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Functional Characterization of Osteoblasts and Osteoclasts from Alkaline Phosphatase Knockout Mice
Abstract
Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.
... Thus, damage to complex processes causes bone loss in bone diseases such as osteoporosis and periodontitis [7,8]. As is well established, the ALP (a key osteoblast differentiation marker) enzyme is required for the formation of hydroxyapatite crystals through the hydrolysis of organic phosphomonoesters and inorganic pyrophosphate [5,6,24,25]. Herein, we demonstrated that TriFs increases ALP levels during osteoblast differentiation and induces osteoblast maturation, leading to mineral apposition. ...
... Herein, we demonstrated that TriFs increases ALP levels during osteoblast differentiation and induces osteoblast maturation, leading to mineral apposition. Alkaline phosphate enzyme activity-mediated hydroxyapatite synthesis is an important process in mineral apposition [24,25]. Alkaline phosphate-deficient mice display bone phenotypes including deformities, fractures, and abnormal mineralization [25]. ...
... Alkaline phosphate enzyme activity-mediated hydroxyapatite synthesis is an important process in mineral apposition [24,25]. Alkaline phosphate-deficient mice display bone phenotypes including deformities, fractures, and abnormal mineralization [25]. Therefore, these findings suggest that TriFs exerts anabolic osteogenic effects by accelerating differentiation and maturation. ...
Gentianae Scabrae Radix is used in traditional medicine and is known to possess bioactive compounds, including secoiridoid glycosides, flavonoids, lignans, and triterpenes. Trifloroside (TriFs) is a secoiridoid glycoside known for its antioxidant activity; however, its other effects have not been studied. In the present study, we investigated the biological effects of TriFs isolated from the roots of Gentianae Scabrae Radix using pre-osteoblast MC3T3E-1 cells. No cellular toxicity was observed with 1 mM TriFs, whereas 5–100 mM TriFs showed a gradual increase in cell viability. Alkaline phosphatase staining and microscopic observations revealed that 1–10 mM TriFs stimulated osteogenic activity during early osteoblast differentiation. Trifloroside also increased mineral apposition during osteoblast maturation. Biochemical analyses revealed that TriFs promoted nuclear RUNX2 expression and localization by stimulating the major osteogenic BMP2-Smad1/5/8-RUNX2 pathway. Trifloroside also increased p-GSK3b, β-catenin, p-JNK, and p-p38, but not Wnt3a, p-AKT, and p-ERK. Moreover, TriFs increased the MMP13 levels and promoted cell migration and adhesion. In contrast, TriFs-induced osteoblast differentiation and maturation had negligible effects on autophagy and necrosis. Our findings suggest that TriFs induces osteogenic effects through differentiation, adhesion, migration, and mineral apposition. Therefore, TriFs is suggested as a potential drug target in osteoblast-mediated bone diseases.
... Osteoblast differentiation stimulates the activity and expression of the ALP enzyme, a well-known early osteoblast differentiation marker that induces the hydrolysis of inorganic pyrophosphate and organic phosphomonoesters [5,6,44,45]. With increased ALP activity, collagen and non-collagenous proteins promote calcium deposition and hydroxyapatite crystallization on the extracellular matrix for mineralization and bone formation [46,47]. ...
... Based on our findings, trifol promotes mineralization during osteoblast maturation, which is consistent with the trifol-induced increase in ALP enzyme activity. ALP knockout animals exhibit abnormal mineral apposition and bone fractures [45]. These findings suggest that trifol exerts anabolic activities that accelerate bone formation and mineral apposition. ...
Plant extracts are widely used as traditional medicines. Sophora flavescens Aiton-derived natural compounds exert various beneficial effects, such as anti-inflammatory, anticancer, antioxidant, and antiregenerative activities, through their bioactive compounds, including flavonoids and alkaloids. In the present study, we investigated the biological effects of an S. flavescens-derived flavonoid, trifolirhizin (trifol), on the stimulation of osteogenic processes during osteoblast differentiation. Trifol (>98% purity) was successfully isolated from the root of S. flavescens and characterized. Trifol did not exhibit cellular toxicity in osteogenic cells, but promoted alkaline phosphatase (ALP) staining and activity, with enhanced expression of the osteoblast differentiation markers, including Alp, ColI, and Bsp. Trifol induced nuclear runt-related transcription factor 2 (RUNX2) expression during the differentiation of osteogenic cells, and concomitantly stimulated the major osteogenic signaling proteins, including GSK3β, β-catenin, and Smad1/5/8. Among the mitogen-activated protein kinases (MAPKs), Trifol activated JNK, but not ERK1/2 and p38. Trifol also increased the osteoblast-mediated bone-forming phenotypes, including transmigration, F-actin polymerization, and mineral apposition, during osteoblast differentiation. Overall, trifol exhibits bioactive activities related to osteogenic processes via differentiation, migration, and mineralization. Collectively, these results suggest that trifol may serve as an effective phytomedicine for bone diseases such as osteoporosis.
... Its main function is to provide inorganic phosphate for the synthesis of hydroxyapatite bone mineral. Another task is the hydrolysis of inorganic pyrophosphate, which serves as a mineralization inhibitor [78,79]. Figure 10 shows the activity of ALP in HeLa cells after 7, 14, and 21 days. ...
... Its main function is to provide inorganic phospha for the synthesis of hydroxyapatite bone mineral. Another task is the hydrolysis of ino ganic pyrophosphate, which serves as a mineralization inhibitor [78,79]. Figure 10 show the activity of ALP in HeLa cells after 7, 14, and 21 days. ...
Calcium phosphate nanoparticles are highly biocompatible and biodegradable in bone regeneration. On the other hand, strontium and magnesium enhance the formation of bone. The substitution of calcium by strontium and magnesium is an efficient way to improve the biological properties of calcium phosphate-based biomaterials. Strontium-doped calcium phosphate nanoparticles and magnesium-doped calcium phosphate nanoparticles with degrees of cation substitution of 5, 10, 15, and 20 mol% with respect to calcium were prepared by precipitation, followed by surface functionalization with polyethyleneimine (PEI, cationic) or carboxymethylcellulose (CMC, anionic). The nanoparticles were characterized by dynamic light scattering (DLS), zeta potential measurement, scanning electron microscopy (SEM), atomic absorption spectrometry (AAS), energy dispersive X-ray analysis (EDX), and X-ray powder diffraction (XRD). The particles were approximately spherical (diameter 40–70 nm). The addition of magnesium and strontium considerably decreased the internal crystallinity, i.e., the doped particles were almost X-ray amorphous. The cell-biological effects were assessed on three different cell lines, i.e., HeLa cells, MG63 cells, and MC3T3 cells. Cell viability tests (MTT) showed a low cytotoxicity, the alkaline phosphatase (ALP) activity was strongly increased, and the nanoparticles were taken up well by the three cell lines.
... Its osteogenic activity hydrolyzes inorganic pyrophosphate and organic phosphomonoesters and causes the synthesis of hydroxyapatite and deposition of minerals, leading to bone formation [34,35]. ALP deletion results in abnormal bone formation and spontaneous fracture [36]. In the present study, we found that Suf-B pro- Figure 5. Effects of Suf-B on adhesion and migration. ...
... Its osteogenic activity hydrolyzes inorganic pyrophosphate and organic phosphomonoesters and causes the synthesis of hydroxyapatite and deposition of minerals, leading to bone formation [34,35]. ALP deletion results in abnormal bone formation and spontaneous fracture [36]. In the present study, we found that Suf-B promoted ALP activ-ity during osteoblast differentiation and subsequently promoted mineralization during osteoblast maturation. ...
Suffruticosol B (Suf-B) is a stilbene found in Paeonia suffruticosa ANDR., which has been traditionally used in medicine. Stilbenes and their derivatives possess various pharmacological effects, such as anticancer, anti-inflammatory, and anti-osteoporotic activities. This study aimed to explore the bone-forming activities and mechanisms of Suf-B in pre-osteoblasts. Herein, >99.9% pure Suf-B was isolated from P. suffruticosa methanolic extracts. High concentrations of Suf-B were cytotoxic, whereas low concentrations did not affect cytotoxicity in pre-osteoblasts. Under zero levels of cytotoxicity, Suf-B exhibited bone-forming abilities by enhancing alkaline phosphatase enzyme activities, bone matrix calcification, and expression levels with non-collagenous proteins. Suf-B induces intracellular signal transduction, leading to nuclear RUNX2 expression. Suf-B-stimulated differentiation showed increases in autophagy proteins and autophagosomes, as well as enhancement of osteoblast adhesion and transmigration on the ECM. These results indicate that Suf-B has osteogenic qualities related to differentiation, autophagy, adhesion, and migration. This also suggests that Suf-B could have a therapeutic effect as a phytomedicine in skeletal disorders.
... Moreover, SoyB promotes bone matrix mineralization during the late stage of osteoblast differentiation. As has been well established, inorganic pyrophosphates and organic phosphomonoesters are hydrolyzed by ALP enzyme activity, leading to the synthesis of hydroxyapatite which is provided for bone matrix mineralization [26,29]. It was reported that ALP-knockout mice showed spontaneous fractures, skeletal deformations, and areas of hypomineralization [29], thereby indicating that SoyB increases osteogenic activities to promote bone matrix mineralization from pre-osteoblasts. ...
... As has been well established, inorganic pyrophosphates and organic phosphomonoesters are hydrolyzed by ALP enzyme activity, leading to the synthesis of hydroxyapatite which is provided for bone matrix mineralization [26,29]. It was reported that ALP-knockout mice showed spontaneous fractures, skeletal deformations, and areas of hypomineralization [29], thereby indicating that SoyB increases osteogenic activities to promote bone matrix mineralization from pre-osteoblasts. ...
Triterpenes are a diverse group of natural compounds found in plants. Soyasapogenol B (SoyB) from Arachis hypogaea (peanut) has various pharmacological properties. This study aimed to elucidate the pharmacological properties and mechanisms of SoyB in bone-forming cells. In the present study, 1–20 μM of SoyB showed no cell proliferation effects, whereas 30–100 μM of SoyB increased cell proliferation in MC3T3-E1 cells. Next, osteoblast differentiation was analyzed, and it was found that SoyB enhanced ALP staining and activity and bone mineralization. SoyB also induced RUNX2 expression in the nucleus with the increased phosphorylation of Smad1/5/8 and JNK2 during osteoblast differentiation. In addition, SoyB-mediated osteoblast differentiation was not associated with autophagy and necroptosis. Furthermore, SoyB increased the rate of cell migration and adhesion with the upregulation of MMP13 levels during osteoblast differentiation. The findings of this study provide new evidence that SoyB possesses biological effects in bone-forming cells and suggest a potentially beneficial role for peanut-based foods.
... Moreover, SoyB promotes bone matrix mineralization during late osteoblast differentiation. As well established, inorganic pyrophosphate and organic phosphomonoesters are hydrolyzed by ALP enzyme activity leading to the synthesis of hydroxyapatite which is provided for bone matrix mineralization [22,25]. It was reported that ALP knockout mice show spontaneous fractures, skeletal deformations, and areas of hypomineralization [25], thereby indicating that SoyB increases osteogenic activities to promote bone matrix mineralization from pre-osteoblasts. ...
... As well established, inorganic pyrophosphate and organic phosphomonoesters are hydrolyzed by ALP enzyme activity leading to the synthesis of hydroxyapatite which is provided for bone matrix mineralization [22,25]. It was reported that ALP knockout mice show spontaneous fractures, skeletal deformations, and areas of hypomineralization [25], thereby indicating that SoyB increases osteogenic activities to promote bone matrix mineralization from pre-osteoblasts. ...
Background: Triterpenoid saponins are a diverse group of natural compounds in plants. A triterpene saponin, Soyasapogenol B (SoyB), from Arachis hypogaea (peanut) has various pharmacological properties. This study aimed to elucidate pharmacological properties and mechanisms of SoyB on bone-forming cells.
Methods: Cell viability adhesion, and migration were analyzed using MTT assay, cell adhesion assay, and Boyden chamber assay. Osteogenic activity and osteogenicity were analyzed using alkaline phosphatase (ALP) staining and activity, and Alizarin Red S (ARS) staining. Cell signaling, protein expression, and autophagy were analyzed using Western blot analysis, immunofluorescence assay, and DAPGreen autophagy detection assay.
Results and Conclusion: In the present study, SoyB (> 99.99% purity), triterpene saponin, was isolated from the fruit of A. hypogaea. At concentrations ranging from 1 to 20 mM, SoyB showed no cell proliferation effects, whereas 30 - 100 mM SoyB increased cell proliferation in MC3T3-E1 cells. Next, osteoblast differentiation was analyzed and found that SoyB enhanced ALP staining and activity and bone mineralization as evidence for early and late osteoblast differentiation. SoyB also induced RUNX2 expression in nucleus with the increased phosphorylation of Smad1/5/8 and JNK2 during osteoblast differentiation. In addition, SoyB-mediated osteoblast differentiation was not associated with autophagy and necroptosis. Furthermore, SoyB increased cell migration and adhesion with the upregulation of MMP13 levels during osteoblast differentiation. The findings of this study provide new evidence that SoyB possesses biological effects on osteogenic activity and osteogenicity in bone-forming cells, and suggest a potentially beneficial role for peanuts foods and drugs containing SoyB in the treatment and prevention of bone diseases.
... Calvarial osteoblasts were isolated from 1-d-old Alpl +/− mice following a standard protocol originated form the methods by Boonekamp et al. 26 The isolation procedure is described in detail by Wennberg et al. 27 The procedure was approved by the Institutional Animal Care and Use Committees at Sanford Burnham Prebys Medical Discovery Institute (SBP). The TNAP knockout line has been maintained by Alpl +/− heterozygous breeding under specific pathogen-free condition in the vivarium of SBP (currently AUF21-002). ...
Children with hemato-oncological diseases may have significant skeletal morbidity, not only during and after treatment but also at the time of diagnosis before cancer treatment. This study was designed to evaluate the vitamin D status and circulating bone metabolic markers and their determinants in children at the time of diagnostic evaluation for hemato-oncological disease.
