Search Results (22)

The addition of dexamethasone in membranes for guided bone regeneration is promising due to its dual effect: (1) anti-inflammatory action and (2) induction of osteogenesis in host stem cells. Electrospun fiber coating with dexamethasone using the layer-by-layer (LBL) technique offers an interesting alternative for the gradual release of the drug, aiming for enhanced osteodifferentiation activity. This study aimed to develop synthetic poly-L-lactide (PLLA) membranes with dexamethasone incorporated into the fibers or coated on their surface, and to evaluate the drug release rate, as well as the material’s ability to promote proliferation, osteoconduction, and osteodifferentiation of human periodontal ligament stem cells (hPDLSCs). PLLA membranes were produced by electrospinning. Dexamethasone was incorporated using three techniques: (A) electrospinning of a co-solution of PLLA with 2.5 w/w% dexamethasone; (B) deposition of four layers on the PLLA membrane using alternating solutions of chitosan and heparin/dexamethasone; (C) deposition of 10 layers on the PLLA membrane using the same solutions. hPDLSC proliferation was measured via CCK-8 at 1, 7, 14, and 21 days. Cellular differentiation was assessed by alkaline phosphatase activity (7 days) and alizarin red staining (21 days) in clonogenic and osteogenic media (ODM). Data were analyzed using one or two-way ANOVA and Tukey test. Electrospun membranes with dexamethasone and those with 4 layers showed immediate drug release within 24 h, whereas 10 layers exhibited gradual release over 14 days. Cumulative drug release was higher for electrospun membranes at 1 and 7 days, similar to 10 layers at 14 and 21 days. The 4 LBL membrane promoted lower hPDLSC proliferation compared to the 10 LBL and electrospun membranes at 21 days but showed increased extracellular matrix mineralization in osteogenic media. No significant differences in alkaline phosphatase expression were observed between materials. Therefore, the addition of dexamethasone in 10 layers, combined with heparin, enables gradual drug release. However, lower drug release in the first 24 h by four LBL membranes improved the material’s osteogenesis properties. None of the materials improved the osteodifferentiation in the clonogenic medium. Full article

Background: Human dental pulp stem cells (HDPSCs) with multi-lineage differentiation potential and migration ability are required for HDPSC-based bone and dental regeneration. Hispidulin is a naturally occurring flavonoid with diverse pharmacological activities, but its effects on biological properties of HDPSCs remain unknown. Therefore, we investigated the effects of hispidulin on the differentiation potential and migration ability of HDPSCs and elucidated their underlying mechanisms. Methods: The osteo/odontogenic capacity of HDPSCs was assessed using the alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. The migration ability of HDPSCs was evaluated using a scratch wound assay. Furthermore, the endothelial differentiation of HDPSCs was examined by using a capillary sprouting assay and by assessing CD31 expression. Results: Hispidulin significantly enhanced the osteo/odontogenic differentiation of HDPSCs with increased expression of osteo/odontogenic differentiation markers. Hispidulin increased the migration of HDPSCs, which was mediated by the upregulation of C-X-C chemokine receptor type 4 (CXCR4). The treatment of HDPSCs with hispidulin enhanced the differentiation of HDPSCs into endothelial cells, as evidenced by increased capillary sprouting and endothelial marker expression. In addition, we demonstrated that hispidulin activated the ERK1/2 signaling, and its inhibition by U0126 significantly suppressed the hispidulin-induced endothelial differentiation of HDPSCs. Conclusions: These findings demonstrate that hispidulin effectively promotes the osteo/odontogenic and endothelial differentiation, and migration of HDPSCs. These results suggest that hispidulin may have potential therapeutic applications in dental pulp regeneration and tissue engineering. Full article

G-protein-coupled receptors (GPCRs) have emerged as critical regulators of bone development and remodeling. In this study, we aimed to identify specific GPCR mutations in osteoporotic patients via next-generation sequencing (NGS). We performed NGS sequencing of six genomic DNA samples taken from osteoporotic patients and two genomic DNA samples from healthy donors. Next, we searched for single-nucleotide polymorphisms (SNPs) in GPCR genes that are associated with osteoporosis. For three osteoporotic patients and one healthy donor, bone biopsies were used to generate patient-specific mesenchymal stem cell (MSC) lines, and their ability to undergo osteodifferentiation was analyzed. We found that MSCs derived from osteoporotic patients have a different response to osteoinductive factors and impaired osteogenic differentiation using qPCR and histochemical staining assays. The NGS analysis revealed specific combinations of SNPs in GPCR genes in these patients, where SNPs in ADRB2 (rs1042713), GIPR (rs1800437), CNR2 (rs2501431, rs3003336), and WLS (rs3762371) were associated with impaired osteogenic differentiation capacity. By integrating NGS data with functional assessments of patient-specific cell lines, we linked GPCR mutations to impaired bone formation, providing a foundation for developing personalized therapeutic strategies. SNP analysis is recognized as a proactive approach to osteoporosis management, enabling earlier interventions and targeted preventive measures for individuals at risk. Furthermore, SNP analysis contributes to the development of robust, holistic risk prediction models that enhance the accuracy of risk assessments across the population. This integration of genetic data into public health strategies facilitates healthcare initiatives. This approach could guide treatment decisions tailored to the patient’s genetic profile and provide a foundation for developing personalized therapeutic strategies. Full article

