Osteogenesis Activity Research Articles

Alendronate-functionalized polymeric micelles target icaritin to bone for mitigating osteoporosis in a rat model

Formulating drugs into nanoparticles that target sites of disease can lead to strong therapeutic effects with lower doses of drugs and lower rates of off-target adverse effects. Few ways to target drugs to bone have been described, hampering the treatment of osteoporosis. Here we exploit the ability of alendronate to bind tightly to hydroxyapatite in bone as a tactic to target polymeric micelles loaded with the plant flavonoid icaritin to osteoporotic lesions. The traditional Chinese medicine icaritin, from Herba Epimedii, has previously been shown to inhibit adipogenesis and enhance osteogenesis by bone mesenchymal stem cells, but the compound on its own persists only briefly in the bloodstream. Our delivery system led to stronger inhibition of adipogenesis and activation of osteogenesis in a rat model of osteoporosis than when the icaritin-loaded micelles lacked alendronate. These results establish the feasibility of using alendronate to target osteogenic phytomolecules to sites of bone injury, which may guide the development of effective therapies against osteoporosis and, by extension, other bone disorders.

Combined use of PRPP, diode laser and piezosurgery device improves reparative osteogenesis previously to dental implants placement

ObjectiveCurrent study evaluated whether use of the platelet-rich powder (PRPP), piezosurgery device and diode laser after removal of jawbones cysts and benign bone formations and extraction of impacted tooth could enhance bone regeneration before dental implantation. MethodsA study was conducted among 200 patients with post-surgical cavities (not exceeding 1.5 cm upon cystectomy, benign bone tumor removal and impacted tooth extraction) who underwent plasma powder, autograft, allograft, xenograft or beta-tricalcium phosphate augmentation procedures. ResultsPRPP implantation in combination with irradiation of bone cavity by diode laser led to the formation of 15.40 % ± 2.1 of new bone. The activity of reparative osteogenesis on the bone surface was BB = 6.71 ± 1.3, which is 3 and 3.5 times higher than the mean value in comparison group after augmentation of xenograft and beta-tricalcium phosphate. The value of bone tissue resorption for main group patients was less pronounced (Ra.Oc = 25.20 % ± 2.1).The ratio between pro- and anti-inflammatory cytokines in the main group after 1, 7 and 30 days since surgical procedure was lower than the ratio of the corresponding cytokines in the comparison groups. ConclusionThe examination of computed tomograms, histological and morphometric analysis of bone tissue after trepanobiopsy, cytological analysis of swabs and enzyme immunoassay to determine local immunity indicated that the proposed method of treatment with the use of PRPP, the piezosurgery device and the diode laser has a positive effect: it helped to optimize reparative osteogenesis after filling post-surgical cavities and restore bone volume.

Composite Mineralized Collagen/Polycaprolactone Scaffold-Loaded Microsphere System with Dual Osteogenesis and Antibacterial Functions.

Biomaterials play an important role in treating bone defects. The functional characteristics of scaffolds, such as their structure, mechanical strength, and antibacterial and osteogenesis activities, effectively promote bone regeneration. In this study, mineralized collagen and polycaprolactone were used to prepare loaded porous scaffolds with bilayer-structured microspheres with dual antibacterial and osteogenesis functions. The different drug release mechanisms of PLGA and chitosan in PLGA/CS microspheres caused differences in the drug release models in terms of the duration and rate of Pac-525 and BMP-2 release. The prepared PLGA(BMP-2)/CS(Pac-525)@MC/PCL scaffolds were analyzed in terms of physical characteristics, bioactivity, and antibacterial properties. The scaffolds with a dimensional porous structure showed similar porosity and pore diameter to cancellous bone. The release curve of the microspheres and scaffolds with high encapsulation rates displayed the two-stage release of Pac-525 and BMP-2 over 30 days. It was found that the scaffolds could inhibit S. aureus and E. coli and then promote ALP activity. The PLGA(BMP-2)/CS(Pac-525)@MC/PCL scaffold could be used as a dual delivery system to promote bone regeneration.

Pharmacological correction of experimentally induced osteoporosis complicated by type 2 diabetes mellitus

BACKGROUND: Osteoporosis remains one of the most important medical problems worldwide with significant economic consequences. The development of new drugs based on unique biologically active compounds can contribute significantly to osteoporosis management. AIM: To evaluate the osteogenesis processes by evaluating bone-remodeling markers in the blood serum during the treatment of experimental osteoporosis complicated by type 2 diabetes mellitus. MATERIALS AND METHODS: The study was performed on an experimental model of osteoporosis, followed by the induction of type 2 diabetes mellitus, using biochemical methods to analyze markers of osteoporosis in the blood serum. RESULTS: The results of the analysis of bone remodeling markers revealed that the antiosteoporotic activity of a composite preparation based on succinic acid salts is dependent on glucose metabolism disorders such as diabetes mellitus. The high efficacy of the new drug in monotherapy and combination with vitamin D3 in the activation of osteogenesis in experimental osteoporosis was balanced by impaired metabolic processes in type 2 diabetes mellitus. CONCLUSIONS: The results indicate the dependence of the pharmacological effectiveness of the antiosteoporosis agent on metabolic disorders, such as type 2 diabetes mellitus, in female rats with experimental osteoporosis.

