Immobilization of alkaline phosphatase on microporous nanofibrous fibrin scaffolds for bone tissue engineering

Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PP_i), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (P_i), an inducer of hydroxyapatite formation. P_i is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with β-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.     

Differential Inducibility of Human and Porcine Dental Pulp-Derived Cells into Odontoblasts

A robust method for generating odontoblasts from cultured dental pulp cells has not been established. In this study, efficient methods for deriving odontoblasts from cultured human and porcine dental pulp-derived cells were investigated with special attention to species differences. Cultured human cells showed relatively low alkaline phosphatase (ALP) activity in the presence of dexamethasone (Dex) and β-glycerophosphate (β-Gly). In contrast, the addition of 1,25-dihydroxyvitaminD₃ (VitD3) significantly increased the ALP activity. In porcine cells, β-Gly alone or a combination of Dex and β-Gly significantly increased ALP activity; however, addition of VitD3 reduced this activity. RT-PCR and Western blotting analysis revealed that the combination of three induction reagents on human cells significantly upregulates the expression of osteocalcin mRNA, and dentin sialoprotein. We propose that the combination of Dex, β-Gly, and VitD3 is critical for differentiation of human dental pulp-derived cells into odontoblasts. In addition, the inducibility of dental pulp-derived cells presented remarkable species differences.     

Effects of pyrophosphate on desmal and endochondral mineralization and TNAP activity in organoid culture

During endochondral and desmal osteogenesis, mineralization of bone and cartilage matrix requires an appropriate solubility product of calcium and phosphate, collagen as a nucleator and deactivation of inhibitors, in order to prevent heterotopic calcification. In the 1960s, Fleisch and coworkers detected pyrophosphate (PPi) as an inhibitor of hydroxyapatite crystal growth, which should be removed by cleavage to tissue non-specific alkaline phosphatase (TNAP) activity. This theory had been established by basic experiments performed with collagen gels and demineralized matrices. In order to investigate the effect of PPi on matrix mineralization in bone and cartilage, calcium content and TNAP activity were measured in organoid cultures of mouse calvarial osteoblasts and limb bud cartilage after treatment with PPi and/or levamisole. In organoid cultures, bone and cartilage develop in a clear histotypical manner. PPi did not induce mineralization. Beta-glycerophosphate (β-GP) and inorganic phosphate (Pi) induced mineralization which could be significantly reduced by PPi. Levamisole, an inhibitor of TNAP, also reduced mineralization; the combination with PPi was additive. TNAP activity was increased after treatment with PPi and levamisole in both osteoblast and cartilage cultures. Mineralization induced by β-GP and Pi decreased TNAP activity in the osteoblast but not in cartilage organoid culture. In this culture system, PPi reduced mineralization as predicted by Fleisch's theory. Indications of cleavage of PPi were indirectly found because inhibition of hydrolysis of PPi by levamisole further reduced mineralization, probably due to the higher amounts of PPi available for binding to hydroxyapatite.     

Cell-specific effects of TNF-α and IL-1β on alkaline phosphatase: implication for syndesmophyte formation and vascular calcification

Tumor necrosis factor (TNF)-α and interleukin (IL)-1β stimulate tissue non-specific alkaline phosphatase (TNAP) activity and mineralization in cultures of vascular smooth muscle cells (VSMCs). They are, therefore, considered as stimulators of vascular calcification in the context of atherosclerosis and diabetes type 2. In contrast, although ankylosing spondylitis (AS) leads to the formation of syndesmophytes, which are ectopic ossifications from entheses (where ligaments, tendons and capsules are attached to bone), anti-TNF-α therapies fail to block bone formation in this disease. In this context, our aims were to compare the effects of TNF-α and IL-1β on TNAP activity and mineralization in entheseal cells and VSMCs. Organotypic cultures of mouse ankle entheses were treated or not with TNF-α and IL-1β for 5 days. Micro-computed tomography was performed to determine trabecular bone parameters, and histology to assess TNAP activity and mineralization. Human mesenchymal stem cells cultured in pellets in chondrogenic conditions and human VSMCs were also used to determine the effects of cytokines on TNAP activity and expression, measured by quantitative PCR. In organotypic cultures, TNF-α and IL-1β significantly reduced the tibia BV/TV ratio. They also inhibited TNAP activity in entheseal chondrocytes in situ, and in mouse and human chondrocytes in vitro. In contrast, TNF-α stimulated TNAP expression and activity in human VSMCs. These differences were likely due to cell-specific effects of peroxisome proliferator-activated receptor γ (PPARγ), which is inhibited by TNF-α. Indeed, in human chondrocytes and VSMCs, the PPARγ inhibitor GW-9662 displayed the same opposite effects as TNF-α on TNAP expression. In conclusion, whereas TNF-α and IL-1β stimulate TNAP activity in VSMCs, they inhibit it in entheseal cells in situ and on chondrocytes in vitro. The identification of PPARγ as a likely mediator of cytokine effects deserves consideration for future research on the mechanisms of ectopic ossification.     

