Bone-derived Cells Research Articles

Application of Periodontal Ligament-Derived Multipotent Mesenchymal Stromal Cell Sheets for Periodontal Regeneration.

Periodontitis is a chronic inflammatory disorder that causes destruction of the periodontal attachment apparatus including alveolar bone, the periodontal ligament, and cementum. Dental implants have been routinely installed after extraction of periodontitis-affected teeth; however, recent studies have indicated that many dental implants are affected by peri-implantitis, which progresses rapidly because of the failure of the immune system. Therefore, there is a renewed focus on periodontal regeneration aroundnatural teeth. To regenerate periodontal tissue, many researchers and clinicians have attempted to perform periodontal regenerative therapy using materials such as bioresorbable scaffolds, growth factors, and cells. The concept of guided tissue regeneration, by which endogenous periodontal ligament- and alveolar bone-derived cells are preferentially proliferated by barrier membranes, has proved effective, and various kinds of membranes are now commercially available. Clinical studies have shown the significance of barrier membranes for periodontal regeneration; however, the technique is indicated only for relatively small infrabony defects. Cytokine therapies have also been introduced to promote periodontal regeneration, but the indications are also for small size defects. To overcome this limitation, ex vivo expanded multipotent mesenchymal stromal cells (MSCs) have been studied. In particular, periodontal ligament-derived multipotent mesenchymal stromal cells are thought to be a responsible cell source, based on both translational and clinical studies. In this review, responsible cell sources for periodontal regeneration and their clinical applications are summarized. In addition, recent transplantation strategies and perspectives about the cytotherapeutic use of stem cells for periodontal regeneration are discussed.

Skeletal-muscle-derived mesenchymal stem/stromal cells from patients with osteoarthritis show superior biological properties compared to bone-derived cells

Mesenchymal stem/stromal cells (MSCs) are being exploited for patient-derived stem-cell therapies. As the biological properties of MSCs derived from skeletal muscle of osteoarthritis patients are poorly understood, the aim of this study was to compare muscle MSCs with well-recognized bone and bone marrow-derived MSCs from these patients.Paired samples of skeletal muscle and trabecular bone tissue were obtained from 21 patients with osteoarthritis. Isolated cells were compared using ex vivo immunophenotyping and detailed in vitro analyses. These included the colony forming unit fibroblast assay, growth kinetics, senescence, multilineage potential, immunophenotyping, and MSC marker gene expression profiling.Freshly isolated MSCs from muscle showed improved viability over bone-derived MSCs, with similar mesenchymal fraction. Muscle-derived MSCs showed superior clonogenicity, higher growth rates, and lower doubling times. Muscle-derived MSCs also showed superior osteogenic and myogenic properties and a positive correlation between CD271 expression and adipogenesis. Senescence rate as well as adipogenic and chondrogenic potentials were similar.Skeletal muscle-derived MSCs of osteoarthritis patients have superior clonogenicity and growth kinetics compared to bone-derived MSCs, making them a good candidate for autologous stem-cell therapies. Moreover, the positive correlation between CD271 and adipogenesis suggest that CD271 expressing muscle MSCs might contribute to muscle steatosis observed in osteoarthritis.

Alterations to the adhesion, growth and osteogenic differentiation of human osteoblast-like cells on nanofibrous polylactide scaffolds with diamond nanoparticles

Nanofibrous scaffolds are promising materials for engineering various types of tissues, including bone tissue. The properties of nanofibers for bone tissue engineering can be improved by adding ceramic or carbon nanoparticles, including diamond nanoparticles (DNPs). In this study, poly(l-lactide) (PLLA) nanofibrous membranes reinforced with detonation DNPs (concentration from 0 to 12.28 wt%), provided by the NanoCarbon Research Institute (Japan) under the name NanoAmando®, were prepared by a needle-less electrospinning process. The biocompatibility of these DNP-reinforced PLLA membranes was tested in vitro using two human lines of osteoblast-like cells, MG-63 and Saos-2. Microscopic and biochemical methods were used. In addition, the expression of cell survival markers, focal adhesion proteins and osteogenic markers were studied in both cell types at mRNA level and at protein level. The mitochondrial activity of both cells types decreased with increasing DNP concentration in the PLLA matrix. At the same time, the expression of cyclin D and survivin, i.e. factors supporting cell growth and survival were downregulated on the DNP-loaded PLLA scaffolds, while KLF6, an antiproliferative factor, was often upregulated, at least in MG-63 cells. Thus, loading the nanofibrous polymeric scaffolds with detonation DNPs had rather adverse effects on the behavior of human bone-derived cell lines. These results are mostly in conflict with our earlier studies on poly(lactide-co-glycolide) (PLGA) scaffolds, loaded with DNPs prepared by etching the diamond layer fabricated by radiofrequency plasma-assisted chemical vapour deposition. The cell behavior on DNP-loaded scaffolds is therefore markedly influenced by the origin and the properties of the DNPs.

The Potential Influence of Bone-Derived Modulators on the Progression of Alzheimer's Disease.

Bone, the major structural scaffold of the human body, has recently been demonstrated to interact with several other organ systems through the actions of bone-derived cells and bone-derived cell secretory proteins. Interestingly, the brain is one organ that appears to fall into this interconnected network. Furthermore, the fact that osteoporosis and Alzheimer's disease are two common age-related disorders raises the possibility that these two organ systems are interconnected in terms of disease pathogenesis. This review focuses on the latest evidence demonstrating the impact of bone-derived cells and bone-derived proteins on the central nervous system, and on how this may be relevant in the progression of Alzheimer's disease and for the identification of novel therapeutic approaches to treat this neurodegenerative disorder.

Spontaneously Formed Spheroids from Mouse Compact Bone-Derived Cells Retain Highly Potent Stem Cells with Enhanced Differentiation Capability.

The results from our recent study showed the presence of two distinct spheroid-forming mechanisms, i.e., spontaneous and mechanical. In this study, we focused on the spontaneously formed spheroids, and the character of spontaneously formed spheroids from mouse compact bone-derived cells (CBDCs) was explored. Cells from (C57BL/6J) mouse leg bones were isolated, and compact bone-derived cells were cultured after enzymatic digestion. Spontaneous spheroid formation was achieved on a culture plate with specific water contact angle as reported. The expression levels of embryonic stem cell markers were analyzed using immunofluorescence and quantitative reverse transcription polymerase chain reaction. Then, the cells from spheroids were induced into osteogenic and neurogenic lineages. The spontaneously formed spheroids from CBDCs were positive for ES cell markers such as SSEA1, Sox2, Oct4, and Nanog. Additionally, the expressions of fucosyltransferase 4/FUT4 (SSEA1), Sox2, and Nanog were significantly higher than those in monolayer cultured cells. The gene expression of mesenchymal stem cell markers was almost identical in both spheroids and monolayer-cultured cells, but the expression of Sca-1 was higher in spheroids. Spheroid-derived cells showed significantly higher osteogenic and neurogenic marker expression than monolayer-cultured cells after induction. Spontaneously formed spheroids expressed stem cell markers and showed enhanced osteogenic and neurogenic differentiation capabilities than cells from the conventional monolayer culture, which supports the superior stemness.

Bone-derived Nestin-positive mesenchymal stem cells improve cardiac function via recruiting cardiac endothelial cells after myocardial infarction

BackgroundBone-derived mesenchymal stem cell (BMSC) transplantation has been reported to be effective for the treatment of ischemic heart disease, but whether BMSCs are the optimal cell type remains under debate. Increasing numbers of studies have shown that Nestin, an intermediate filament protein, is a potential marker for MSCs, which raises the question of whether Nestin+ cells in BMSCs may play a more crucial role in myocardial repair.MethodsNestin+ cells were isolated using flow cytometry by gating for CD45− Ter119− CD31− cells from the compact bone of Nestin-GFP transgenic mice, expressing GFP driven by the Nestin promoter. Colony-forming and proliferative curve assays were conducted to determine the proliferative capacity of these cells, while qRT-PCR was used to analyze the mRNA levels of relative chemokines and growth factors. Cardiac endothelial cell (CEC) recruitment was assessed via a transwell assay. Moreover, permanent ligation of the left anterior descending (LAD) coronary artery was performed to establish an acute myocardial infarction (AMI) mouse model. After cell transplantation, conventional echocardiography was conducted 1 and 4 weeks post-MI, and hearts were harvested for hematoxylin-and-eosin (HE) staining and immunofluorescence staining 1 week post-MI. Further evaluation of paracrine factor levels and administration of a neutralizing antibody (TIMP-1, TIMP-2, and CXCL12) or a CXCR4 antagonist (AMD3100) in MI hearts were performed to elucidate the mechanism involved in the chemotactic effect of Nestin+ BMSCs in vivo.ResultsCompared with Nestin− BMSCs, a greater proliferative capacity of Nestin+ BMSCs was observed, which further exhibited moderately high expression of chemokines instead of growth factors. More CEC recruitment in the Nestin+ BMSC-cocultured group was observed in vitro, while this effect was obviously abolished after treatment with neutralizing antibodies against TIMP-1, TIMP-2, or CXCL12, and more importantly, blocking the CXCL12/CXCR4 axis with a AMD3100 significantly reduced the chemotactic effect of Nestin+ BMSCs. After transplantation into mice exposed to myocardial infarction (MI), Nestin+ BMSC-treated mice showed significantly improved survival and left ventricular function compared with Nestin− BMSC-treated mice. Moreover, endogenous CECs were markedly increased, and chemokine levels were significantly higher, in the infarcted border zone with Nestin+ BMSC treatment. Meanwhile, neutralization of each TIMP-1, TIMP-2, or CXCL12 in vivo could reduce the left ventricular function at 1 and 4 weeks post-MI; importantly, the combined use of these three neutralizing antibodies could make a higher significance on cardiac function. Finally, blocking the CXCL12/CXCR4 axis with AMD3100 significantly reduced the left ventricular function and greatly inhibited Nestin+ BMSC-induced CEC chemotaxis in vivo.ConclusionsThese results suggest that Nestin+ BMSC transplantation can improve cardiac function in an AMI model by recruiting resident CECs to the infarcted border region via the CXCL12/CXCR4 chemokine pathway. And we demonstrated that Nestin+BMSC-secreted TIMP-1/2 enhances CXCL12(SDF1α)/CXCR4 axis-driven migration of endogenous Sca-1+ endothelial cells in ischemic heart post-AMI. Taken together, our results show that Nestin is a useful marker for the identification of functional BMSCs and indicate that Nestin+ BMSCs could be a better therapeutic candidate for cardiac repair.

Fatty acids from fish or vegetable oils promote the adipogenic fate of mesenchymal stem cells derived from gilthead sea bream bone potentially through different pathways.

Fish are rich in n-3 long-chain polyunsaturated fatty acids (LC-PUFA), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, thus they have a great nutritional value for human health. In this study, the adipogenic potential of fatty acids commonly found in fish oil (EPA and DHA) and vegetable oils (linoleic (LA) and alpha-linolenic (ALA) acids), was evaluated in bone-derived mesenchymal stem cells (MSCs) from gilthead sea bream. At a morphological level, cells adopted a round shape upon all treatments, losing their fibroblastic form and increasing lipid accumulation, especially in the presence of the n-6 PUFA, LA. The mRNA levels of the key transcription factor of osteogenesis, runx2 significantly diminished and those of relevant osteogenic genes remained stable after incubation with all fatty acids, suggesting that the osteogenic process might be compromised. On the other hand, transcript levels of the main adipogenesis-inducer factor, pparg increased in response to EPA. Nevertheless, the specific PPARγ antagonist T0070907 appeared to suppress the effects being caused by EPA over adipogenesis. Moreover, LA, ALA and their combinations, significantly up-regulated the fatty acid transporter and binding protein, fatp1 and fabp11, supporting the elevated lipid content found in the cells treated with those fatty acids. Overall, this study has demonstrated that fatty acids favor lipid storage in gilthead sea bream bone-derived MSCs inducing their fate into the adipogenic versus the osteogenic lineage. This process seems to be promoted via different pathways depending on the fatty acid source, being vegetable oils-derived fatty acids more prone to induce unhealthier metabolic phenotypes.

Temperature Affects Musculoskeletal Development and Muscle Lipid Metabolism of Gilthead Sea Bream (Sparus aurata).

World population is expected to increase to approximately 9 thousand million people by 2050 with a consequent food security decline. Besides, climate change is a major challenge that humanity is facing, with a predicted rise in mean sea surface temperature of more than 2°C during this century. This study aims to determine whether a rearing temperature of 19, 24, or 28°C may influence musculoskeletal development and muscle lipid metabolism in gilthead sea bream juveniles. The expression of growth hormone (GH)/insulin-like growth factors (IGFs) system-, osteogenic-, myogenic-, and lipid metabolism-related genes in bone and/or white muscle of treated fish, and the in vitro viability, mineralization, and osteogenic genes expression in primary cultured cells derived from bone of the same fish were analyzed. The highest temperature significantly down-regulated igf-1, igf-2, the receptor igf-1ra, and the binding proteins igfbp-4 and igfbp-5b in bone, and in muscle, igf-1 and igf-1ra, suggesting impaired musculoskeletal development. Concerning myogenic factors expression, contrary responses were observed, since the increase to 24°C significantly down-regulated myod1 and mrf4, while at 28°C myod2 and myogenin were significantly up-regulated. Moreover, in the muscle tissue, the expression of the fatty acid transporters cd36 and fabp11, and the lipases lipa and lpl-lk resulted significantly increased at elevated temperatures, whereas β-oxidation markers cpt1a and cpt1b were significantly reduced. Regarding the primary cultured bone-derived cells, a significant up-regulation of the extracellular matrix proteins on, op, and ocn expression was found with increased temperatures, together with a gradual decrease in mineralization along with fish rearing temperature. Overall, these results suggest that increasing water temperature in this species appears to induce unfavorable growth and development of bone and muscle, through modulating the expression of different members of the GH/IGFs axis, myogenic and osteogenic genes, while accelerating the utilization of lipids as an energy source, although less efficiently than at optimal temperatures.

Enhanced osteogenic differentiation of human bone-derived mesenchymal stem cells in 3-dimensional printed porous titanium scaffolds by static magnetic field through up-regulating Smad4.

The reconstruction of large bone defects remains a significant challenge for orthopedists. Three-dimensional-printed (3DP) scaffold is considered a promising repair material. Static magnetic field (SMF) treatment is an effective and noninvasive therapeutic method to improve bone regeneration. However, the osteogenic effect of SMF on human bone-derived mesenchymal stem cells (hBMSCs) in 3DP scaffolds, as well as its potential mechanism, are unclear. In this study, the osteogenic effect of SMF on hBMSCs in a 3DP scaffold was investigated in vitro and in vivo. In addition, the potential mechanism for promoting osteogenesis was investigated by proteomic analysis. The results showed that SMF promoted osteogenic differentiation of hBMSCs in vitro. A total of 185 differential proteins were identified under SMF conditions by proteomic analysis. The osteogenic effect might be associated with bone morphogenetic protein-Smad1/5/8-signaling pathway and increased transport of phosphorylated Smad1/5/8 and phosphorylated Smad2/3 to the nucleus by up-regulating Smad4 under SMF conditions. The in vivo experiment showed that bone regeneration and osseointegration was enhanced by SMF in the rat model of bone defect. In conclusion, moderate SMF was a safe and effective method for enhancing osteogenesis in 3DP scaffolds in vitro and in vivo.-He, Y., Yu, L., Liu, J., Li, Y., Wu, Y., Huang, Z., Wu, D., Wang, H., Wu, Z., Qiu, G. Enhanced osteogenic differentiation of human bone-derived mesenchymal stem cells in 3-dimensional printed porous titanium scaffolds by static magnetic field through up-regulating Smad4.

Association between chronic kidney disease and carotid intima-media thickness in relation to circulating CD34-positive cell count among community-dwelling elderly Japanese men

Background and aimsEndothelial injury is well-known as a process that can lead to chronic kidney disease (CKD) and atherosclerosis. Hematopoietic activity is known to be associated inversely with CKD and positively with atherosclerosis. Since bone-derived progenitor cells (CD34-positive cells) contribute to endothelial repair (including the progression of atherosclerosis), understanding the association between CKD and carotid intima-media thickness (CIMT), in relation to circulating CD34-positive cell count, may be an efficient means of clarifying the mechanisms underlying endothelial activity. MethodsWe conducted a cross-sectional study of 570 elderly Japanese men aged 60–69 years, who underwent a general health check-up. Participants were stratified as per a median circulating CD34-positive cell count (1.01 cells/μL). ResultsIndependent of the known cardiovascular risk factors, CIMT was found to be positively associated with CKD in the participants with high circulating CD34-positive cell counts but not in participants with low counts. Odds ratios were 1.40 (1.04, 1.89) for participants with high and 1.01 (0.72, 1.43) for participants with low circulating CD34-positive cell counts after adjustment for known cardiovascular risk factors at 95% confidence intervals for CKD with one standard deviation increment of CIMT. ConclusionsA positive association between CIMT and CKD was observed among participants with high circulating CD34-positive cell counts but not among participants with low counts. Endothelial repair activity might determine the association between CKD and CIMT.

Jagged1 promotes mineralization in human bone-derived cells

ObjectivesThe present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined. MethodsGene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively. ResultsFrom bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells. ConclusionNotch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration.

A novel long noncoding RNA AK016739 inhibits osteoblast differentiation and bone formation.

The incidence of postmenopausal osteoporosis research 50% in middle-aged and older women, however, effects of existing therapy are not ideal. Emerging evidence have proved that long noncoding RNAs (lncRNAs) was correlated with multiple physiological and pathology processes including development, carcinogenesis, and osteogenesis. However, reports on lncRNAs regulating bone formation were relatively limited. In this study, we screened osteogenic lncRNAs through mRNA/lncRNA microarray combined with gene coexpression analysis. The biological function of the screened lncRNA was assessed both in vitro and in vivo. The effects of the lncRNA on osteogenic transcription factors were also evaluated. We identified AK016739, which was correlated with osteogenic differentiation and enriched in skeletal tissues of mice. The expression levels of AK016739 in bone-derived mesenchymal stem cells were increased with age and negatively correlated with osteogenic differentiation marker genes. Experiments showed that AK016739 inhibited osteoblast differentiation, and in vivo inhibition of AK016739 by its small interfering RNA would rescue bone formation in ovariectomized osteoporosis mice model. In addition, AK016739 suppressed both expression levels and activities of osteogenic transcription factors. This newly identified lncRNA AK016739 has revealed a new mechanism of osteogenic differentiation and provided new targets for treatment of skeletal disorders.

Very Long-Chain C24:1 Ceramide Is Increased in Serum Extracellular Vesicles with Aging and Can Induce Senescence in Bone-Derived Mesenchymal Stem Cells.

Extracellular vesicles (EVs), including exosomes and microvesicles, function in cell-to-cell communication through delivery of proteins, lipids and microRNAs to target cells via endocytosis and membrane fusion. These vesicles are enriched in ceramide, a sphingolipid associated with the promotion of cell senescence and apoptosis. We investigated the ceramide profile of serum exosomes from young (24–40 yrs.) and older (75–90 yrs.) women and young (6–10 yrs.) and older (25–30 yrs.) rhesus macaques to define the role of circulating ceramides in the aging process. EVs were isolated using size-exclusion chromatography. Proteomic analysis was used to validate known exosome markers from Exocarta and nanoparticle tracking analysis used to characterize particle size and concentration. Specific ceramide species were identified with lipidomic analysis. Results show a significant increase in the average amount of C24:1 ceramide in EVs from older women (15.4 pmol/sample) compared to those from younger women (3.8 pmol/sample). Results were similar in non-human primate serum samples with increased amounts of C24:1 ceramide (9.3 pmol/sample) in older monkeys compared to the younger monkeys (1.8 pmol/sample). In vitro studies showed that primary bone-derived mesenchymal stem cells (BMSCs) readily endocytose serum EVs, and serum EVs loaded with C24:1 ceramide can induce BMSC senescence. Elevated ceramide levels have been associated with poor cardiovascular health and memory impairment in older adults. Our data suggest that circulating EVs carrying C24:1 ceramide may contribute directly to cell non-autonomous aging.

Copy number gain of ZEB1 mediates a double-negative feedback loop with miR-33a-5p that regulates EMT and bone metastasis of prostate cancer dependent on TGF-β signaling.

Background: The reciprocal repressive loop between ZEB1 and miRNAs has been extensively reported to play an important role in tumor progression and metastasis of various human tumor types. The aim of this study was to elucidate the role and the underlying mechanism of the double-negative feedback loop between ZEB1and miR-33a-5p in bone metastasis of prostate cancer (PCa).Methods: miR-33a-5p expression was examined in 40 bone metastatic and 165 non-bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-33a-5p expression and clinicopathological characteristics, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-33a-5p in bone metastasis of PCa were investigated both by EMT and the Transwell assay in vitro, and by a mouse model of left cardiac ventricle inoculation in vivo. siRNA library, real-time PCR and chromatin immunoprecipitation (ChIP) were used to identify the underlying mechanism responsible for the decreased expression of miR-33a-5p in PCa. Bioinformatics analysis, Western blotting and luciferase reporter analysis were employed to examine the relationship between miR-33a-5p and its potential targets. Clinical correlation of miR-33a-5p with its targets was examined in human PCa tissues and primary PCa cells.Results: miR-33a-5p expression was downregulated in PCa tissues with bone metastasis and bone-derived cells, and low expression of miR-33a-5p strongly and positively correlated with advanced clinicopathological characteristics, and shorter overall and bone metastasis-free survival in PCa patients. Upregulating miR-33a-5p inhibited, while silencing miR-33a-5p promoted EMT, invasion and migration of PCa cells. Importantly, upregulating miR-33a-5p significantly repressed bone metastasis of PC-3 cells in vivo. Our results further revealed that recurrent ZEB1 upregulation induced by copy number gains transcriptionally inhibited miR-33a-5p expression, contributing to the reduced expression of miR-33a-5p in bone metastatic PCa tissues. In turn, miR-33a-5p formed a double negative feedback loop with ZEB1 in target-independent manner, which was dependent on TGF-β signaling. Finally, the clinical negative correlations of miR-33a-5p with ZEB1 expression and TGF-β signaling activity were demonstrated in PCa tissues and primary PCa cells.Conclusion: Our findings elucidated that copy number gains of ZEB1-triggered a TGF-β signaling-dependent miR-33a-5p-mediated negative feedback loop was highly relevant to the bone metastasis of PCa.

Surface modification of ultrafine-grained titanium: Influence on mechanical properties, cytocompatibility, and osseointegration potential.

The main objective of this study was to demonstrate that dental implants made from ultrafine-grain titanium (UFG-Ti) can be created that replicate state of the art surfaces of standard coarse-grain titanium (Ti), showing excellent cytocompatibility and osseointegration potential while also providing improved mechanical properties. UFG-Ti was prepared by continuous equal channel angular processing (ECAP), and surfaces were treated by sandblasting and acid etching. Mechanical properties (tensile and fatigue strength), wettability, and roughness parameters were evaluated. Human trabecular bone-derived osteoblast precursor cells (HBCs) were cultured on all samples to examine cytocompatibility and mineralization after 4 and 28days, respectively. Biomechanical pull-out measurements were performed in a rabbit in vivo model 4weeks after implantation. Both yield and tensile strength as well as fatigue endurance were higher for UFG-Ti compared to Ti by 40%, 45%, and 34%, respectively. Fatigue endurance was slightly reduced following surface treatment. Existing surface treatment protocols could be applied to UFG-Ti and resulted in similar roughness and wettability as for standard Ti. Cell attachment and spreading were comparable on all samples, but mineralization was higher for the surfaces with hydrophilic treatment with no significant difference between UFG-Ti and Ti. Pull-out tests revealed that osseointegration of surface-treated UFG-Ti was found to be similar to that of surface-treated Ti. It could be demonstrated that existing surface treatments for Ti can be translated to UFG-Ti and, furthermore, that dental implants made from surface-treated UFG-Ti exhibit superior mechanical properties while maintaining cytocompatibility and osseointegration potential.

Enhanced Osteogenic Differentiation of Human Bone-Derived Mesenchymal Stem Cells in Three-Dimensional Printed Porous Titanium Scaffolds by Static Magnetic Field Through Up-Regulating Smads

Background: The reconstruction of large bone defects remains a significant challenge for orthopedists. 3D printed porous titanium scaffold (3DP scaffold) is considered a promising material for repairing defects. However, its application is limited owing to its poor bioactivity. Static magnetic field (SMF) treatment is effective and non-invasive with the potential for clinical translation, and has been shown to improve bone regeneration. However, the osteogenic effect of SMF on human bone-derived mesenchymal stem cells (hBMSCs) in 3DP scaffolds, as well as its potential mechanism, are unclear. Methods: The osteogenic effect of SMF on hBMSCs in a 3DP scaffold was investigated in vitro and in vivo. In addition, the potential mechanism for promoting osteogenesis was investigated by proteomic analysis and verified in vitro. Findings: The data showed that the cell viability and proliferation of hBMSCs were not significantly affected by SMF. SMF promoted the cellular morphology, adhesion, and interaction of hBMSCs, and enhanced osteogenic differentiation. A total of 185 differential peptides were identified under SMF conditions by proteomic analysis. The transport of pSmad1/5/8 and pSmad2/3 to the nucleus by up-regulating Smad4 under SMF conditions was demonstrated. The in vivo experiment showed that bone regeneration was enhanced by SMF. Interpretation: Moderate SMF was a safe and effective method for enhancing osteogenesis in 3DP scaffolds in vitro and in vivo. This effect might be associated with up-regulating Smads and increased intranuclear translocation of pR-Smads. The details of the potential mechanism should be validated by further work. Funding Statement: This work was supported by the Fundamental Research Funds for Central Public Welfare Research Institutes, Chinese Academy of Medical Sciences (no. 2016ZX310177-7), and the National High-Tech R&D Program of China (863 Program, 2015AA020316, 2015AA033601). Declaration of Interests: The authors state: None. Ethics Approval Statement: The regulations of the Administration of Affairs Concerning Experimental Animals (China, 2014), the National Institutes of Health Guide for the Care and Use of Laboratory Animals (GB14925-2010), and the Laboratory Animal Center of Peking Union Medical College Hospital (PUMCH) were strictly adhered to. Animal experiments were approved by the Institutional Animal Care and Use Committee of PUMCH.

The Effect of Vancomycin on the Viability and Osteogenic Potential of Bone-Derived Mesenchymal Stem Cells.

Traditionally, methicillin-resistant Staphylococcus aureus (MRSA) is treated with vancomycin, administrated intravenously or applied directly onto infected tissue. The effect of direct (as opposed to systemic) vancomycin treatment on bone formation and remodelling is largely unknown. The minimal inhibitory concentration (MIC) of vancomycin was determined by adding 200μL of different concentrations (1-20μg/mL) to actively growing cultures of S. aureus Xen 31 (methicillin-resistant) and S. aureus Xen 36 (methicillin-sensitive), respectively, and recording changes in optical density over 24h. Bone marrow-derived and proximal femur-derived mesenchymal stem cells (bmMSCs and pfMSCs) from rat femora were exposed to 1 × MIC (5μg/mL) and 4 × MIC (20μg/mL) of vancomycin for 7days. Cell viability was determined by staining with crystal violet and MTT (3-(4,5- di methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), respectively, and osteogenic differentiation by staining with Alizarin Red S. Vancomycin had no effect on the viability of bmMSCs and pfMSCs, even at high levels (20μg/mL). The osteogenic differentiation of pfMSCs was partially inhibited, while osteogenesis in bmMSCs was not severely affected. The direct application of vancomycin to infected bone tissue, even at excessive levels, may preserve the viability of resident MSC populations.

Around 90° Contact Angle of Dish Surface Is a Key Factor in Achieving Spontaneous Spheroid Formation.

Following the discovery of the primary culture of neural stem cells, the spheroid culture has been recognized as one of the selective culture methods for somatic stem cells. Since then, various methods were reported to generate spheroids, which can enrich the potent stem cell population. However, the fundamental factors affecting spheroid formation remain unclear. In this study, we focused on the surface property of the culture dishes, in particular, hydrophobicity. Primary mouse skin culture cells were prepared with conventional two-dimensional culture, and then, the cells were transferred to culture dishes with varying hydrophobicity, which was confirmed with the water contact angles. Of these, a culture dish possessing an almost 90° water contact angle was the only one that successfully exhibited spheroid formation. The spheroid formation was spontaneous, efficient, and stable. Since this outcome was achieved with a conventional culture medium with serum, but without any additives such as epidermal growth factor, basic fibroblast growth factor, and B27, the spheroid formation from this process was not affected by serum and was also not dependent on additives. The results from immunofluorescence and quantitative real-time polymerase chain reaction testing showed the expression of embryonic stem cell markers such as SSEA-1, SOX2, OCT4, and Nanog, which confirmed that the spheroids with this method are comparable to those from other methods. This outcome was reproducible and could be applied not only to skin-derived cells but also to oral mucosa-derived cells, cortical bone-derived cells, and 3T3 cells, also suggesting the generality and robustness of this phenomenon.

FasL Modulates Expression of Mmp2 in Osteoblasts

FasL is a well-known actor in the apoptotic pathways but recent reports have pointed to its important novel roles beyond cell death, as observed also for bone cells. This is supported by non-apoptotic appearance of FasL during osteogenesis and by significant bone alterations unrelated to apoptosis in FasL deficient (gld) mice. The molecular mechanism behind this novel role has not yet been revealed. In this report, intramembranous bone, where osteoblasts differentiate directly from mesenchymal precursors without intermediary chondrogenic step, was investigated. Mouse mandibular bone surrounding the first lower molar was used as a model. The stage where a complex set of bone cells (osteoblasts, osteocytes, osteoclasts) is first present during development was selected for an initial examination. Immunohistochemical staining detected FasL in non-apoptotic cells at this stage. Further, FasL deficient vs. wild type samples subjected to osteogenic PCR Array analysis displayed a significantly decreased expression of Mmp2 in gld bone. To examine the possibility of this novel FasL–Mmp2 relationship, intramembranous bone-derived osteoblastic cells (MC3T3-E1) were treated with anti-FasL antibody or rmFasL. Indeed, the FasL neutralization caused a decreased expression of Mmp2 and rmFasL added to the cells resulted in the opposite effect. Since Mmp2-/- mice display age-dependent alterations in the intramembranous bone, early stages of gld mandibular bone were examined and age-dependent phenotype was confirmed also in gld mice. Taken together, the present in vivo and in vitro findings point to a new non-apoptotic function of FasL in bone development associated with Mmp2 expression.

A bone mineralization defect in the Pahenu2 model of classical phenylketonuria involves compromised mesenchymal stem cell differentiation

Osteopenia is observed in some patients affected by phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU). Bone density studies, in diverse PKU patient cohorts, have demonstrated bone disease is neither fully penetrant nor uniform in bone density loss. Biochemical assessment has generated a muddled perspective regarding mechanisms of the PKU bone phenotype where the participation of hyperphenylalaninemia remains unresolved. Osteopenia is realized in the Pahenu2 mouse model of classical PKU; although, characterization is incomplete. We characterized the Pahenu2 bone phenotype and assessed the effect of hyperphenylalaninemia on bone differentiation. Employing Pahenu2 and control animals, cytology, static and dynamic histomorphometry, and biochemistry were applied to further characterize the bone phenotype. These investigations demonstrate Pahenu2 bone density is decreased 33% relative to C57BL/6; bone volume/total volume was similarly decreased; trabecular thickness was unchanged while increased trabecular spacing was observed. Dynamic histomorphometry demonstrated a 25% decrease in mineral apposition. Biochemically, control and PKU animals have similar plasma cortisol, adrenocorticotropic hormone, and 25-hydroxyvitamin D. PKU animals show moderately increased plasma parathyroid hormone while plasma calcium and phosphate are reduced. These data are consistent with a mineralization defect. The effect of hyperphenylalaninemia on bone maturation was assessed in vitro employing bone-derived mesenchymal stem cells (MSCs) and their differentiation into bone. Using standard culture conditions, PAH deficient MSCs differentiate into bone as assessed by in situ alkaline phosphatase activity and mineral staining. However, PAH deficient MSCs cultured in 1200 μM PHE (metric defining classical PKU) show significantly reduced mineralization. These data are the first biological evidence demonstrating a negative impact of hyperphenylalaninemia upon bone maturation. In PAH deficient MSCs, expression of Col1A1 and Rankl are suppressed by hyperphenylalaninemia consistent with reduced bone formation and bone turnover. Osteopenia is intrinsic to PKU pathology in untreated Pahenu2 animals and our data suggests PHE toxicity participates by inhibiting mineralization in the course of MSC bone differentiation.