Osteogenic Biomarkers Research Articles

Folic acid pretreatment and its sustained delivery for chondrogenic differentiation of MSCs

Dietary uptake of folic acid (FA) improves cartilage regeneration. In this work, we discovered that three days of FA treatment is highly effective for promoting chondrogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs). In a three-dimensional pellet culture, the levels of typical chondrogenic biomarkers, sulfated glycosaminoglycan, proteoglycan, type II collagen (COL II), SRY box transcription factor 9 (SOX 9), cartilage oligomeric matrix protein (COMP), and aggrecan (ACAN) increased significantly in proportion to FA concentration up to 30 μM. At the mRNA expression level, COL II, SOX 9, COMP, and ACAN increased 3.6–6.0-fold with FA treatment at 30 μM compared with the control system that did not receive FA treatment, and the levels with FA treatment were 1.6–2.5 times greater than those in the kartogenin-treated positive control system. FA treatment did not increase type I collagen α1 (COL I α1), an osteogenic biomarker which is a concern with most chondrogenic promoters. At the high FA concentration of 100 μM, significant decreases in chondrogenic biomarkers were observed, which might be related to DNA methylation. A thermogel system incorporating TMSCs and FA provided sustained release of FA over several days, similar to the FA treatment. The thermogel system confirmed the efficacy of FA in promoting chondrogenic promotion of TMSCs. The increased nuclear translocation of core-binding factor β subunit (CBFβ) and the runt-related transcription factor 1 (RUNX1) expression after FA treatment, together with molecular docking studies, suggest that the chondrogenic enhancement mechanism of FA is mediated by CBFβ and RUNX1.

Pralatrexate Sustainably Released from Polypeptide Thermogel Is Effective for Chondrogenic Differentiation of Mesenchymal Stem Cells.

Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor β subunit (CBFβ) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFβ and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.

Teriparatide with Octacalcium Phosphate Collagen Composite Stimulates Osteogenic Factors.

Octacalcium phosphate and collagen composite (OCPcol) promotes osteogenic differentiation and angiogenesis, thereby enhancing bone regeneration. Although a newly developed freeze-dried composite of OCPcol and teriparatide (OCPcolTPTD) reinforced bone regeneration more than OCPcol, the mechanism of bone regeneration remains unresolved. In this study, disks containing OCPcolTPTD, OCPcol, or β-tricalcium phosphate (β-TCP) col were inserted into rodents with calvarial bone defects, before euthanasia 4 weeks later. Immunohistochemical and histochemical analyses were performed on bone samples to evaluate bone matrix development, angiogenesis, and osteoclast and osteoblast localization. In the OCPcolTPTD and OCPcol groups, bone regeneration was observed at the surface of calvarial dura mater and around acidophilic granular cells with abundant collagenous fiber-containing cells. Furthermore, the newly formed bone in the OCPcolTPTD group showed a larger total area and individual separated area than the other groups. Various osteogenic proteins were detected in the regenerated bone and peri-bone tissues by histochemistry and immunohistochemistry. Although the expression of several osteogenic biomarkers in the OCPcolTPTD group after 4 weeks of implantation was significantly lower than that in the OCPcol group, new bone formation by OCPcolTPTD in the center of the defect, where bone regeneration is difficult, tended to be superior to that by OCPcol. These results suggest that OCPcolTPTD enhanced bone regeneration more evenly and homogenously than OCPcol. Impact statement Our study suggests that octacalcium phosphate and collagen (OCPcol) together with a TPTD enhances bone regeneration in rodents with calvarial bone defects. Furthermore, we believe that composite of OCPcol and teriparatide (OCPcolTPTD) could be developed into novel clinical technique for the regeneration or repair of bone.

