Sol-Gel Synthesis, in vitro Behavior, and Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation and Proliferation of Bioactive Glass 58S.

Background: Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. Methods: The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. Results: The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. Conclusion: The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering.

Icariin inhibits proliferation, migration, and invasion of medulloblastoma DAOY cells by regulation of SPARC.

Icariin (ICA) is obtained from Epimedium brevicornu maxim and exploited to remedy miscellaneous cancers. But the role of ICA in medulloblastoma remains hazy. The research delved into the antitumor activity of ICA in medulloblastoma DAOY cells. ICA with diverse concentrations was utilized to stimulate DAOY cells, and the biological functions of ICA in medulloblastoma DAOY cells were examined. Then, the relative SPARC expression was determined in ICA-managed DAOY cells, and the pc-SPARC vector was transfected into DAOY cells to further probe the influence of SPARC and JAK1/STAT3 and PI3K/AKT pathways in ICA-managed DAOY cells. A xenograft model was established to investigate the function of ICA in vivo. ICA restrained cell viability, expedited apoptosis, prohibited cell migration and invasion, and meanwhile affected the associative factors expression in DAOY cells. Additionally, SPARC expression was declined in ICA-stimulated DAOY cells. Overexpressed SPARC reversed the functions of ICA in above-involved cell behaviors of DAYO cells and the correlative protein levels. Besides, ICA notably frustrated JAK1/STAT3 and PI3K/AKT activations in DAOY cells. Beyond that, ICA prohibited tumor formation in vivo. The results concluded that ICA exhibited the antitumor activity in DAOY cells via decreasing SPARC and inactivating JAK1/STAT3 and PI3K/AKT pathways.

Secreted protein acidic and rich in cysteine and bioenergetics: Extracellular matrix, adipocytes remodeling and skeletal muscle metabolism.

The extracellular matrix (ECM) remodeling plays important roles in both adipocytes shape/expansion remodeling and the skeletal muscle (SM) metabolism. Secreted protein acidic and rich in cysteine (SPARC) is expressed in divers tissues including adipose tissue (AT) and SM where it impacts a variety of remodeling as well as metabolic functions. SPARC, also known as osteonectin or BM-40, is a glycoprotein associated with the ECM. Numerous researches attempted to elucidate the implications of SPARC in these two key metabolic tissues under different conditions. Whereas SPARC deficiency tends to shape the remodeling of the adipocytes and the fat distribution, this deficiency decreases SM metabolic properties. On the other hand, SPARC seems to be an enhancer of the metabolism and a mediator of the exercise-induced adaptation in the SM and as well as an adipogenesis inhibitor. Some findings about the SPARC effects on AT and SM seem "contradictory" in terms of tissue development and energy profile therefore highlighting the mechanistic role of SPARC in both is a priority. Yet, within this review, we expose selected researches and compare the results. We conclude with explanations to "reconcile" the different observations, hypothesize the feedback and regulatory character of SPARC and put its roles within the energetic and structural maps of both adipocytes and myocytes in homeostasis and in situations such as obesity or exercise. These properties explain the modifications and the remodeling seen in AT and SM undergoing adaptive changes (obesity, exercise, etc.) and represent a starting point for precise therapeutic targeting of SPARC-related pathways is conditions such as obesity, sarcopenia and diabetes.

Effects of Huangkui Capsule on the Expression of SPARC in the Kidney Tissue of a Rat Model with Diabetic Nephropathy.

OBJECTIVE: The objective of the research is to investigate the effects of Huangkui capsule on the expression of SPARC in the kidney tissues of diabetic nephropathy. METHODS: SD rats were divided into three groups: normal control group, untreated DN group and HKC-treated DN group. The therapeutic effects and underlying molecular mechanism of HKC on DN rats induced by streptozotocin were evaluated by the levels of serum creatinine, blood urea nitrogen, 24-hour urinary protein and the expression of SPARC. Pathological changes in kidney tissues were observed through hematoxylin-eosin (HE) staining. Moreover, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to detect the variation of SPARC. RESULTS: This study was performed to investigate the effects of HKC on DN in SD rats model and its molecular mechanism. Our results showed that the rats treated with HKC had an improved general state and reduced creatinine, blood urea nitrogen and 24-hour urinary protein levels. The deterioration of renal function was delayed due to treatment with HKC. HE staining was utilized to observe that HKC can improve histopathological findings in the kidney tissues of DN rats, including kidney fibrosis. Results of western blot and qRT-PCR showed that HKC can inhibit the expressions of SPARC in the rat model of DN. CONCLUSION: The present findings demonstrated that HKC inhibited SPARC level and had significant therapeutic effects on DN.