This cross-sectional study included 165 children (91 males, median age 6.9 years range 0.2–17.7 years). Of them, 76 patients were diagnosed with extracranial or intracranial solid tumors, 83 with leukemia, and 6 with bone marrow failure. Bone metabolism was assessed by measuring serum 25-hydroxyvitamin D, parathyroid hormone, bone alkaline phosphatase, intact N-terminal propeptide of type I procollagen, and C-terminal cross-linked telopeptide of type I collagen.
Vitamin D deficiency was found in 30.9% of children. Lower 25-hydroxyvitamin D levels were associated with older age, lack of vitamin D supplementation, season outside summer, and a country of parental origin located between latitudes -45° and 45°. Children diagnosed with leukemia had lower levels of markers of bone formation and bone resorption than those who had solid tumors or bone marrow failure.
In conclusion, vitamin D deficiency was observed in one-third of children with newly diagnosed cancer. Bone turnover markers were decreased in children with leukemia, possibly because of the suppression of osteoblasts and osteoclasts by leukemic cells. The identification of patients with suboptimal vitamin D status and compromised bone remodeling at cancer diagnosis may aid in the development of supportive treatment to reduce the adverse effects of cancer and its treatment.
... Calvarial osteoblasts were isolated from 1-d-old Alpl +/− mice following a standard protocol originated form the methods by Boonekamp et al. 26 The isolation procedure is described in detail by Wennberg et al. 27 The procedure was approved by the Institutional Animal Care and Use Committees at Sanford Burnham Prebys Medical Discovery Institute (SBP). The TNAP knockout line has been maintained by Alpl +/− heterozygous breeding under specific pathogen-free condition in the vivarium of SBP (currently AUF21-002). ...
Tissue-nonspecific alkaline phosphatase (TNALP) is a glycoprotein expressed by osteoblasts that promotes bone mineralization. TNALP catalyzes the hydrolysis of the mineralization inhibitor inorganic pyrophosphate (PPi) and ATP to provide inorganic phosphate (Pi), thus controlling the PPi/Pi ratio to enable the growth of hydroxyapatite crystals. N-linked glycosylation of TNALP is essential for protein stability and enzymatic activity and is responsible for the presence of different bone isoforms of TNALP associated with functional and clinical differences. The site-specific glycosylation profiles of TNALP are, however, elusive. TNALP has five potential N-glycosylation sites located at the asparagine (N) residues 140, 230, 271, 303 and 430. The objective of this study was to reveal the presence and structure of site-specific glycosylation in TNALP expressed in osteoblasts. Calvarial osteoblasts derived from Alpl+/− expressing SV40 Large T antigen were transfected with soluble epitope-tagged human TNALP. Purified TNALP was analyzed with a lectin microarray, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography with tandem mass spectrometry (LC/MS–MS). The results showed that all sites (n = 5) were fully occupied predominantly with complex-type N-glycans. High abundance of galactosylated biantennary N-glycans with various degrees of sialylation was observed on all sites, as well as glycans with no terminal galactose and sialic acid. Furthermore, all sites had core fucosylation except site N271. Modelling of TNALP, with the protein structure prediction software ColabFold, showed possible steric hindrance by the adjacent side chain of W270, which could explain the absence of core fucosylation at N271. These novel findings provide evidence for N-linked glycosylation on all five sites of TNALP, as well as core fucosylation on four out of five sites. We anticipate that this new knowledge can aid in the development of functional and clinical assays specific for the TNALP bone isoforms.
... Alkaline phosphate is known as an osteoblastic mineralization marker, e.g. [34][35][36]. The WNT gene family is pivotal in regulating osteoblast differentiation and bone formation [37]. ...
Background:
Bone damage has welfare and economic impacts on modern commercial poultry and is known as one of the major challenges in the poultry industry. Bone damage is particularly common in laying hens and is probably due to the physiological link between bone and the egg laying process. Previous studies identified and validated quantitative trait loci (QTL) for bone strength in White Leghorn laying hens based on several measurements, including bone composition measurements on the cortex and medulla of the tibia bone. In a previous pedigree-based analysis, bone composition measurements showed heritabilities ranging from 0.18 to 0.41 and moderate to strong genetic correlations with tibia strength and density. Bone composition was measured using infrared spectroscopy and thermogravimetry. The aim of this study was to combine these bone composition measurements with genotyping data via a genome-wide association study (GWAS) to investigate genetic markers that contribute to genetic variance in bone composition in Rhode Island Red laying hens. In addition, we investigated the genetic correlations between bone composition and bone strength.
Results:
We found novel genetic markers that are significantly associated with cortical lipid, cortical mineral scattering, medullary organic matter, and medullary mineralization. Composition of the bone organic matter showed more significant associations than bone mineral composition. We also found interesting overlaps between the GWAS results for tibia composition traits, particularly for cortical lipid and tibia strength. Bone composition measurements by infrared spectroscopy showed more significant associations than thermogravimetry measurements. Based on the results of infrared spectroscopy, cortical lipid showed the highest genetic correlations with tibia density, which was negative (- 0.20 ± 0.04), followed by cortical CO3/PO4 (0.18 ± 0.04). Based on the results of thermogravimetry, medullary organic matter% and mineral% showed the highest genetic correlations with tibia density (- 0.25 ± 0.04 and 0.25 ± 0.04, respectively).
Conclusions:
This study detected novel genetic associations for bone composition traits, particularly those involving organic matter, that could be used as a basis for further molecular genetic investigations. Tibia cortical lipids displayed the strongest genetic associations of all the composition measurements, including a significantly high genetic correlation with tibia density and strength. Our results also highlighted that cortical lipid may be a key measurement for further avian bone studies.
... Fig. 9D illustrated the up-regulated or down-regulated genes in the 7 cell clusters. The dot plots showed the proportion of cells expressing LYZ (cancer marker) [47], CD3D (T cell marker) [48], ALPL (bone remodeling indicator or marker of osteoblast activity) [49], and ACP5 (progression and metastasis-related oncogene) [50] and their scaled relative expression level in 7 cell clusters (Fig. 10A). Specifically, LYZ, ALPL, and ACP5 was highly expressed in OS cell clusters, indicating highly malignant behaviors and osteogenic activities of these tumor cells. ...
Background
Osteosarcoma (OS) is a common malignant tumor in osteoarticular system, the 5-year overall survival of which is poor. Enhancer RNAs (eRNAs) have been implicated in the tumorigenesis of various cancer types, whereas their roles in OS tumorigenesis remains largely unclear.
Methods
Differentially expressed eRNAs (DEEs), transcription factors (DETFs), target genes (DETGs) were identified using limma (Linear Models for Microarray Analysis) package. Prognosis-related DEEs were accessed by univariate Cox regression analysis. A multivariate model was constructed to evaluate the prognosis of OS samples. Prognosis-related DEEs, DETFs, DETGs, immune cells, and hallmark gene sets were co-analyzed to construct an regulatory network. Specific inhibitors were also filtered by connectivity Map analysis. External validation and scRNA-seq analysis were performed to verify our key findings.
Results
3,981 DETGs, 468 DEEs, 51 DETFs, and 27 differentially expressed hallmark gene sets were identified. A total of Multivariate risk predicting model based on 18 prognosis-related DEEs showed a high accuracy (area under curve (AUC) = 0.896). GW-8510 was the candidate inhibitor targeting prognosis-related DEEs (mean = 0.670, p < 0.001). Based on the OS tumorigenesis-related regulation network, we identified that CCAAT enhancer binding protein alpha (CEBPA, DETF) may regulate CD8A molecule (CD8A, DEE), thereby promoting the transcription of CD3E molecule (CD3E, DETG), which may affect allograft rejection based on CD8+ T cells.
Conclusion
We constructed an eRNA-based prognostic model for predicting the OS patients’ prognosis and explored the potential regulation network for OS tumorigenesis by an integrated bioinformatics analysis, providing promising therapeutic targets for OS patients.
... There is no evidence to suggest that osteoblasts from TNAP mutant mice respond differently to PTH at the PTH receptor signaling level. A study by Wennberg et al. found no differences when comparing the functional response to PTH in osteoblasts from TNAP+/+, +/-, and -/-mice, as well as equivalent in vitro bone resorption responses to PTH in calvarial bones [32]. Certainly, the osteoblast production of alkaline phosphatase is compromised and it would be of interest to know what serum calcium and serum PTH levels are in the mutant mice. ...
Hypophosphatasia, the rare heritable disorder caused by TNAP enzyme mutations, presents wide-ranging severity of bone hypomineralization and skeletal abnormalities. Intermittent PTH (1-34) increased long bone volume in Alpl-/- mice but did not alter the skull phenotype. PTH may have therapeutic value for adults with TNAP deficiency-associated osteoporosis.
Introduction:
Hypophosphatasia is the rare heritable disorder caused by mutations in the tissue non-specific alkaline phosphatase (TNAP) enzyme leading to TNAP deficiency. Individuals with hypophosphatasia commonly present with bone hypomineralization and skeletal abnormalities. The purpose of this study was to determine the impact of intermittent PTH on the skeletal phenotype of TNAP-deficient Alpl-/- mice.
Methods:
Alpl-/- and Alpl+/+ (wild-type; WT) littermate mice were administered PTH (1-34) (50 µg/kg) or vehicle control from days 4 to 12 and skeletal analyses were performed including gross measurements, micro-CT, histomorphometry, and serum biochemistry.
Results:
Alpl-/- mice were smaller with shorter tibial length and skull length compared to WT mice. Tibial BV/TV was reduced in Alpl-/- mice and daily PTH (1-34) injections significantly increased BV/TV and BMD but not TMD in both WT and Alpl-/- tibiae. Trabecular spacing was not different between genotypes and was decreased by PTH in both genotypes. Serum P1NP was unchanged while TRAcP5b was significantly lower in Alpl-/- vs. WT mice, with no PTH effect, and no differences in osteoclast numbers. Skull height and width were increased in Alpl-/- vs. WT mice, and PTH increased skull width in WT but not Alpl-/- mice. Frontal skull bones in Alpl-/- mice had decreased BV/TV, BMD, and calvarial thickness vs. WT with no significant PTH effects. Lengths of cranial base bones (basioccipital, basisphenoid, presphenoid) and lengths of synchondroses (growth plates) between the cranial base bones, plus bone of the basioccipitus, were assessed. All parameters were reduced (except lengths of synchondroses, which were increased) in Alpl-/- vs. WT mice with no PTH effect.
Conclusion:
PTH increased long bone volume in the Alpl-/- mice but did not alter the skull phenotype. These data suggest that PTH can have long bone anabolic activity in the absence of TNAP, and that PTH may have therapeutic value for individuals with hypophosphatasia-associated osteoporosis.
... Alkaline phosphatase enzymatic activity is inhibited by partial gravity Alkaline phosphatase is a widely used marker for osteoblast maturation and an important regulator of osteoblast mineralization [37][38][39][40] . To quantify the impairment of osteogenic differentiation by SPG, we measured ALP activity in 7F2 preosteoblasts, cultured for up to 6 days in osteogenic media in Earth (control) and SPG conditions (Mars, Moon, Micro). ...
The multifaceted adverse effects of reduced gravity pose a significant challenge to human spaceflight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts’ function are less well understood. Utilizing the software-driven newer version of the Random Positioning Machine (RPMSW), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon (0.16 G), and microgravity (Micro, ~10−3 G). Short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. Our data suggest that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration.
... Together with OCL, enhanced expression of ALP improves bone mineralization. Recently, Wennerg et al. showed that mineralization of osteoblasts was significantly delayed in alkaline phosphatase (TNAP) knockout mice as well as phenotypic abnormalities of infantile hypophosphatasia occurring in null mice [20]. Therefore, enhanced expression of ALP, OCL together with OPN indicate the beneficial effect of PRHD@MnFe 2 O 4 40/60 on preosteoblasts activity modulating their survival, fate and mineralization (Figures 8 and 9). ...
Osteoporosis is characterized by the reduction of bone mineral density and the weakness of the bone strength leading to fractures. Searching for new compounds that stimulate bone activity and their ability to reconstruct seems to be a promising tool in osteoporosis treatment. Here, we performed analyses comparing the impact of polyrhodanine (PRHD) and its derivatives on the viability (anti-proliferative tests), morphology and mitochondrial network (confocal microscopy) towards pre-osteoblasts (MC3T3-E1 cell line) and osteoclasts (4B12 cell line). Moreover, we assessed the expression of genes associated with the apoptosis, inflammation and osteogenic differentiation by qPCR technique. Our results clearly demonstrated that PRHD and its modification at ratio 10/90 significantly improves the pre-osteoblast’s proliferative abilities, while reducing osteoclast function. The observed effects were strongly correlated with the cytoskeleton and mitochondrial network development and arrangement. Additionally, the expression profile of genes revealed enhanced apoptosis of osteoclasts in the case of PRHD and its modification at ratio 10/90. Moreover, in this case we also observed strong anti-inflammatory properties demonstrated by decreased expression of Il1b, Tnfa and Tgfb in pre-osteoblasts and osteoclasts. On the other hand, enhanced expression of the markers associated with bone remodeling, namely, osteopontin (OPN), osteocalcin (OCL) and alkaline phosphatase (ALP), seem to confirm the role of PRHD@MnFe2O4 in the promotion of differentiation of pre-osteoblasts through the ALP-OPN-OCL axis. Based on these observations, PRHD@MnFe2O4 could be a potential agent in osteoporosis treatment in future, however, further studies are still required.
... Calcifying VSMCs from the aortic arch are more prone to expressing ALP in comparison with VSMCs from the abdominal aorta [53]. Experimental studies have demonstrated a role for ALP in the propagation of tissue calcification [54,55], and we have previously described the putative greater importance of B/I and B1x, in comparison with B1 and B2, in this process [11]. Isoform B2 has the greatest potential to degrade the calcification inhibitor pyrophosphate [20] and also has the highest binding capacity to collagen I [21]. ...