Modulatory activity of a bovine hydrolyzed collagen-hydroxyapatite food complex on human primary osteoblasts after simulating its gastrointestinal digestion and absorption

Introduction: osteoporosis is the most prevalent bone disease and one of the main causes of chronic disability in middle and advanced ages. Conventional pharmacological treatments are still limited, and their prolonged use can cause adverse effects that motivate poor adherence to treatment. Nutritional strategies are traditionally based on supplementing the diet with calcium and vitamin D. Recent studies confirm that the results of this supplementation are significantly improved if it is accompanied by the intake of oral hydrolyzed collagen. Objective: to evaluate the possible in vitro osteogenic activity of a peptide-mineral complex formed by bovine hydrolyzed collagen and bovine hydroxyapatite (Phoscollagen®, PHC®). Methods: the digestion and absorption of PHC® were simulated using the dynamic gastrointestinal digester of AINIA and Caco-2 cell model, respectively. Primary cultures of human osteoblasts were treated with the resulting fraction of PHC® and changes were evaluated in the proliferation of preosteoblasts and in the mRNA expression of osteogenic biomarkers at different stages of osteoblast maturation: Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OC) and type I collagen (ColA1). Results: an increase in preosteoblastic proliferation was observed (p ≤ 0,05). No changes were detected in the biomarkers of osteoblasts with 5 days of differentiation, but were with 14 days, registering an increase in Runx2 (p = 0.0008), ColA1 (p = 0.035), OC (p = 0.027) and ALP (without significance). Conclusion: these results show that the PHC® peptide-mineral complex stimulates the activity of mature osteoblasts, being capable of promoting bone formation.

Methyl-CpG-binding protein 2 promotes osteogenic differentiation of bone marrow mesenchymal stem cells through regulating forkhead box F1/Wnt/β-Catenin axis

ABSTRACT Postmenopausal osteoporosis is characterized by inadequate bone formation of osteoblasts and excessive bone resorption of osteoclasts. Bone marrow mesenchymal stem cells (BMSCs), with the potential of osteogenic differentiation, have been widely used in the bone tissues engineering for the treatment of bone diseases, including postmenopausal osteoporosis. Methyl-CpG-binding protein 2 (MECP2) has been reported to be implicated in bone formation during the development of Rett syndrome. However, the influence of MeCP2 on osteogenic differentiation of BMSCs during osteoporosis remains unclear. Firstly, mice model with estrogen deficiency-induced osteoporosis was established through ovariectomy (OVX). MeCP2 was found to be down-regulated in bone tissues and BMSCs of OVX-induced osteoporosis mice. Secondly, over-expression of MeCP2 enhanced the calcium deposition of BMSCs isolated from the OVX-induced osteoporosis mice. Moreover, expression of osteogenic biomarkers including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), collagen type I alpha 1 (COL1A1), and osteocalcin (OCN) was increased in BMSCs by overexpression of MeCP2. Thirdly, over-expression of MeCP2 reduced protein expression of forkhead box F1 (FOXF1) and adenomatous polyposis coli (APC), while enhanced Wnt5a and β-catenin expression in BMSCs. Over-expression of FOXF1 attenuated MeCP2 over-expression-induced decrease of FOXF1 and APC, as well as increase of Wnt5a and β-catenin. Finally, the increased calcium deposition, protein expression of ALP, RUNX2COL1A1 and OCN induced by concomitant overexpression of MeCP2 were also restored by FOXF1 over-expression. In conclusion, MeCP2 promoted osteogenic differentiation of BMSCs through regulating FOXF1/Wnt/β-Catenin axis to attenuate osteoporosis. MeCP2 over-expression reduced FOXF1 to promote the activation of Wnt5a/β-Catenin and promote osteogenic differentiation of BMSCs during the prevention of postmenopausal osteoporosis.

Biofunctional phosphorylated magnetic scaffold for bone tissue engineering

The development of highly bioactive engineered scaffolds is required to promote bone regeneration and the success of bone tissue engineering treatment approaches. This study attempts to fabricate a biofunctional magnetic scaffold based on new phosphorylated polycaprolactone combined with gelatin (MNPs-PCL-P/gelatin). Phosphorylated polymer and magnetic nanoparticles (MNPs) were synthesized and characterized by NMR, FT-IR, TEM, and DLS instruments. The synthetic polymer, MNPs, and biopolymer were mixed then freeze-dried to prepare a porous scaffold. Physiochemical assessments showed that a scaffold with well-developed porous morphology, and stable structure was obtained. MNPs-PCL-P/gelatin scaffold had no toxicity on human dental pulp stem cells (hDPSCs). The use of phosphorous-containing polymer resulted in improvement of the scaffold’s osteoconductivity to support proper cell attachment and promote cell proliferation. Phosphate group by mimicking function of bone phosphate groups stimulate bone mineralization that reflected by alizarin red S staining assay. The presence of MNPs resulted in higher ALP activity and increased expression level of RUNX2, BMP2 osteogenic biomarkers. Also, phosphorylation enhanced osteoinductivity of scaffold and upregulate RUNX2, BMP2, COL1A1, and OCN genes in phosphors-containing scaffold test groups. It seems that biocompatible MNPs-PCL-P/gelatin scaffold possesses the potential of applications in bone tissue engineering.