Correlating blood-based DNA methylation markers and prostate cancer risk in African-American men.

The objective of this work was to investigate the clinical significance of promoter gene DNA methylation changes in whole blood from African-American (AA) men with prostate cancer (PCa). We used high throughput pyrosequencing analysis to quantify percentage DNA methylation levels in a panel of 8 genes (RARß2, TIMP3, SPARC, CDH13, HIN1, LINE1, CYB5R2 and DRD2) in blood DNA obtained from PCa and non-cancerous controls cases. Correlations of methylation status and various clinicopathological features were evaluated. Six genes tested achieved significant difference in DNA methylation levels between the PCa compared to control cases (P < 0.05). The TIMP3 loci demonstrated significant correlation of DNA methylation with age for all cases analyzed (p < 0.05). We observed an inverse correlation between CDH13 methylation (p = 0.045; r = -0.21) and serum vitamin D level whereas TIMP3 methylation (p = 0.021; r = -0.24) and DRD2 methylation (p = 0.056; r = -0.201) showed inverse correlation with supplementary vitamin D in the cancer cases. We also observed a direct correlation between methylation of RARß2 (p = 0.0036; r = 0.293) and SPARC (p = 0.0134; r = 0.20) loci with PSA level in the controls but not the cancer cases. In addition, alcohol cases significantly correlated with higher RARß2 methylation (p = 0.0314) in comparison with non-alcohol cases. Furthermore, we observed an inverse correlation of DRD2 methylation (p = 0.0349; r = -0.343) and Gleason score. Our data suggests that promoter methylation occurred more frequently in the blood of AA PCa and is associated with various clinicopathological features in AA men with PCa.

Targeted Pth4-expressing cell ablation impairs skeletal mineralization in zebrafish.

Skeletal development and mineralization are essential processes driven by the coordinated action of neural signals, circulating molecules and local factors. Our previous studies revealed that the novel neuropeptide Pth4, synthesized by hypothalamic cells, was involved in bone metabolism via phosphate regulation in adult zebrafish. Here, we investigate the role of pth4 during skeletal development using single-cell resolution, two-photon laser ablation of Pth4:eGFP-expressing cells and confocal imaging in vivo. Using a stable transgenic Pth4:eGFP zebrafish line, we identify Pth4:eGFP-expressing cells as post-mitotic neurons. After targeted ablation of eGFP-expressing cells in the hypothalamus, the experimental larvae exhibited impaired mineralization of the craniofacial bones whereas cartilage development was normal. In addition to a decrease in pth4 transcript levels, we noted altered expression of phex and entpd5, genes associated with phosphate homeostasis and mineralization, as well as a delay in the expression of osteoblast differentiation markers such as sp7 and sparc. Taken together, these results suggest that Pth4-expressing hypothalamic neurons participate in the regulation of bone metabolism, possibly through regulating phosphate balance during zebrafish development.

Characterization of Collagen Peptides in Elaphuri Davidiani Cornu Aqueous Extract with Proliferative Activity on Osteoblasts Using Nano-Liquid Chromatography in Tandem with Orbitrap Mass Spectrometry.

First documented in Shennong Bencao Jing (about 200 B.C.-200 A.D.), Elaphuri Davidiani Cornu (EDC) has been recorded for its effects in strengthening bones and balancing other aspects of overall health for approximately 2000 years. In the present study, our aim was to investigate which are the components of the active EDC fraction by a peptidomic strategy. We explored the extent to which EDC increases the proliferation of osteoblasts by measuring the elevations in osteonectin and type I collagen mRNA levels and characterized it using nano-flow liquid chromatography in tandem with orbitrap mass spectrometry. In total, 272 peptide sequences from collagens were determined. "Hot regions" in parent proteins determined by peptide heat maps which indicated that amino acid sequences in the regions might undergo proteolysis easily and generate peptides. Among the identified peptides, 90.2% were hydrophilic, and the molecular weight of 97.1% of identified peptides was lower than 2000 Da. According to these results, EDC collagen-derived peptides were easily analyzed and identified. Moreover, this methodology is feasible to characterize the active peptides matrices originated from collagen hydrolysates or some other animal horn- derived TCMs.