Circulating alkaline phosphatase (ALP) is an independent cardiovascular risk marker. Serum bone ALP (BALP) isoforms indicate bone turnover and comprise approximately 50% of total circulating ALP. In chronic kidney disease (CKD), mortality is highest in patients with increased ALP and BALP and low bone turnover. However, not all low bone turnover states are associated with increased mortality. Chronic inflammation and oxidative stress, features of protein energy wasting syndrome, induce cardiovascular BALP activity and fibro-calcification, while bone turnover is suppressed. Circulating BALP isoform B1x is associated with low ALP and low bone turnover and has been exclusively detected in CKD. We investigated the association of serum B1x with survival, abdominal aortic calcification (AAC) score, and aortic pulse wave velocity (PWV) in CKD. Serum ALP, BALP isoforms, parathyroid hormone (PTH), PWV, and AAC were measured repeatedly over 2 years in 68 prevalent dialysis patients. Mortality was assessed after 5 years. B1x was detected in 53 patients. A competing risk analysis revealed an association of B1x with improved 5-year survival; whereas, baseline PWV, but not AAC score, predicted mortality. However, PWV improved in 26 patients (53%), and B1x was associated with variation of PWV over time (p = 0.03). Patients with B1x had lower PTH and total ALP, suggesting an association with lower bone turnover. In conclusion, B1x is associated with time-varying PWV, lower circulating ALP, and improved survival in CKD, and thus may be an indicator of a reduced cardiovascular risk profile among patients with low bone turnover.
... The LEPR + CD45 -BM-MSCs were divided into six distinct groups by an unbiased clustering analysis (Figure 1C and S2D). Based on known cell markers or functional genes, the different subtypes of BM-MSCs were annotated as: 1) osteoblast precursor (cluster C1, expressing osteogenic markers including collagen 1 and ALPL [36,37]); 2) adipocyte precursor (cluster C2, expressing adiponectin and MGP [38,39]); 3) chondrocyte precursor (cluster C6, expressing CD56 and WIF1 [14,40]); and 4) terminal-stage cells that do not express differentiation markers (clusters C3-C5) (Figure 1D). ...
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.
... Physiologically, TNAP hydrolyzes inorganic pyrophosphate, which is an inhibitor of HA formation during mineralization, and provides inorganic phosphate for HA formation [19,88,89]. Alpl −/− osteoblasts expressed osteopontin (OPN), OCN, COL1, Runx2, and other osteogenic markers, but do not initiate mineralization in vitro [90]. Meanwhile, Alpl −/− mice exhibited bone defects [91]. ...
Bone defects and periodontal disease are pathological conditions that may become neglected diseases if not treated properly. Hydroxyapatite (HA), along with tricalcium phosphate and bioglass ceramic, is a biomaterial widely applied to orthopedic and dental uses. The in vivo performance of HA is determined by the interaction between HA particles with bone cells, particularly the bone mineralizing cells osteoblasts. It has been reported that HA-induced osteoblastic differentiation by increasing the expression of osteogenic transcription factors. However, the pathway involved and the events that occur in the cell membrane have not been well understood and remain controversial. Advances in gene editing and the discovery of pharmacologic inhibitors assist researchers to better understand osteoblastic differentiation. This review summarizes the involvement of extracellular signal-regulated kinase (ERK), p38, Wnt, and bone morphogenetic protein 2 (BMP2) in osteoblastic cellular regulation induced by HA. These advances enhance the current understanding of the molecular mechanism of HA as a biomaterial. Moreover, they provide a better strategy for the design of HA to be utilized in bone engineering.
... Expression of ALP marker at the mRNA level at 14 days of culture determined by qPCR showed that ALP reaches its highest expression in the presence of osteogenic factors as 0.4-fold more elevated than the control media (Supplementary Figure S1). These differences in the assay showed that the effect of the osteogenic factors has a positive impact on the early phase of osteoblast differentiation, and this is in agreement with studies that report that ALP is a marker of osteoblasts phenotype, that confirms the maturation period and the beginning of osteoblasts differentiation and extracellular matrix mineralization (Wennberg et al., 2000;Kim et al., 2012;Martins et al., 2014;Sharma et al., 2014). ...
Skeletal reconstruction is necessary in cases of bone defects created by tumors, trauma, and abnormalities. Regeneration of bone defects remains a critical problem, and current approaches are based on biocompatible scaffolds. Spheroids represent a simple 3D system since no supporting material is required for cell growth. Different techniques are used to generate spheroids, such as hanging drop, low-attachment plates, and magnetic nanoparticles. The idea of using magnetic nanoparticles is to cross-link through cell membrane overnight to create complex 3D cellular spheroid by using magnets to guide the cellular response. Herein, the current study aimed to achieve 3D human fetal osteoblast (hFOB) spheroid under magnetic levitation. Formation of 3D spheroid culture under magnetic levitation was evaluated by cell viability at 3, 7, and 14 days. Morphology of the 3D hFOB spheroid was analyzed by SEM and fluorescence microscopy and the differentiation towards mineralized lineage by ALP assay, qPCR, and alizarin red staining. The cell viability indicated that the 3D hFOB spheroid still viable after 14 days of culture. ALP assay, qPCR analysis expression of Col1, ALP, and Itg-β1 molecules, and calcium deposition with alizarin red showed a high level of bioactivity of the 3D hFOB spheroid. SEM images allowed the morphological analysis of the 3D microtissue-like spheroid with the presence of matrix deposition. These results indicate that magnetic levitation culture enables 3D stable osteoblast spheroids and could be a promising strategy for engineering application in the 3D construct in surgery regeneration of mineralized tissue.
... Moreover, the expression of matrix proteins Col I, BGLAP and OPN in cl1-Ob-Lpar1 −/− cells and Dmp1 in Lpar1ΔOb bone explants are significantly down-regulated. Interestingly, the temporal shift of increase in ALP and BSP transcripts and decay in the level of bone matrix proteins (Col I, OPN, Dmp1) is found in aging and senescent osteoblasts [47,48]. Our data suggest that Lpar1-deficient osteoblasts are prematurely engaged in an aging program. ...
L'os est un organe dynamique qui se renouvelle constamment grâce à une activité coordonnée des ostéoblastes et des ostéocytes qui forment la matrice osseuse nouvelle, et des ostéoclastes qui la dégradent. De la naissance à la fin de l'adolescence, le squelette subit une croissance rapide, médiée par une activité de formation osseuse intense, encore appelée modelage osseux. Cette acquisition de masse osseuse est finement régulée grâce à l'action de nombreux facteurs systémiques et locaux. L'acide lysophosphatidique (LPA) est un médiateur lipidique naturel dont les fonctions biologiques affectent de nombreux organes et de nombreux types cellulaires, y compris les cellules osseuses, ostéoblastes, ostéocytes ostéoclastes. L'action du LPA sur le tissu osseux a été mise en évidence pour la première fois dans notre laboratoire, dans un modèle murin dans lequel l'expression de son récepteur LPA1 a été désactivée de façon globale (Lpar1-/-). Ces animaux présentent un défaut de croissance ainsi que des anomalies osseuses sévères. Cependant, en raison de l'expression ubiquitaire du récepteur LPA1, ce modèle ne permet pas de déterminer l'effet spécifique du LPA dans cellules ostéoblastiques dont l'action est majoritaire au cours de la croissance. L'objectif de mon travail de thèse a été d'étudier le rôle spécifique du LPA et de son récepteur LPA1 dans la lignée ostéoblastique, afin de déterminer son importance pendant le modelage de l'os. Pour ce faire, j'ai étudié des souris Lpar1-ΔOb, générées au laboratoire, et dont l'expression du récepteur LPA1 a été spécifiquement supprimée dans la lignée ostéoblastique. Ces souris ont présenté une baisse de la minéralisation osseuse et une réduction de l'épaisseur de l'os corticale, ainsi qu'une augmentation de la porosité osseuse. In vitro, les ostéoblastes primaires Lpar1-ΔOb et cl1-Ob-Lpar1-/- immortalisés ont révélé une prolifération cellulaire réduite, une altération de la différentiation et une activité de minéralisation réduite. De plus, j'ai mis en évidence chez les souris Lpar1-ΔOb une nette altération de la fonction des ostéocytes. Enfin, j'ai pu montrer in vitro, à la fois dans les ostéoblastes primaires Lpar1-ΔOb et immortalisés cl1-Ob-Lpar1-/-, que l'absence du récepteur LPA1 induit une altération importante de la formation des dendrites, processus précoce et déterminant dans leur différenciation en ostéocyte (ostéocytogenèse). Ces résultats suggèrent un nouveau rôle pour le LPA dans le contrôle de la masse osseuse via la minéralisation osseuse et la fonction des ostéocytes. Ils représentent une piste intéressante à explorer en physiopathologies pour résoudre des problématiques de minéralisation. Ils pourraient également ouvrir des perspectives dans l'exploration de certaines anomalies d'acquisition de masse osseuse comme la scoliose idiopathique de l'adolescent, qui est une pathologie rare dans laquelle des défauts de fonction ostéocytaire similaires à ceux des souris Lpar1-ΔOb ont été rapportés.
... The increased ALP activity of the cells by CORM-3 provides beneficial conditions for the mineralization procedure which enhances the differentiation into osteoblasts. 39 The data of this study demonstrated that CORM-3 notably increased the mRNA and protein expression of ALP, OPN and Runx2, enhanced the ALP activity during the osteogenic induction of the hPDLSCs by releasing CO, which eventually resulted in the increased osteogenic differentiation. ...
Purpose:
Limited intrinsic regeneration capacity following bone destruction remains a significant medical problem. Multiple regulatory effects of carbon monoxide releasing molecule-3 (CORM-3) have been reported. The aim of this study was to investigate the effect of CORM-3 on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) during osteogenesis.
Patients and methods:
hPDLSCs obtained from healthy periodontal ligament tissues were cultured and identified with specific surface antigens by flow cytometry. Effect of CORM-3 on the proliferation of hPDLSCs was determined by CCK-8 assay. Alizarin red staining and alkaline phosphatase (ALP) activity were used to assess the osteogenic differentiation of hPDLSCs. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to detect the expression of the indicated genes. Critical-sized skull defect was made in Balb/c-nude mice, microcomputed tomography (Micro-CT) and Masson trichrome staining were used to assess the new bone regeneration in mice.
Results:
CORM-3 (400 μmol/l) significantly promoted the proliferation of hPDLSCs. CORM-3 pretreatment not only notably enhanced the mRNA and protein expression of osteo-specific marker OPN, Runx2 and ALP, but also increased mineral deposition and ALP activity by the release of CO on day 3, 7 and 14 (P<0.05). Degassed CORM-3 did not show the same effect as CORM-3. In animal model, application of CORM-3 with hPDLSCs transplantation highly increased new bone formation in skull defect region.
Conclusion:
CORM-3 promoted osteogenic differentiation of hPDLSCs, and increased hPDLSCs-induced new bone formation in mice with critical-sized skull defect, which suggests an efficient and promising strategy in the treatment of disease with bone defect.
... In osteoblastic precursor cells, expression of ALPL correlates with the start of mineralization in a time-dependent manner [21]. Cultures of calvaria-derived osteoblasts from ALPL-deficient mice form organized nodules but fail to carry out the process of mineralization [22]. Overall, serum ALPL levels correlate with osteoblastic differentiation and activity and are used to support clinical diagnosis, as they are elevated in diseases with higher levels of bone remodeling, such as Paget's disease of bone and hyperparathyroidism. ...
Bone tissue in vertebrates is essential to performing movements, to protecting internal organs and to regulating calcium homeostasis. Moreover, bone has also been suggested to contribute to whole-body physiology as an endocrine organ, affecting male fertility; brain development and cognition; and glucose metabolism. A main determinant of bone quality is the constant remodeling carried out by osteoblasts and osteoclasts, a process consuming vast amounts of energy. In turn, clinical conditions associated with impaired glucose metabolism, including type I and type II diabetes and anorexia nervosa, are associated with impaired bone turnover. As osteoblasts are required for collagen synthesis and matrix mineralization, they represent one of the most important targets for pharmacological augmentation of bone mass. To fulfill their function, osteoblasts primarily utilize glucose through aerobic glycolysis, a process which is regulated by various molecular switches and generates adenosine triphosphate rapidly. In this regard, researchers have been investigating the complex processes of energy utilization in osteoblasts in recent years, not only to improve bone turnover in metabolic disease, but also to identify novel treatment options for primary bone diseases. This review focuses on the metabolism of glucose in osteoblasts in physiological and pathophysiological conditions.
... From the osteogenic differentiation study, the released rhBMP-2 plays different roles to regulate the expression of osteogenic markers (Fig. 7(c)). As expected, the released rhBMP-2 can significantly increase the expression of Alp which is a promising indicator reflecting the degrees of cell differentiation and functional status of osteoblasts [37], and therefore exhibited the potential use in promoting the bone regeneration. However, for another typical gene-Col I, there were no significant difference in its expression among these three groups, which may possibly be attributed to the related cells have not yet progressed to secretory stage [38]. ...
As generally accepted, inhibiting the bacterial invasion at initial stage and promoting the behavior of related osteogenesis cells afterwards is crucial to achieve favorable osteointegration after dental implantation. In this study, a novel combined structured hydrogel composed of chitosan and pore-closed poly(lactic-co-glycolic acid) microparticles was prepared and characterized. In vitro and in-vivo studies have identified that this biocompatible material can rapidly release vancomycin at initial 2 days and then sustainedly release recombinant human bone morphogenetic protein-2 for about 12 days, thus respectively accomplish antibacterial and osteogenesis functions. This sequential drug release system can be used as a promising coating material to improve the surface conditions of dental implant to enhance the osteointegration after surgery.
... [108] The critical importance of TNAP in proper skeletal mineralization has been extensively studied and reviewed in the literature. [109][110][111][112][113] Studies have shown that TNAP deficiency is associated with hypophosphatasia affecting a range of mineralizing tissues, such as craniofacial and long bones as well as teeth. [114,115] The presence of TNAP in mineralizing tissues such as bone, including rat tibia and femur, and teeth has previously been reported. ...
Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca10(PO4)6(OH)2) within the organic matrix of the tissue. The ability of specific vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing), and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work provided a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then provides generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein (MGP) were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicate that osteocalcin (OCN) is closely involved in mineralization but do not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons (TLTs) as an animal model for vertebrate mineralization.