Microfibrillar-associated protein 5 regulates osteogenic differentiation by modulating the Wnt/\u03b2-catenin and AMPK signaling pathways

BackgroundDysfunctional osteogenesis of bone marrow mesenchymal stem cells (BMSCs) plays an important role in osteoporosis occurrence and development. However, the molecular mechanisms of osteogenic differentiation remain unclear. This study explored whether microfibrillar-associated protein 5 (MFAP5) regulated BMSCs osteogenic differentiation.MethodsWe used shRNA or cDNA to knock down or overexpress MFAP5 in C3H10 and MC3T3-E1 cells. AR-S- and ALP-staining were performed to quantify cellular osteogenic differentiation. The mRNA levels of the classical osteogenic differentiation biomarkers Runx2, Col1α1, and OCN were quantified by qRT-PCR. Finally, we employed Western blotting to measure the levels of Wnt/β-catenin and AMPK signaling proteins.ResultsAt days 0, 3, 7, and 14 after osteogenic induction, AR-S- and ALP-staining was lighter in MFAP5 knockdown compared to control cells, as were the levels of Runx2, Col1α1 and OCN. During osteogenesis, the levels of β-catenin, p-GSK-3β, AMPK, and p-AMPK were upregulated, while that of GSK-3β was downregulated, indicating that Wnt/β-catenin and AMPK signaling were activated. The relevant molecules were expressed at lower levels in the knockdown than control group; the opposite was seen for overexpressing cell lines.ConclusionsMFAP5 regulates osteogenesis via Wnt/β‑catenin- and AMPK-signaling; MFAP5 may serve as a therapeutic target in patients with osteoporosis.

Combination of parathyroid hormone pretreatment and mechanical stretch promotes osteogenesis of periodontal ligament fibroblasts

Parathyroid hormone (PTH) potentiates the mechanical loading induced bone formation in fracture healing and orthodontics. This study aimed to gain insight into the underlying mechanisms in periodontal ligament fibroblasts (PDLFs). Human PDLFs were cultured and subjected to uniaxial cyclic stretch at 0.5Hz and 2000μ for 0, 6, 12, and 24hours, respectively. 10nM PTH was preadministered for 30minutes before loading. The expression of PTH1R and osteogenic biomarkers Runx2, osteopontin, collagen type 1, alkaline phosphatase was assessed via immunofluorescence staining, quantitative polymerase chain reaction, or Western blot. Transfection of siPTH1R was applied, and alterations of osteogenic biomarkers were examined by Western blot. The expression of essential Wnt signal components Wnt3a, β-catenin, low-density lipoprotein receptor-related protein 5, Wnt5a, receptor tyrosine kinase-like orphan receptor 2 were examined, and the influence of dickkopf-related protein 1 on osteogenic biomarkers was evaluated. The expression of PTH1R was instantaneously upregulated with PTH pretreatment and maintained a gradual increase until 24hours. PTH synergistically enhanced the increase of Runx2, osteopontin, collagen type 1, and alkaline phosphatase under cyclic stretch, which was substantially attenuated by siPTH1R transfection. As for Wnt signal components, synergistic upregulation was detected on Wnt3a, β-catenin, and low-density lipoprotein receptor-related protein 5, whereas Wnt5a and receptor tyrosine kinase-like orphan receptor 2 increased relatively mildly. Blockage of the canonical Wnt/β-catenin pathway by dickkopf-related protein 1 impaired the boost of osteogenic biomarkers under the combined action of PTH and cyclic stretch. The combination of PTH pretreatment and cyclic stretch promotes osteogenesis of PDLFs synergistically, and the canonical Wnt/β-catenin pathway is crucially involved in the underlying mechanism.

Network Pharmacology-Based Strategy for the Investigation of the Anti-Osteoporosis Effects and Underlying Mechanism of Zhuangguguanjie Formulation.