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Probiotics (Bifidobacterium longum) Increase Bone Mass Density and Upregulate Sparc and Bmp-2 Genes in Rats with Bone Loss Resulting from Ovariectomy.

Probiotics are live microorganisms that exert beneficial effects on the host, when administered in adequate amounts. Mostly, probiotics affect the gastrointestinal (GI) tract of the host and alter the composition of gut microbiota. Nowadays, the incidence of hip fractures due to osteoporosis is increasing worldwide. Ovariectomized (OVX) rats have fragile bone due to estrogen deficiency and mimic the menopausal conditions in women. Therefore, this study aimed to examine the effects of Bifidobacterium longum (B. longum) on bone mass density (BMD), bone mineral content (BMC), bone remodeling, bone structure, and gene expression in OVX rats. The rats were randomly assigned into 3 groups (sham, OVX, and the OVX group supplemented with 1 mL of B. longum 10(8)-10(9) colony forming units (CFU)/mL). B. longum was given once daily for 16 weeks, starting from 2 weeks after the surgery. The B. longum supplementation increased (p < 0.05) serum osteocalcin (OC) and osteoblasts, bone formation parameters, and decreased serum C-terminal telopeptide (CTX) and osteoclasts, bone resorption parameters. It also altered the microstructure of the femur. Consequently, it increased BMD by increasing (p < 0.05) the expression of Sparc and Bmp-2 genes. B. longum alleviated bone loss in OVX rats and enhanced BMD by decreasing bone resorption and increasing bone formation.

Secreted protein acidic and rich in cysteine antagonizes bufalin-induced apoptosis in gastric cancer cells.

Bufalin is an active compound in the traditional Chinese medicine Chan Su, which has been shown to induce apoptosis in a range of cancer cell types. However, certain gastric cancer cells are known to be resistant to bufalin. Intracellular secreted protein acidic and rich in cysteine (SPARC) regulates proliferation and apoptosis. This study aimed to evaluate the role of SPARC in bufalin-induced apoptosis in SGC7901 and MGC803 gastric cancer cells. SGC7901 cells with high SPARC expression were more resistant to bufalin than MGC803 cells with low SPARC expression. This resistance was significantly reversed by small interfering (si)RNA-mediated knockdown of SPARC. Furthermore, it was shown that SPARC negatively regulated bufalin-induced intrinsic apoptosis by protecting mitochondrial integrity, decreasing the release of cytoplasmic cytochrome c and increasing the ratio of Bcl-2/Bax. In addition, SPARC overcame bufalin-induced G2/M phase arrest by increasing levels of Cyclin B1 and Cyclin A protein expression. SPARC also activated cellular survival signals, including Src and Akt, but not extracellular signal-regulated kinase. This study demonstrated that SPARC antagonizes bufalin-induced apoptosis via inhibition of the intrinsic apoptosis pathway, inhibition of cell cycle arrest and activation of certain pathways involved in proliferation. This provides novel evidence for SPARC as a potential target by which to sensitize gastric cancer cells to bufalin.

Pharmacological regulation of SPARC by lovastatin in human trabecular meshwork cells.

PURPOSE: Statins have been shown to increase aqueous outflow facility. The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is a critical mediator of aqueous outflow and intraocular pressure (IOP). Here, we examine the effects of lovastatin on SPARC expression in trabecular meshwork (TM) cells, exploring the molecular mechanisms involved. METHODS: Primary cultured human TM cells were incubated for 24, 48, and 72 hours with 10 µM lovastatin. In separate cultures, media was supplemented with either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) for the duration of the 72-hour time point experiment. Trabecular meshwork cells were also pretreated for 24 hours with lovastatin followed by 24-hour stimulation with 3 ng/mL TGF-ß2. Cell lysates and media were harvested and relative mRNA and protein level changes were determined. Krüppel-like factor 4 (KLF4) localization in normal human anterior segments was examined by immunofluorescence. Adenovirus expressing human KLF4 was used and relative changes in SPARC mRNA and protein levels were assessed. RESULTS: Incubating TM cells with lovastatin suppressed SPARC mRNA and protein levels. This effect was reversed upon media supplementation with GGPP but not FPP. Pretreating cells with lovastatin inhibited TGF-ß2 induction of SPARC. The KLF4 transcription factor was expressed throughout the TM and the inner and outer walls of Schlemm's canal. Lovastatin treatment upregulated KLF4 mRNA and protein levels. Overexpression of KLF4 downregulated SPARC expression. CONCLUSIONS: Collectively, our data identify lovastatin as an important pharmacological suppressor of SPARC expression in TM cells, and provide further insight into the molecular mechanisms mediating statin enhancement of aqueous outflow facility.

