Extracellular Matrix Mineralization Research Articles

Dental Pulp Stem Cell Conditioned Medium Enhance Osteoblastic Differentiation and Bone Regeneration.

Cell-free approaches, utilizing mesenchymal stem cell secretome, have promising prospects in various fields of regenerative medicine. In this study, we examined in vitro and in vivo the potential of dental pulp stem cell-conditioned medium (DPSC-CM) for bone regeneration. The secretome of undifferentiated stem cells from dental pulp were collected, and the effects of this DPSC-CM were assessed for osteodifferentiation of osteoblast-like cells (MG-63) and osteoblasts deriving from DPSC. Cell proliferation, alkaline phosphatase (ALP) activity, gene expression of Runt-related transcription factor 2 (Runx2), Bone Sialoprotein (BSP), Osteocalcin (OCN), and extracellular matrix mineralization were evaluated. The rat caudal vertebrae critical size defect model was to investigate the effect of DPSC-CM in vivo. Results showed that DPSC-CM induced cell growth, and increased ALP activity and the expression of key marker genes at an early stage of osteoblastic differentiation compared to control. A rat bone defect model was used to illustrate the effect of DPSC-CM in vivo. The bone density within the defects were improved using conditioned medium, even though there was no significant difference between the control and DPSC-CM groups. The analysis of DPSC-CM by human growth factor antibody array revealed the presence of several factors involved in osteogenesis. Taken together, these findings indicate that DPSC-CM is a promising therapeutic candidate for bone regenerative therapy, accelerating the maturation of osteoblastic cells. And even though safety and efficiency of DPSC-CM have to be confirmed in preclinical studies, these results represent a first step toward clinical application.

Investigating the potential effect of Holothuria scabra extract on osteogenic differentiation in preosteoblast MC3T3-E1 cells

The present medical treatments of osteoporosis come with adverse effects. It leads to the exploration of natural products as safer alternative medical prevention and treatment. The sea cucumber, Holothuria scabra, has commercial significance in Asian countries with rising awareness of its properties as a functional food. This study aims to investigate the effects of the inner wall (IW) extract isolated from H. scabra on extracellular matrix maturation, mineralization, and osteogenic signaling pathways on MC3T3-E1 preosteoblasts. The IW showed the expression of several growth factors. Molecular docking revealed that H. scabra BMP2/4 binds specifically to mammal BMP2 type I receptor (BMPR-IA). After osteogenic induction, the viability of cells treated with IW extract was assessed and designated with treatment of 0.1, 0.5, 1, and 5 µg/ml of IW extract for 21 consecutive days. On days 14 and 21, treatments with IW extract at 1 and 5 µg/ml showed increased alkaline phosphatase (ALP) activity and calcium deposit levels in a dose-dependent manner compared to the control group. Moreover, the transcriptomic analysis of total RNA of cells treated with 5 µg/ml of IW extract exhibited upregulation of TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways at days 14. This study suggests that IW extract from H. scabra exhibits the potential to enhance osteogenic differentiation and mineralization of MC3T3-E1 preosteoblasts through TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways. Further investigation into the molecular mechanisms underlying the effect of IW extract on osteogenesis is crucial to support its application as a naturally derived supplement for prevention or treatment of osteoporosis.

Matrix Gla protein suppresses osteoblast senescence and promotes osteogenic differentiation by the PI3K-AKT signaling pathway

Age-related bone loss in mice is associated with senescent cell accumulation and reduced bone formation by osteoblasts. Matrix Gla protein (MGP), secreted by osteoblasts, is pivotal in regulating the bone extracellular matrix mineralization. Previous research has demonstrated that Mgp null mice exhibit osteopenia and fractures, and ultimately die prematurely. To elucidate the mechanisms underlying MGP’s role of MGP in bone metabolism, we generated osteoblast-specific Mgp knockout (Mgp cKO) mice by crossing Mgpfl/fl mice with Bglap-Cre mice. The study revealed that in 3-month-old Mgp cKO male mice, trabecular bone volume decreased, and the senescence marker protein p21 increased. Primary osteoblasts from Mgp cKO mice exhibited markers of DNA damage and senescence, such as increased γH2AX foci, p21, and senescence-associated β-galactosidase staining, as well as attenuated cellular proliferation and osteogenic differentiation abilities. In addition, bone marrow stromal cells’ colony formation and spontaneous osteogenic ability were impaired in Mgp cKO mice, whereas osteoclastogenesis was enhanced. In vitro treatment with recombinant human MGP promotes osteogenesis in osteoblasts derived from Mgp cKO mice via the PI3K-AKT signaling pathway. Thus, our results suggest that MGP is protective by suppressing osteoblast senescence, offering new insights into potential therapeutic strategies for age-related osteoporosis.