Background: Pathological bone fracturing is an escalating problem driven by increasing aging and obesity. Bioceramics, particularly tricalcium-phosphate-based materials (TCP), are renowned for their exceptional biocompatibility, osteoconductivity, and ability to promote biomineralization. In the present study, we designed and characterized TCP porous granules doped with strontium (Sr) and copper (Cu) (CuSr TCP). Sr²⁺ ions were selected as Sr plays a crucial role in early bone formation, osteogenesis, and angiogenesis; Cu²⁺ ions possess antibacterial properties. Materials: The synthesized CuSr TCP granules were characterized by X-ray diffraction. Cytotoxicity and cell proliferation analyses’ assays were performed through the lactate dehydrogenase (LDH) activity and CCK-8 viability tests in rat bone marrow-derived mesenchymal stem cells (BM-MSCs). Hemolytic activity was carried out with human red blood cells (RBCs). Early and late osteogenesis were assessed with alkaline phosphatase (ALP) and Alizarin Red S activity in human osteoblast progenitor cells and rat BM-MSCs. The influence of CuSr TCP on angiogenesis was investigated in human umbilical vein endothelial cells (HUVECs). Results: We have demonstrated that media enriched with CuSr TCP in concentrations ranging from 0.1 mg/mL to 1 mg/mL were not cytotoxic and did not significantly affect cell proliferation rate motility. Moreover, a concentration of 0.5 mg/mL showed a 2.5-fold increase in the migration potential of BM-MSCs. We also found that CuSr TCP-enriched media slightly increased early osteogenesis. We also found that Sr and Cu substitutions in TCP particles significantly enhanced the measured angiogenic parameters compared to control and unsubstituted TCP granules. Conclusion: Our results demonstrate that TCP porous granules doped with Sr and Cu are biocompatible, promote osteodifferentiation and angiogenesis, and could be recommended for further in vivo studies. Full article

Human osteosarcoma (OS) is a rare tumor predominantly affecting long bones and characterized by a poor prognosis. Currently, the first line of intervention consists of the surgical resection of primary tumors combined with radiotherapy and chemotherapy, with a profound impact on the patient’s life. Since the surgical removal of OS frequently results in a large resection of bones, the use of biomaterials to sustain the stability of the remaining tissue and to stimulate bone regeneration is challenging. Moreover, residual neoplastic cells might be responsible for tumor recurrence. Here, we explored the potential of tellurium-ion-doped bioactive glass as a novel therapeutic intervention to both eradicate residual malignant cells and promote bone regeneration. Bioactive glass (BAG) has been extensively studied and employed in the field of regenerative medicine due to its osseointegration properties and ability to improve bone tissue regeneration. We found that the incorporation of tellurium (Te) in BAG selectively kills OS cells through ferroptosis while preserving the viability of hBMSCs and stimulating their osteodifferentiation. However, the mechanism of Te toxicity is still unclear: (i) Te-BAG generates lipid-ROS through LOXs activity but not iron overload; (ii) Te-dependent ferroptosis is mediated by GPX4 down-regulation; and (iii) the anti-ferroptotic activity of FSP1 is abrogated, whose expression confers the resistance of OS to the canonical induction of ferroptosis. Overall, our data show that Te-doped bioglass could represent an interesting biomaterial with both pro-ferroptotic activity towards residual cancer cells and pro-osteoregenerative activity. Full article