DYNAMICS OF BIOCHEMICAL MARKERS OF HUMORAL IMMUNITY AND METABOLISM IN MANDIBULAR BONE TISSUE UNDER INFLUENCE ON REPARATIVE OSTEOGENESIS

The scientific and practical significance of our study lies in the application of various therapeutic measures aimed at accelerating the processes of reparative osteogenesis in the presence of perforated defects in the bone tissue of the mandible using hydroxyapatite-containing osteotropic material “Biomin GT bone graft» and thymaline (injections into the surrounding soft tissues) analyzed in our article.
 The aim of the study is to investigate the dynamics of secretory immunoglobulin A content and activity of acid and alkaline phosphatases in the oral fluid of rats under the conditions of influence on the processes of reparative osteogenesis when filling the bone defect with osteotropic material and injecting thymaline into the surrounding soft tissues at different study periods.
 Materials and methods. Experimental studies were conducted on 30 mature WAG population rats weighing 160-180 g, which were divided into five groups. The control group consisted of 6 intact rats, the first group included rats with a simulated hole defect of the mandible, the second group included rats with a simulated hole defect followed by closure of the bone defect with hydroxyapatite-containing osteotropic material, the third group included rats with a mandibular defect after thymaline injections into the surrounding soft tissues, and the fourth group included animals with a defect after filling the bone defect with osteotropic material and injections of thymaline into the surrounding soft tissues. The concentration of sIgA in the oral fluid of rats was determined by enzyme-linked immunosorbent assay using «The IgA Saliva ELISA kit» (Diametra, Italy). The activity of alkaline (ALP) and acid phosphatase (AP) in the biomaterial was measured using a set of reagents “Granum» Ukraine, Kharkiv. The optical density was measured using a STAT-FAX 303+ immunoenzyme analyzer.
 Results and discussion. A decrease in the content of sIgA in the oral fluid of rats of groups I-IV was found both on day 3 and day 7 of the study compared with the control group. On the 14th day, an increase (by 16.7%) in the content of sIgA in the oral fluid of rats of group IV was determined compared to the first group. The content of sIgA in the oral fluid of rats of groups I and III remained below the control by 29.6%, 32.7%, 29.9%, respectively. On the 28th day, only in rats of group IV, sIgA exceeded the value of its content in rats of the control group and was 52.6% higher compared to rats of group I. On the 3rd day, an increase in the activity of ALP from 102% to 111%, respectively, was observed in the oral fluid of rats of groups I-IV compared to intact animals. On the 7th day, the greatest (18.9%) decrease in the activity of ALP was observed in the oral fluid of rats of group IV; on the 14th day, only rats of group IV showed a decrease in the activity of ALP (by 38.1%) compared to the 3rd day. On the 28th day, a decrease in the activity of ALP (by 43.12%) was observed in the oral fluid of rats of group IV compared to day 3 and was equal to the control. On the third day, there was an increase in the activity of AP in the oral fluid of all rats. When comparing the values of AP activity in the oral fluid of rats of groups I and IV, a difference of 17.3% was determined. In rats of group IV on day 14: 25.6% decrease in AP activity compared to day 3 of the study, although it differed from the control group. However, statistically significant decrease in AP activity was observed in rats of group IV compared to groups I-III.
 Conclusions. In rats with a simulated mandibular perforation defect on the third and seventh days of observation, a deficiency of sIgA in the oral fluid was observed, indicating a decrease in the functioning of the humoral link of local immunity. In rats of group IV, on the 14th and 28th day of the study, the sIgA content was normalized. There is an activation of reparative osteogenesis in the bone tissue of the mandible in rats with a simulated hole defect under the conditions of its subsequent closure with hydroxyapatite-containing osteotropic material and injections of thymaline into the surrounding soft tissues. The determination of biochemical markers of bone metabolism, in particular alkaline and acid phosphatase in the oral fluid in mandibular fractures, can be used in clinical practice to improve the efficiency of diagnosing reparative osteogenesis in the jaw bones.