Ascorbic acid induces osteoblast differentiation of human suspension mononuclear cells.

BACKGROUND AIMS: Suspension mononuclear cells (MNCs) can be differentiated into osteoblasts with the induction of ascorbic acid and ß-glycerophosphate. The aim of this study was to determine the ability of suspension MNCs to differentiate into osteoblasts using ascorbic acid only. METHODS: Suspension MNCs were obtained by a combination of gradient centrifugation and culture selection. Suspension MNCs were subjected to differentiation assay by culturing them inside proliferation medium supplemented with 10 µg/mL, 30 µg/mL, 50 µg/mL, 60 µg/mL, 90 µg/mL and 500 µg/mL of ascorbic acid. Proliferation medium supplemented with 50 µg/mL ascorbic acid and 10 mmol/L ß-glycerophosphate was used as a positive control for osteoblast induction, and proliferation medium without ascorbic acid was used as a negative control. Differentiation analysis was performed using alkaline phosphatase (ALP) assay, von Kossa staining and expression of osteoblast-related genes. RESULTS: With all concentrations of ascorbic acid used, there was a significant increase (P < 0.05) in ALP-specific activity and mineralized nodule formation throughout the differentiation course compared with negative control. Ascorbic acid was also able to activate the expression of osteopontin (SPP1), osteonectin (SPARC) and runt-related transcription factor 2 (RUNX2) messenger RNA in positive control and ascorbic acid-induced MNCs (30 µg/mL and 90 µg/mL) but not in negative control. CONCLUSIONS: Ascorbic acid alone was sufficient to induce osteoblast differentiation from suspension MNCs; 30-90 µg/mL of ascorbic acid was found to be the optimal concentration. Ascorbic acid can be used as a nutritional supplement for cellular therapy of bone-related disease.

Differential survival trends of stage II colorectal cancer patients relate to promoter methylation status of PCDH10, SPARC, and UCHL1.

Surgical excision of colorectal cancer at early clinical stages is highly effective, but 20-30% of patients relapse. Therefore, it is of clinical relevance to identify patients at high risk for recurrence, who would benefit from adjuvant chemotherapy. The objective of this study was to identify prognostic and/or predictive methylation markers in stage II colorectal cancer patients. Therefore, we selected six gene promoters (FZD9, PCDH10 (protocadherin 10), SFRP2, SPARC (secreted protein acidic and rich in cysteine), UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), and WIF1) for methylation analysis in formalin-fixed, paraffin-embedded primary tumor samples of colorectal cancer patients (n=143) who were enrolled in a prospective randomized phase III trial of the Austrian Breast and Colorectal cancer Study Group. Patients were randomized to adjuvant chemotherapy with 5-fluorouracil and leucovorin or surveillance only. Survival analyses revealed that combined evaluation of three promoters (PCDH10, SPARC, and UCHL1) showed differential effects with regard to disease-free survival and overall survival in the two treatment groups (significance level 0.007). In the chemotherapy arm, a statistically insignificant trend for patients without methylation toward longer survival was observed (P=0.069 for disease-free survival and P=0.139 for overall survival). Contrary, patients in the surveillance arm without methylation in their gene promoters had shorter disease-free survival and overall survival (P=0.031 for disease-free survival and P=0.003 for overall survival), indicating a prognostic effect of methylation in this group (test for interaction, P=0.006 for disease-free survival and P=0.018 for overall survival). These results indicate that promoter methylation status of PCDH10, SPARC, and UCHL1 may be used both as prognostic and predictive molecular marker for colorectal cancer patients and, therefore, may facilitate treatment decisions for stage II colorectal cancer.

