted the senescence of DPSCs, while metformin treatment or Lenti-CAB39 transfection inhibited cellular senescence. In conclusion, these results indicated that metformin could alleviate the senescence of DPSCs by downregulating miR-34a-3p and upregulating CAB39 through the AMPK/mTOR signaling pathway. This study elucidates on the inhibitory effect of metformin on DPSC senescence and its potential as a therapeutic target for senescence treatment.

The microenvironment, or niche, regulates stem cell fate and improves differentiation efficiency. Human umbilical cord mesenchymal stem cells (hUC-MSCs) are ideal cell source for bone tissue engineering. However, the role of the microenvironments in hUC-MSC-based bone regeneration is not yet fully understood. This study is aimed at investigating the effects of the in vitro culture microenvironment (hUC-MSCs, nano-hydroxyapatite/collagen/poly (L-lactide) (nHAC/PLA), osteogenic media (OMD), and recombinant human bone morphogenetic protein-7 (rhBMP-7)) and the in vivo transplanted microenvironment (ectopic and orthotopic) on bone regeneration ability of hUC-MSCs. The isolated hUC-MSCs showed self-renewal potential and MSCs characteristics. In the in vitro two-dimensional culture microenvironment, OMD or OMD with rhBMP-7 significantly enhanced hUC-MSCs osteocalcin immunofluorescence staining, alkaline phosphatase, and Alizarin red staining; OMD with rhBMP-7 exhibited the highest ALP secretion and mineralized matrix formation. In the in vitro three-dimensional culture microenvironment, nHAC/PLA supported hUC-MSCs adhesion, proliferation, and differentiation; the microenvironment containing OMD or OMD and rhBMP-7 shortened cell proliferation progression and made osteogenic differentiation progression advance; rhBMP-7 significantly attenuated the inhibiting effect of OMD on hUC-MSCs proliferation and significantly enhanced the promoting effect of OMD on gene expression and protein secretion of osteogenic differentiation markers, calcium and phosphorous concentration, and mineralized matrix formation. The in vitro three-dimensional culture microenvironment containing OMD and rhBMP-7 induced hUC-MSCs to form the most new bones in ectopic or orthotopic microenvironment as proved by microcomputed tomography and hematoxylin and eosin staining, but bone formation in orthotopic microenvironment was significantly higher than that in ectopic microenvironment. The results indicated that the combination of in vitro hUC-MSCs+nHAC/PLA+OMD+rhBMP-7 microenvironment and in vivo orthotopic microenvironment provided a more optimized niche for bone regeneration of hUC-MSCs. This study elucidates that hUC-MSCs and their local microenvironment, or niche, play an important role in hUC-MSC-based bone regeneration. The endogenously produced BMP may serve an important regulatory role in the process.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a promising alternative source of mesenchymal stem cells (MSCs) that are enormously attractive for clinical use. This study was designed to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or osteogenic media (OMD) on bone regeneration of hUC-MSCs seeded on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA) in a rabbit model. The characteristics of stem cells were analyzed by plastic adherence, cell phenotype, and multilineage differentiation potential. Cell proliferation was examined using cell counting kit-8 assay. Osteogenic differentiation was evaluated by quantitative Ca²⁺ concentration, PO₄³⁻ concentration, alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralized matrix formation. Bone regeneration was investigated in jaw bone defect repair in rabbit by microcomputed tomography, fluorescent labeling, and hematoxylin and eosin staining. Except for initial stress response, OMD and OMD + rhBMP-7 inhibited the proliferation of hUC-MSCs seeded on nHAC/PLA; rhBMP-7 inhibited cell proliferation in the nonlogarithmic phase and attenuated the inhibitory effect of OMD on cell proliferation. The inhibitory effects of OMD, rhBMP-7, and OMD + rhBMP-7 on cell proliferation were ranked as OMD > OMD + rhBMP-7 > rhBMP-7. OMD, rhBMP-7, and OMD + rhBMP-7 promoted Ca²⁺ concentration, PO₄³⁻ concentration, ALP activity, OCN secretion, and mineralized matrix formation of hUC-MSCs seeded on nHAC/PLA. The promoting effects of OMD, rhBMP-7, and OMD+rhBMP-7 on Ca²⁺ concentration, PO₄³⁻ concentration, ALP activity, OCN secretion, and mineralized matrix formation were ranked as rhBMP-7 > OMD > OMD + rhBMP-7, OMD > OMD + rhBMP-7 > rhBMP-7, OMD > rhBMP-7 > OMD + rhBMP-7, rhBMP-7 > OMD + rhBMP-7 > OMD, and OMD > rhBMP-7 > OMD + rhBMP-7, respectively. In rabbit jaw bone defect repair, OMD, rhBMP-7, and OMD + rhBMP-7 enhanced bone regeneration of hUC-MSCs seeded on nHAC/PLA, but the largest bone mineral apposition rate and bone formation were presented in cultures with rhBMP-7. These findings suggested that the combined use of rhBMP-7 and OMD may have no ideal synergistic effect on bone regeneration of hUC-MSCs seeded on nHAC/PLA in rabbit jaw bone defect.

This study aimed to assess the clinical effectiveness of the sausage technique with simultaneous implant placement in the aesthetic zone. A total of 28 patients with horizontal bone defects and consecutive loss of multiple teeth in the maxillary anterior region were included in this retrospective study. Bone augmentation was performed using the sausage technique, and the conventional guided bone regeneration (GBR) technique served as the control. Cone-beam computed tomography (CBCT) was performed preoperatively, postoperatively, and after 6 months of bone healing. The horizontal bone width and bone volume were measured from CBCT images. The changes in horizontal width and bone volume, along with the corresponding resorption rates, were calculated. After 6 months of bone healing, the average bone width increased from 3.18 mm to 7.82 mm, with an increase of 4.64 mm in the sausage technique group. Meanwhile, the horizontal bone width, horizontal bone gain, and bone augmentation volume were significantly higher in the sausage technique group than in the conventional GBR group (P<0.05). The sausage technique resulted in more horizontal bone gain compared with the conventional technique in the cases with horizontal bone defects and consecutive loss of multiple teeth in the anterior maxillary region. When combined with simultaneous implant placement for horizontal bone augmentation, the sausage technique achieves favorable clinical outcomes in the aesthetic zone.