3D-printed piezoelectric scaffolds composed of uniform core-shell structured BaTiO3@ bioactive glasses particles for bone regeneration

The piezoelectric property and biocompatibility of barium titanate (BTO) is exceptional, whereas its biological activity is limited. Bioactive glasses (BG) are widely recognized for osteoinductive bioactivities. Hence, it is meaningful to explore the reasonable design of BTO and BG combinations to achieve bioactive piezoelectric tissue engineering scaffolds that mimic natural bone. In this study, homogeneous core-shell particles of BTO wrapped in BG layer were firstly fabricated via a sol-gel process. Various BG weight ratios (5 wt%, 10 wt%, and 15 wt%) in BTO@BG particles have been able to achieve the shell thickness in the range of 4 nm–16 nm. Furthermore, the composite particles were three-dimensionally printed into porous scaffolds through gas extrusion using polyvinyl pyrrolidone as binders. The composite scaffolds have been extensively characterized with respect to their physical and functional properties. Desirable piezoelectric scaffolds (BTO@15BG-S) with compressive strength at ∼66 MPa, and piezoelectric constant (d33) of ∼1.1 pC/N were obtained after sintering and polarization. Our findings indicated that higher sintering temperatures led to denser scaffolds with reduced porosity, increased compressive strength and higher d33 constant. Additionally, an increase in the amount of BG coating promoted scaffold calcination via melt filling in pores, while reduced the piezoelectric constant d33. Among all scaffolds, BTO@15BG-S exhibited superior ability to induce apatite formation when sintered at 1250 °C and soaked in simulated body fluid for 7 days. In vitro culture rat bone marrow mesenchymal stem cells (rBMSCs) and cellular assessments confirmed that polarized piezoelectric ceramic scaffolds could effectively enhance cell adhesion, proliferation, and osteogenic differentiation compared to unpolarized scaffolds. An increase in BG coating amount enhanced the capacity of piezoelectric ceramic scaffolds to upgrade osteogenesis activities of rBMSCs. These results rendered BTO@BG-S composite scaffolds promising candidate in the field of bone repair.

Reparative osteogenesis in mandible in cases of filling a bone defect with hydroxyapatite-containing osteotropic material and injecting the surrounding soft tissues with thymalin: experimental and morphological study.

Aim of the study was to identify the morphological features of reparative osteogenesis in the lower jaw bone of rats in cases of filling a bone defect with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT") and injecting the surrounding soft tissues with thymalin. Materials and Methods: An experiment was conducted on 48 mature rats of the WAG population weighing 160-180 grams which were divided into four groups. Group 1 included 12 rats with a simulated holey defect in the lower jaw. Group 2 included 12 rats with a simulated holey defect in the lower jaw followed by its closure with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT"). Group 3 included 12 rats with a simulated holey defect in the lower jaw with injecting the surrounding soft tissues with thymalin. Group 4 included 12 rats with a simulated holey defect in the lower jaw followed by its closure with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT") and injecting the surrounding soft tissues with thymalin. The material for the morphological study was a fragment of the lower jaw from the area of the simulated holey defect. Histological, morphometric and statistical research methods were used. Results: In this study, it was shown by the authors an activation of reparative osteogenesis in the lower jaw under conditions of simultaneous filling the bone defect with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT") and injection the surrounding bone defect soft tissue with thymalin. Stimulation of reparative osteogenesis in the lower jaw of rats occurred due to rapid cleaning of the bone defect cavity from necrotic tissues and hematoma fragments; a decrease in the number of neutrophil leukocytes, an increase in the number and morphofunctional state of monocytes, macrophages, lymphocytes, cells of fibroblastic differon; balanced change (increase or decrease) in the number and morphofunctional state of bone forming osteoblasts and bone resorbing osteoclasts depending on the stage of reparative osteogenesis; activation of hematopoietic processes in lamellar bone tissue from the regenerate; activation of bone tissue mineralization processes. Conclusions: Thymalin injection in the soft tissues surrounding the bone defect in the lower jaw, filled with hydroxyapatite-containing osteotropic material (bone graft "Biomin GT"), significantly stimulates the process of reparative osteogenesis, which makes it possible to recommend this technique in dentistry for treatment the patients with mandible bone tissue defects.

(TiZrNbTaMo)N nanocomposite coatings embedded with silver nanoparticles: imparting mechanical, osteogenic and antibacterial traits to dental implants