... It is important to note that while results from this study show that TNAP deficiency decreases trabecular bone volume and promotes bone marrow stromal cell osteoblast and adipocyte differentiation, while decreasing proliferation, we interpret our results to indicate that TNAP is essential for trabecular bone osteogenesis. This is based upon prior studies of Alpl −/− mice that showed significant differences in osteoblast but not osteoclast function between Alpl −/− and Alpl +/+ mice [31]. A limitation of this study is that we did not study osteoclastogenesis or function in Alpl −/− implanted ossicles or tibia. ...
Tissue nonspecific alkaline phosphatase (TNAP/Alpl) is associated with cell stemness; however, the function of TNAP in mesenchymal progenitor cells remains largely unknown. In this study, we aimed to establish an essential role for TNAP in bone and muscle progenitor cells. We investigated the impact of TNAP deficiency on bone formation, mineralization, and differentiation of bone marrow stromal cells. We also pursued studies of proliferation, mitochondrial function and ATP levels in TNAP deficient bone and muscle progenitor cells. We find that TNAP deficiency decreases trabecular bone volume fraction and trabeculation in addition to decreased mineralization. We also find that Alpl−/− mice (global TNAP knockout mice) exhibit muscle and motor coordination deficiencies similar to those found in individuals with hypophosphatasia (TNAP deficiency). Subsequent studies demonstrate diminished proliferation, with mitochondrial hyperfunction and increased ATP levels in TNAP deficient bone and muscle progenitor cells, plus intracellular expression of TNAP in TNAP+ cranial osteoprogenitors, bone marrow stromal cells, and skeletal muscle progenitor cells. Together, our results indicate that TNAP functions inside bone and muscle progenitor cells to influence mitochondrial respiration and ATP production. Future studies are required to establish mechanisms by which TNAP influences mitochondrial function and determine if modulation of TNAP can alter mitochondrial respiration in vivo.
... To further investigate the influence of drug released from Ica-SH/BCP on osteogenic differentiation of OVX-rBMSCs, the expressions of osteogenic specific proteins -ALP, BMP2 and OPN were also analyzed. As a ubiquitous membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters at the basic pH value [44], ALP is usually highly expressed during the initial phase of osteogenesis, and thus ALP is generally considered as an important marker of osteoblastic differentiation, which plays an important role in the initiation of mineralization [45,46]. As shown in Fig. 7A, the quantitative results of ALP activity showed that BCP and Ica-SH/BCP groups displayed the similar ALP activity at day 4 (p>0.05), ...
The regeneration of bone defects in osteoporotic patients presents a big challenge. Icariin (Ica), which is the main active component of traditional Chinese medicine - herba epimedii, has been widely loaded to scaffolds for the treatment of bone fracture and osteoporosis. To increase drug loading capacity, and meanwhile achieve a stable and safe drug delivery, in present study, a novel icariin derivative - thiolated icariin (Ica-SH) molecule with good cytocompatibility and bioactivity was incorporated into biphasic calcium phosphate (BCP) ceramic scaffold through Ica-SH intermolecular disulfide bonds (abbr., Ica-SH/BCP) to form film coating. Compared with Ica-loaded BCP scaffolds via physical adsorption (abbr., Ica/BCP), the formation of Ica-SH intermolecular disulfide bonds in Ica-SH/BCP scaffolds led to a decreased real-time drug release concentration, and a reduced drug release rate. The released drug from Ica-SH/BCP scaffolds could promote the proliferation, migration, and osteoblastic differentiation of ovariectomized rat bone mesenchymal stem cells (OVX-rBMSCs), and up-regulate the angiogenic gene expression in human umbilical vein endothelial cells (HUVECs) in vitro. The OVX-rat femoral condyle defect model also demonstrated that Ica-SH/BCP scaffolds promoted a superior bone regeneration in osteoporotic animals than BCP ones. These findings suggest that Ica-SH is a promising drug, which can be facilely incorporated into a variety of scaffolds for repairing bone defects of patients, in particular the ones with osteoporosis.
... The hypomineralisation is caused by accumulation of inorganic pyrophospate (PPi) in the extracellular matrix, which inhibits hydroxyapatite crystals' formation and affects the cementum in the outside faces of teeth root (Foster et al. 2012). Indeed, osteoblasts from tnsalp null mice differentiate normally but are unable to initiate mineralisation in vitro while osteoclastogenesis and bone resorption are not altered (Wennberg et al. 2000). ...
Bone remodelling is a complex mechanism regulated by osteoclasts and osteoblasts and perturbation of this process leads to the onset of diseases, which may be characterized by altered bone erosion or formation. In this review we will describe some bone formation-related disorders as Sclerosteosis, Van Buchem disease, Hypophosphatasia and Camurati-Engelmann disease. In the past decades the research focused on these rare disorders offered the opportunity to understand important pathways regulating bone formation. Thus, the identification of the molecular defects behind the etiopathology of these diseases will open the way for new therapeutic approaches applicable also to the management of more common bone diseases including osteoporosis.
... Our studies of the BALP isoforms have demonstrated functional differences in kinetic properties and collagen binding abilities. The study by Wennberg et al. [167] demonstrated that media supplemented with recombinant human TNALP restored mineralization in osteoblast cultures from TNALP KO mice. ...
... The phosphate is thought to be from the collagen processing method, which might have led to a strong and specific signal induction of SPP1. SPP1 expression is directly correlated with the enzymatic activity of alkaline phosphatase [19,29,67] and levels of inorganic phosphate in the medium [68]. In addition, increased intracellular inorganic phosphate increases SPP1 expression and stimulates mineralization [69]. ...
OssiMend® Bioactive (Collagen Matrix Inc., NJ) is a three-component porous composite bone graft device of 45S5 Bioglass/carbonate apatite/collagen. Our in vitro studies showed that conditioned media of the dissolution products of OssiMend Bioactive stimulated primary human osteoblasts to form mineralized bone-like nodules in vitro in one week, in basal culture media (no osteogenic supplements). Osteoblast differentiation was followed by gene expression analysis and a mineralization assay. In contrast, the dissolution products from commercial OssiMend (Bioglass-free carbonate apatite/collagen scaffolds), or from 45S5 Bioglass particulate alone, did not induce the mineralization of the extracellular matrix, but did induce osteoblast differentiation to mature osteoblasts, evidenced by the strong upregulation of BGLAP and IBSP mRNA levels. The calcium ions and soluble silicon species released from 45S5 Bioglass particles and additional phosphorus release from OssiMend mediated the osteostimulatory effects. Medium conditioned with OssiMend Bioactive dissolution had a much higher concentration of phosphorus and silicon than media conditioned with OssiMend and 45S5 Bioglass alone. While OssiMend and OssiMend Bioactive led to calcium precipitation in cell culture media, OssiMend Bioactive produced a higher concentration of soluble silicon than 45S5 Bioglass and higher dissolution of phosphorus than OssiMend. These in vitro results suggest that adding 45S5 Bioglass to OssiMend produces a synergistic osteostimulation effect on primary human osteoblasts.
In summary, dissolution products of a Bioglass/carbonate apatite/collagen composite scaffold (OssiMend® Bioactive) stimulate human osteoblast differentiation and mineralization of extracellular matrix in vitro without any osteogenic supplements. The mineralization was faster than for dissolution products of ordinary Bioglass.
... CXCL12-CXCR4 signaling regulates osteoblast formation by promoting alkaline phosphatase (ALP)-activity in human (Hosogane et al., 2010;Li et al., 2017), and murine osteoblasts in vitro. ALP-activity is often used as a marker of osteoblastic development (Stein and Lian, 1993), and ALP KO mice demonstrate defects in bone mineralization (Wennberg et al., 2000). Mesenchymal stem cells exposed to osteogenic medium with CXCL12 demonstrated higher ALP-activity, supporting a role for CXCL12 in osteogenic differentiation in vitro (Kortesidis et al., 2005). ...
Cranial neural crest cells (CNCCs), identified by expression of transcription factor Sox9, migrate to the first branchial arch and undergo proliferation and differentiation to form the cartilage and bone structures of the orofacial region, including the palatal bone. Sox9 promotes osteogenic differentiation and stimulates CXCL12-CXCR4 chemokine-receptor signaling, which elevates alkaline phosphatase (ALP)-activity in osteoblasts to initiate bone mineralization. Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion. Since we earlier demonstrated chemokine-receptor mediated signaling by the MES, we hypothesized that chemokine CXCL12 is expressed by the disintegrating MES to promote the formation of an osteogenic center by CXCR4-positive osteoblasts. Disturbed migration of CNCCs by excess oxidative and inflammatory stress is associated with increased risk of cleft lip and palate (CLP). The cytoprotective heme oxygenase (HO) enzymes are powerful guardians harnessing injurious oxidative and inflammatory stressors and enhances osteogenic ALP-activity. By contrast, abrogation of HO-1 or HO-2 expression promotes pregnancy pathologies. We postulate that Sox9, CXCR4, and HO-1 are expressed in the ALP-activity positive osteogenic regions within the CNCCs-derived palatal mesenchyme. To investigate these hypotheses, we studied expression of Sox9, CXCL12, CXCR4, and HO-1 in relation to palatal osteogenesis between E15 and E16 using (immuno)histochemical staining of coronal palatal sections in wild-type (wt) mice. In addition, the effects of abrogated HO-2 expression in HO-2 KO mice and inhibited HO-1 and HO-2 activity by administrating HO-enzyme activity inhibitor SnMP at E11 in wt mice were investigated at E15 or E16 following palatal fusion. Overexpression of Sox9, CXCL12, CXCR4, and HO-1 was detected in the ALP-activity positive osteogenic regions within the palatal mesenchyme. Overexpression of Sox9 and CXCL12 by the disintegrating MES was detected. Neither palatal fusion nor MES disintegration seemed affected by either HO-2 abrogation or inhibition of HO-activity. Sox9 progenitors seem important to maintain the CXCR4-positive osteoblast pool to drive osteogenesis. Sox9 expression may facilitate MES disintegration and palatal fusion by promoting epithelial-to-mesenchymal transformation (EMT). CXCL12 expression by the MES and the palatal mesenchyme may promote osteogenic differentiation to create osteogenic centers. This study provides novel evidence that CXCL12-CXCR4 interplay facilitates palatal osteogenesis and palatal fusion in mice.
... As per previous work, we confirmed osteogenic differentiation in vitro by staining for ALP activity [47][48][49][50][51][52][53] . ALP is a phospho-ester, imperative to the mineralisation process 54,55 . Fluorescent microscopy (405 nm) was used to image Hoechst stained cells, with 15 images taken from each of 5 wells at 4× magnification. ...
The pathogenesis of declining bone mineral density, a universal feature of ageing, is not fully understood. Somatic mitochondrial DNA (mtDNA) mutations accumulate with age in human tissues and mounting evidence suggests that they may be integral to the ageing process. To explore the potential effects of mtDNA mutations on bone biology, we compared bone microarchitecture and turnover in an ageing series of wild type mice with that of the PolgAmut/mut mitochondrial DNA ‘mutator’ mouse. In vivo analyses showed an age-related loss of bone in both groups of mice; however, it was significantly accelerated in the PolgAmut/mut mice. This accelerated rate of bone loss is associated with significantly reduced bone formation rate, reduced osteoblast population densities, increased osteoclast population densities, and mitochondrial respiratory chain deficiency in osteoblasts and osteoclasts in PolgAmut/mut mice compared with wild-type mice. In vitro assays demonstrated severely impaired mineralised matrix formation and increased osteoclast resorption by PolgAmut/mut cells. Finally, application of an exercise intervention to a subset of PolgAmut/mut mice showed no effect on bone mass or mineralised matrix formation in vitro. Our data demonstrate that mitochondrial dysfunction, a universal feature of human ageing, impairs osteogenesis and is associated with accelerated bone loss.
... The animals also develop epileptic seizures usually dying before weaning [44][45][46]. Osteoblasts isolated from knockout animals differentiate normally but fail to mineralise the deposited matrix because of the increased ePP i levels; this further illustrates the importance of TNSALP in regulating ePP i levels [47]. ...
Extracellular pyrophosphate (ePPi) was first identified as a key endogenous inhibitor of mineralisation in the 1960's by Fleisch and colleagues. The main source of ePPi seems to be extracellular ATP which is continually released from cells in a controlled way. ATP is rapidly broken down by enzymes including ecto-nucleotide pyrophosphatase/phosphodiesterases to produce ePPi. The major function of ePPi is to directly inhibit hydroxyapatite formation and growth meaning that this simple molecule acts as the body's own "water softener". However, studies have also shown that ePPi can influence gene expression and regulate its own production and breakdown. This review will summarise our current knowledge of ePPi metabolism and how it acts to prevent pathological soft tissue calcification and regulate physiological bone mineralisation.
... TNSALP is essential for skeletal mineralization, which is verified by hypophosphatasia caused by deactivating mutations in the TNSALP gene (Whyte, 2010), a rare heritable inborn error of metabolism characterized by deficiency of bone mineralization associated with low serum ALP activity (Henthorn and Whyte, 1992;Whyte, 2016). The physiological role of TNSALP in bone mineralization was further demonstrated in clinical and research laboratories (Anderson et al., 2004;Hessle et al., 2002;Liu et al., 2014;Nielson et al., 2012;Wennberg et al., 2000;Whyte, 2010). The primary function of TNSALP in mineralizing tissues is to act in concert with plasma cell membrane glycoprotein-1 to fine-tune inorganic pyrophosphate (PPi) concentrations to maintain the optimal concentration of PPi for controlled mineralization (Hessle et al., 2002). ...
Tributyltin (TBT), a proven endocrine disrupter, was widely used in industry and agriculture. Previous research showed that TBT could alter the balance between osteogenesis and adipogenesis, which may have significant consequences for bone health. Herein, we exposed male rats to TBT chloride (TBTCl) to evaluate the deleterious effects of TBT on bone. Exposure to 50 μg kg-1 TBT resulted in a significant decrease in bone mineral density (BMD) at the femur diaphysis region in the rat. A dose-dependent increase in lipid accumulation and adipocyte number was observed in the bone marrow (BM) of the femur. Meanwhile, TBTCl treatment significantly enhanced the expression of PPARγ and attenuated the expression of Runx2 and β-catenin in BM. In addition, serum ALP activity of TBT-exposed rats also showed a dose-dependent decrease. These results suggest that TBT could reduce BMD via inhibition of the Wnt/β-catenin pathway and skew the adipo-osteogenic balance in the BM of rats.