As the society is aging, the increasing prevalence of osteoporosis has generated huge social and economic impact, while the drug therapy for osteoporosis is limited due to multiple targets involved in this disease. Zhuangguguanjie formulation (ZG) is extensively used in the clinical treatment of bone and joint diseases, but the underlying mechanism has not been fully described. This study aimed to examine the therapeutic effect and potential mechanism of ZG on postmenopausal osteoporosis. The ovariectomized (OVX) mice were treated with normal saline or ZG for 4 weeks after ovariectomy following a series of analyses. The bone mass density (BMD) and trabecular parameters were examined by micro-CT. Bone remodeling was evaluated by the bone histomorphometry analysis and ELISA assay of bone turnover biomarkers in serum. The possible drug–disease common targets were analyzed by network pharmacology. To predict the potential biological processes and related pathways, GO/KEGG enrichment analysis was performed. The effects of ZG on the differentiation phenotype of osteoclasts and osteoblasts and the predicted pathway were verified in vitro. The results showed that ZG significantly improved the bone mass and micro-trabecular architecture in OVX mice compared with untreated OVX mice. ZG could promote bone formation and inhibit bone resorption to ameliorate ovariectomy-induced osteoporosis as evidenced by increased number of osteoblast (N.Ob/Tb.Pm) and decreased number of osteoclast (N.Oc/Tb.Pm) in treated group compared with untreated OVX mice. After identifying potential drug–disease common targets by network pharmacology, GO enrichment analysis predicted that ZG might affect various biological processes including osteoblastic differentiation and osteoclast differentiation. The KEGG enrichment analysis suggested that PI3K/Akt and mTOR signaling pathways could be the possible pathways. Furthermore, the experiments in vitro validated our findings. ZG significantly down-regulated the expression of osteoclast differentiation markers, reduced osteoclastic resorption, and inhibited the phosphorylation of PI3K/Akt, while ZG obviously up-regulated the expression of osteogenic biomarkers, promoted the formation of calcium nodules, and hampered the phosphorylation of 70S6K1/mTOR, which can be reversed by the corresponding pathway activator. Thus, our study suggested that ZG could inhibit the PI3K/Akt signaling pathway to reduce osteoclastic bone resorption as well as hamper the mTORC1/S6K1 signaling pathway to promote osteoblastic bone formation.

Nanohydroxyapatite, Nanosilicate-Reinforced Injectable, and Biomimetic Gelatin-Methacryloyl Hydrogel for Bone Tissue Engineering.

PurposeGiven that autologous bone graft for bone defects is limited by insufficient supply and morbidity at the donor site, developing biomimetic graft materials as an alternative has gained consistent attention. However, obstacles in designing bone-mimetic materials that could integrate the biomimetic nature of the bone extracellular matrix, osteogenic cells, and osteoinductive ingredients with a fast and convenient strategy still exist.MethodsThis study designed and fabricated a mesenchymal stem cell (MSC)-laden, nanohydroxyapatite (HAP), and nanosilicate (SN)-loaded bone mimetic and injectable gelatin-methacryloyl hydrogel (GelMA-HAP-SN) system for bone tissue engineering, and systemically investigated the osteogenic capacity of GelMA-HAP-SN in vitro and in vivo.ResultsIntroducing HAP enhanced the compositional similarity to the natural bone extracellular matrix, and SN loading endowed the hydrogel with injectable and osteogenic ability. As a result, the GelMA-HAP-SN hydrogel demonstrated an increase in cellular viability, proliferation, and spreading behavior. The GelMA-HAP-SN hydrogel also amplified the embedded MSCs’ osteogenic biomarkers’ expression and matrix mineralization. Furthermore, the MSC-encapsulated GelMA-HAP-SN hydrogel was injected into rats’ critical-sized calvaria defect, and micro-CT and histomorphometry staining results further confirmed its excellent bone regeneration ability.ConclusionThese MSC-loaded GelMA-HAP-SN hydrogels are potential graft materials for bone defect treatment.

The RNA demethylase ALKBH5 promotes osteoblast differentiation by modulating Runx2 mRNA stability

AlkB homolog 5 (ALKBH5) has been reported as a key m6A demethylase that is involved in development and diseases; however, the function of ALKBH5 in osteogenesis remains unknown. In this study, we report that ALKBH5 mRNA and protein expression were upregulated during osteoblast differentiation and that ALKBH5 knockdown suppressed osteoblast differentiation, mineralization, and the expression of osteogenic biomarkers. Conversely, ALKBH5 overexpression promoted osteogenesis. Moreover, the expression of wild-type ALKBH5, but not the m6A-modified active site mutant ALKBH5, could rescue ALKBH5 knockdown-induced osteogenesis inhibition. Furthermore, knockdown of ALKBH5 significantly impaired the mRNA stability of the transcription factor Runx2, which plays a key role in osteoblast differentiation. Taken together, our results suggest that ALKBH5 promotes osteogenesis through modulating Runx2 mRNA stability.