First identification of a gene defect for hypophosphatasia: evidence that alkaline phosphatase acts in skeletal mineralization

Hypophosphatasia is a heritable disorder characterized by defective osteogenesis and deficient liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Severe forms of the disease are inherited in an autosomal recessive fashion. We examined cultured skin fibroblasts from twelve patients with severe hypophosphatasia. All were deficient in L/B/K ALP activity, yet produced normal levels of the corresponding mRNA. Sequence analysis of L/B/K ALP cDNA isolated from one of the patient-derived fibroblast lines revealed a point mutation that converted amino acid 162 of mature L/B/K ALP from alanine to threonine. The patient was homozygous and the parents, who are second cousins, heterozygous for this mutation. Introduction of the mutation into an otherwise normal cDNA disrupted the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene resulted in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.     

First Identification of a Gene Defect for Hypophosphatasia: Evidence That Alkaline Phosphatase Acts in Skeletal Mineralization

Hypophosphatasia is a heritable disorder characteriazed by defective osteogenesis and deficient liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Severe forms of the disease are inherited in an autosomal recessive fashion. We examined cultured skin fibroblasts from twelve patients with severe hypophosphatasia. All were deficient in L/B.K ALP activity, yet produced normal levels of the corresponding mRNA. Sequence analysis of L/B/K ALP cDNA isolated from one of the patient-derived fibroblast lines revealed a point mutation that converted amino acid 162 of mature L/B/K ALP from alanine to threonine. The patient was homozygous and the parents, who are second cousins, heterozygous for this mutation. Introduction of the mutation into an otherwise normal cDNA disrupted the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene resulted in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.     

Regulation of Matrix Vesicle Metabolism by Vitamin D Metabolites

Matrix vesicles are membrane organelles found in the extracellular matrix of calcifying cells. Vitamin D-responsive alkaline phosphatase specific activity has been localized to matrix vesicles in chondrocyte and osteoblast cultures. The effect of hormone is both metabolite and cell specific. Alkaline phosphatase in matrix vesicles produced by resting zone chondrocytes is stimulated by 24, 25(OH)₂D₃ whereas alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is responsive to 1,25(OH)₂D₃. However, mesenchymal cell cultures, which exhibit a chondrogenic phenotype when exposed to bone inductive proteins in vitro, produce vesicles with alkaline phosphatase activity that is unaffected by either 1,25(OH)₂D₃ or 24, 25(OH)₂D₃. Incorporation and release of arachidonic acid into phosphatidylethanolamine is also differentially regulated by 1,25(OH)₂D₃ and 24, 25(OH)₂D₃ in chondrocytes. These data suggest that vitamin D metabolites may regulate endochondral ossification by altering matrix vesicle enzyme activities, perhaps through changes in membrane phospholipid metabolism.     

Alkaline and acid phosphatases in bone cells serve as phosphohydrolases at physiological pH in vivo: a histochemical implication

Net activity of tissue-nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) remains to be determined since enzyme histochemistry has adopted biochemically determined optimal pH, which is not likely to represent local pH in vivo. The present study aimed to evaluate TNAP and ACP activities associated with bone cells at physiological pH. At the physiological pH of tissue fluid, intense phosphatase reactions were demonstrable in osteoblasts and osteoclasts as well as the bone matrix associated with osteoclasts. In fresh-frozen and freeze-substituted specimens, intense phosphatase reactions appeared at both alkaline and neutral pH along the entire surface of osteoblasts including the osteoidal surface, where TNAP was shown to be absent by immunohistochemistry. Combined specificity tests suggested that TNAP and ACP in bone cells can serve as phosphohydrolases at pH 7.3 and that reactions along the osteoidal surface of osteoblasts differ from that of TNAP and represent novel enzyme.     

Alkaline and Acid Phosphatases in Bone Cells Serve as Phosphohydrolases at Physiological pH In Vivo: A Histochemical Implication

Net activity of tissue-nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) remains to be determined since enzyme histochemistry has adopted biochemically determined optimal pH, which is not likely to represent local pH in vivo. The present study aimed to evaluate TNAP and ACP activities associated with bone cells at physiological pH. At the physiological pH of tissue fluid, intense phosphatase reactions were demonstrable in osteoblasts and osteoclasts as well as the bone matrix associated with osteoclasts. In fresh-frozen and freeze-substituted specimens, intense phosphatase reactions appeared at both alkaline and neutral pH along the entire surface of osteoblasts including the osteoidal surface, where TNAP was shown to be absent by immunohistochemistry. Combined specificity tests suggested that TNAP and ACP in bone cells can serve as phosphohydrolases at pH 7.3 and that reactions along the osteoidal surface of osteoblasts differ from that of TNAP and represent novel enzyme.     