... While minor differences in HPP severity were documented (possibly due to genetic background), both models replicated clinical manifestations of severe infantile HPP. The Millán lab employed the LJ HPP mouse model extensively over the next two decades, exploring the role of TNSALP in bone mineralization, vitamin B metabolism, and other organ systems, as well as elucidating interactions of TNSALP with other mineralization regulators including ANK and ENPP1 (Anderson et al. 2004(Anderson et al. , 2005Cruz et al. 2017;Harmey et al. 2004Harmey et al. , 2006Hessle et al. 2002;Johnson et al. 2000;Narisawa et al. 2001Narisawa et al. , 2003Sebastian-Serrano et al. 2016;Shao et al. 2000;Street et al. 2013;Wennberg et al. 2000). ...
Hypophosphatasia (HPP) is an inherited disorder that affects bone and tooth mineralization characterized by low serum alkaline phosphatase. HPP is caused by loss-of-function mutations in the ALPL gene encoding the protein, tissue-nonspecific alkaline phosphatase (TNSALP). TNSALP is expressed by mineralizing cells of the skeleton and dentition and is associated with the mineralization process. Generalized reduction of activity of the TNSALP leads to accumulation of its substrates, including inorganic pyrophosphate (PPi) that inhibits physiological mineralization. This leads to defective skeletal mineralization, with manifestations including rickets, osteomalacia, fractures, and bone pain, all of which can result in multi-systemic complications with significant morbidity, as well as mortality in severe cases. Dental manifestations are nearly universal among affected individuals and feature most prominently premature loss of deciduous teeth. Management of HPP has been limited to supportive care until the introduction of a TNSALP enzyme replacement therapy (ERT), asfotase alfa (AA). AA ERT has proven to be transformative, improving survival in severely affected infants and increasing overall quality of life in children and adults with HPP. This chapter provides an overview of TNSALP expression and functions, summarizes HPP clinical types and pathologies, discusses early attempts at therapies for HPP, summarizes development of HPP mouse models, reviews design and validation of AA ERT, and provides up-to-date accounts of AA ERT efficacy in clinical trials and case reports, including therapeutic response, adverse effects, limitations, and potential future directions in therapy.
... On the basis of our previous studies and literature, the expression of OCN demonstrates osteogenesis, ALP provides phosphonic acid for the deposition of hydroxyapatite crystals, and COL I is essential for bone formation and bone strength. These three markers may represent different stages of bone formation [2,25]. ...
Ossification of the posterior longitudinal ligament (OPLL) manifests as ectopic bone formation in spinal ligament tissue. As revealed by in vitro studies, fibroblasts from patients with OPLL or healthy ligament fibroblasts undergo mechanical stress (MS). We previously demonstrated that a cell-cell junction protein, connexin 43 (Cx43), is significantly up-regulated in OPLL cells and previous data indicated that some proteins related to the endoplasmic reticulum (ER) stress response are elevated during the development of OPLL. The present study utilized gain- and loss-of-function tools to delineate the contribution of the ER stress response within ligament fibroblasts under OPLL-inducing stimuli and the crosstalk between Cx43 signaling and the ER stress response. The ER stress process was augmented by the induction of Cx43 expression in OPLL cells or cells under MS. Cx43 over-expression also promoted ER stress and ossification in OPLL cells. Moreover, the activation of ER stress was accompanied with increased oxidative stress, which was inhibited by Cx43 gene silencing. Cx43 knockdown also improved ER stress-related ossification in OPLL cells. The blockage of ER stress using a chemical compound or small interfering RNA was sufficient to overcome MS-induced ossification in OPLL cells. These findings were further validated in patients with OPLL, as the mRNA levels of Cx43 and PKR-like endoplasmic reticulum kinase (a single-pass type I ER membrane protein kinase), a major transducer of ER stress, were significantly increased compared with non-OPLL subjects. In conclusion, this study demonstrates that ER stress participates in Cx43-related OPLL.
Extracellular pyrophosphate (PPi) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PPi decreased osteoclast formation and resorptive activity by ≤50 %. These inhibitory actions were associated with reduced expression of genes involved in osteoclastogenesis (Tnfrsf11a, Dcstamp) and bone resorption (Ctsk, Car2, Acp5). In osteoblasts, PPi present for the entire (0-21 days) or latter stages of culture (7-21/14-21 days) decreased bone mineralisation by ≤95 %. However, PPi present for the differentiation phase only (0-7/0-14 days) increased bone formation (≤70 %). Prolonged treatment with PPi resulted in earlier matrix deposition and increased soluble collagen levels (≤2.3-fold). Expression of osteoblast (RUNX2, Bglap) and early osteocyte (E11, Dmp1) genes along with mineralisation inhibitors (Spp1, Mgp) was increased by PPi (≤3-fold). PPi levels are regulated by tissue non-specific alkaline phosphatase (TNAP) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). PPi reduced NPP1 expression in both cell types whereas TNAP expression (≤2.5-fold) and activity (≤35 %) were increased in osteoblasts. Breakdown of extracellular ATP by NPP1 represents a key source of PPi. ATP release from osteoclasts and osteoblasts was decreased ≤60 % by PPi and by a selective TNAP inhibitor (CAS496014-12-2). Pertussis toxin, which prevents Gαi subunit activation, was used to investigate whether G-protein coupled receptor (GPCR) signalling mediates the effects of PPi. The actions of PPi on bone mineralisation, collagen production, ATP release, gene/protein expression and osteoclast formation were abolished or attenuated by pertussis toxin. Together these findings show that PPi, modulates differentiation, function and gene expression in osteoblasts and osteoclasts. The ability of PPi to alter ATP release and NPP1/TNAP expression and activity indicates that cells can detect PPi levels and respond accordingly. Our data also raise the possibility that some actions of PPi on bone cells could be mediated by a Gαi-linked GPCR.
We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3-4 month) and old (18-19 month) mice, age correlated with reduced mRNA of mineral transport proteins: alkaline phosphatase (ALP), ankylosis (ANK), the Cl-/H+ exchanger ClC3, and matrix proteins collagen 1 (Col1) and osteocalcin (BGLAP). Some proteins including the neutral phosphate transporter2 (NPT2) were not reduced. These are predominately osteoblast proteins, but in mixed cell populations. Remarkably, in osteoblasts differentiated from preparations of stromal stem cells (SSC) made from bone marrow cells in young and old mice, differentiated in vitro on perforated polyethylene terephthalate membranes, mRNA confirmed decreased expression with age of most transport-related and bone matrix proteins. Additional mRNAs in osteoblasts in vitro included ENPP1, unchanged, and ENPP2, reduced with age. Decrease with age in ALP activity and protein by Western Blot were also significant. Transport protein findings correlated with micro-computed tomography of lumbar vertebra showing that trabecular bone of old mice is osteopenic relative to young mice, consistent with other studies. Pathway analysis of osteoblasts differentiated in vitro showed that cells from old animals had reduced Erk1/2 phosphorylation and decreased Smad 2 mRNA, consistent with TGF-β pathway, and reduced β-catenin mRNA consistent with WNT pathway regulation. Our results show that decline in bone density with age reflects, selective changes resulting effectively in a phenotype modification. Reduction of matrix and mineral transport protein expression with age is regulated by multiple signaling pathways.
Aligned submicron fibers have played an essential role in inducing stem cell proliferation and differentiation in tissue engineering. However, it remains challenging to fully understand and modify the differences between stem cell proliferation and differentiation on aligned-random fibers. Therefore, in this study, we aimed to identify the differential causes of stem cell proliferation and differentiation between BMSCs on aligned-random fibers with different elastic modulus, and to change the differential levels between them through a regulatory mechanism mediated by BCL-6 and miR-126-5p. The results showed that the most significant PIP2 alterations were found in the aligned fibers compared with the random fibers, which had a regular and oriented structure, excellent elastic modulus and cytocompatibility, regular cytoskeleton and movement pattern, high differentiation potential, and the most differentiated PIP2 gene. The same trend was actual for the aligned fibers with a lower elastic modulus. The level of proliferative differentiation genes in cells on random fibers and high elastic modulus aligned fibers was altered by BCL-6 and miR-126-5p mediated regulatory mechanisms to make the apparent cell distribution nearly consistent with the cell state on low elastic modulus aligned fibers. This work demonstrates the reason for the difference of cells between the two kinds of fibers and on fibers with different elastic modulus. It provides more insights for understanding the gene-level regulation of cell growth in tissue engineering. These findings will greatly help the application and design of 3D tissue culture for tissue engineering. This article is protected by copyright. All rights reserved.
Purpose
Baduanjin (BDJ) exercise is a traditional exercise that combines breathing, body movement, meditation and awareness to help delay the onset and progression of senile degenerative musculoskeletal diseases, such as osteoporosis (OP). The aim of this meta-analysis is to evaluate the efficacy of BDJ exercise, and preliminarily infer its effective mechanism in the treatment of OP.
Methods
We identified relevant randomized controlled trials (RCTs) through eight databases, and compared BDJ exercise with the control groups (including blank control and conventional treatment intervention). The main outcome measure was bone mineral density (BMD), the additional outcome measures were visual analogue scale (VAS), Berg balance scale (BBS), serum Calcium (Ca), serum Phosphorus (P), serum Alkaline phosphatase (ALP), and serum bone gla protein (BGP). Meta-analysis and trial sequence analysis (TSA) were performed using RevMan 5.4, Stata 16.0, and TSA 0.9.
Results
In total, 13 RCTs involving 919 patients were included in the analysis. For postmenopausal osteoporosis, BDJ exercise alone and BDJ exercise combined with conventional treatment can improve the BMD of lumbar spine. BDJ exercise alone can influence serum Ca and ALP. BDJ exercise combined with conventional treatment can improve balance (BBS) and influence serum BGP. For senile osteoporosis, BDJ exercise alone and BDJ exercise combined with conventional treatment can improve balance (BBS). BDJ exercise combined with conventional treatment can improve the BMD of hip and pain relieve (VAS). For primary osteoporosis, BDJ exercise combined with conventional treatment can improve the BMD of lumbar spine and femoral neck.
Conclusion
Baduanjin exercise may be beneficial to improve BMD, relieve pain, improve balance ability, influence serum BGP and serum ALP in patients with OP, but differences occur due to various types of OP. Due to the low quality of research on the efficacy and mechanism of BDJ exercise in the treatment of OP, high-quality evidence-based research is still needed to provide reliable supporting evidence.
Systematic Review Registration
[ http://www.crd.york.ac.uk/PROSPERO ], identifier [CRD42022329022].
Polyetheretherketone (PEEK) is a candidate material for bone implants as an alternative to metals. However, PEEK exhibits poor osseointegration and low endothelial compatibility. In the present study, we demonstrated the photo‐tethering of collagen onto PEEK surfaces to facilitate osteoblastic and vascular endothelial performance. In particular, collagen with methacryloyl groups was covalently tethered to the PEEK surface via surface‐initiated photopolymerization. This process is simpler than the conventional method of collagen‐tethering and can be extended to the surface‐patterning treatment of collagen. The Collagen was confirmed to be tethered to the PEEK surface using attenuated total reflection Fourier transform infrared measurements, bicinchoninic acid assays, and atomic force microscopic observations. When human bone marrow‐derived mesenchymal stem cells (HbmMSCs) were cultured on collagen‐tethered PEEK (COL‐PEEK) surfaces, the cells favorably adhered and proliferated. After inducing osteogenic differentiation, the cells on the COL‐PEEK surfaces showed higher expression levels of osteoblast‐related genes and mineralization than those on the PEEK surface. Moreover, the tethering of collagen greatly improved endothelial proliferation. The COL‐PEEK surfaces promoted endothelial networking in co‐culture with HbmMSCs. These results suggest that COL‐PEEK is highly compatible with both osteoblasts and vascular endothelial cells. COL‐PEEK is a promising implant that induces osteogenesis and angiogenesis to repair bone tissues. This article is protected by copyright. All rights reserved
Polyozellus multiplex, a wild mushroom belonging to Thelephoracea, is distributed in Korea, Japan, and China. Thelephoric acid (Thel), a terphenylquinone found in P. multiplex, reportedly exhibits beneficial effects, including antioxidant, anticancer, and neuroprotective activities. Herein, we extracted Thel from P. multiplex (>95.8% purity) and examined its osteogenic effects on pre-osteoblasts. Thel exerted no cytotoxicity in pre-osteoblasts. Based on alkaline phosphatase staining and activity, Thel promoted osteoblast differentiation and bone matrix mineralization. Biochemical investigations revealed that Thel enhanced nuclear runt-related transcription factor 2 (RUNX2) expression and accumulation via Smad1/5/8 phosphorylation, while exhibiting synergistic activities following cotreatment with bone morphogenetic protein 2 or fibroblast growth factor 2 in pre-osteoblast cultures. Thel increased cell adhesion and migration in differentiating osteoblasts by upregulating matrix metalloproteinase 13 (MMP13). Collectively, these findings indicate that Thel induced osteogenic properties by regulating RUNX2 and MMP13 expression, suggesting that Thel could be further developed to treat bone disorders.
Mechanical loading as a result of load‐bearing physical activity is an important regulator of both bone mass and bone microarchitecture. Sex hormone receptor signaling involves several separate signaling cascades in a tissue‐specific manner, affecting osteoclastic resorption, osteoblastic bone formation, bone modeling and remodeling, and is also an important determinant of bone mass. Interestingly, estrogen receptors play an important role in load‐induced modeling of bone, an effect surprisingly found to be independent of estrogen. Modeling of jaw bones in response to orthodontic tooth movement is dependent on several factors where reshaping of bone by osteoclast and osteoblast activities are of major importance. Although not much studied, loading, sex hormones, and their receptors are likely to affect orthodontic tooth movement. This chapter gives an overview of bone cell differentiation and function and describes the role of sex hormone receptors in mechanical loading, and their possible role in orthodontic tooth movement.