Designing highly osteogenic and effective antibacterial biomaterials with a prolonged service life for oral implants remains an ongoing challenge. In this work, the multi-element (TiZrTaNbMo)N high entropy nitride (HEN), with different concentrations of Ag, was co-deposited onto surgical grade commercially pure titanium by a reactive sputter-deposition technique using a double cathode glow discharge plasma system. The effects of Ag concentration on the microstructure and mechanical properties of the HEN-Ag nanocomposite coatings were investigated by various testing methods. Potentiodynamic polarization measurements were used to evaluate the corrosion resistance of the coatings in simulated body fluid. The antibacterial activity of the HEN-Ag nanocomposite coatings against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) strains was examined based on the bacterial inhibitory zones and bacterial killing efficiency. The cytocompatibility and osteogenesis activity of the HEN nanocomposite coatings using MC3T3-E1 cells were evaluated by a CCK-8 assay and an alkaline phosphatase quantitative assay. These studies demonstrate that the Ag-free HEN coating was composed of a face-centered-cubic (FCC) nitride phase, which displays good mechanical properties, anti-corrosion performance and osteogenic function, but no antibacterial properties were observed. In contrast, the HEN0.9Ag0.1 coating, composed of amorphous/nanocrystalline high-entropy nitride embedded with silver nanoparticles ∼10 nm in diameter, displays good mechanical and anti-corrosion performance, together with the excellent cell compatibility and antibacterial performance, which offer potential for providing outstanding protective efficiency for dental implants. Although the HEN0.8Ag0.2 nanocomposite coating exhibited superior antibacterial efficacy, potential for in vitro cytotoxicity was identified.

Gallium-Doped Hydroxyapatite: Shape Transformation and Osteogenesis Activity.

In this study, we employed a chemical precipitation method to successfully synthesize nanoparticles of gallium-doped hydroxyapatite (Ga-HAp). The microstructure of Ga-HAp was precisely tailored by modulating the concentration of gallium ions. Our findings unequivocally demonstrate that gallium ions exert a pronounced inhibitory influence on the growth of HAp crystals, and this inhibitory potency exhibits a direct correlation with the concentration of gallium. Furthermore, gallium ions facilitate the metamorphosis of HAp nanoparticles, transitioning them from nanoneedles to nanosheets. It is worth noting, however, that gallium ions exhibit a limited capacity to substitute for calcium ions within the crystal lattice of HAp, with the maximum substitution rate capped at 4.85%. Additionally, gallium plays a pivotal role in constraining the release of ions from HAp, and this behavior remains consistent across samples with varying Ga doping concentrations. Our in vitro experiments confirm that Ga-doped HAp amplifies both the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

Neurotransmission, Vasculogenesis, and Osteogenesis Activities are Altered in the Aging Temporomandibular Joint of the Senescence-Accelerated Prone 8 Mouse Model

BackgroundAlterations in neurotransmission, vasculogenesis, and osteogenesis pathways that may play pivotal roles in age-related changes in the temporomandibular joint (TMJ) are poorly understood. PurposeThis study aimed to measure the associations between gene and protein profiles in senescence-accelerated prone 8 (SAMP8) mice. Study designThe investigators designed and utilized three groups of two mouse models: 1) early aging SAMP8 at 24 weeks of age and control SAMR1 at 12 and 24 wks (each stage n=12). Predictor/exposure/independent variableThe independent variable was investigated using 3 mouse models: an early aging mouse model and a control mouse model (12 wks and 24 wks). Main outcome variable(s)The primary outcome variables were CGRP, VEGF-A, CD31, LYVE-1, osteocalcin, osteopontin, type I and II collagen, and MMP-2. The secondary outcome variables were histological characteristics. CovariatesNot applicable AnalysesThe gene and protein expression profiles of neurotransmitters, vasculogenesis, and osteogenesis were identified by quantitative real-time polymerase chain reaction and dot blot analysis, respectively. The cellular localization of these events was verified by in situ hybridization and immunohistochemistry. Bivariate statistics were computed for each of the outcome variables. Statistical significance was set to a P value < 0.05. ResultsThe expression of CGRP mRNA in the bony mandibular condyle (BMC) of SAMP8 mice (SAMP8, 3.3 ±0.39 vs. SAMR1, 0.001±0.0001) was high at 24 weeks of age (24 wks) (p<0.001). Higher numbers of cells positive for CGRP (MF, 1.97±0.13 vs. 0.35±0.05; CMC, 1.86±0.15 vs. 0.62±0.08; BMC, 1.32±0.10 vs. 0.28±0.06;) and VEGF-A (MF, 2.95±0.22 vs. 0.85±0.06; MC, 11.29±0.37 vs. 9.78 ±0.35; CMC, 12.4±0.53 vs. 1.41±1.25; BMC 1.97±0.15 vs. 0.63±0.06) antibodies were found in the TMJ (p<0.01). Conclusions and relevanceThe neurotransmitter, vasculogenesis, and osteogenesis pathways are associated with TMJ aging in the SAMP8 mouse model. In the future, the SAMP8 mouse model may prove to be a robust model for identifying molecular and biochemical events underlying the effects of feeding, occlusal changes, and tooth loss in the aging TMJ.

Divalent Anion-Induced Biohydrogels with High Strength, Anti-swelling, and Bioactive Capability for Enhanced Skull Bone Regeneration.

