Cellular and extracellular matrix of bone, with principles of synthesis and dependency of mineral deposition on cell membrane transport

Bone differs from other connective tissues; it is isolated by a layer of osteoblasts that are connected by tight and gap junctions. This allows bone to create dense lamellar type I collagen, control pH, mineral deposition, and regulate water content forming a compact and strong structure. New woven bone formed after degradation of mineralized cartilage is rapidly degraded and resynthesized to impart structural order for local bone strength. Ossification is regulated by thickness of bone units and by patterning via bone morphogenetic receptors including activin, other bone morphogenetic protein receptors, transforming growth factor-β receptors, all part of a receptor superfamily. This superfamily interacts with receptors for additional signals in bone differentiation. Important features of the osteoblast environment were established using recent tools including osteoblast differentiation in vitro. Osteoblasts deposit matrix protein, over 90% type I collagen, in lamellae with orientation alternating parallel or orthogonal to the main stress axis of the bone. Into this organic matrix, mineral is deposited as hydroxyapatite. Mineral matrix matures from amorphous to crystalline hydroxyapatite. This process includes at least two-phase changes of the calcium–phosphate mineral as well as intermediates involving tropocollagen fibrils to form the bone composite. Beginning with initiation of mineral deposition, there is uncertainty regarding cardinal processes, but the driving force is not merely exceeding the calcium-phosphate solubility product. It occurs behind a epithelial-like layer of osteoblasts, which generate phosphate and remove protons liberated during calcium-phosphate salt deposition. The forming bone matrix is discontinuous from the general extracellular fluid. Required adjustment of ionic concentrations and water removal from bone matrix are important details remaining to be addressed.

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Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein.

The Journal of Cell Biology ◽

10.1083/jcb.131.2.539 ◽

1995 ◽

Vol 131 (2) ◽

pp. 539-549 ◽

Cited By ~ 191

Author(s):

S L Dallas ◽

K Miyazono ◽

T M Skerry ◽

G R Mundy ◽

L F Bonewald

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Staining Pattern ◽

Binding Protein ◽

Type I ◽

Tgf Beta ◽

Fetal Rat ◽

Structural Matrix

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.

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EFFECT OF COLLAGEN ON THE MORPHOLOGY AND STRUCTURE OF CALCIUM PHOSPHATE NANOPARTICLES

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s1016237214500616 ◽

2014 ◽

Vol 26 (05) ◽

pp. 1450061

Author(s):

Hoda Salemi ◽

Aliasghar Behnamghader ◽

Mohamadreza Baghaban Eslaminejad ◽

Mohammad Ataei

Keyword(s):

Calcium Phosphate ◽

Electrostatic Interactions ◽

Type I Collagen ◽

Bone Matrix ◽

Control Sample ◽

Molar Ratio ◽

Type I ◽

Sem Images ◽

Collagen Concentration ◽

Calcium Phosphate Nanoparticles

Collagen and noncollagenous proteins have an important role in the formation of mineral constituent of bone matrix. In this research, the morphology and phase characteristics of calcium phosphate nanoparticles in presence of collagen were investigated. The synthesis reaction was initiated by mixing H 3 PO 4 as phosphorous source and CaCl 2 as calcium source and type I collagen. Collagen concentration in suspension and Ca to P ratio was 1% and 1.67, respectively. The samples (with collagen and without collagen), were heat treated at 600°C and characterized by X-Ray diffraction (XRD), Fourier transformation infrared (FTIR) and scanning electron microscopy (SEM). More smaller and flake-like shape particles were observed in the SEM images of sample synthesized in the presence of collagen compared to the control sample which was constituted of larger granular particles. The XRD results revealed that the synthesized mineral powders with collagen were composed of hydroxyapatite and octacalcium phosphate. P – O and OH characteristic peaks were identified in FTIR spectra. In hybrid sample, the shift of amides band, revealed the electrostatic interactions between calcium phosphate ions and carboxyl or amino groups of collagen fibrils. The Ca to P molar ratio for sample with collagen was 1.9. It was found that the sample synthesized in the presence of collagen has a similar microstructure to natural bone.

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Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells

Current Issues in Molecular Biology ◽

10.3390/cimb43030102 ◽

2021 ◽

Vol 43 (3) ◽

pp. 1451-1459

Author(s):

Kengo Kato ◽

Manami Ozaki ◽

Kumiko Nakai ◽

Maki Nagasaki ◽

Junya Nakajima ◽

...

Keyword(s):

Matrix Protein ◽

Type I Collagen ◽

Inflammatory Diseases ◽

Bone Matrix ◽

Nodule Formation ◽

Type I ◽

Alizarin Red ◽

Odontogenic Infections ◽

Low Concentrations

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

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Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.3.c810 ◽

1997 ◽

Vol 273 (3) ◽

pp. C810-C815 ◽

Cited By ~ 327

Author(s):

I. Owan ◽

D. B. Burr ◽

C. H. Turner ◽

J. Qiu ◽

Y. Tu ◽

...