High-fat diet exacerbates periodontitis: is it because of dysbacteriosis or stem cell dysfunction?

Previous studies have shown that high-fat diet (HFD) may aggravate periodontitis, however the underlining mechanism remains to be further clarified. This study aims to explore whether HFD promotes periodontitis by inducing periodontal microbiota dysbiosis or stem cell dysfunction. A high-fat diet was given to four-week-old male Sprague-Dawley rats for 12 weeks. Periodontitis was induced during the latter 4 weeks. At the end of the 12th week, samples were collected after euthanasia. Maxillae were harvested for histological or microbial analysis. The microbial 16S rRNA gene sequencing was performed with the Illumina MiSeq platform. The data was analyzed through RDP Classifier against the SILVA database. The mandible molars were harvested for isolating periodontal ligament stem cells (PDLSCs). The protein level of p27, p21, and p16, which are negative regulators of the cell cycle, in PDLSCs were detected. Markers of osteogenic differentiation and pro-inflammatory mediators were detected by real-time polymerase chain reaction. Activation of pro-inflammatory signaling pathways was detected by Western blotting. We found that HFD significantly increased ligature-induced alveolar bone loss. HFD resulted in a less diverse periodontal microbiota, with increased proportions of Lactococcus, Bacillus, Alloprevotella, Carnobacterium, and Exiguobacterium and decreased proportion of Nitrospira. HFD increased the protein levels of p27, p16, and p21, and upregulated the expression of osteogenic biomarkers, IL-1β and IL-10 with the ERK1/2 signaling pathway activated in PDLSCs.

MyomiR-OsteomiR crosstalk induced by different modes and intensities of exercise training and its role in controlling osteogenic differentiation in old male Wistar rats

The crosstalk between skeletal muscles and other tissues such as bones is typically established via the secretion of myokines and myomiRs induced by exercise training (ET). The present study aimed at evaluating the relationship between changes made by different ET modes and intensities in myomiRs, osteomiRs, and other myogenic and osteogenic biomarkers in old male Wistar rats. To this end, a total number of 50 old (23months of age) male Wistar rats were randomly assigned to four experimental groups, namely, moderate-intensity endurance training (MIET), high-intensity endurance training (HIET), moderate-intensity resistance training (MIRT), high-intensity resistance training (HIRT), and control (CON), each one comprised of 10 subjects. The study findings revealed positive correlations between myomiRs (i.e., miR-1) and myomiR-204a (r=0.725; p=0.042), myomiR-1, and runt-related transcription factor 2 (RUNX2) osteogenic marker (r=0.869; p=0.025) in the HIET group, myomiR-206 and peroxisome proliferator-activated receptor gamma (PPARγ) (r=0.908; p=0.012) in the MIRT group, myomiR-133a and osteomiR-133a (r=0.971; p=0.005) in the MIET group, myomiR-133a and osteomiR-204a in the MIRT group (r=0.971; p=0.004), and myomiR-133a and RUNX2 gene expression in the HIET group (r=0.861; p=0.027). It was concluded that myomiRs involved in myoblast-osteoblast differentiation might not alone regulate the myogenic and osteogenic targets in response to different modes and intensities of ET treatments.

Interplay between surface chemistry and osteogenic behaviour of sulphate substituted nano-hydroxyapatite

Surface potential and chemical compositions of the bioceramics are the core of therapeutic effect and are key factors to trigger the interfacial interactions with surrounding hard and soft tissues to repair and regeneration. Ionic substitution in hydroxyapatite (Hap) lattice significantly influences the zeta potential from −16.46 ± 0.66 mV to −6.01 ± 0.68 mV as well as an average nano-rod length from ~40 nm to ~26 nm with respect to SO42− ion content. Moreover, the surface chemistry of Hap is mainly inter-related to SO42− substitution rate at PO42− site. Specifically, nano-sized feature with lowered negative surface potential influences the protein adsorption via their weak repulsive or attractive forces. Bovine serum albumin (BSA) and lysozyme (LSZ) adsorption studies confirmed the increased affinity to active binding sites of Hap's surface. Further, SO42− ion substituted Hap (SNHA) showed improved in vitro biomineralization activity and alkaline phosphatase activity. Expression of osteogenic biomarkers such as collagen I, V, osteopontin and osteocalcin were evaluated in Saos-2 and MC3T3-E1 cells. Gene expression of these markers was influenced by SO42− ion content in Hap (maximum with 10SNHA). Altogether, these data emphasizes that chemical composition and surface properties are dominant aspect in bioceramic development towards bone regeneration.