Increased intracranial pressure after traumatic brain injury (TBI) is an urgent problem in clinical practice. A pliable hydrogel is preferred for cranioplasty applications after TBI since it can protect brain tissue and promote bone healing. Nevertheless, biohydrogels for cranial bone regeneration still face challenges of poor mechanical properties, large swelling ratios, and low osteogenesis activity. Herein, inspired by Hofmeister effects, biopolymer hydrogels composed of protein and polysaccharides were treated with a Hofmeister series including a series of monovalent and divalent anions. Our results reveal that the divalent anion-cross-linked biohydrogels exhibit stronger mechanical properties and lower swelling ratios compared with monovalent-anion treated gels. Intriguingly, the divalent HPO42- anion induced biohybrid hydrogels with excellent mechanical behaviors (3.7 ± 0.58 MPa, 484 ± 76.7 kPa, and 148.3 ± 6.85 kJ/m3), anti-swelling capability (16.7%), and gradual degradation ability, significantly stimulating osteogenic differentiation and in vivo cranial bone regeneration. Overall, this study may provide new insights into the design of biomimetic hydrogels for treating cranial bone defects after TBI.

Varying degrees of spontaneous osteogenesis of Masquelet’s induced membrane: experimental and clinical observations

BackgroundMasquelet’s induced membrane (IM) has osteogenesis activity, but IM spontaneous osteogenesis (SO) has not been described previously.ObjectivesTo report on varying degrees of IMSO and analyze its possible causes.MethodsTwelve eight-week-old male Sprague-Dawley rats with 10 mm right femoral bone defects who received the first stage of IM technique (IMT) were used to observe the SO. In addition, clinical data from patients with bone defects who received the first stage of IMT with an interval of > 2 months post-operatively and exhibited SO between January 2012 and June 2020 were retrospectively analyzed. The SO was divided into four grades according to the amount and characteristics of the new bone formation.ResultsAt twelve weeks, grade II SO was observed in all rats, and more new bone was formed in the IM near the bone end forming an uneven margin. Histology revealed bone and cartilage foci in the new bone. Four of the 98 patients treated with the first stage of IMT exhibited IMSO, including one female and three males with a median age of 40.5 years (range 29–52 years). The bone defects were caused by severe fractures and infection in two cases and by infection or tumor in one case each. Partial or segmental defects occurred in two cases. The time interval between inserting a cement spacer and diagnosis of SO ranged from six months to nine years. Two cases were grade I, and one case each of grades III and IV.ConclusionVarying degrees of SO confirm the existence of the IMSO phenomenon. Bioactive bone tissue or local inflammation and a long time interval are the primary reasons underlying enhancement of the osteogenic activity of IM and leading to SO, which tends to take place as endochondral osteogenesis.

Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling

Eurycomanone (EN) is a diterpenoid compound isolated from the roots of Eurycoma longifolia (E. longifolia). Previous studies have confirmed that E. longifolia can enhance bone regeneration and bone strength. We previously isolated and identified ten quassinoids from E. longifolia, and the result displayed that five aqueous extracts have the effects on promotion of bone formation, among whom EN showed the strongest activity. However, the molecular mechanism of EN on bone formation was unknown, and we further investigated in this study. After the verification of purity of extracted EN, following experiments were conducted. Firstly, the pharmacologic action of EN on normal bone mineralization and the therapeutic effect of EN on Dex-induced bone loss using zebrafish larvae. The mineralization area and integral optical density (IOD) were evaluated using alizarin red staining. Then the vital signaling pathways of EN relevant to OP was identified through network pharmacology analysis. Eventually in vitro, the effect of EN on cell viability, osteogenesis activities were investigated in human bone marrow mesenchymal stem cells (hMSCs) and C3H10 cells, and the molecular mechanisms by which applying AKT inhibitor A-443654 in hMSCs. In zebrafish larvae, the administration in medium of EN (0.2, 1, and 5 μM) dramatically enhanced the skull mineralization area and integral optical density (IOD), and increased mRNA expressions of osteoblast formation genes (ALP, RUNX2a, SP7, OCN). Meanwhile, exposure of EN remarkably alleviated the inhibition of bone formation induced by dexamethasone (Dex), prominently improved the mineralization, up-regulated osteoblast-specific genes and down-regulated osteoclast-related genes (CTSK, RANKL, NFATc1, TRAF6) in Dex-treated bone loss zebrafish larvae. Network pharmacology outcomes showed the MAPK and PI3K-AKT signaling pathways are closely associated with 10 hub genes (especially AKT1), and AKT/GSK-3β/β-catenin was selected as the candidate analysis pathway. In hMSCs and C3H10 cells, results showed that EN at appropriate concentrations of 0.008–5 μM effectively increased the cell proliferation. In addition, EN (0.04, 0.2, and 1 μM) significantly stimulated osteogenic differentiation and mineralization as well as significantly increased the protein phosphorylation of AKT and GSK-3β, and expression of β-catenin, evidencing by the results of ALP and ARS staining, qPCR and western blotting. Whereas opposite results were presented in hMSCs when treated with AKT inhibitor A-443654, which effectively inhibited the pro-osteogenesis effect induced by EN, suggesting EN represent powerful potential in promoting osteogenesis of hMSCs, which may be closely related to the AKT/GSK-3β/β-catenin signaling pathway. Altogether, our findings indicate that EN possesses remarkable effect on bone formation via activating AKT/GSK-3β/β-catenin signaling pathway in most tested concentrations. This study demonstrates EN is a new effective monomer in promoting bone formation, which may be a promising anabolic agent for osteoporosis (OP) treatment.