Potential of Cranberry to Stimulate Osteogenesis: An In Vitro Study

This study investigated the potential of Cranberry extract to stimulate osteogenesis in vitro. The total phenolic and monomeric anthocyanin contents in the Cranberry were determined. Liquid chromatography coupled with mass spectrometry was used to identify the Cranberry’s constituents. To assess the Cranberry’s cytotoxicity, a thiazolyl blue tetrazolium bromide assay was employed. Concerning the osteogenesis potential of Cranberry, alkaline phosphatase (ALP) activity, bone morphogenetic protein 2 (BMP-2) expression, and extracellular matrix mineralization were evaluated. The total phenolic content was 522.72 ± 9.80 mg GAE g-1 ES and 364.95 ± 12.49 mg GAE/g detected by the Fast Blue BB and Folin–Ciocalteu method, respectively. For monomeric anthocyanin, the content was 460 ± 30 mg ECG g-1 ES. Moreover, Cranberry concentrations ranged from 62.5 to 500 mg/mL and were found to be biocompatible with osteoblasts and mesenchymal stromal cells. Regarding osteogenesis, 20 mg/mL of Cranberry promoted 2-fold more ALP activity and almost 1.5-fold more BMP-2 than compared to the positive control group. Additionally, 200 mg/mL of Cranberry stimulated a 1.7-fold increase in extracellular matrix mineralization compared to the positive control group. In conclusion, Cranberry displayed potential in stimulating early and late markers of osteogenesis. Its ability to promote osteogenesis and its biocompatibility at higher concentrations hold promise for future application into biomaterials for bone regeneration.

Extracorporeal Magnetotransduction Therapy as a New Form of Electromagnetic Wave Therapy: From Gene Upregulation to Accelerated Matrix Mineralization in Bone Healing.

Electromagnetic field therapy is gaining attention for its potential in treating bone disorders, with Extracorporeal Magnetotransduction Therapy (EMTT) emerging as an innovative approach. EMTT offers a higher oscillation frequency and magnetic field strength compared to traditional Pulsed Electromagnetic Field (PEMF) therapy, showing promise in enhancing fracture healing and non-union recovery. However, the mechanisms underlying these effects remain unclear. This study demonstrates that EMTT significantly enhances osteoblast bone formation at multiple levels, from gene expression to extracellular matrix mineralization. Key osteoblastogenesis regulators, including SP7 and RUNX2, and bone-related genes such as COL1A1, ALPL, and BGLAP, were upregulated, with expression levels surpassing those of the control group by over sevenfold (p < 0.001). Enhanced collagen synthesis and mineralization were confirmed by von Kossa and Alizarin Red staining, indicating increased calcium and phosphate deposition. Additionally, calcium imaging revealed heightened calcium influx, suggesting a cellular mechanism for EMTT's osteogenic effects. Importantly, EMTT did not compromise cell viability, as confirmed by live/dead staining and WST-1 assays. This study is the first to show that EMTT can enhance all phases of osteoblastogenesis and improve the production of critical mineralization components, offering potential clinical applications in accelerating fracture healing, treating osteonecrosis, and enhancing implant osseointegration.