Composite Hydrogel of Methacrylated Hyaluronic Acid and Fragmented Polycaprolactone Nanofiber for Osteogenic Differentiation of Adipose-Derived Stem Cells.

Composite hydrogels with electrospun nanofibers (NFs) have recently been used to mimic the native extracellular matrix. In this study, composite hydrogels of methacrylated hyaluronic acid containing fragmented polycaprolactone NFs were used for bone tissue engineering. The composite (NF/hydrogel) was crosslinked under ultraviolet (UV) light. The incorporation of fragmented polycaprolactone NFs increased the compression modulus from 1762.5 to 3122.5 Pa. Subsequently, adipose-derived stem cells incorporated into the composite hydrogel exhibited a more stretched and elongated morphology and osteogenic differentiation in the absence of external factors. The mRNA expressions of osteogenic biomarkers, including collagen 1 (Col1), alkaline phosphatase, and runt-related transcription factor 2, were 3–5-fold higher in the composite hydrogel than in the hydrogel alone. In addition, results of the protein expression of Col1 and alizarin red staining confirmed osteogenic differentiation. These findings suggest that our composite hydrogel provides a suitable microenvironment for bone tissue engineering.

Novel osteoprotective nanocochleate formulation: A dual combination therapy-codelivery system against glucocorticoid induced osteoporosis

Phosphatidylserine nanocochleates (Nanocochs) are novel delivery systems that may play a prominent osteoprotective role with their cargo, vitamin D3 (Vit-D3), against osteoporosis. Therefore, this study was conducted to characterize a Nanococh containing vitamin D3 (Nanococh-D3) and investigate its potential role in improving GIO in a rat model. Roll-shaped Nanococh-D3 particles were obtained in a size range of 320 nm with a sustained release performance. Oral Nanococh-D3 significantly increased the bioavailability of Vit-D3, enhanced bone mechanical strength, and improved osteogenic biomarkers including B-ALP, osteocalcin, Ca, and OPG in GIO rats. This formulation markedly suppressed gene expression of RANK and RANKL in treated rats. Histomorphometric analysis showed significant repairs in bone tissues and TRAP staining indicated a significant decrease in osteoclasts using Nanococh-D3 in osteoporotic rats. Nanococh alone similar to Nanococh-D3 acted better than AL as a standard anti-osteoporotic drug in the improvement of bone strength. In conclusion, our results established the potential role of Nanococh-D3 against osteoporosis in rats.

Thermogelling Inclusion Complex System for Fine-Tuned Osteochondral Differentiation of Mesenchymal Stem Cells.

How to control osteochondral differentiation of mesenchymal stem cells at a proper stage is a key issue for articular cartilage regeneration. To solve this problem, injectable scaffolds with different chemical functional groups were designed by introducing one equivalent of α-cyclodextrin (α-CD) carboxylate and α-CD phosphate along poly(ethylene glycol)-poly(l-alanine) (PEG-L-PA) block copolymers. Dynamic light scattering, transmission electron microscopy images, and two-dimensional NMR spectra indicated that the PEG-L-PA block copolymers formed inclusion complexes with α-CD derivatives. Aqueous solutions of PEG-L-PA block copolymers (P), α-CD carboxylate/PEG-L-PA block copolymers (PCC), and α-CD phosphate/PEG-L-PA block copolymers (PCP) underwent sol-to-gel transition as the temperature increased. The storage moduli of P, PCC, and PCP gels ranged from 1000 to 1300 Pa at 37 °C. Tonsil-derived mesenchymal stem cells (TMSCs) were incorporated in situ in the gel during thermogelation of P, PCC, and PCP, which became the three-dimensional cell culture systems with different functional groups. After 21 days of incubation of TMSCs in the P, PCC, and PCP systems, the chondrogenic differentiation biomarker of type II collagen significantly increased in the P system, whereas the osteogenic biomarkers of osteocalcin and runt-related transcription factor 2 significantly increased in the PCP system. Both chondrogenic and osteogenic biomarkers were highly expressed in the PCC system. This study proved that thermogelling inclusion complex systems consisting of PEG-L-PA block copolymers and α-CD derivatives could be an excellent injectable matrix for fine-controlling osteochondral differentiation of mesenchymal stem cells.