Repurposing sarecycline for osteoinductive therapies: an in vitro and ex vivo assessment.

Tetracyclines (TCs) embrace a class of broad-spectrum antibiotics with unrelated effects at sub-antimicrobial levels, including an effective anti-inflammatory activity and stimulation of osteogenesis, allowing their repurposing for different clinical applications. Recently, sarecycline (SA)-a new-generation molecule with a narrower antimicrobial spectrum-was clinically approved due to its anti-inflammatory profile and reduced adverse effects verified with prolonged use. Notwithstanding, little is known about its osteogenic potential, previously verified for early generation TCs. Accordingly, the present study is focused on the assessment of the response of human bone marrow-derived mesenchymal stromal cells (hBMSCs) to a concentration range of SA, addressing the metabolic activity, morphology and osteoblastic differentiation capability, further detailing the modulation of Wnt, Hedgehog, and Notch signaling pathways. In addition, an ex vivo organotypic bone development system was established in the presence of SA and characterized by microtomographic and histochemical analysis. hBMSCs cultured with SA presented a significantly increased metabolic activity compared to control, with an indistinguishable cell morphology. Moreover, RUNX2 expression was upregulated 2.5-fold, and ALP expression was increased around sevenfold in the presence of SA. Further, GLI2 expression was significantly upregulated, while HEY1 and HNF1A were downregulated, substantiating Hedgehog and Notch signaling pathways' modulation. The ex vivo model developed in the presence of SA presented a significantly enhanced collagen deposition, extended migration areas of osteogenesis, and an increased bone mineral content, substantiating an increased osteogenic development. Summarizing, SA is a promising candidate for drug repurposing within therapies envisaging the enhancement of bone healing/regeneration.

Surfactin suppresses osteoclastogenesis via the NF-κB signaling pathway, promotes osteogenic differentiation in vitro, and inhibits oestrogen deficiency-induced bone loss in vivo.

Fractures caused by osteoporosis (OP) are one of the main causes of death in the elderly, bringing a heavy burden to the country and society. The imbalance between osteoblast-mediated osteogenesis and osteoclast-mediated bone resorption is an important cause of OP. Therefore, finding drugs that can regulate this dynamic balance can be an important way to treat osteoporosis. Surfactin is a highly effective biosurfactant derived from Bacillus subtilis and it has been proven to have various pharmacological effects in previous studies, but its effect on bone metabolism remains unknown. Here, we performed a study on the role and mechanism of Surfactin in inhibiting osteoclastogenesis and its possible mechanism as well as the role in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). We investigated the effect of Surfactin on osteoclast differentiation and osteogenic differentiation in vitro and in vivo. The effect of Surfactin on the activity of osteoclastogenesis and osteogenesis was verified by CCK-8 assay, quantitative Real-time polymerase chain reaction (qPCR) and Western blotting analysis were used to verify the effect of Surfactin on osteoclast and osteogenic differentiation-specific genes and proteins. The effect of Surfactin on TRAP、ALP activity and mineral deposition was verified by TRAP、ALP and ARS staining. We then used an ovariectomy-induced osteoporosis mice model to observe the effect of Surfactin in vivo. Surfactin is noncytotoxic to BMMs, RAW264.7, and BMSCs. And it can effectively inhibit osteoclastogenesis and promote osteogenic differentiation. Moreover, we found that Surfactin can inhibit the differentiation of osteoclasts through the NF-κB signaling pathway. Surfactin can also alleviate bone loss in ovariectomy-induced osteoporosis mice. Our results suggest that Surfactin can inhibit osteoclastogenesis through the NF-κB signaling pathway, promote the osteogenic differentiation of BMSCs, and also can effectively alleviate bone loss in ovariectomy-induced osteoporosis mice.