Sequential release of vancomycin and BMP-2 from chitosan/nano-hydroxyapatite thermosensitive hydrogel for the treatment of chronic osteomyelitis

In this study, we developed scaffolds materials with microspheres to form a double sustained release system.Chitosan/nano-hydroxyapatite (CS-HA) was used as a drug carrier to construct a sustained-release system for Bone morphogenetic protein-2(BMP-2) and Vancomycin (VAN). Furthermore, VAN and BMP-2 loaded microspheres (Ms) were prepared by the emulsion ultrasonic method.The resultant composites were characterized by Scanning electron microscope (SEM), compressive strength, porosity, and biodegradation. The characterization results showed uniform porous and rough surface, enhanced thermal stability, and highest compressive strength ((1.912 ± 0.012) Kpa, the surface of the two microspheres was slightly folded and showed a regular spherical shape.The loading rate of BMP-2 was (59.611 × 10-4 ± 0.023 × 10-4)% and the encapsulation rate was (6.022 ± 0.005)%. The release rate of vancomycin and BMP-2 was 57.194% and 12.968% respectively. Osteogenic differentiation of Bone marrow mesenchymal stem cells (BMSCs) was confirmed by alkaline phosphatase quantification. The deposition of late osteogenic markers (calcium phosphates) detected by Alizarin red, which indicated extracellular matrix mineralization. The results showed that BMP-2/VAN in CS-HA hydrogel successfully achieved the sequential release of the double drugs, which could benefit bone regeneration.

Combined Effects of Cyclic Hypoxic and Mechanical Stimuli on Human Bone Marrow Mesenchymal Stem Cell Differentiation: A New Approach to the Treatment of Bone Loss.

Background: The prevention and treatment of bone loss and osteoporotic fractures is a public health challenge. Combined with normobaric hypoxia, whole-body vibration has a high clinic potential in bone health and body composition. The effect of this therapy may be mediated by its action on bone marrow mesenchymal stem cells (MSCs). Objectives: Evaluate the effects of cyclic low-vibration stimuli and/or hypoxia on bone marrow-derived human MSC differentiation. Methods: MSCs were exposed four days per week, two hours/day, to hypoxia (3% O2) and/or vibration before they were induced to differentiate or during differentiation into osteoblasts or adipocytes. Gene and protein expression of osteoblastic, adipogenic, and cytoskeletal markers were studied, as well as extracellular matrix mineralization and lipid accumulation. Results: early osteoblastic markers increased in undifferentiated MSCs, pretreated in hypoxia and vibration. This pretreatment also increased mRNA levels of osteoblastic genes and beta-catenin protein in the early stages of differentiation into osteoblasts without increasing mineralization. When MSCs were exposed to vibration under hypoxia or normoxia during osteoblastic differentiation, mineralization increased with respect to cultures without vibrational stimuli. In MSCs differentiated into adipocytes, both in those pretreated as well as exposed to different conditions during differentiation, lipid formation decreased. Changes in adipogenic gene expression and increased beta-catenin protein were observed in cultures treated during differentiation. Conclusions: Exposure to cyclic hypoxia in combination with low-intensity vibratory stimuli had positive effects on osteoblastic differentiation and negative ones on adipogenesis of bone marrow-derived MSCs. These results suggest that in elderly or frail people with difficulty performing physical activity, exposure to normobaric cyclic hypoxia and low-density vibratory stimuli could improve bone metabolism and health.

Biocalcification in porcelaneous foraminifera.

Living organisms control the formation of mineral skeletons and other structures through biomineralization. Major phylogenetic groups usually consistently follow a single biomineralization pathway. Foraminifera, which are very efficient marine calcifiers, making a substantial contribution to global carbonate production and global carbon sequestration, are regarded as an exception. This phylum has been commonly thought to follow two contrasting models of either in situ 'mineralization of extracellular matrix' attributed to hyaline rotaliid shells, or 'mineralization within intracellular vesicles' attributed to porcelaneous miliolid shells. Our previous results on rotaliids along with those on miliolids in this paper question such a wide divergence of biomineralization pathways within the same phylum of Foraminifera. We have found under a high-resolution scanning electron microscopy (SEM) that precipitation of high-Mg calcitic mesocrystals in porcelaneous shells takes place in situ and form a dense, chaotic meshwork of needle-like crystallites. We have not observed calcified needles that already precipitated in the transported vesicles, what challenges the previous model of miliolid mineralization. Hence, Foraminifera probably utilize less divergent calcification pathways, following the recently discovered biomineralization principles. Mesocrystalline chamber walls in both models are therefore most likely created by intravesicular accumulation of pre-formed liquid amorphous mineral phase deposited and crystallized within the extracellular organic matrix enclosed in a biologically controlled privileged space by active pseudopodial structures. Both calcification pathways evolved independently in the Paleozoic and are well conserved in two clades that represent different chamber formation modes.