Circular RNA YAP1 attenuates osteoporosis through up-regulation of YAP1 and activation of Wnt/β-catenin pathway

BackgroundOsteoporosis is a systemic bone disease resulting from decreased bone mass and bone microstructure degeneration. Yes-associated protein 1 (YAP1) belongs to YAP family and plays a significant part in controlling bone quality. Aim of the studyPresent study aimed to study the function and up-stream mechanism of YAP1 in the differentiation of BMSCs (bone marrow stromal cells) and MC3T3-E1. MethodsALP staining, alizarin red staining and western blot analysis of osteogenic biomarkers determined osteogenic differentiation in BMSCs and MC3T3-E1. Mechanistic assays including luciferase reporter assay, RIP assay and RNA pull down assay disclosed the interplays between RNAs. ResultsYAP1 promoted osteogenic differentiation of BMSCs and MC3T3-E1. Circ_0024097 originated from YAP1 sponged miR-376b-3p to elevate YAP1 expression in BMSCs and MC3T3-E1. Further, YAP1 mediated circ_0024097- promoted effects on osteogenic differentiation. Moreover, circ_0024097 activated Wnt/β-catenin pathway to facilitate osteogenic differentiation. ConclusionIt was firstly uncovered in present study that circ_0024097 attenuated osteoporosis through promoting osteogenic differentiation via miR-376b-3p/YAP1 axis and Wnt/β-catenin pathway.

Mineral Distribution Spatially Patterns Bone Marrow Stromal Cell Behavior on Monolithic Bone Scaffolds

Interfaces between soft tissue and bone are characterized by transitional gradients in composition and structure that mediate substantial changes in mechanical properties. For interfacial tissue engineering, scaffolds with mineral gradients have shown promise in controlling osteogenic behavior of seeded bone marrow stromal cells (bMSCs). Previously, we have demonstrated a ‘top-down’ method for creating monolithic bone-derived scaffolds with patterned mineral distributions similar to native tissue. In the present work, we evaluated the ability of these scaffolds to pattern osteogenic behavior in bMSCs in basic, osteogenic, and chondrogenic biochemical environments. Immunohistochemical (IHC) and histological stains were used to characterize cellular behavior as a function of local mineral content. Alkaline phosphatase, an early marker of osteogenesis, and osteocalcin, a late marker of osteogenesis, were positively correlated with mineral content in basic, osteogenic, and chondrogenic media. The difference in bMSC behavior between the mineralized and demineralized regions was most pronounced in an basic biochemical environment. In the mineralized regions of the scaffold, osteogenic markers were clearly present as early as 4 days in culture. In osteogenic media, osteogenic behavior was observed across the entire scaffold, whereas in chondrogenic media, there was an overall reduction in osteogenic biomarkers. Overall, these results indicate local mineral content of the scaffold plays a key role in spatially patterning bMSC behavior. Our results can be utilized for the development of interfacial tissue engineered scaffolds and understanding the role of local environment in determining bMSC behavior. Statement of significanceSoft tissue-to-bone interfaces, such as tendon-bone, ligament-bone, and cartilage-bone, are ubiquitous in mammalian musculoskeletal systems. These interfacial tissues have distinct, hierarchically-structured gradients of cellular, biochemical, and materials components. Given the complexity of the biological structures, interfacial tissues present unique challenges for tissue engineering. Here, we demonstrate that material-derived cues can spatially pattern osteogenic behavior in bone marrow stromal cells (bMSCs). Specifically, we observed that when the bMSCs are cultured on bone-derived scaffolds with mineral gradients, cells in contact with higher mineral content display osteogenic behavior at earlier times than those on the unmineralized substrate. The ability to pattern the cellular complexity found in native interfaces while maintaining biologically relevant structures is a key step towards creating engineered tissue interfaces.