Bone mineralization‐dependent craniosynostosis and craniofacial shape abnormalities in the mouse model of infantile hypophosphatasia

Background: Inactivating mutations in tissue‐nonspecific alkaline phosphatase (TNAP) cause hypophosphatasia (HPP), which is commonly characterized by decreased bone mineralization. Infants and mice with HPP can also develop craniosynostosis and craniofacial shape abnormalities, although the mechanism by which TNAP deficiency causes these craniofacial defects is not yet known. Manifestations of HPP are heterogeneous in severity, and evidence from the literature suggests that much of this variability is mutation dependent. Here, we performed a comprehensive analysis of craniosynostosis and craniofacial shape variation in the Alpl^?/? mouse model of murine HPP as an initial step toward better understanding penetrance of the HPP craniofacial phenotype. Results: Despite similar deficiencies in alkaline phosphatase, Alpl^?/? mice develop craniosynostosis and a brachycephalic/acrocephalic craniofacial shape of variable penetrance. Only those Alpl^?/? mice with a severe bone hypomineralization defect develop craniosynostosis and an abnormal craniofacial shape. Conclusions: These results indicate that variability of the HPP phenotype is not entirely dependent upon the type of genetic mutation and level of residual alkaline phosphatase activity. Additionally, despite a severity continuum of the bone hypomineralization phenotype, craniofacial skeletal shape abnormalities and craniosynostosis occur only in the context of severely diminished bone mineralization in the Alpl^?/? mouse model of HPP. Developmental Dynamics 245:175–182, 2016. © 2015 Wiley Periodicals, Inc.     

Periostin promotes migration,proliferation, and differentiation of human periodontal ligament mesenchymal stem cells

Overview: Periostin (POSTN) is critical to bone and dental tissue morphogenesis, postnatal development, and maintenance; however, its roles in tissue repair and regeneration mediated by human periodontal ligament mesenchymal stem cells (PDLSCs) remain unclear. The present study was designed to evaluate the effects of POSTN on hPDLSCs in vitro. Materials and Methods: hPDLSCs were isolated and characterized by their expression of the cell surface markers CD44, CD90, CD105, CD34, and CD45. Next, 100 ng/mL recombinant human POSTN protein (rhPOSTN) was used to stimulate the hPDLSCs. Lentiviral POSTN shRNA was used to knockdown POSTN. The cell counting kit-8 (CCK8) and scratch assay were used to analyze cell proliferation and migration, respectively. Osteogenic differentiation was investigated using an alkaline phosphatase (ALP) activity assay, alizarin staining, and quantitative calcium analysis and related genes/protein expression assays. Results: Isolated hPDLSCs were positive for CD44, CD90, and CD105 and negative for CD34 and CD45. In addition, 100 ng/mL rhPOSTN significantly accelerated scratch closure, and POSTN-knockdown cells presented slower closure at 24 h and 48 h. Furthermore, the integrin inhibitor Cilengitide depressed the scratch closure that was enhanced by POSTN at 24 h. The CCK8 assay showed that 100 ng/mL rhPOSTN promoted hPDLSC proliferation. Moreover, 100 ng/mL rhPOSTN increased the expression of RUNX2, OSX, OPN, OCN, and VEGF and enhanced ALP activity and mineralization. POSTN silencing decreased the expression of RUNX2, OSX, OPN, OCN, and VEGF and inhibited ALP activity and mineralization. Conclusions: POSTN accelerated the migration, proliferation, and osteogenic differentiation of hPDLSCs.     

Evidence of differential renal dysfunctions during exercise in men

Post-exercise proteinuria is a common phenomenon in healthy subjects. Previous studies have used albumin (Alb) and β₂-microglobulin (β₂-m) molecules as representatives of high- and low-molecular-weight proteins. Recently, more specific markers of the human kidney proximal tubule have been used to identify the precise site of alterations. Active male subjects underwent two strenuous runs, one 400-m run and one 3000-m run. Urine was collected from the subjects before and after each event. Total protein (TP), Alb, α₁-microglobulin (α₁-m), β₂-m, intestinal alkaline phosphatase (IAP), tissue-nonspecific alkaline phosphatase (TNAP) and N-acetyl-β-d-glucosaminidase (NAG) were determined for each sample. The short-distance run (400?m) resulted in the largest increases (P?≤?0.05) in TP (31-fold), Alb (100-fold) and β₂-m (164-fold) as compared to the long-distance run (3000-m). The α₁-m excretion rates were increased to a lesser extent by the exercises. The IAP activity was slightly increased (+90%) by the 400-m run while the TNAP and NAG activities showed a 6.8-fold and a 3.6-fold increase, respectively, after this event. Smaller increases were recorded for the long-distance run (P?=?0.05). To conclude, the present investigation showed that: (1) post-exercise proteinuria is related to the absolute intensity of exercise; (2) the impairment of protein reabsorption is revealed better by changes in Alb and β₂-m; (3) changes in TNAP and NAG activities could reveal biochemical modifications that occur in the proximal tubule, particularly at the S1-S2 segment.     