Mechanical properties and biological behavior of refractory TiZrNbTa medium-entropy and TiZrHfNbTa high-entropy alloy nanofilms on AISI 316L for bone implants

To investigate the feasibility of high-entropy alloys as the coating materials for the application of bone implants, TiZrNbTa refractory medium-entropy alloy (RMEA) and TiZrHfNbTa refractory high-entropy alloy (RHEA) nanofilms were prepared on AISI 316L stainless steel by DC magnetron sputtering and post-annealing treatment. Then, the microstructure, mechanical properties, wear resistance, corrosion resistance, biocompatibility and osteogenic activity of the nanofilms were systematically studied. The results showed that both the films exhibited uniform and dense nanostructures. Post-annealing treatment not only improved the adhesion strength, but also formed an oxidized layer. Notably, owing to the enhanced high-entropy and lattice distortion effects, the RHEA nanofilm showed finer grain sizes, higher hardness (14 GPa), H/E, H3/E2 and better wear resistance. The in vitro experiments proved that RHEA nanofilm had the highest Ecorr (−0.127 V), with the Icorr value was two orders of magnitude lower than substrate. Its osteoblast proliferative capacity was 1.5 times higher than substrate (after culturing for 5 days), and the collagen secretion and extracellular matrix mineralization reached an optimal level, showing excellent osteogenic activity. Therefore, RHEA nanofilm coated biomaterials can be a more attractive candidate for bone replacement.

Effects of photobiomodulation on different phases of in vitro osteogenesis.

The present study aimed to evaluate the effect of photobiomodulation therapy (PBM) on different stages of osteogenesis in vitro. For this, osteoblastic-like cells (Saos-2 cell lineage) were irradiated in two different periods: during the Proliferation phase (PP; from the second to the fourth day) and during the Differentiation phase (DP; from the seventh to the ninth day). The energy density used in the study was 1.5J/ cm2. The following parameters were evaluated: 1) quantification ofcollagen type 1 (COL 1), osteopontin (OPN), and bone morphogenetic protein 2 (BMP-2); 2) quantification of alkaline phosphatase (ALP) activity; and 3) quantification of extracellular matrix (ECM)mineralization. Non-irradiated cultures were used as controls. The data were analyzed using the Student's t-test or one-way ANOVA, considering a significance level of 5%. The results indicated that COL 1 and BMP-2 quantification was higher in Saos-2 irradiated during the DP in relation to the control group at day 10 (p < 0.05). No differences were observed for other comparisons at this time point(p>0.05).OPN expression was greater in PP compared with the other experimental groups at day 10 (p < 0.05). Irradiation did not affect ALP activity in Saos-2 regardless of the exposure phase and the time point evaluated (p > 0.05). At day 14, ECMmineralization was higher in Saos-2 cultures irradiated during the DP in relation to the PP (p < 0.05). In conclusion, the results suggested that the effects of PBM on osteoblastic cells may be influenced by the stage of cell differentiation.

Copper nanoparticles incorporated visible light-curing chitosan-based hydrogel membrane for enhancement of bone repair

Alveolar bone defects caused by tumor, trauma and inflammation can lead to the loss of oral function and complicate denture restoration. Currently, guided bone regeneration (GBR) barrier membranes for repairing bone defect cannot effectively promote bone regeneration due to their unstable degradation rate and poor antibacterial properties. Furthermore, they require additional tailoring before implantation. Therefore, this study developed a visible light-curing hydrogel membrane (CF–Cu) comprising methacrylated carboxymethyl chitosan (CMCS-MA), silk fibroin (SF), and copper nanoparticles (Cu NPs) to address these shortcomings of commercial membranes. The CF-Cu hydrogel, characterized by scanning electron microscopy (SEM), a universal testing machine, and swelling and degradation tests, demonstrated a smooth porous network structure, suitable swelling ratio, biodegradability, and enhanced mechanical strength. Cytotoxicity and hemolysis tests in vitro demonstrated excellent cyto- and hemo-compatibility of the CF-Cu hydrogel extracts. Additionally, evaluation of antibacterial properties in vitro, including colony forming unit (CFU) counts, MTT assays, and live/dead fluorescence staining, showed that the CF-Cu hydrogel exhibited excellent antibacterial properties, inhibiting over 80% of S. aureus, S. mutans, and P. gingivalis with CF-1Cu hydrogel compared to the control group. Moreover, evaluation of osteogenic differentiation of rBMSCs in vitro suggested that the CF-1Cu hydrogel significantly improved alkaline phosphatase (ALP) activity and the mineralization of extracellular matrix, up-regulating the expressions of osteogenesis-related genes (Runx2, ALP, Col-1, OPN and BSP). In summary, these results indicated that CF-1Cu hydrogel exhibited excellent cytocompatibility, antibacterial and osteogenic properties in vitro. Therefore, the CF-1Cu hydrogel holds potential as a viable material for application in GBR procedures aimed at addressing bone defects.