Ultralong hydroxyapatite nanowires-incorporated dipeptide hydrogel with enhanced mechanical strength and superior in vivo osteogenesis activity

Self-assembling peptide hydrogels have attracted considerable interest in tissue engineering for their biocompatibility, biodegradability, low immunogenicity, facile modification, and similarity to the native extracellular matrix. However, they still suffer from weak mechanical strength and lack of specific bioactivity. Ultralong hydroxyapatite nanowires (UHANWs) are synthesized to work as a reinforcement agent similar to the steel bars in the reinforced concrete to co-assemble with dipeptide nanofibers, endowing the hybrid hydrogel with enhancing and controllable mechanical properties. Furthermore, additional factors BMP-2 can be introduced into the hydrogel to improve its bioactivity. The experimental results show that the incorporation of UHANWs into the hydrogel matrix leads to an obvious increase in the storage modulus of scaffolds, exhibiting good biocompatibility and bioactivity. The hybrid hydrogels can facilitate the proliferation and osteogenetic differentiation of cells on their surface. The in vivo evaluation reveals that bone defects implanted with the as-designed hydrogel scaffolds show enhanced bone regeneration activity, implying their great potential in biomedical fields.

Tripeptide IRW (Ile-Arg-Trp) as a Potential Nutraceutical Intervention in Osteoporosis.

Bone health is an important medical concern in rapidly aging demographics worldwide. Excessive bone resorption, due to enhanced activity of osteoclasts, is a major underlying cause of bone disorders such as osteoporosis. Inflammation and oxidative stress are key factors contributing to increased osteoclastic activity. Like increased activity of osteoclasts, depletion of osteoblasts also contributes to weakened structural integrity of bone. Considering the epidemiology of bone disorders and aging demographics there is a substantial need for novel bone health therapeutics. IRW (Ile-Arg-Trp), an egg-derived tripeptide, exhibits a spectrum of pharmacological activity. In our recent work, we have shown that IRW inhibits osteoclastogenesis and promotes osteogenesis in the mouse macrophage RAW 264.7 and MC3T3-E1 cells. IRW treatment (25 and 50 μM) significantly inhibited osteoclastogenesis-associated factors [TRAF6 (TNF Receptor Associated Factor 6), Fos Proto-Oncogene (c-Fos), Nuclear Factor of Activated T Cells 1 (NFATc1), and cathepsin K] and upregulated osteogenesis-associated factors [RUNX2 (Runt-related transcription factor 2) and RANKL (Receptor activator of nuclear factor kappa-B ligand)] in the two cell lines. Currently, we are conducting studies to analyze the impact of IRW on Angiotensin II (Ang II)-induced stress in vitro and in vivo. In summary, our recent work presents the ability of IRW to prevent LPS-induced inflammatory bone resorption and activation of osteogenesis activity via multiple signaling pathways.

Titania-Nanotube-Coated Titanium Substrates Promote Osteogenesis and Suppress Osteoclastogenesis via Integrin ανβ3.

The balance of bone turnover mediated by osteoclastogenesis and osteogenesis implants that could suppress osteoclastogenesis and promote osteogenesis is an appropriate treatment strategy for osteoporosis patients. Titanium is one of the most applied materials in implants. In this study, titania nanotubes (Ti-NTs) were produced by anodization at 10, 40, and 60 V. We found that Ti-NTs were nontoxic to bone marrow mesenchymal stem cells (BMSCs). Ti-NTs suppressed osteoclast formation and function in a diameter dependent manner in vitro. Furthermore, Ti-NTs enhanced the activity of osteogenesis, expressions of osteogenesis-related marker genes were increased and β-Catenin pathway was active. Alkaline phosphatase (ALP) activity and matrix mineralization were also promoted in vitro. To explore the possible mechanisms, we performed a series of experiments to indicate the effects of Ti-NTs on cytoskeletal organization and integrin ανβ3 expression of osteoclasts and osteoblasts. The results demonstrated that 90-nm-diameter Ti-NTs could suppress the expression of integrin ανβ3 in osteoclast precursor cells. Interestingly, it revealed an opposite effect on BMSCs. Moreover, 90 nm-diameter Ti-NTs prevented ovariectomy (OVX)-induced bone loss. These findings indicated that Ti-NTs could inhibit osteoclastogenesis and enhance osteogenesis; it was mediated via regulation of integrin ανβ3─90 nm-diameter Ti-NT revealed a good biological ability especially suited for osteoporosis treatment.