Effects of tricalcium silicate cements on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells in vitro

Tricalcium silicate cements have been successfully employed in the biomedical field as bioactive bone and dentin substitutes, with widely acclaimed osteoactive properties. This research analyzed the effects of different tricalcium silicate cement formulations on the temporal osteoactivity profile of human bone marrow-derived mesenchymal stem cells (hMW-MSCs). These cells were exposed to four commercially available tricalcium silicate cement formulations in osteogenic differentiation medium. After 1, 3, 7 and 10 days, quantitative real-time polymerase chain reaction and Western blotting were performed to detect expression of the target osteogenic markers ALP, RUNX2, OSX, OPN, MSX2 and OCN. After 3, 7, 14 and 21 days, alkaline phosphatase assay was performed to detect changes in intracellular enzyme level. An Alizarin Red S assay was performed after 28 days to detect extracellular matrix mineralization. In the presence of tricalcium silicate cements, target osteogenic markers were downregulated at the mRNA and protein levels at all time points. Intracellular alkaline phosphatase enzyme levels and extracellular mineralization of the experimental groups were not significantly different from the untreated control. Quantitative polymerase chain reaction results showed increases in downregulation of RUNX2, OSX, MSX2 and OCN with increasing time of exposure to the tricalcium silicate cements, while ALP showed peak downregulation at day 7. For Western blotting, OSX, OPN, MSX2 and OCN showed increased downregulation with increased exposure time to the tested cements. Alkaline phosphatase enzyme levels generally declined after day 7. Based on these results, it is concluded that tricalcium silicate cements do not induce osteogenic differentiation of hBM-MSCs in vitro.     

SV-40 Large-T Immortalization of Embryonic Bone Cells: Establishment of Osteoblastic Clonal Cell Lines

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC₅₀, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)₂ vitamin D₃ treatment.

The other cell line (RCT-1). derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 mm retinoic acid (RA). ALP activity increased from 0.003 to 0.25 m mole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC₅₀, 10 nM). Osteopontin mRNA was induced by 1.25 (OH)₂D₃.

This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.     

1,25-Dihydroxyvitamin D3 stimulates odontoblastic differentiation of human dental pulp-stem cells in vitro

Background: 1,25-Dihydroxyvitamin D₃ (1,25-OH D₃) plays an important role in mineralized tissue metabolism, including teeth. However, few studies have addressed its role in odontoblastic differentiation of human dental pulp-stem cells (hDPSCs). Aim: This study aimed to understand the influence of various concentrations of 1,25-OH D₃ on the proliferation capacity and early dentinogenesis responses of hDPSCs. Materials and Methods: hDPSCs were obtained from the impacted third molar teeth. Monolayer cultured cells were incubated with a differentiation medium containing different concentrations of 1,25-OH D₃ (0.001, 0.01, and 0.1 µM). All groups were evaluated by S-phase rate [immunohistochemical (IHC) bromodeoxyuridine (BrdU) staining], STRO-1 and dentin sialoprotein (DSP)+ levels (IHC), and alkaline phosphatase (ALP, enzyme-linked immunosorbent assay (ELISA)) levels. Results: The number of cells that entered the S-phase was determined to be the highest and lowest in the control and 0.001 µM 1,25-OH D₃ groups, respectively. The 0.1 µM vitamin D₃ group had the highest increase in DSP+ levels. The highest Stro-1 levels were detected in the control and 0.1 µM 1,25-OH D₃ groups, respectively. The 0.1 µM 1,25-OH D₃ induced a mild increase in ALP activity. Conclusions: This study demonstrated that 1,25-OH D₃ stimulated odontoblastic differentiation of hDPSCs in vitro in a dose-dependent manner. The high DSP + cell number and a mild increase in ALP activity suggest that DPSCs treated with 0.1 μM 1,25-OH D₃ are in the later stage of odontoblastic differentiation. The results confirm that 1,25-OH D₃-added cocktail medium provides a sufficient microenvironment for the odontoblastic differentiation of hDPSCs in vitro.     

Expression of SP7/osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen huoxue decoction北大核心

BACKGROUND: Bushen Huoxue Decoction (BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits. OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase (ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD. METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing (blank control group), Cbfal/RUNX2 gene silencing lentivirus (silencing group), and negative viral vector (negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group. RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity (P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly (P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.