Jaboticaba Peel Extract Attenuates Ovariectomy-Induced Bone Loss by Preserving Osteoblast Activity.

Therapies to prevent osteoporosis are relevant since it is one of the most common non-communicable human diseases in the world and the most prevalent bone disorder in adults. Since jaboticaba peel extract (JPE) added to the culture medium enhanced the osteogenic potential of mesenchymal stem cells (MSCs) derived from osteoporotic rats, we hypothesized that JPE prevents the development of ovariectomy-induced osteoporosis. Ovariectomized rats were treated with either JPE (30 mg/kg of body weight) or its vehicle for 90 days, starting 7 days after the ovariectomy. Then, the femurs were subjected to microcomputed tomography and histological analyses, and the osteoblast and adipocyte differentiation of MSCs was evaluated. JPE attenuated ovariectomy-induced bone loss, as evidenced by higher bone volume/total volume and trabecular number, along with lower trabecular separation and bone marrow adiposity. These protective effects of JPE on bone tissue are due to its ability to prevent the imbalance between osteoblast and adipocyte differentiation of MSCs, since, compared with MSCs derived from ovariectomized rats treated with vehicle, MSCs treated with JPE exhibited higher gene and protein expression of osteogenic markers and extracellular matrix mineralization, as well as lower gene expression of adipogenic markers. These data highlight the potential therapeutic use of JPE to prevent osteoporosis.

β-glycerophosphate, not low magnitude fluid shear stress, increases osteocytogenesis in the osteoblast-to-osteocyte cell line IDG-SW3

ABSTRACT Aim As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3. Methods Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (β-glycerophosphate (βGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker. Results βGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. βGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing βGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition. Conclusions This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.

Hepatocyte growth factor and insulin-like growth factor-1 were used to repair bone-cartilage defects in bone marrow mesenchymal stem cells in a rabbit model of postmenopausal

In postmenopausal osteoporosis (PMOP), an imbalance exists in the differentiation of bone marrow mesenchymal stem cells (BMSCs), with a decrease in osteogenic differentiation and an increase in adipogenic differentiation. This imbalance leads to bone marrow adiposity, bone loss, bone fragility, and a substantial rise in fracture risk. After a patient experiences an osteochondral defect due to trauma, it struggles to heal naturally, presenting a clinical challenge for treatment. Our study delved into the abnormal differentiation of BMSCs in PMOP by conducting transcriptome sequencing on BMSCs from a PMOP model (PMOP-BMSCs) and a healthy control model (Normal-BMSCs). We identified insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) genesas significantly low-expressed protein-coding genes during the osteogenic cartilage differentiation process of PMOP-BMSCs. Due to the downregulation of its expression, it leads to the deletion of the proteins it encodes IGF-1 and HGF. In order to verify the sequencing results, the feasibility of co-culture the above two growth factors with PMOP-BMSCs to repair osteochondral defects was discussed. The findings indicated that the inclusion of elements enhanced the DNA replication activity and extracellular matrix mineralization of PMOP-BMSCs. It also promoted the construction of tissue-engineered bone in vitro and the up- regulation of Runx2, BMP4, OCN, ACAN, collagen type Ⅰ, II, and Sox9 osteochondral differentiation markers. In the rabbit model of knee osteochondral injury with PMOP, the group treated with both growth factors and PMOP-BMSCs showed superior outcomes in repairing cartilage and subchondral bone defects compared to the other groups. We suggest that the addition of HGF and IGF-1 increases the expression of osteoblast and cartilage-related genes and proteins, promoting the proliferation and differentiation of osteochondrous bone in PMOP-BMSCs. These findings could offer a novel cell therapy strategy for treating postmenopausal osteoporosis, utilizing growth factors.