The low level laser therapy effect on the remodeling of bone extracellular matrix.

The low level laser therapy (LLLT) has been used as an option to accelerate the regeneration of bone tissue. In this study, both femurs of male Wistar rats (30 animals) were injured with a drill and the effect of LLLT using a laser diode (100 mW at 660 nm) in the bone matrix on the left paw measured. LLLT effect on the healing bone tissue matrix was evaluated by a combination of immunohistochemical histomorphometry, confocal immunofluorescence microscopy and isolation and characterization of glycosaminoglycans. Histomorphometric analysis showed that LLLT increased bone matrix and showing more organized. Alcian Blue and PAS staining seems to suggest differential glycosaminoglycans and glycoproteins. The data showed increased expression of chondroitin sulfate and hyaluronic acid, after reduction as the LLLT and mature bone, resembling the expression of osteonectin and biglycan. The difference in expression of siblings (DMP-1, OPN and BSP) is in accordance with the repair accelerated bone formation after the application of LLLT as compared with control. The expression of osteonectin and osteocalcin supports their role in bone mineralization protein, indicating that LLLT accelerates this process. The overall data show that LLLT bone changes dynamic array, shortening the time period involved in the bone repair.

SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS.

SPARC-like 1 (SC1) is a member of the SPARC family of matricellular proteins that has been implicated in the regulation of processes such as cell migration, proliferation, and differentiation. Here we show that SC1 exhibits remarkably diverse and dynamic expression in the developing and adult nervous system. During development, SC1 localizes to radial glia and pial-derived structures, including the vasculature, choroid plexus, and pial membranes. SC1 is not downregulated in postnatal development, but its expression shifts to distinct time windows in subtypes of glia and neurons, including astrocytes, large projection neurons, Bergmann glia, Schwann cells, and ganglionic satellite cells. In addition, SC1 expression levels and patterns are not altered in the SPARC null mouse, suggesting that SC1 does not compensate for the absence of SPARC. We conclude that SC1 and SPARC may share significant homology, but are likely to have distinct but complementary roles in nervous system development.

Effects of dicalcium silicate coating ionic dissolution products on human mesenchymal stem-cell proliferation and osteogenic differentiation.

This study investigated the effects of ionic dissolution products released from dicalcium silicate (DS) coatings on human mesenchymal stem cells (hMSC), cultured in the presence or absence of the dissolution products, with or without osteogenic supplements (OS). DS(+) medium promoted cell proliferation during the first 4 days, but then inhibited proliferation. DS(+)OS(-) medium increased alkaline phosphatase (ALP) activity on day 14, and upregulated runt-related transcription factor 2 and osteonectin mRNA on days 7 and 14, respectively. The addition of osteogenic supplements (DS(+)OS(+)) led to a significant increase in ALP activity from days 7 to 21, upregulation of osteogenic markers on day 14, and formation of more mineralized nodules on day 28. The results demonstrated that the ionic dissolution products from DS coating alone can partly induce osteogenic differentiation of hMSC, and that the addition of osteogenic supplements further enhances osteoblast-specific gene expression and mineralization in hMSC.

Molecular cloning and functional analysis of an orange-spotted grouper (Epinephelus coioides) secreted protein acidic and rich in cysteine (SPARC) and characterization of its expression response to nodavirus.

Mammalian secreted protein acidic and rich in cysteine (SPARC) is the primary regulator of cell shape and cell adhesion to fibronectin. We, for the first time, report the complete sequencing of SPARC cDNA from orange-spotted grouper. Despite the difference in the lengths of the SPARC transcripts, all of the SPARC molecules encoded a signal peptide, follistain-like copper binding sequence (KGHK) domain, and extracellular domain. The grouper SPARC gene was differentially expressed in vivo and contributed differently to high-level expression of SPARC in muscle. Immunohistochemical staining demonstrated a decreased level of SPARC in nodavirus-infected grouper compared with healthy grouper. Comparative real-time polymerase chain reaction analyses of eye tissues of viral nervous necrosis grouper and healthy grouper were performed. Recombinant SPARC produced changes in grouper cell shape 24 h after treatment. The results provide new insight into the pathogenesis of nodavirus, and demonstrate an experimental rationale for SPARC characterization in nodavirus-infected grouper.