Changes in the Main Bone Marrow Cell Subpopulations May Potentially Contribute to the Initiation of Acute Myeloid Leukemia

Introduction: In acute myeloid leukemia (AML), differentiation and proliferation of early progenitors is inhibited, leading to blast accumulation in the bone marrow (BM) and in the peripheral blood (PB). Hematopoietic stem cells (HSCs) reside in specialized BM microenvironment (ME), mainly containing osteocytes and adipocytes, derived from BM mesenchymal stem cells (MSCs). Recent studies suggest a possible role of the ME in various hematological malignancies, that either serves as a potential source of an altered milieu or mediates abnormal interaction with HSCs. BM ME contributes to disease pathogenesis, chemotherapy resistance and recurrence in certain types of hematological cancers, but there is limited evidence that BM cells themselves may initiate AML. BM derived MSCs are characterized by a potential to mainly differentiate to adipocytes, osteocytes or chondrocytes. Our experiments have revealed an imbalance between osteocytes and adipocytes in the BM of AML patients that is not observed in the BM of healthy donors. We hypothesize that this imbalance could result in the development of leukemic stem cells/pre-leukemic cells, leading to leukemia initiation. The current study aimed to characterize the composition of AML BM derived adipocytes and osteocytes and evaluate the ability of these subpopulations to support AML cell viability. Methods: Secreted levels of adipogenic and osteogenic biomarkers in the BM plasma were measured using ELISA. The potential of AML MSCs to differentiate to adipocytes and osteocytes was assessed using FACS analysis. Exome sequencing was applied for genetic alteration screening in AML MSCs. Obtained adipocytes and osteocytes were separately co-cultured with AML cells and colony forming unit (CFU) assays were used to evaluate AML cell viability. ProcartaPlex immunoassay was used to evaluate levels of cytokines and other potential mediators secreted to the conditioned media. Results: The study included BM samples of 10 AML patients and 13 healthy donors. Levels of secreted FABP4 and osteocalcin biomarkers were reduced in AML plasma (279.7±164 pg/ml and 0.00057±0.017 pg/ml, respectively) compared to that of healthy controls (1230±1502 pg/ml and 0.0016±0.00052 pg/ml, respectively), with P=0.02 for FABP4 and P=0.017 for osteocalcin. According to adipocyte cell differentiation, patients were divided into two clusters. Cluster-1 demonstrated a higher expression level of FABP4 (mean 65.2±11.4%) compared to healthy controls (mean: 47.5±19.2 %; P=0.05), while cluster-2 exhibited a lower level of FABP4 expression (mean 30.78±6.1%) than healthy controls (mean 47.6±19.2%; P=0.01). A nonsynonymous somatic mutation in the AHNAK2 gene (V3209L) was found in patients' mesenchymal stromal cells. A statistically significant reduction in AML cell proliferation (P<0.01) was revealed following co-culture with adipocyte cells of cluster-1 patients. Of the 10 cytokines tested, only ANG1 levels were significantly elevated in the co-culture of adipocytes from cluster-1 (189.5±17.3pg/ml), cluster-2 (258.08±34.33pg/ml) or osteocytes (392.04±58.3pg/ml) with AML cells of the same patients compared to the levels observed in controls (0.002±0.052pg/ml for adipocytes and 168.663pg/ml for osteocytes; P<0.01). Conclusions: The current study has demonstrated that changes in the secretion of stromal specific biomarkers and in the MSC ability to differentiate to adipocytes and osteocytes indicate impaired functional properties of BM MSCs in AML. Within the adipocyte subpopulation in the AML BM, clusters with either increased or inhibited adipogenesis have been revealed. These variations in adipocyte and osteocyte cells reflect interpatient heterogeneity in AML BM subpopulations. The AHNAK2 gene mutation found in AML MSCs in our study and the previously suggested role of this gene in adipocyte differentiation regulation, may explain in part the phenotype diversity observed in AML patients. Adipocyte cells, derived from an AML patient, were found to inhibit normal hematopoiesis in HSCs of the same patient. This specific crosstalk may be related to the elevated levels of ANG1 mediating abnormal signals from adipocytes to HSCs. Overall, the findings of this study may support the contribution of abnormal composition of the main BM subpopulations to leukemogenesis and could ultimately pave the way to novel therapeutic strategies targeting the BM niche. Disclosures No relevant conflicts of interest to declare.