Keyword(s):

Fluid Flow ◽

Type I Collagen ◽

Bone Matrix ◽

Mechanical Strain ◽

Extracellular Fluid ◽

Mechanical Stretch ◽

Type I ◽

Physiological Strain ◽

Fluid Forces ◽

Strain Magnitude

Mechanical force applied to bone produces two localized mechanical signals on the cell: deformation of the extracellular matrix (substrate strain) and extracellular fluid flow. To study the effects of these stimuli on osteoblasts, MC3T3-E1 cells were grown on type I collagen-coated plastic plates and subjected to four-point bending. This technique produces uniform levels of physiological strain and fluid forces on the cells. Each of these parameters can be varied independently. Osteopontin (OPN) mRNA expression was used to assess the anabolic response of MC3T3-E1 cells. When fluid forces were low, neither strain magnitude nor strain rate was correlated with OPN expression. However, higher-magnitude fluid forces significantly increased OPN message levels independently of the strain magnitude or rate. These data indicate that fluid forces, and not mechanical stretch, influence OPN expression in osteoblasts and suggest that fluid forces induced by extracellular fluid flow within the bone matrix may play an important role in bone formation in response to mechanical loading.

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Support of bone mineral deposition by regulation of pH

AJP Cell Physiology ◽

10.1152/ajpcell.00056.2018 ◽

2018 ◽

Vol 315 (4) ◽

pp. C587-C597 ◽

Cited By ~ 7

Author(s):

Harry C. Blair ◽

Quitterie C. Larrouture ◽

Irina L. Tourkova ◽

Li Liu ◽

Jing Hao Bian ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Extracellular Space ◽

Type I Collagen ◽

Bone Matrix ◽

Membrane Vesicles ◽

Bone Collagen ◽

Type I ◽

Mineral Deposition ◽

The Matrix ◽

Ph Dependent

Osteoblasts secrete collagen and isolate bone matrix from extracellular space. In the matrix, alkaline phosphatase generates phosphate that combines with calcium to form mineral, liberating 8 H+ per 10 Ca+2 deposited. However, pH-dependent hydroxyapatite deposition on bone collagen had not been shown. We studied the dependency of hydroxyapatite deposition on type I collagen on pH and phosphate by surface plasmon resonance in 0–5 mM phosphate at pH 6.8–7.4. Mineral deposition saturated at <1 mM Ca2+ but was sensitive to phosphate. Mineral deposition was reversible, consistent with amorphous precipitation; stable deposition requiring EDTA removal appeared with time. At pH 6.8, little hydroxyapatite deposited on collagen; mineral accumulation increased 10-fold at pH 7.4. Previously, we showed high expression Na+/H+ exchanger (NHE) and ClC transporters in osteoblasts. We hypothesized that, in combination, these move protons across osteoblasts to the general extracellular space. We made osteoblast membrane vesicles by nitrogen cavitation and used acridine orange quenching to characterize proton transport. We found H+ transport dependent on gradients of chloride or sodium, consistent with apical osteoblast ClC family Cl−,H+ antiporters and basolateral osteoblast NHE family Na+/H+ exchangers. Little, if any, active H+ transport, supported by ATP, occurred. Major transporters include cariporide-sensitive NHE1 in basolateral membranes and ClC3 and ClC5 in apical osteoblast membranes. The mineralization inhibitor levamisole reduced bone formation and expression of alkaline phosphatase, NHE1, and ClC5. We conclude that mineral deposition in bone collagen is pH-dependent, in keeping with H+ removal by Cl−,H+ antiporters and Na+/H+-exchangers. Periodic orientation hydroxyapatite is organized on type I collagen-coiled coils.

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Collagen-the Ultrastructural Element of the Bone Matrix

Revista de Chimie ◽

10.37358/rc.18.7.6400 ◽

2018 ◽

Vol 69 (7) ◽

pp. 1706-1709

Author(s):

Nicoleta Dumitru ◽

Andra Cocolos ◽

Andra Caragheorgheopol ◽

Constantin Dumitrache ◽

Ovidiu Gabriel Bratu ◽

...

Keyword(s):

Type I Collagen ◽

Bone Microarchitecture ◽

Complex Structure ◽

Bone Matrix ◽

Organic Matrix ◽

Bone Diseases ◽

Bone Collagen ◽

Type I ◽

New Therapeutic Approaches ◽

And Function

There is an increased interest and more studies highlight the fact that bone strength depends not only on bone tissue quantity, but also on its quality, which is characterized by the geometry and shape of bones, trabecular bone microarchitecture, mineral content, organic matrix and bone turnover. Fibrillar type I collagen is the major organic component of bone matrix, providing form and a stable template for mineralization. The biomedical importance of collagen as a biomaterial for medical and cosmetic purposes and the improvement of the molecular, cellular biology and analytical technologies, led to increasing interest in establishing the structure of this protein and in setting of the relationships between sequence, structure, and function. Bone collagen crosslinking chemistry and its molecular packing structure are considered to be distinct features. This unique post-translational modifications provide to the fibrillar collagen matrices properties such as tensile strength and viscoelasticity. Understanding the complex structure of bone type I collagen as well as the dynamic nature of bone tissues will help to manage new therapeutic approaches to bone diseases.