Optimization of culture conditions for osteogenically-induced mesenchymal stem cells in ß-tricalcium phosphate ceramics with large interconnected channels.

The aim of this study was to optimize culture conditions for human mesenchymal stem cells (hMSCs) in ß-tricalcium phosphate ceramics with large interconnected channels. Fully interconnected macrochannels comprising pore diameters of 750 µm and 1400 µm were inserted into microporous ß-tricalcium phosphate (ß-TCP) scaffolds by milling. Human bone marrow-derived MSCs were seeded into the scaffolds and cultivated for up to 3 weeks in both static and perfusion culture in the presence of osteogenic supplements (dexamethasone, ß-glycerophosphate, ascorbate). It was confirmed by scanning electron microscopic investigations and histological staining that the perfusion culture resulted in uniform distribution of cells inside the whole channel network, whereas the statically cultivated cells were primarily found at the surface of the ceramic samples. It was also determined that perfusion with standard medium containing 10% fetal calf serum (FCS) led to a strong increase (seven-fold) of cell numbers compared with static cultivation observed after 3 weeks. Perfusion with low-serum medium (2% FCS) resulted in moderate proliferation rates which were comparable to those achieved in static culture, although the specific alkaline phosphatase (ALP) activity increased by a factor of more than 3 compared to static cultivation. Gene expression analysis of the ALP gene also revealed higher levels of ALP mRNA in low-serum perfused samples compared to statically cultivated constructs. In contrast, gene expression of the late osteogenic marker bone sialoprotein II (BSPII) was decreased for perfused samples compared to statically cultivated samples.

Amniotic fluid-derived mesenchymal stem cells lead to bone differentiation when cocultured with dental pulp stem cells.

Mesenchymal stem cells are present in many tissues of the human body, including amniotic fluid (AF) and dental pulp (DP). Stem cells of both AF and DP give rise to a variety of differentiated cells. In our experience, DP stem cells (DPSCs) display a high capacity to produce bone. Therefore, our aim was to investigate if AF-derived stem cells (AFSCs) were able to undergo bone differentiation in the presence of DPSCs. AFSCs were seeded under three different conditions: (i) cocultured with DPSCs previously differentiated into osteoblasts; (ii) cultured in the conditioned medium of osteoblast-differentiated DPSCs; (iii) cultured in the osteogenic medium supplemented with vascular endothelial growth factor and bone morphogenetic protein-2 (BMP-2). Results showed that AFSCs were positive for mesenchymal markers, and expressed high levels of Tra1-60, Tra1-80, BMPR1, BMPR2, and BMP-2. In contrast, AFSCs were negative for epithelial and hematopoietic/endothelial markers. When AFSCs were cocultured with DPSCs-derived osteoblasts, they differentiated into osteoblasts. A similar effect was observed when AFSCs were cultured in the presence of a conditioned medium originated from DPSCs. We found that osteoblasts derived from DPSCs released large amounts of BMP-2 and vascular endothelial growth factor into the culture medium and that those morphogens significantly upregulate RUNX-2 gene, stimulating osteogenesis. This study highlights the mechanisms of osteogenesis and strongly suggests that the combination of AFSCs with DPSCs may provide a rich source of soluble proteins useful for bone engineering purposes.

Wnt5a promotes differentiation of human dental papilla cells.

AIM: To investigate the role of Wnt5a in the process of differentiation of human dental papilla cells (HDPCs). METHODOLOGY: Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of Wnt5a on the differentiation of HDPCs. The effect of Wnt5a on HDPCs differentiation was determined by ALP activity assay, ALP staining and mineral induction assay. Mineralization-related gene expressions were assessed by RT-PCR. RESULTS: Immunostaining revealed Wnt5a expression in the odontoblast layer and dental papilla tissue. Over-expression of Wnt5a by transfecting HDPCs with an Wnt5a-carrying construct increased ALPase activity and the formation of mineralized nodules of HDPCs. RT-PCR analysis showed that the expressions of mineralization-related genes, such as bone sialoprotein, collagen type I, osteonectin, osteopontin (OCN), dentine matrix protein-1 were up-regulated by Wnt5a. CONCLUSIONS: Wnt5a promoted differentiation of HDPCs.