Preparation of Ag@3D-TiO2 Scaffolds and Determination of its Antimicrobial Properties and Osteogenesis-promoting Ability.

The micro-nano structure of 3D-printed porous titanium (Ti) alloy with excellent performance in avoiding stress shielding and promoting bone tissue differentiation provides a new opportunity for the development of bone implants, but it necessitates higher requirements for bone tissue differentiation and the antibacterial properties of bone implants in clinical practice. This study investigated the preparation, antimicrobial properties, and osteogenesis-promoting ability of the 3D printed porous Ti alloy anodic oxidized Ag-carrying (Ag@3D-TiO2) scaffolds. The 3D printed porous Ti alloy (3D-Ti), anodized 3D printed porous Ti alloy (3D-TiO2), and Ag@3D-TiO2 scaffolds were synthesized using electron beam melting. The antimicrobial properties of the scaffolds were examined using antibacterial tests and their cytocompatibility was assessed using a cell proliferation assay and acridine orange/ethidium bromide (AO/EB) staining. In vitro cellular assays were used to investigate the effects of the scaffold microstructural features on cell activity, proliferation, and osteogenesis-related genes and proteins. In vivo animal experiments were used to evaluate the anti-inflammatory and osteogenesis-promoting abilities of the scaffolds. The Ag@3D-TiO2 scaffolds exhibited sustained anti-microbial activity over time, enhanced cell proliferation, facilitated osteogenic differentiation, and increased extracellular matrix mineralization. In addition, alkaline phosphatase (ALP), collagen type I (COL-I), and osteocalcin (OCN)-related genes and proteins were upregulated. In vivo animal implantation experiments, the anti-inflammatory effect of the Ag@3D-TiO2 scaffolds were observed using histology, and a large amount of fibrous connective tissue was present around it; the Ag@3D-TiO2 scaffolds were more bio-compatible with the surrounding tissues compared with 3D-Ti and 3D-TiO2; a large amount of uniformly distributed neoplastic bone tissue existed in their pores, and the chronic systemic toxicity test showed that the 3D-Ti, 3D-TiO2, and Ag@3D-TiO2 scaffolds are biologically safe. The goal of this study was to create a scaffold that exhibits antimicrobial properties and can aid bone growth, making it highly suitable for use in bone tissue engineering.

Pluronic F127 hydrogel-loaded extracellular vesicles from adipose-derived mesenchymal stem cells promote tracheal cartilage regeneration via SCNN1B delivery

Extracellular vesicles (EVs) derived from adipose-derived mesenchymal stem cells (AMSC-EVs) have been highlighted as a cell-free therapy due to their regenerative capability to enhance tissue and organ regeneration. Herein, we aimed to examine the mechanism of PF127-hydrogel@AMSC-EVs in promoting tracheal cartilage defect repair. Based on bioinformatics methods, SCNN1B was identified as a key gene for the osteogenic differentiation of AMSCs induced by AMSC-EVs. EVs were isolated from rat AMSCs and then loaded onto thermo-sensitive PF-127 hydrogel to develop PF127-hydrogel@AMSC-EVs. It was established that PF127-hydrogel@AMSC-EVs could effectively deliver SCNN1B into AMSCs, where SCNN1B promoted AMSC osteogenic differentiation. The promotive effect was evidenced by enhanced ALP activity, extracellular matrix mineralization, and expression of s-glycosaminoglycan, RUNX2, OCN, collagen II, PERK, and ATF4. Furthermore, the in vivo experiments revealed that PF127-hydrogel@AMSC-SCNN1B-EVs stimulated tracheal cartilage regeneration in rats through PERK/ATF4 signaling axis activation. Therefore, PF127-hydrogel@AMSC-SCNN1B-EVs may be a novel cell-free biomaterial to facilitate tracheal cartilage regeneration and cartilage injury repair.

Organic Matrices of Calcium Carbonate Biominerals Improve Osteoblastic Mineralization.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO3 biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

Methodology and Characterization of a 3D Bone Organoid Model Derived from Murine Cells.