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Modulation of EndMT by Hydrogen Sulfide in the Prevention of Cardiovascular Fibrosis

Antioxidants ◽

10.3390/antiox10060910 ◽

2021 ◽

Vol 10 (6) ◽

pp. 910

Author(s):

Lara Testai ◽

Vincenzo Brancaleone ◽

Lorenzo Flori ◽

Rosangela Montanaro ◽

Vincenzo Calderone

Keyword(s):

Hydrogen Sulfide ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Type I ◽

Vascular Endothelial ◽

Independent Manner ◽

Mesenchymal Transition ◽

Adult Age ◽

Cardiovascular Fibrosis

Endothelial mesenchymal transition (EndMT) has been described as a fundamental process during embryogenesis; however, it can occur also in adult age, underlying pathological events, including fibrosis. Indeed, during EndMT, the endothelial cells lose their specific markers, such as vascular endothelial cadherin (VE-cadherin), and acquire a mesenchymal phenotype, expressing specific products, such as α-smooth muscle actin (α-SMA) and type I collagen; moreover, the integrity of the endothelium is disrupted, and cells show a migratory, invasive and proliferative phenotype. Several stimuli can trigger this transition, but transforming growth factor (TGF-β1) is considered the most relevant. EndMT can proceed in a canonical smad-dependent or non-canonical smad-independent manner and ultimately regulate gene expression of pro-fibrotic machinery. These events lead to endothelial dysfunction and atherosclerosis at the vascular level as well as myocardial hypertrophy and fibrosis. Indeed, EndMT is the mechanism which promotes the progression of cardiovascular disorders following hypertension, diabetes, heart failure and also ageing. In this scenario, hydrogen sulfide (H2S) has been widely described for its preventive properties, but its role in EndMT is poorly investigated. This review is focused on the evaluation of the putative role of H2S in the EndMT process.

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Targeting lncRNA H19/miR-29b/COL1A1 Axis Impedes Myofibroblast Activities of Precancerous Oral Submucous Fibrosis

International Journal of Molecular Sciences ◽

10.3390/ijms22042216 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2216

Author(s):

Cheng-Chia Yu ◽

Yi-Wen Liao ◽

Pei-Ling Hsieh ◽

Yu-Chao Chang

Keyword(s):

Type I Collagen ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Oral Submucous Fibrosis ◽

Ectopic Expression ◽

Collagen Gel ◽

Type I ◽

Migration Ability ◽

Potentially Malignant ◽

Submucous Fibrosis

Oral submucous fibrosis (OSF) is known as a potentially malignant disorder, which may result from chemical irritation due to areca nuts (such as arecoline). Emerging evidence suggests that fibrogenesis and carcinogenesis are regulated by the interaction of long noncoding RNAs (lncRNAs) and microRNAs. Among these regulators, profibrotic lncRNA H19 has been found to be overexpressed in several fibrosis diseases. Here, we examined the expression of H19 in OSF specimens and its functional role in fibrotic buccal mucosal fibroblasts (fBMFs). Our results indicate that the aberrantly overexpressed H19 contributed to higher myofibroblast activities, such as collagen gel contractility and migration ability. We also demonstrated that H19 interacted with miR-29b, which suppressed the direct binding of miR-29b to the 3′-untranslated region of type I collagen (COL1A1). We showed that ectopic expression of miR-29b ameliorated various myofibroblast phenotypes and the expression of α-smooth muscle actin (α-SMA), COL1A1, and fibronectin (FN1) in fBMFs. In OSF tissues, we found that the expression of miR-29b was downregulated and there was a negative correlation between miR-29b and these fibrosis markers. Lastly, we demonstrate that arecoline stimulated the upregulation of H19 through the transforming growth factor (TGF)-β pathway. Altogether, this study suggests that increased TGF-β secretion following areca nut chewing may induce the upregulation of H19, which serves as a natural sponge for miR-29b and impedes its antifibrotic effects.

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The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms22041861 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1861

Author(s):

Jemima Seidenberg ◽

Mara Stellato ◽

Amela Hukara ◽

Burkhard Ludewig ◽

Karin Klingel ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Cardiac Fibrosis ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Beclin 1 ◽

Type I ◽

Alpha Smooth Muscle Actin

Background: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. Methods and Results: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-β. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-β-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. Conclusion: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-β-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

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