Biomineralized dipeptide self-assembled hydrogel with ultrahigh mechanical strength and osteoinductivity for bone regeneration

Peptide self-assembled hydrogels are ideal scaffolds for three-dimentional cell culture and tissue engineering due to their native extracellular matrix enabling the transport of nutrients as well as the cells growth and expansion. However, the use of peptide hydrogels in the hard tissues regeneration such as alveolar and skull bone has been largely unexplored, hindered by the insufficient mechanical strength and osteogenesis activity. Herein, peptide self-assembled nanostructures are used as templates to emulate natural biomineralization process. The vaterite nanoparticles loaded with thermolysin act as a role of mature osteoblasts secreting collagen fibrils to initiate dipeptide self-assembly as biomimetic extracellular matrix. Meanwhile, the vaterite nanoparticles with a high specific surface area form strong intermolecular and interfacial interaction with hydrogel networks, acting as a cross-linker to achieve extrafibrillar mineralization. Moreover, vaterite nanoparticles function as a mineral reservoir to release Ca2+, inducing intrafibrillar mineralization, further enhancing the mechanical property. The resulting hydrogel exhibits an unprecedently high value of storage modulus (473 kPa), excellent biocompatibility and osteoinductivity. In rat calvarial defect model, the hydrogel significantly accelerates regeneration of osteogenesis tissue, thus indicating the potential of such peptide hydrogels for application in bone regeneration.

The Functional and Molecular Alterations in Human Bone Marrow Mesenchymal Stem Cells in Chronic Myelomonocytic Leukemia

Background: Crosstalk between the bone marrow microenvironment and leukemia cells plays a pivotal role in pathogenesis. Mesenchymal stem cells (MSCs) acquire defects during natural ageing, with reduced regenerative potential and functional impairment. Since chronic myelomonocytic leukemia (CMML) mainly affects older adults, defective MSCs could play a critical and targetable role in disease initiation and progression. Identifying molecular abnormalities associated with CMML MSCs would reveal novel therapeutic targets for patient benefit. Methods: Phenotypes of age-matched healthy and CMML MSCs were compared by a range of functional approaches, including senescence, multilineage differentiation and hematopoietic stem cell maintenance assays. To identify altered epigenetic and transcriptional networks we performed RNA-Seq (n=18 CMML/10 healthy) and ATAC-Seq (n=12 CMML/5 healthy) on cultured MSCs (CD44+73+90+105+146+), integrating datasets to explore gene regulatory networks operational within CMML MSCs. To investigate direct crosstalk between MSCs and malignant cells we performed RNA-Seq on healthy MSCs following 7 days co-culture with CMML blood mononuclear cells (MNCs). Functional validation was performed through inhibitor studies using apoptosis, senescence, osteogenesis, and stem cell maintenance as readouts. Results: CMML MSCs displayed consistent and profound intrinsic growth defects, with higher senescence and apoptosis, and lower proliferation and clonogenic capacity. CMML MSCs also displayed a marked differentiation bias, with increased osteogenic lineage potential but reduced adipogenicity and chondrogenicity. Importantly, CMML MSCs demonstrated reduced stem cell maintenance capability in co-culture experiments, confirming a functional consequence on hematopoiesis. Transcriptomic analysis showed activation of novel pathways including mTOR, PI3K-Akt signaling, and oxidative phosphorylation in CMML MSCs, in addition to Wnt pathway upregulation as shown previously (Figure 1A). Consistent with functional readouts, CMML MSCs were transcriptionally primed towards the osteo-lineage, with high expression of osteogenic promoting factors (CORIN, LTF, TREM2, SMAD5, ALX5) and downregulation of osteogenic inhibitors (RBPJ, CLU). In line with transcriptomic analysis, ATAC-Seq of CMML MSCs highlighted aberrantly accessible chromatin regions linked to genes involved in mTOR, PI3K-Akt and Wnt signaling as well as promotion of osteogenesis (Figure 1A). Integration of datasets identified downregulated expression and activity of the transcription factor EBF1, where reduced expression is associated with self-renewal defects, decreased adipogenesis, cellular senescence and defective stem cell maintenance ability, compatible with our observations in CMML MSCs. Similarly, we identified downregulated activity of the transcription factor RBPJ, potentially mediating the preferential enhancement of osteogenesis observed in CMML MSCs. Following co-culture with CMML MNCs, healthy MSCs showed activation of mTOR, PI3K-Akt and osteogenesis, verifying a direct influence of leukemic cells on MSCs. Remarkably, integration of all datasets identified consistently upregulated genes (RGS4, TPI1) which positively regulate mTOR and PI3K-Akt signaling. Inhibiting these aberrantly active pathways in CMML MSCs using small molecule modulators significantly reversed the phenotypic abnormalities with a reduction in apoptosis, senescence and osteogenesis and restoration of their ability to support and maintain stem cells (Figure 1B). Discussion: Our integrated analysis identified altered gene regulatory networks that are linked to abnormal pathways active in CMML MSCs, which can be targeted to restore their normal function. These results highlight novel pathogenic roles for non-clonal MSCs in the CMML microenvironment, and potential adjunctive strategies for targeting microenvironmental alongside cell intrinsic vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal