The Role of Neutrophil Elastase in Aortic Valve Calcification

Background: Calcific aortic valve disease (CAVD) is the most commonly valvular disease in the western countries initiated by inflammation and abnormal calcium deposition. Currently, there is no clinical drugs for CAVD. Neutrophil elastase(NE) plays a causal role in inflammation and participates actively in cardiovascular diseases. However, the effects of NE on valve calcification remains unclear. So we next explore whether it is involved in valve calcification and the molecular mechanisms involved.Methods: NE expression and activity in calcific aortic valve stenosis (CAVS) patients (n=58) and healthy patients (n=30) were measured by enzyme-linked immunosorbent assay (ELISA), western blot and immunohistochemistry (IHC). Porcine aortic valve interstitial cells (pVICs) were isolated and used in vitro expriments. The effects of NE on pVICs inflammation, apoptosis and calcification were detected by hochest 33258 staining, MTT assay, reverse transcription polymerase chain reaction (RT-PCR) and western blot. The effects of NE knockdown and NE activity inhibitor Alvelestat on pVICs inflammation, apoptosis and calcification under osteogenic medium induction were also detected by RT-PCR, western blot, alkaline phosphatase staining and alizarin red staining. Changes of Intracellular signaling pathways after NE treatment were measured by western blot.Results: The level and activity of NE were evaluated in patients with CAVS and calcified valve tissues. NE promoted inflammation, apoptosis and phenotype transition in pVICs in the presence or absence of osteogenic medium. Under osteogenic medium induction, NE silencing or NE inhibitor Alvelestat both suppressed the osteogenic differentiation of pVICs. Mechanically, NE played its role in promoting osteogenic differentiation of pVICs by activating the NF-κB and AKT signaling pathway.Conclusions: Collectively, NE is highly involved in the pathogenesis of valve calcification. Targeting NE such as Alvelestat may be a potential treatment for CAVD.

Nutraceuticals Synergistically Promote Osteogenesis in Cultured 7F2 Osteoblasts and Mitigate Inhibition of Differentiation and Maturation in Simulated Microgravity

Microgravity is known to impact bone health, similar to mechanical unloading on Earth. In the absence of countermeasures, bone formation and mineral deposition are strongly inhibited in Space. There is an unmet need to identify nutritional countermeasures. Curcumin and carnosic acid are phytonutrients with anticancer, anti-inflammatory, and antioxidative effects and may exhibit osteogenic properties. Zinc is a trace element essential for bone formation. We hypothesized that these nutraceuticals could counteract the microgravity-induced inhibition of osteogenic differentiation and function. To test this hypothesis, we cultured 7F2 murine osteoblasts in simulated microgravity (SMG) in a Random Positioning Machine in the presence and absence of curcumin, carnosic acid, and zinc and evaluated cell proliferation, function, and differentiation. SMG enhanced cell proliferation in osteogenic medium. The nutraceuticals partially reversed the inhibitory effects of SMG on alkaline phosphatase (ALP) activity and did not alter the SMG-induced reduction in the expression of osteogenic marker genes in osteogenic medium, while they promoted osteoblast proliferation and ALP activity in the absence of traditional osteogenic media. We further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space.

Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.

SUMO1 modification of IGF-1R combining with SNAI2 inhibited osteogenic differentiation of PDLSCs stimulated by high glucose

Background Periodontal disease, an oral disease characterized by loss of alveolar bone and progressive teeth loss, is the sixth major complication of diabetes. It is spreading worldwide as it is difficult to be cured. The insulin-like growth factor 1 receptor (IGF-1R) plays an important role in regulating functional impairment associated with diabetes. However, it is unclear whether IGF-1R expression in periodontal tissue is related to alveolar bone destruction in diabetic patients. SUMO modification has been reported in various diseases and is associated with an increasing number of biological processes, but previous studies have not focused on diabetic periodontitis. This study aimed to explore the role of IGF-1R in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in high glucose and control the multiple downstream damage signal factors. Methods PDLSCs were isolated and cultured after extraction of impacted teeth from healthy donors or subtractive orthodontic extraction in adolescents. PDLSCs were cultured in the osteogenic medium with different glucose concentrations prepared by medical 5% sterile glucose solution. The effects of different glucose concentrations on the osteogenic differentiation ability of PDLSCs were studied at the genetic and cellular levels by staining assay, Western Blot, RT-PCR, Co-IP and cytofluorescence. Results We found that SNAI2, RUNX2 expression decreased in PDLSCs cultured in high glucose osteogenic medium compared with that in normal glucose osteogenic medium, which were osteogenesis-related marker. In addition, the IGF-1R expression, sumoylation of IGF-1R and osteogenic differentiation in PDLSCs cultured in high glucose osteogenic medium were not consistent with those cultured in normal glucose osteogenic medium. However, osteogenic differentiation of PDLCSs enhanced after adding IGF-1R inhibitors to high glucose osteogenic medium. Conclusion Our data demonstrated that SUMO1 modification of IGF-1R inhibited osteogenic differentiation of PDLSCs by binding to SNAI2 in high glucose environment, a key factor leading to alveolar bone loss in diabetic patients. Thus we could maximize the control of multiple downstream damage signaling factors and bring new hope for alveolar bone regeneration in diabetic patients.

The Effect of Advanced Glycation End Products (AGEs) on Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) with regard to Osteogenesis and Calcification

Background: Diabetes Mellitus is a systemic disease characterized by an increase in blood glucose which, in the long term, enhances advanced glycation end product and leads to impaired osteogenesis. In prosthodontics, the osteogenic process plays an important role in successful treatment. Purpose: The purpose of this study is to determine the effect of Advanced Glycation End products (AGEs) present in Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) on osteogenesis and calcification. Materials and Methods: MSCs isolated from human umbilical cord were cultured and underwent expansion up to passage 5. The research sample was divided into two sub-groups; a treatment group (osteogenic medium+AGE-BSA medium) and a control group (osteogenic medium) each of which underwent three replications. Samples were examined immunocytochemically on days 1, 3, 7, 8, 9, 12, 14 and 21 to quantify the level of RUNX2 expression. Alizarin red staining was performed on day 21. Results: In the treatment group, RUNX2 expression increased on day 3, reaching a peak on days 7 and 14. That expression decreased on day 8. In the control group, the expression of RUNX2 reached its peak on day 8 before decreasing on day 9. The presence of alizarin red indicated calcification in the control medium, but less mineralization in the treatment group. Conclusion: The research indicated that AGE-BSA enhances the production of RUNX2 expression in hUCMSCs at both the initial and maturation stages. This finding was supported by the results of alizarin red staining which indicated that increased levels of RUNX2 produced less mineralization.

Epigenetic Mechanism of TGF-β1 Promoting Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in Osteoarthritis

Objectives: It had been proved that TGF-β1 was correlated with onset of osteoarthritis in vitro and vivo. Here, This study was to elucidate the epigenetic mechanism of TGF-β1 promoting osteogenic differentiation in osteoarthritis. Methods: hBMSCs surface antigens were assayed by flow cytometry tests. qRT-PCR was performed to detect hBMSCs mRNA levels of RUNX2, PPARγ and SOX9. hBMSCs were stained by osteoalkaline phosphatase and alizarin red. The qRT-PCR and Western blot were both used to detect the expression levels of methylases, demethylases and osteogenic transcription factor RUNX2 after hBMSCs were cultrued in osteogenic medium coincubated with TGF-β1 solution. Results: hBMSCs were identified by over expressions of CD90, CD105 and CD44, as well as the positive multi-diffenentiation potential tests. hBMSCs bone alkaline phosphatase and alizarin red staining were observed to deepen in TGF-β1 group compared with the osteogenic culture group. The mRNA expression levels of EZH1, KDM2B, KDM4A/4B/4C/4D, and KDM6A /6B were increased in hBMSCs cultured in osteogenic medium. The expression levels of KDM6A/6B were shown increasement when TGF-β1 was co-incubated with osteogenic medium. Furthermore, the mRNA and protein levels of KDM6A/6B were significantly decreased after SB431542 was added in the medium. RUNX2 was significantly inhibited by the addition of GSK-J4 solution, while KDM6A/6B expression level did not change significantly. Conclusion: The osteogenic differentiation of hBMSCs was related to the enhanced expressions of EZH1, KDM2B, KDM4A-4D, KDM6A/6B. The expression levels of demethylase KDM6A/6B were positively regulated by the TGF-β/Smad signaling pathway, which promoted the osteogenic differentiation of hBMSCs.

Functional tissue-engineered bone-like graft made of a fibrin scaffold and TG2 gene-modified EMSCs for bone defect repair

The transplantation of tissue-engineered scaffolds with stem cells is a promising therapeutic approach for bone defect repair. To improve the therapeutic efficacy of this approach, in this study, a novel biofunctional live tissue-engineered bone-like graft was designed and constructed using a fibrin scaffold loaded with TG2 gene-modified ectomesenchymal stem cells (TG2-EMSCs) derived from nasal respiratory mucosa for bone defect repair. Autocalcification of the cell-free fibrin gel in osteogenic medium with additional alkaline phosphatase (ALP) and the osteogenic differentiation of TG2-EMSCs on the fibrin scaffold were assessed in vitro. The results indicated that the cell-free fibrin gel could autocalcify in the osteogenic medium with ALP and that the overexpression of TG2 by TG2-EMSCs could promote the osteogenic differentiation of these stem cells in the fibrin scaffold. Moreover, TG2 could enhance the deposition of extracellular matrix proteins in the fibrin scaffold, followed by calcification of the bone matrix in vitro. After transplantation into critical-sized cranial defects in rats, the functional tissue-engineered bone-like grafts improved bone regeneration. These results indicate that this tissue-engineered bone-like graft could improve the process of bone defect repair.

Concentrated Growth Factors (CGF) Induce Osteogenic Differentiation in Human Bone Marrow Stem Cells

Bone regeneration is a complex process regulated by several factors that control overlapping biological processes, coordinating interactions among distinct cell populations. There is a great interest in identifying new strategies for inducing osteogenesis in a safe and efficient manner. Concentrated Growth Factor (CGF) is an autologous blood derived product obtained by centrifugation of venous blood following the procedure set on the Silfradent device. In this study the effects of CGF on osteogenic differentiation of human Bone Marrow Stem Cells (hBMSC) in vitro have been investigated; hBMSC were cultured with CGF or osteogenic medium, for 21 days. The osteogenic differentiation was evaluated measuring alkaline phosphatase (ALP) enzyme activity, matrix mineralization by alizarin red staining and through mRNA and protein quantification of osteogenic differentiation markers by Real-time PCR and Western blotting, respectively. The treatment with CGF stimulated ALP activity and promoted matrix mineralization compared to control and seems to be more effective than osteogenic medium. Also, hBMSC lost mesenchymal markers and showed other osteogenic features. Our study showed for the first time that CGF alone is able to induce osteogenic differentiation in hBMSC. The application of CGF on hBMSC osteoinduction might offer new clinical and biotechnological strategies in the tissue regeneration field.

Osteogenic potential of human adipose derived stem cells (hASCs) seeded on titanium trabecular spinal cages

Spine degenerative conditions are becoming increasingly prevalent, affecting about 5.7% of the population in Europe, resulting in a significant reduction of life’s quality. Up to now, many materials have been used in manufacturing cage implants, used as graft substitutes, to achieve immediate and long-term spinal fixation. Particularly, titanium and its alloys are emerging as valuable candidates to develop new types of cages. The aim of this in vitro study was to evaluate the adhesion, proliferation and osteogenic differentiation of adipose derived mesenchymal stem cells (ASCs) seeded on trabecular titanium cages. ASCs adhered, proliferated and produced an abundant extracellular matrix during the 3 weeks of culture. In the presence of osteogenic medium, ASCs differentiated into osteoblast-like cells: the expression of typical bone genes, as well as the alkaline phosphatase activity, was statistically higher than in controls. Furthermore, the dispersive spectrometry microanalysis showed a marked increase of calcium level in cells grown in osteogenic medium. Plus, our preliminary data about osteoinduction suggest that this titanium implant has the potential to induce the ASCs to produce a secretome able to trigger a shift in the ASCs phenotype, possibly towards the osteogenic differentiation, as illustrated by the qRT-PCR and ALP biochemical assay results. The trabecular porous organization of these cages is rather similar to the cancellous bone structure, thus allowing the bone matrix to colonize it efficiently; for these reasons we can conclude that the architecture of this cage may play a role in modulating the osteoinductive capabilities of the implant, thus encouraging its engagement in in vivo studies for the treatment of spinal deformities and diseases.

Direct Conversion of Human Fibroblasts into Osteoblasts Triggered by Histone Deacetylase Inhibitor Valproic Acid

The generation of functional osteoblasts from human somatic cells could provide an alternative means of regenerative therapy for bone disorders such as osteoporosis. In this study, we demonstrated the direct phenotypic conversion of human dermal fibroblasts (HDFs) into osteoblasts by culturing them in osteogenic medium supplemented with valproic acid (VPA), a histone deacetylase (HDAC) inhibitor. HDFs cultured with the VPA in osteogenic medium exhibited expression of alkaline phosphatase and deposition of mineralized calcium matrices, which are phenotypical characteristics of functional osteoblasts. They also expressed osteoblast-specific genes such as alkaline phosphatase, osteopontin, and bone sialoprotein, which demonstrated their direct conversion into osteoblasts. In addition, co-treatment with VPA and a specific inhibitor for activin-like kinase 5 (ALK5i II) had a synergistic effect on direct conversion. It is considered that the inductive effect of VPA on the conversion into osteoblast-lineage is due to the opening of the nucleosome structure by HDAC inhibitor, which facilitates chromatin remodeling and cellular reprogramming. Our findings provide a novel insight into the direct conversion of human somatic cells into transgene-free osteoblasts with small chemical compounds, thus making bone regeneration using cellular reprogramming strategy more clinically feasible.