Bone formation occurs throughout life to support growth and bone remodeling in response to mechanical forces, metabolic needs, and tissue repair upon injury. The physiological requirement for continuous bone renewal to accommodate remodeling necessitates recruitment, proliferation, and differentiation of bone-forming osteoblasts to counteract the activity of bone-resorbing osteoclasts. Numerous developmental regulatory pathways control transcriptional and posttranscriptional epigenetic mechanisms during multiple stages of osteoblast growth and differentiation. These mechanisms modulate expression of genes associated with the biosynthesis, organization, and mineralization of the bone extracellular matrix, as well as metabolic responses. This chapter focuses on the molecular phenotype and physiological functions of osteoblasts in bone formation and bone homeostasis. Our understanding of the molecular mechanisms that control osteoblast lineage progression permits consideration of new therapies for stimulating bone formation in the aging skeleton, fracture repair, pathologies of metabolic bone diseases, and orthopedic implant stability.
Bone turnover markers (BTMs) are divided into the markers of bone formation and those of bone resorption. N-terminal collagen type I extension propeptide and serum C-terminal cross-linking telopeptide of type I collagen have been selected as the reference markers of bone formation and resorption, respectively. BTM levels are influenced by numerous factors. BTM cannot be used for the diagnosis of osteoporosis or to predict accelerated bone loss. The utility of BTMs for fracture prediction in postmenopausal women requires further studies. Bone resorption inhibitors decrease BTM levels. Changes in BTMs after their withdrawal vary according to their mechanisms of action. Bone formation–stimulating drugs increase the concentrations of bone formation markers. BTM may be used for dose-finding studies, bridging studies, and, hopefully, for monitoring the antiosteoporotic treatment. Sequential therapy trials, head-to-head comparisons, and combination therapy studies improve our understanding of the mechanisms of action of antiosteoporotic drugs on bone metabolism.
PurposeThis research evaluated clinical, histological, and radiological osseous regeneration in a critical-sized bilateral cortico-medullary osseous defect in model rabbits from New Zealand after receiving a hydroxyapatite matrix and polylactic polyglycolic acid (HA/PLGA) implanted with human dental pulp stem cells (DPSCs).Methods
Eight New Zealand rabbits with bilateral mandibular critical-sized defects were performed where one side was treated with an HA/PLGA/DPSC matrix and the other side only with an HA/PLGA matrix for 4 weeks.ResultsAn osseointegration was clinically observed as well as a reduction of 70% of the surgical lumen on one side and a 35% on the other. Histologically, there was neo-bone formation in HA/PLGA/DPSC scaffold and angiogenesis. A bone radiodensity (RD) of 80% was radiologically observed achieving density levels similar to mandibular bone, while the treatment with HA/PLGA matrix achieves RD levels of 40% on its highest peaks.ConclusionsHA/PLGA/DPSC scaffold was an effective in vivo method for mandibular bone regeneration in critical-sized defects induced on rabbit models.
Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in the calcification sites of the skeletal tissue. It promotes hydroxyapatite crystal formation by degrading inorganic pyrophosphate (PPi) and increasing inorganic phosphate (Pi) concentration. However, abnormalities in Alpl-/- mouse-derived osteoblasts are poorly understood, and the involvement of TNAP in osteoblast differentiation remains unclear. Therefore, in this study, we aimed to investigate the precise role of TNAP in osteoblast differentiation. TNAP inhibition by levamisole, a reversible TNAP inhibitor, suppressed the expression of osteoblast differentiation marker genes in wild-type osteoblastic cells. Alpl overexpression increased the expression of master osteoblast transcription factor genes runt-related transcription factor 2 (Runx2) and Sp7 and the mature osteoblast and osteocyte marker genes, bone γ-carboxyglutamate protein 2 (Bglap2) and dentin matrix protein 1 (Dmp1), respectively in Alpl-deficient osteoblastic cells. TNAP regulated Runx2 expression, which in turn regulated the expression of all other osteoblast markers, except Dmp1. Dmp1 expression was independent of RUNX2 but was dependent on extracellular Pi concentration in Runx2-deficient osteogenic cells. These results suggest that TNAP functions as an osteogenic differentiation regulator either by regulating Runx2 expression or by controlling extracellular Pi concentration.
Hypophosphatasia (HPP) is a rare genetic disease, characterized by the defective production of tissue-non-specific alkaline phosphatase (TNSALP). Six subtypes of the disease – affecting neonates (beginning in utero), infants, children, or adults – are recognized: perinatal lethal, prenatal benign, infantile, childhood, adult, and odontohypophosphatasia. The clinical presentation of these subtypes is very different and the severity ranges from mild to lethal. This chapter, after an overview of the genetics, epidemiology, classification, and clinical presentation of the different forms of HPP, will review the current experience with enzyme replacement therapy (ERT).
Hypophosphatasia is a heritable form of rickets/osteomalacia with extremely variable clinical expression. Severe forms are inherited in an autosomal recessive fashion; the mode of transmission of mild forms is uncertain. The biochemical hallmark of hypophosphatasia is deficient activity of the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Previously, we demonstrated in one inbred infant that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal disease. We have now examined TNSALP cDNAs from four unrelated patients with the severe perinatal or infantile forms of hypophosphatasia. Each of the eight TNSALP alleles from these four individuals contains a different point mutation that causes an amino acid substitution. These base changes were not detected in at least 63 normal individuals and, thus, appear to be causes of hypophosphatasia in the four patients. (Two additional base substitutions, found in one allele from each of the four patients, are linked polymorphisms.) Twenty-three unrelated patients (of 50 screened), who reflect the entire clinical spectrum of hypophosphatasia, possess one of our of the above eight mutations. In two of these additional patients, mild forms of the disease are also inherited in an autosomal recessive fashion. Our findings indicate that hypophosphatasia can be caused by a number of different missense mutations and that the specific interactions of different TNSALP mutant alleles are probably important for determining clinical expression. Severe forms, perinatal and infantile disease, are largely the result of compound heterozygosity for different hypophosphatasia alleles. At least some cases of childhood and adult hypophosphatasia are inherited as autosomal recessive traits.
Uptake of mineral ions by isolated matrix vesicles (MV) incubated in synthetic cartilage lymph follows a consistent pattern. After an initial lag period, MV rapidly accumulate large amounts of Ca2+ and Pi before the appearance of crystalline mineral. The ability of MV to accumulate Ca2+ is readily destroyed by proteases, indicating that proteins are important in Ca2+ accumulation. Since MV contain significant amounts of phosphatidylserine (PS), an acidic phospholipid with affinity for Ca2+, it seemed probable that this lipid might also contribute to Ca2+ binding. The development of methods for reproducible isolation of pure active MV enabled us to search for factors responsible for the rapid accumulation of Ca2+. Reported here are studies which reveal that a set of intensely staining MV proteins, extractable with EGTA, selectively bind to Ca2+, but only in the presence of acidic phospholipids. These 30-36-kDa proteins form readily sedimentable insoluble ternary complexes of protein, Ca2+, and lipid in the presence of low levels of Ca2+. With liposomes composed of PS, alone or in combination with phosphatidylethanolamine, submicromolar levels of Ca2+ or certain other divalent cations, but not Mg2+, are sufficient to form the complexes. The physical and chemical properties of these MV proteins appear to be like those of the calpactin family of membrane-associated proteins. In fact, these MV proteins were found to cross-react with antibodies to calpactin II. Thus, calpactins appear to be important protein constituents of avian growth plate MV. This finding helps explain the enrichment in PS previously noted in MV and may also point to the mechanism by which MV rapidly accumulate Ca2+.
Fourier transform infrared spectroscopy (FTIR) was used to characterize the organic and mineral phases present during the induction of mineral formation by collagenase-released matrix vesicles (CRMV) during incubation in a synthetic cartilage lymph in vitro. CRMV mineralization, which occurs in the absence of alkaline phosphatase organic phosphate substrates, is characterized by an initial short lag period of limited Ca2+ accumulation, followed by a period of rapid Ca2+ uptake, and finally, by a plateau period during which Ca2+ accumulation continued at a slower rate. FTIR spectra taken at timed intervals during the induction of mineralization revealed the presence of absorptions characteristic of protein, phospholipid, and mineral components in the CRMV. These became progressively more intense with time. To reveal underlying changes occurring during the successive stages of Ca2+ accumulation, FTIR spectra of nascent (or demineralized) CRMV were computer-subtracted from subsequent spectra, nulling on the C-H stretch modes characteristic of the lipid acyl chains. These difference spectra showed little change during early Ca2+ loading, revealing that mineral ions initially accumulated in a form similar to that present in nascent matrix vesicles (MV). During the period of rapid Ca2+ uptake prior to appearance of crystalline mineral, difference spectra revealed subtle changes in the carbonyl and amide nitrogen stretch modes indicative of protein conformational changes. The first definable mineral phase appeared late in the rapid Ca2+ uptake period and was a distinct, crystalline octacalcium phosphate (OCP)-like phase. With time, the OCP-like precursor became more apatitic in character. There was no evidence that any amorphous calcium phosphate phase formed during the MV mineralization sequence. The mature MV mineral phase closely resembled hydroxyapatite formed via an OCP precursor and was similar to other biological apatites that show a substantial incorporation of carbonate.
Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Clinical severity is variable, ranging from death in utero (due to severe rickets) to pathologic fractures first presenting in adult life. Affected siblings, however, are phenotypically similar. Severe forms of the disease are inherited in an autosomal recessive fashion; heterozygotes often show reduced serum ALP activity. The specific gene defects in hypophosphatasia are unknown but are thought to occur either at the L/B/K ALP locus or within another gene that regulates L/B/K ALP expression. We used the polymerase chain reaction to examine L/B/K ALP cDNA from a patient with a perinatal (lethal) form of the disease. We observed a guanine-to-adenine transition in nucleotide 711 of the cDNA that converts alanine-162 of the mature enzyme to threonine. The affected individual, whose parents are second cousins, is homozygous for the mutant allele. Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolishes the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.
Vesicles have been identified within the cartilage matrix of the upper tibial epiphyseal plate of normal mice. They were seen at all levels within the plate and usually did not appear to be in contact with cartilage cells. Vesicles were concentrated within the matrix of the longitudinal septa from the proliferative zone downward. They varied considerably in size ( approximately 300 A to approximately 1 micro) and in shape. They were bounded by unit membranes, and contained materials of varying density including, rarely, ribosomes. A close association was demonstrated between matrix vesicles and calcification: in the lower hypertrophic and calcifying zones of the epiphysis, vesicles were found in juxtaposition to needle-like structures removed by demineralization with ethylenediaminetetraacetate and identified by electron diffraction as hydroxyapatite and/or fluorapatite crystal structure-the former being indistinguishable from the latter for most cases in which electron diffraction methods are employed. Decalcification also revealed electron-opaque, partially membrane-bounded structures within previously calcified cartilage of the epiphyseal plate and underlying metaphysis which corresponded in size and distribution to matrix vesicles. It is suggested that matrix vesicles are derived from cells and that they may play a role in initiating calcification at the epiphysis.
In humans, deficiency of the tissue non-specific alkaline phosphatase (TNAP) gene is associated with defective skeletal mineralization. In contrast, mice lacking TNAP generated by homologous recombination using embryonic stem (ES) cells have normal skeletal development. However, at approximately two weeks after birth, homozygous mutant mice develop seizures which are subsequently fatal. Defective metabolism of pyridoxal 5'-phosphate (PLP), characterized by elevated serum PLP levels, results in reduced levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the brain. The mutant seizure phenotype can be rescued by the administration of pyridoxal and a semi-solid diet. Rescued animals subsequently develop defective dentition. This study reveals essential physiological functions of TNAP in the mouse.
Hypophosphatasia, a heritable disease characterized by deficient activity of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP), results in rickets and osteomalacia. Although identification of TNSALP gene defects in hypophosphatasia establishes a role of ALP in skeletal mineralization, the precise function remains unclear. The initial site of mineralization (primary mineralization) normally occurs within the lumen of TNSALP-rich matrix vesicles (MVs) of growth cartilage, bone, and dentin. We investigated whether defective calcification in hypophosphatasia is due to a paucity and/or a functional failure of MVs secondary to TNSALP deficiency. Nondecalcified autopsy bone and growth plate cartilage from five patients with perinatal (lethal) hypophosphatasia were studied by nondecalcified light and electron microscopy to assess MV numbers, size, shape, and ultrastructure and whether hypophosphatasia MVs contain apatite-like mineral, as would be the case if these MVs retained their ability to concentrate calcium and phosphate internally despite a paucity of TNSALP in their investing membranes. We found that hypophosphatasia MVs are present in approximately normal numbers and distribution and that they are capable of initiating internal mineralization. There is retarded extravesicular crystal propagation. Thus, in hypophosphatasia the failure of bones to calcify appears to involve a block of the vectorial spread of mineral from initial nuclei within MVs, outwards, into the matrix. We conclude that hypophosphatasia MVs can concentrate calcium and phosphate internally despite a deficiency of TNSALP activity.
Hypophosphatasia, a rare inherited disorder characterized by defective bone mineralization, is highly variable in its clinical expression. The disease is due to various mutations in the tissue-non-specific alkaline phosphatase ( TNSALP ) gene. We report here the use of clinical data, site-directed mutagenesis and computer-assisted modelling to propose a classification of 32 TNSALP gene mutations found in 23 European patients, 17 affected with lethal hypophosphatasia and six with non-lethal hypophosphatasia. Transfection studies of the missense mutations found in non-lethal hypophosphatasia showed that six of them allowed significant residual in vitro enzymatic activity, suggesting that these mutations corresponded to moderate alleles. Each of the six patients with non-lethal hypophosphatasia carried at least one of these alleles. The three-dimensional model study showed that moderate mutations were not found in the active site, and that most of the severe missense mutations were localized in crucial domains such as the active site, the vicinity of the active site and homodimer interface. Some mutations appeared to be organized in clusters on the surface of the molecule that may represent possible candidates for regions interacting with the C-terminal end involved in glycosylphosphatidylinositol (GPI) attachment or with other dimers to form tetramers. Finally, our results show a good correlation between clinical forms of the disease, mutagenesis experiments and the three-dimensional structure study, and allowed us to clearly distinguish moderate alleles from severe alleles. They also confirm that the extremely high phenotypic heterogeneity observed in patients with hypophosphatasia was due mainly to variable residual enzymatic activities allowed by missense mutations found in the human TNSALP gene.