Objectives: This study developed a sol–gel tricalcium silicate/graphene oxide (TCS-GO) composite and examined its physicochemical properties, antimicrobial activity, and osteo/odontogenic effect on dental pulp stem cells. Methods: Tricalcium silicate was synthesized and combined with graphene oxide at three different concentrations, namely 0.02%, 0.04%, and 0.08% w/w, while tricalcium silicate and mineral trioxide aggregate served as controls. The setting time, compressive strength, pH, and calcium ion release of the composites were evaluated, as well as antimicrobial properties against Streptococcus mutans and Lactobacillus acidophilus. Additionally, the viability of dental pulp stem cells; apatite forming ability; and the gene expression of Alkaline phosphatase, Dentin sialophosphoprotein, and Runt-related transcription factor 2 were assessed. Results: TCS-GO (0.08%) showed a significantly shorter setting time and higher compressive strength when compared to MTA (p < 0.05). Additionally, tricalcium silicate and TCS-GO groups showed a higher release of Ca ions than MTA, with no significant difference in pH values among the different groups. TCS-GO (0.08%) also demonstrated a significantly stronger antimicrobial effect against Lactobacillus acidophilus compared to MTA (p < 0.05). ALP expression was higher in TCS-GO (0.08%) than MTA on days 3 and 7, while DSPP expression was higher in TCS-GO (0.08%) than MTA on day 3 but reversed on day 7. There was no significant difference in RUNX2 expression between TCS-GO (0.08%) and MTA on days 3 and 7. Conclusions: The TCS-GO (0.08%) composite demonstrated superior physicochemical characteristics and antimicrobial properties compared to MTA. Moreover, the early upregulation of ALP and DSPP markers in TCS-GO (0.08%) indicates that it has the potential to promote and enhance the osteo/odontogenic differentiation of DPSCs. Full article

Bone morphogenetic proteins (BMPs) have tremendous therapeutic potential regarding the treatment of bone and musculoskeletal disorders due to their osteo-inductive ability. More than twenty BMPs have been identified in the human body with various functions, such as embryonic development, skeleton genesis, hematopoiesis, and neurogenesis. BMPs can induce the differentiation of MSCs into the osteoblast lineage and promote the proliferation of osteoblasts and chondrocytes. BMP signaling is also involved in tissue remodeling and regeneration processes to maintain homeostasis in adults. In particular, growth factors, such as BMP-2 and BMP-7, have already been approved and are being used as treatments, but it is unclear as to whether they are the most potent BMPs that induce bone formation. According to recent studies, BMP-9 is known to be the most potent inducer of the osteogenic differentiation of mesenchymal stem cells, both in vitro and in vivo. However, its exact role in the skeletal system is still unclear. In addition, research results suggest that the molecular mechanism of BMP-9-mediated bone formation is also different from the previously known BMP family, suggesting that research on signaling pathways related to BMP-9-mediated bone formation is actively being conducted. In this study, we performed a phosphorylation array to investigate the signaling mechanism of BMP-9 compared with BMP-2, another influential bone-forming growth factor, and we compared the downstream signaling system. We present a mechanism for the signal transduction of BMP-9, focusing on the previously known pathway and the p53 factor, which is relatively upregulated compared with BMP-2. Full article

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O₂) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs’ and norm-naïve EVs’ parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes. Full article

Angiogenesis plays an important role in the development of bone and bone regeneration to provide the required molecules. Mesenchymal stem cells (MSCs) are pluripotent, self-renewing, and spindle-shaped cells, which can differentiate into multiple lineages such as chondrocytes, osteocytes, and adipocytes. MSCs derived from bone marrow (BMMSCs), adipose tissue (ADMSCs), and Wharton’s jelly (UCMSCs) are popular in the field of tissue regeneration. MSCs have been proposed that can promote bone regeneration by enhancing vascularization. In this study, the angiogenic potential of secretomes of undifferentiated and osteo-differentiated BMMSCs, ADMSCs, and UCMSCs seeded on human decellularized allogeneic bone were compared. Human umbilical vein endothelial cells (HUVECs) were treated with MSC secretomes. Cell growth, cell migration, and angiogenesis of HUVECs were analyzed by MTT, wound healing, and tube formation assays. Angiogenic gene expression levels of MSCs were evaluated using real-time quantitative PCR. Antibody neutralization was performed to validate the candidate target. Our study demonstrates that the angiogenic gene expression profile is tissue-dependent and the angiogenic ability of secretomes is independent of the state of differentiation. We also explore that IL-1b is important for MSC angiogenic potential. Taken together, this study proves that IL-1b in the secretomes plays a vital role in angiogenesis. Full article

We questioned the relevance of evaluating residual cell viability in human amniotic membrane (hAM) after its cryopreservation since cell survival is controversial and its ability to act as a matrix (including the presence of growth factors and cytokines) appears to be most important for tissue regeneration purposes. We also discussed the usefulness of osteodifferentiating amniotic cells in whole hAM for bone repair applications. We have evidence that determining residual cell viability after cryopreservation and hAM osteodifferentiation is not justified. Full article