Here, we report on the development of a cost-effective, well-characterized three-dimensional (3D) model of bone homeostasis derived from commonly available stocks of immortalized murine cell lines and laboratory reagents. This 3D murine-cell-derived bone organoid model (3D-mcBOM) is adaptable to a range of contexts and can be used in conjunction with surrogates of osteoblast and osteoclast function to study cellular and molecular mechanisms that affect bone homeostasis in vitro or to augment in vivo models of physiology or disease. The 3D-mcBOM was established using a pre-osteoblast murine cell line, which was seeded into a hydrogel extracellular matrix (ECM) and differentiated into functional osteoblasts (OBs). The OBs mineralized the hydrogel ECM, leading to the deposition and consolidation of hydroxyapatite into bone-like organoids. Fourier-transform infrared (FTIR) spectroscopy confirmed that the mineralized matrix formed in the 3D-mcBOM was bone. The histological staining of 3D-mcBOM samples indicated a consistent rate of ECM mineralization. Type I collagen C-telopeptide (CTX1) analysis was used to evaluate the dynamics of OC differentiation and activity. Reliable 3D models of bone formation and homeostasis align with current ethical trends to reduce the use of animal models. This functional model of bone homeostasis provides a cost-effective model system using immortalized cell lines and easily procured supplemental compounds, which can be assessed by measuring surrogates of OB and OC function to study the effects of various stimuli in future experimental evaluations of bone homeostasis.

Design and characterization of 3D printed pore gradient hydrogel scaffold for bone tissue engineering

Macroporous hydrogel scaffolds are widely used in tissue engineering to promote cell growth and proliferation. Aiming to enhance cell seeding efficiency and facilitate the osteodifferentiation of mesenchymal stem cells, this study demonstrates the fabrication of pore gradient biodegradable hydrogel scaffolds inspired by natural bone structure for bone tissue engineering applications. The scaffolds were fabricated via extrusion-based 3D printing, using sequential deposition of three customized Gelatin/Oxidized Alginate - based inks with subsequent cryogenic crosslinking for permanent structure fixation. The resulting constructs were characterized and featured a continuous gradient morphology with pore sizes ranging from 10 to 300 μm. The gradient scaffolds exhibited improved mechanical stability, with a compression resistance of 149 kPa, as opposed to the non-gradient scaffold's 116 kPa at 70 % strain, and a sustained degradation rate with only a 10 % loss of its initial weight within three weeks. Gradient scaffolds demonstrated a doubling of cell seeding efficiency to 47 % with dense and homogeneously distributed cellular layers, as evidenced by confocal and electron microscopy. Furthermore, the gradient scaffolds demonstrated superior osteodifferentiation, with significantly higher ALP and DMP1 production and enhanced extracellular matrix mineralization compared to gradientless macroporous scaffolds. This study provides insights into the design of macroporous scaffolds and emphasizes the advantages of pore gradient over homogeneous gradientless morphologies.

An Injectable Magnesium-Based Cement Stimulated with NIR for Drug-Controlled Release and Osteogenic Potential.

Magnesium phosphate bone cement (MPC) has gained widespread usage in orthopedic implantation due to its fast-setting and high initial strength benefits. However, the simultaneous attainment of drug-controlled release and osteogenic potential in MPC remains a significant challenge. Herein, a strategy to create a smart injectable cement system using nanocontainers and chondroitin sulfate is proposed. It employs nanocontainers containing alendronate-loaded mesoporous silica nanoparticles, which are surface-modified with polypyrrole to control drug release in response to near-infrared (NIR) stimulation. The alendronate-incorporated cement (ACMPC) exhibits improved compressive strength (70.6 ± 5.9 MPa), prolonged setting time (913 s), and exceptional injectability (96.5% of injection rate and 242 s of injection time). It also shows the capability to prevent degradation, thus preserving mechanical properties. Under NIR irradiation, the cement shows good antibacterial properties due to the combined impact of hyperthermia, reactive oxygen species, and alendronate. Furthermore, the ACMPC (NIR) group displays good biocompatibility and osteogenesis capabilities, which also lead to an increase in alkaline phosphatase activity, extracellular matrix mineralization, and the upregulation of osteogenic genes. This research has significant implications for developing multifunctional biomaterials and clinical applications.