A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in "tiptoe walking" (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12-14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-beta and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited approximately 80-90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent 45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.
Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (-/-) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of approximately 50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at approximately 10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.
With the ageing population in most countries, disorders of bone and mineral metabolism are becoming increasingly relevant to every day clinical practice. Consequently, the interest in, and the need for effective measures to be used in the screening, diagnosis and follow-up of such pathologies have markedly grown. Together with clinical and imaging techniques, biochemical tests play an important role in the assessment and differential diagnosis of metabolic bone disease. In recent years, the isolation and characterisation of cellular and extracellular components of the skeletal matrix have resulted in the development of molecular markers that are considered to reflect either bone formation or bone resorption. These biochemical indices are non-invasive, comparatively inexpensive and, when applied and interpreted correctly, helpful tools in the diagnostic and therapeutic assessment of metabolic bone disease. This review provides an overview of the current evidence regarding the clinical use of biochemical markers of bone remodelling in bone disease, with an emphasis on osteoporosis.
The first application of atomic absorption spectra to chemical analysis was made over 100 years ago by Kirchhoff [1], who demonstrated the presence of various elements in the solar atmosphere. Together with Bunsen he demonstrated shortly afterwards that atomic spectra, whether in emission or absorption, could be the basis of a powerful method of chemical analysis [2–4].
Using primer sets specific for mouse N-cadherin and rat testicular RNA for RT-PCR, a full-length complementary DNA (cDNA) coding for rat testicular N-cadherin was isolated. The deduced amino acid sequence of rat N-cadherin yielded a 883-amino acid polypeptide that displayed a 98.6% identity with the mouse homolog. N-Cadherin was found to be expressed by Sertoli and germ cells in the rat testis by RT-PCR. Using Sertoli-germ cell cocultures, it was found that the N-cadherin expression increased with time in culture. To assess whether this is due to a soluble factor(s) released from germ cells that affects Sertoli cell N-cadherin expression, germ cell-conditioned media (GCCM) were fractionated by preparative anion-exchange HPLC, and the resulting fractions were divided into 14 pools. Pool 4 was found to contain a factor(s) that induced a dose-dependent stimulation on Sertoli cell N- cadherin expression with a maximal stimulation at 2μ g protein/dish/4.5 × 10⁶ Sertoli cells. At higher doses between 12 and 32 μg protein/dish, this pool relinquished its effect on Sertoli cell N-cadherin expression suggestive of a biphasic effect. This biphasic effect was confirmed using increasing doses of crude GCCM on Sertoli cell cultures. Since nonviable germ cells failed to stimulate Sertoli cell N-cadherin expression, it illustrates the observed stimulatory effect by GCCM is likely to be mediated via a soluble factor(s) releasing from viable germ cells. These results reveal the presence of a stimulatory factor(s) in GCCM that can modulate Sertoli cell N-cadherin expression in vitro. Since N-cadherin plays a crucial role in facilitating invasive capacity of metastatic tumor cells, the observation of germ cell-released factor(s) in affecting Sertoli cell N-cadherin expression may suggest its possible role in facilitating germ cell migration during spermatogenesis.
We report the inactivation, via homologous recombination, of two of the three active mouse alkaline phosphatase genes, i.e., embryonic (EAP) and tissue nonspecific (TNAP). Whereas expression of the EAP isozyme was abolished in all tissues that express EAP developmentally (such as the preimplantation embryo, thymus, and testis), the EAP knock-out mice show no obvious phenotypic abnormalities. They reproduce normally and give birth to live offspring, indicating the nonessential role of EAP during embryonic development. Mice deficient in the TNAP gene mimic a severe form of hypophosphatasia. These TNAP −/− mice are growth impaired, develop epileptic seizures and apnea, and die before weaning. Examination of the tissues indicates abnormal bone mineralization and morphological changes in the osteoblasts, aberrant development of the lumbar nerve roots, disturbances in intestinal physiology, increased apoptosis in the thymus, and abnormal spleens. Our results indicate that, in the mouse, TNAP appears not to be essential for the initial events leading to bone mineral deposition but that TNAP seems to play a role in the maintenance of this process after birth. The other phenotypic manifestations may be a consequence of the lack of TNAP in the developing neural tube between stages E8.5 and E13.5 of embryogenesis. We hypothesize that the autonomic nervous system is compromised in these TNAP −/− mice. Dev. Dyn. 208:432–446, 1997. © 1997 Wiley-Liss, Inc.
Using primer sets specific for mouse N-cadherin and rat testicular RNA for RT-PCR, a full-length complementary DNA (cDNA) coding for rat testicular N-cadherin was isolated. The deduced amino acid sequence of rat N-cadherin yielded a 883-amino acid polypeptide that displayed a 98.6% identity with the mouse homolog. N-Cadherin was found to be expressed by Sertoli and germ cells in the rat testis by RT-PCR. Using Sertoli-germ cell cocultures, it was found that the N-cadherin expression increased with time in culture. To assess whether this is due to a soluble factor(s) released from germ cells that affects Sertoli cell N-cadherin expression, germ cell-conditioned media (GCCM) were fractionated by preparative anion-exchange HPLC, and the resulting fractions were divided into 14 pools. Pool 4 was found to contain a factor(s) that induced a dose-dependent stimulation on Sertoli cell N-cadherin expression with a maximal stimulation at 2 microg protein/dish/4.5 x 10(6) Sertoli cells. At higher doses between 12 and 32 microg protein/dish, this pool relinquished its effect on Sertoli cell N-cadherin expression suggestive of a biphasic effect. This biphasic effect was confirmed using increasing doses of crude GCCM on Sertoli cell cultures. Since nonviable germ cells failed to stimulate Sertoli cell N-cadherin expression, it illustrates the observed stimulatory effect by GCCM is likely to be mediated via a soluble factor(s) releasing from viable germ cells. These results reveal the presence of a stimulatory factor(s) in GCCM that can modulate Sertoli cell N-cadherin expression in vitro. Since N-cadherin plays a crucial role in facilitating invasive capacity of metastatic tumor cells, the observation of germ cell-released factor(s) in affecting Sertoli cell N-cadherin expression may suggest its possible role in facilitating germ cell migration during spermatogenesis.
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Investigation of the kindred of a 58-year-old woman with all of the features of "adult" hypophosphatasia revealed 12 individuals in 3 generations with subnormal circulating total alkaline phosphatase (AP) activity. The pattern of inheritance suggested autosomal dominant transmission, with incomplete penetrance of the trait particularly in the young males. Hypophosphatasic individuals other than the proposita were clinically well but had loss of permanent teeth, showing that dental abnormalities could be the only clinical manifestation of the disorder. Radiographic investigation of the proposita revealed that completion of stress fractures was necessary for healing; maturation of incomplete fractures resulted in stable Looser zones. Skeletal survey and radionuclide bone imaging were unremarkable in hypophosphatasic individuals without fracture. Subclinical osteopenia was found in several affected women by metacarpal cortical width and bone densitometric measurements. Laboratory studies showed increased plasma and urinary phosphoethanolamine levels in affected individuals. Phosphoethanolamine and phosphoserine appeared to be natural subtrates for AP since a negative correlation existed between each substrate and circulating total AP activity. Phosphoethanolamine and phosphoserine levels were greatest in the clinically affected proposita; furthermore, only she showed absence of leukocyte AP activity. Heat fractionation of her total circulating AP activity suggested severe reduction in the bone isoenzyme. Hypophosphatasic children had higher levels of total circulating AP than affected adults; the increase was apparently secondary to increased bone isoenzyme. Iliac crest bone biopsies showed greater abnormality in affected women. Osteoidosis was particularly pronounced in the proposita's younger affected sister and hypophosphatasic daughter. Histomorphometric analyses of the biopsies revealed a paucity of osteoblasts despite increased quantities of unmineralized matrix. The finding that hypophosphatasic children in this kindred had higher circulating total AP activity than adults and were able to model their skeleton normally, together with observations that the bone biopsy in adults had a paucity of osteoblasts, suggests that some factor(s) during growth is able to induce both AP activity and osteoblast function, or, that this disorder is an "abiotrophy" with deficient osteoblastic formation and/or accelerated destruction in adult life.
Rat calvaria (RC) cells grown in medium containing ascorbic acid form nodules of osteoid and cells. When 10 mM beta-Glycerophosphate (beta-GP) is added, the osteoid mineralizes in two phases: an initiation phase that is dependent upon alkaline phosphatase activity and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require added beta-GP (Bellows et al., Bone Miner 1991;14:27-40). The present experiments were performed to determine whether beta-GP is converted to inorganic phosphate (Pi) during the initiation phase of the mineralization process and whether increased Pi can replace beta-GP in the initiation phase. Measurements of Pi concentrations in the culture medium showed that during the first 8 h of the initiation phase of mineralization, 10 mM beta-GP was rapidly degraded resulting in Pi concentrations of 9-10 mM. The production rate of Pi from beta-GP was linear (r = 0.996) and the alkaline phosphatase activity in the same cultures indicated a potential for conversion of beta-GP to Pi that was greater than the actual conversion rate. The addition of 2-5 mM Pi in the absence of beta-GP also initiated mineralization. Mineralization initiated by either beta-GP or Pi progressed in the absence of added beta-GP or Pi. 100 microM Levamisole inhibited the initiation of beta-GP-induced mineralization and the conversion of beta-GP to Pi, but did not affect Pi-induced initiation of mineralization. The addition of 1-5 mM Pi to cultures in which mineralization had been initiated by 10 mM beta-GP had no significant effect on the progression phase of mineralization. Neither beta-BP nor Pi initiated 45Ca uptake in cultures without nodules (RC population I) and the histological appearance of the mineralized tissue in either phosphate source appeared identical. The present experiments show that beta-GP is rapidly and virtually completely degraded to Pi during the initiation phase of mineralization and that the addition of increased concentrations of Pi can replace beta-GP in the initiation phase of mineralization in the absence of non-specific 45Ca uptake or apparent cellular toxicity.
Osteoid nodules form but do not mineralize in fetal rat calvaria cell cultures grown in alpha-minimal essential medium with 10% fetal bovine serum in the absence of Na beta-glycerophosphate (beta-GP). To study factors involved in the initiation and progression of mineralization, cultures were treated with beta-GP and radiolabelled with 0.1-0.2 microCi/ml 45Ca after nodules had formed (17-19 days in medium without beta-GP). Concentrations of beta-GP from 1 to 14 mM induced a dose-dependent increase in 45Ca uptake. 45Ca uptake was restricted to nodule-containing cultures and did not occur in cultures without nodules. Continuous labelling over 72 h compared with 2 h pulses over the same time period showed that little mineralization occurred over the first 8-12 h and that the rate of mineralization was maximal and constant after 24 h exposure to beta-GP. Calcium uptake from medium was slow during the first 12 h of beta-GP exposure but increased rapidly thereafter until the medium calcium concentration reached a steady state of between 0.5 and 0.6 mM. Measurement of calcium concentration in the medium after mineralization had been initiated (24 h after beta-GP exposure) showed a linear calcium uptake into nodules (r = 0.990) over a 7 h period at a rate of 9.2 micrograms calcium/h/culture. Initiation of mineralization was prevented by 100 microM levamisole, but not by 100 microM dexamisole. When 100 microM levamisole was added 24 h after mineralization had been initiated by the addition of beta-GP, the progression of mineralization was unaffected. Similarly, after mineralization had been initiated for 24 h by 10 mM beta-GP, mineralization continued independent of the presence of beta-GP. The data show that the initiation and progression of mineralization are separate phenomena and that organic phosphate and alkaline phosphatase play a crucial role in the initiation of mineralization but are not required for the continuation of mineralization of bone nodules.
A review of the human alkaline phosphatases dealing specifically with (1) the gene loci, (2) characterization and discrimination of the various enzymes, (3) polymorphism at the enzyme level, (4) cDNA and gene structures, (5) membrane binding, (6) the carbohydrate moieties, (7) hypophosphatasia, (8) alkaline phosphatases in malignancies, (9) function.
The relation between the level of cyclic AMP and bone resorption was studied in a bone organ culture system, using calvaria from newborn mice. Two methylxanthines, iso-butyl-methylxanthine and theophylline and two non-xanthine inhibitors of cyclic AMP phosphodiesterase, Ro 20-1724 and rolipram, stimulated the release of [45Ca] and [3H] from bones prelabelled in vivo with [45Ca]- and [3H]proline, respectively. The release occurred after a delay of more than 24 hr. In 120-hr cultures, theophylline, IBMX, rolipram and Ro 20-1724, all stimulated the release of stable calcium, inorganic phosphate and the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase from mouse calvarial bones. In addition, all four phosphodiesterase inhibitors decreased the amount of hydroxyproline in the bones at the end of the culture period. The release of minerals and the decrease of hydroxyproline was abolished by indomethacin. In short-term cultures (24 hr), rolipram and Ro 20-1724 did not reduce PTH-stimulated mineral mobilization, whereas the two methylxanthines, and dibutyryl cyclic AMP and 8-bromo cyclic AMP, did cause a reduction of PTH-stimulated mineral release during the first 24 hr. All four phosphodiesterase inhibitors increased the accumulation of cyclic AMP in the calvaria and inhibited cyclic AMP hydrolysis in extracts of calvarial bone. There was a correlation between the magnitude of the initial rise in cyclic AMP and the delayed stimulation of bone resorption. However, much lower concentrations of the PDE inhibitors were sufficient to produce a delayed increase in bone resorption than to block phosphodiesterase and significantly raise cyclic AMP levels. It is suggested that the elevation of cyclic AMP in a subset of bone cells results in an acute reduction of bone mobilization and the cAMP elevation in another subset to a delayed rise in bone resorption.