Background: Dental stem cells, which originate from the neural crest, due to their easy accessibility might be good candidates in neuro-regenerative procedures, along with graphene-based nanomaterials shown to promote neurogenesis in vitro. We aimed to explore the potential of liquid-phase exfoliated graphene (LPEG) film to stimulate the neuro-differentiation of stem cells from apical papilla (SCAP). Methods: The experimental procedure was structured as follows: (1) fabrication of graphene film; (2) isolation, cultivation and SCAP stemness characterization by flowcytometry, multilineage differentiation (osteo, chondro and adipo) and quantitative PCR (qPCR); (3) SCAP neuro-induction by cultivation on polyethylene terephthalate (PET) coated with graphene film; (4) evaluation of neural differentiation by means of several microscopy techniques (light, confocal, atomic force and scanning electron microscopy), followed by neural marker gene expression analysis using qPCR. Results: SCAP demonstrated exceptional stemness, as judged by mesenchymal markers’ expression (CD73, CD90 and CD105), and by multilineage differentiation capacity (osteo, chondro and adipo-differentiation). Neuro-induction of SCAP grown on PET coated with graphene film resulted in neuron-like cellular phenotype observed under different microscopes. This was corroborated by the high gene expression of all examined key neuronal markers (Ngn2, NF-M, Nestin, MAP2, MASH1). Conclusions: The ability of SCAPs to differentiate toward neural lineages was markedly enhanced by graphene film. Full article

The slow proliferation rate and poor osteodifferentiation ability of inflammatory periodontal membrane stem cells extracted from periodontitis tissues (i-PDLSCs) account for poor efficiency in treating inflammatory bone loss. Exosomes reportedly have inducible and relatively stable components, allowing them to promote inflammatory bone repair, but obtaining i-PDLSCs exosomes with the ability to promote osteodifferentiation is challenging. In the present study, i-PDLSCs were extracted from periodontal membrane tissues of patients with severe periodontitis, and in vitro induction with gallic acid (GA) significantly promoted the proliferative activity of i-PDLSCs at a concentration of 10 mM, with TC₀ of 11.057 mM and TC₅₀ of 67.56 mM for i-PDLSCs. After mRNA sequencing, we found that GA could alleviate oxidative stress in i-PDLSCs and increase its mitochondrial membrane potential and glucose aerobic metabolism level, thus promoting the osteodifferentiation of i-PDLSCs. After exosomes of i-PDLSCs after GA induction (i-EXO-GA) were isolated by differential centrifugation, we found that 200 ug/mL of i-EXO-GA could remarkably promote the osteodifferentiation of i-PDLSCs. Overall, our results suggest that GA induction can enhance the proliferation and osteodifferentiation in primary cultures of i-PDLSCs in vitro, mediated by alleviating oxidative stress and glycometabolism levels in cells, which further influences the osteodifferentiation-promoting ability of i-EXO-GA. Overall, we provide a viable cell and exosome induction culture method for treating inflammatory alveolar defects associated with periodontitis. Full article

Implantable biomaterials play a key role for the success of orthopedic surgery procedures. However, infections remain one of the most damaging post-operative complications that lead to the implant failure. Recently, several approaches have been proposed to avoid or manage implant-associated infections. Among these, an appropriate surface functionalization to confer intrinsic antibacterial properties preserving the osteo-integration ability represents an appealing strategy for the development of innovative implant materials. Titanium and its alloys are the most used materials for manufacturing of both articular and bone skull prostheses as well as dental implants. However, to date there is still a significant clinical need to improve their bioactivity, osseointegration and antibacterial activity. In this study, titanium biomimetic scaffolds are prepared by nano-functionalization with TiO₂ (Ti_TiO₂) and γFe₂O₃ (Ti_γFe₂O₃). Both cytocompatibility and antibacterial activity have been evaluated. Data show that both nano-functionalized scaffolds exhibit a good antibacterial activity towards Staphylococcus aureus, reducing colony number to 99.4% (Ti_TiO₂) and 99.9% (Ti_γFe₂O₃), respectively. In addition, an increase of both human adipose-derived mesenchymal stem cells (hADSCs) cell proliferation (up to 4.3-fold for Ti_TiO₂ and 3.7-fold for Ti_γFe₂O₃) and differentiation has been observed. These data suggest that these nano-functionalized titanium substrates represent promising prototypes for new antimicrobial and osteoconductive biomaterials to be used in the orthopedic field to reconstruct significant bone defect. Full article

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16–20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP–Smad signaling pathway. Full article

Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimulation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials. Full article