Matrix vesicles (MV) induce mineralization by compartmentalization of ion accumulation and crystal nucleation within membrane-enclosed extracellular microstructures. MV derive from cell surface microvilli by processes that cause selective enrichment of specific proteins, enzymes, lipids, and electrolytes. Incubated in synthetic cartilage lymph (SCL), MV accumulate Ca2+ and Pi, inducing mineral formation in a sequence of stages that can be altered by specific affectors. Rapid uptake of mineral ions by MV precedes formation of the first crystalline phase, octacalcium phosphate (OCP), which later converts to apatite (HAP). Early uptake of Ca2+ and Pi by MV is pH and protease sensitive, and is stimulated by o-phenanthroline (OP), a Zn2+ chelator. Recent studies reveal that a quantitatively major group of MV proteins bind to Ca2+ with high affinity in a lipid-dependent manner. These MV proteins appear to be involved in transport and accumulation of Ca2+ and Pi by MV, and may catalyze nucleation of the first mineral phase.
The addition of the organic phosphate, beta-glycerophosphate, to the culture media of osteoid forming cells or tissues will induce formation of mineralized bone. The mechanisms behind this phenomenon are not clearly understood. In order to gain new understanding of organic phosphate induced mineralization of bone it was decided to attempt to inhibit this process in two fundamentally different ways. Firstly, the reversible inhibitor of alkaline phosphatase, Levamisole, was used to help define the role of alkaline phosphatase in mineralization in vitro. Secondly, inorganic pyrophosphate, a known inhibitor of hydroxyapatite (HA) formation was also used. It was hypothesized that inorganic pyrophosphate, in addition to its ability to block HA formation, might also interfere with organic phosphate access to alkaline phosphatase and thereby prevent mineralization. The data show that mineralization is blocked when alkaline phosphatase activity is inhibited by Levamisole prior to but not after osteoid maturation. Inorganic pyrophosphate blocks organic phosphate induced mineralization whether added before or after osteoid formation. Organic phosphate effects on alkaline phosphatase activity are reversed by the addition of inorganic pyrophosphate either before or after osteoid formation. These findings suggest a role for alkaline phosphatase in organic phosphate induced mineralization. The data show further that inorganic pyrophosphate may effect mineralization of bone not only by blocking apatite formation but possibly by modulating organic phosphate metabolism.
Studies of bone cells in culture have raised two salient questions: are the findings representative of the in vivo situation and can the conflicting data from different cell models be reconciled? Review of the literature indicates that all osteoblastic cells, defined by their origin or by their ability to produce mineralized matrix, have a few common properties: production of type I collagen; increased alkaline phosphatase activity; and parathyroid hormone-stimulated adenylate cyclase. Other features, such as osteocalcin and prostaglandin E production and the response to prostaglandin E, are selectively expressed by certain cell types. Pilot studies on mRNA levels of 'bone proteins' in developing calvaria suggest that such differences may reflect stages in osteoblastic differentiation. Immortalization of calvaria-derived cells using a SV40 large T antigen vector, which may freeze the cells in their particular state of differentiation (as proposed for leukaemia cells), yields phenotypes consistent with that hypothesis. Immortal cell lines may thus help to characterize osteoblastic differentiation. The diversity of osteoblast responses in culture to hormones and growth factors could be due to these phenotype differences but could also represent a subspecialization of differentiated cells. In addition, in the organism regulatory agents act in concert on a heterogeneous interactive cell population. Nonetheless cell cultures can be useful in screening for and predicting in vivo responses, as was shown by the 1,25-(OH)2D3 stimulation of osteocalcin, and for studying the molecular mechanisms of regulatory effects. Cell lines are also convenient for the production of specific proteins and cDNA libraries, and for the expression of specific genes.
The effect of a preincubation period, in basic medium or in medium with inhibitors of prostaglandin biosynthesis, on the response to different stimulators of bone resorption has been studied in an organ culture system using calvarial bones from neonatal mice. Bone resorption was assessed either by the release of 45Ca or by the release of 3H from [3H]-proline labeled bones. Preincubated bones were cultured for 18-24 hr in medium, with and without indomethacin, hydrocortisone, and dexamethasone, and then extensively washed before being transferred to culture medium containing different stimulators of bone resorption. Preincubation in medium containing indomethacin or corticosteroids resulted in an increased response to parathyroid hormone (PTH), prostaglandin E2 (PGE2), 1-alpha-hydroxyvitamin-D3 and thrombin as compared to the response in bones which were exposed to the stimulants directly after dissection. Preincubation in basic medium did not enhance the subsequent response to PTH. By using a preincubation period in indomethacin, the dose-response curves for the stimulatory effect of PTH and PGE2 on mineral mobilization could be sensitized as compared to the curves obtained with fresh bones. Thus, the concentration of agonists causing 50% stimulation of 45Ca release was decreased by a factor of 10. The threshold for actions of PTH and PGE2 on 45Ca release was 0.01-0.03 and 1-3 nmol/l, respectively.
This article presents detailed clinical and laboratory investigations of six hypophosphatasia kindreds. Serum alkaline phosphatase and urinary phosphoethanolamine comparisons between the affected population and a normal control population demonstrate these parameters routinely identify the heterozygous individual when age and sex variations are accounted for. Using clinical data from the kindred population and a detailed review of the literature, the type and frequency of clinical findings for both the homozygous and heterozygous genotype are enumerated. The clinical and biochemical phenotypes were subjected to segregation analysis. When the results of these analyses are viewed in light of their mathematical limitations and the genetic precepts of autosomal dominant and recessive inheritance, hypophosphatasia is best described as an autosomal dominant disorder with 85% penetrance and homozygous lethality.
I. Introduction A FUNCTIONAL relationship between cell growth and the initiation and progression of events associated with differentiation has been a fundamental question challenging developmental biologists for more than a century. In the case of bone, as observed with other cells and tissue, the relationship of growth and differentiation must be maintained and stringently regulated, both during development and throughout the life of the organism, to support tissue remodeling. For many years, bone was defined anatomically and examined largely in a descriptive manner by ultrastructural analysis and by biochemical and histochemical methods. These studies provided the basis for our understanding of bone tissue organization and orchestration of the progressive recruitment, proliferation, and differentiation of the various cellular components of bone tissue. Now, complemented by an increased knowledge of molecular mechanisms that are associated with and regulate expression of genes encoding phenotypic compone...
The ability of Levamisole to decrease mineralization in skeletal tissue is usually related to its effect on alkaline phosphatase (ALP). However, Levamisole is also suspected to diminish mineralization by an additional mechanism which is unrelated to the ALP control of apatite crystal growth. To delineate the time in differentiation during which Levamisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells were propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0.2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. This suggested the existence of a Levamisole-sensitive mechanism for mineralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Levamisole decreased Day 21 mineralization when added on Days 0, 3, 5, and 7, but not when added on Day 9. The Levamisole-induced inhibition of mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to mineralize is determined during the first week of culture.(ABSTRACT TRUNCATED AT 250 WORDS)
The permissive effect of beta-GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto-ALP at physiological pH range. Beta-GP present in the culture medium for 8 days exerted a stimulatory effect on 45Ca incorporation into matrix cell layers while the ecto-ALP activity level measured on intact cells with a saturating concentration of pNPP was similar for cells grown either in the presence or absence of beta-GP. In both types of cultures, beta-GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of beta-GP on 45Ca incorporation and generation of Pi was similar (k phi = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and beta-GP hydrolyses, which were likely catalyzed by the same ecto-enzyme. The rate of 45Ca incorporation into matrix cell layers, which was high (0.90 mumol/4h/mg cell protein) in cells grown in the absence of beta-GP, was inhibited by 50% by levamisole. In cells grown in the absence of beta-GP, the 45Ca incorporation rate increased progressively after beta-GP addition, reaching after 12 h the value of cultures grown in the presence of beta-GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of beta-GP-derived Pi rapidly led to high 45Ca incorporation rate which was unaffected by levamisole. beta-GP removal from cultures grown in its presence reduced by 50% the 45Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto-ALP to hydrolyze beta-GP in cultured fetal calvaria cells, yet it influenced the beta-GP-stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels.
MC3T3-E1 cells grown with ascorbic acid express sequentially osteoblastic marker proteins such as alkaline phosphatase (ALPase) and then form a mineralized extracellular matrix (ECM) as a consequence of osteoblastic differentiation. To explore the functional roles of ALPase in the process of osteoblastic maturation, an inducible expression vector for antisense ALPase RNA was constructed and stably transfected into MC3T3-E1 cells. The expression of antisense ALPase RNA in the differentiated MC3T3-E1 transfectants reduced markedly the ALPase activity, which resulted in a significant decrease in the deposition of minerals upon prolonged culture. These findings demonstrated directly that ALPase participated in the mineralization of ECM.
The tissue non-specific alkaline phosphatase (TNAP) knock-out mouse is a model of infantile hypophosphatasia displaying impaired bone mineralization, epileptic seizures, apnoea, abnormal apoptosis in the thymus, abnormal lumbar nerve roots, and postnatal death. Administration of vitamin B6 suppresses the epileptic seizures in TNAP-/- mice. This paper examines to what extent the diverse abnormalities seen in these mice are due to impaired utilization of vitamin B6, using two complementary approaches: administration of vitamin B6 to TNAP null mice and deprivation of vitamin B6 in wild-type and TNAP heterozygous mice. Administration of exogenous pyridoxal HCl delayed the onset of epileptic attacks and increased the life span of TNAP-/- mice. The episodes of apnoea ceased and the appearance of lumbar nerve roots improved, but hypomineralization and accumulation of osteoid continued to worsen with age. Control mice fed a vitamin B6-depleted diet developed epileptic seizures indistinguishable from those observed in TNAP-/- mice, abnormal apoptosis in the thymus, and thinning of the nerve roots, but showed no evidence of bone mineralization abnormalities. Depletion of vitamin B6 did not affect the ability of primary cultures of osteoblasts to deposit bone mineral in vitro. While abnormal metabolism of vitamin B6 explains many of the abnormalities in this mouse model of infantile hypophosphatasia, it is not the basis of the abnormal mineralization that characterizes this disease.
An enzyme is present in the ossifying cartilage of young rats and rabbits, which rapidly hydrolyses hexosemonophosphoric acid, yielding free phosphoric acid. In respect of this enzyme the kidney is considerably less active (about 50%) than an equal weight of epiphyseal cartilage. Other tissues contain the enzyme in a very much lower degree, muscle and blood being almost inactive. Non-ossifying cartilage shows less than one-tenth of the hydrolytic power of ossifying cartilage. The same tissues in approximately the same order also hydrolyse glycerophosphoric acid. One of the two phosphoric acid groups of hexosediphosphoric acid is very readily hydrolysed by almost all tissues (including muscle and non-ossifying cartilage) except blood. The possible significance of this enzyme in the process of ossification in the animal body is discussed and certain preliminary experiments in various directions are briefly described, by the further prosecution of which it is hoped to obtain more information on this subject.
Atomic absorption spectrometry Analytical Flame Spectroscopy
- Willis
- Jb
Willis JB 1970 Atomic absorption spectrometry. In: Mavrod-ineanu R (ed.) Analytical Flame Spectroscopy. Eindhoven, Philips Technical Library, Macmillan, London, U.K., pp. 525– 594.
The RT-PCRs were repeated three times per experiment, and we extracted RNA from three separate experiments, all yielding identical RT-PCR patterns
- ј-Ctt Cct Gtg Ctc Cgt Gct G-3ј Cbfa
- ј-Cac Tac Ctt Att Gcc Ctc Ctg Ctt Gga C-3ј ; Osteopontin ( Op ), 5ј-Gct Tct Gag Cat Gcc Ctc Tga Tcag Ga-3ј
Cbfa1, 5Ј-CTT CCT GTG CTC CGT GCT G-3Ј and 5Ј-GAG GAA TGC GCC CTA AAT CAC-3Ј; ␣ 1 -procollagen I, 5Ј-TCC TGA AGG TGC TAT TTA AGA A-3Ј and 5Ј-TTG GGT CAT TTC CAC ATG C-3Ј; osteocalcin (OC), 5Ј-GTC CTC CTG GTT CAT TTC TTT GGG TAA C-3Ј and 5Ј-CAC TAC CTT ATT GCC CTC CTG CTT GGA C-3Ј; osteopontin (OP), 5Ј-GCT TCT GAG CAT GCC CTC TGA TCAG GA-3Ј and 5Ј-GTG CAG AAG CTT TTG GTT ACA ACG GTT G-3Ј. The RT-PCRs were repeated three times per experiment, and we extracted RNA from three separate experiments, all yielding identical RT-PCR patterns. Protein synthesis Osteoblasts were isolated from TNAPϩ/ϩ, TNAPϩ/Ϫ, and TNAPϪ/Ϫ mice, seeded at a density of 1 ϫ 10
Comparative study of the effects of cyclic nucleotide phosphodiesterase inhibitors on bone resorption and cyclic AMP formation in vitro
- Ransjö U M Lerner
- Fredholm
- Bb
Lerner U, Ransjö M, Fredholm BB 1986 Comparative study of the effects of cyclic nucleotide phosphodiesterase inhibitors on bone resorption and cyclic AMP formation in vitro. Biochem Pharmacol 35:4177– 4189.
Osteoblast tissue-nonspecific alkaline phosphatase (TNAP) functions to both antagonize and regulate the miner-alization inhibitor PC-1
- Johnson Ka L Hessle
- S Vaingankar
- Mauro C S Wennberg
- S Narisawa
- Goding Jw
- K Sano
- Millá Jl
- Terkeltaub
Johnson KA, Hessle L, Vaingankar S, Wennberg C, Mauro S, Narisawa S, Goding JW, Sano K, Millá JL, Terkeltaub R 2000 Osteoblast tissue-nonspecific alkaline phosphatase (TNAP) functions to both antagonize and regulate the miner-alization inhibitor PC-1. Am J Physiol (in press).
Nature's window on al-kaline phosphatase function in man Principles of Bone Biology
- Whyte
Whyte MP 1996 Hypophosphatasia: Nature's window on al-kaline phosphatase function in man. In: Bilezikian JP, Raisz LG, Rodan GA (eds.) Principles of Bone Biology. Academic Press, San Diego, CA, U.S.A., pp. 951–968.