Formation Of Organic Matrix Research Articles

The salivary peptide histatin-1 enhances bone repair in vivo

The salivary peptide histatin-1 was recently described as a novel osteogenic factor that stimulates cell adhesion, migration, and differentiation in bone-lineage cells. Since these cell responses collectively contribute to bone regeneration, we hypothesized that histatin-1 harbors the capacity to enhance bone tissue repair at the preclinical level. By using a model of monocortical bone defect, we explored the effects of histatin-1 in tibial mineralization and organic matrix formation in vivo. To this end, different amounts of histatin-1 were embedded in one-mm3 collagen sponges and then applied to tibial monocortical defects in C57bl/6 mice. After seven days, mice were euthanized, and samples were processed for subsequent analysis. Micro-computed tomography screening showed that histatin-1 increased intraosseous mineralization, and this phenomenon was accompanied by augmented collagen matrix deposition and closure of cortical defect edges, as determined by Hematoxylin-Eosin and Masson's Trichrome staining. Moreover, immunohistochemical analyses showed that histatin-1 increased the expression of the osteogenic marker alkaline phosphatase, which was accompanied by augmented blood vessel formation. Collectively, our findings show that histatin-1 itself promotes bone regeneration in an orthotopic model, proposing this molecule as a therapeutic candidate for use in bone regenerative medicine.

Rifampicin restores extracellular organic matrix formation and mineralization of osteoblasts after intracellular Staphylococcus aureus infection.

AimsBone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy.MethodsThe human osteoblast-like Saos-2 cells infected with S. aureus EDCC 5055 strain and treated with 8 µg/ml RMP underwent osteogenic stimulation for up to 21 days. Test groups were Saos-2 cells + S. aureus and Saos-2 cells + S. aureus + 8 µg/ml RMP, and control groups were uninfected untreated Saos-2 cells and uninfected Saos-2 cells + 8 µg/ml RMP.ResultsThe S. aureus-infected osteoblasts showed a significant number of intracellular bacteria colonies and an unusual higher metabolic activity (p < 0.005) compared to uninfected osteoblasts. Treatment with 8 µg/ml RMP significantly eradicated intracellular bacteria and the metabolic activity was comparable to uninfected groups. The RMP-treated infected osteoblasts revealed a significantly reduced amount of mineralized extracellular matrix (ECM) at seven days osteogenesis relative to uninfected untreated osteoblasts (p = 0.007). Prolonged osteogenesis and RMP treatment at 21 days significantly improved the ECM mineralization level. Ultrastructural images of the mineralized RMP-treated infected osteoblasts revealed viable osteoblasts and densely distributed calcium crystal deposits within the extracellular organic matrix. The expression levels of prominent bone formation genes were comparable to the RMP-treated uninfected osteoblasts.ConclusionIntracellular S. aureus infection impaired osteoblast metabolism and function. However, treatment with low dosage of RMP eradicated the intracellular S. aureus, enabling extracellular organic matrix formation and mineralization of osteoblasts at later stage.Cite this article: Bone Joint Res 2022;11(5):327–341.

New Insights on the Diurnal Mechanism of Calcification in the Stony Coral, Stylophora pistillata

Scleractinian corals are evolutionary-successful calcifying marine organisms, which utilize an endo-symbiotic relationship with photosynthetic dinoflagellate algae that supply energy products to their coral hosts. This energy further supports a higher calcification rate during the day in a process known as light enhanced calcification. Although this process has been studied for decades, the mechanisms behind it are still unknown. However, photosynthesis and respiration also cause daily fluctuations in oxygen and pH levels, resulting in the coral facing highly variable conditions. Here we correlated gene expression patterns with the physiological differences along the diel cycle to provide new insights on the daily dynamic processes, including circadian rhythm, calcification, symbiosis, cellular arrangement, metabolism, and energy budget. During daytime, when solar radiation levels are highest, we observed increased calcification rate combined with an extensive up-regulation of genes associated with reactive oxygen species, redox, metabolism, ion transporters, skeletal organic matrix, and mineral formation. During the night, we observed a vast shift toward up-regulation of genes associated with cilia movement, tissue development, cellular movement, antioxidants, protein synthesis, and skeletal organic matrix formation. Our results suggest that light enhanced calcification is related to several processes that occur across the diel cycle; during nighttime, tissue might elevate away from the skeleton, extending the calcifying space area to enable the formation of a new organic framework template. During daytime, the combination of synthesis of acid-rich proteins and a greater flux of ions to the sites of calcification facilitate the conditions for extensive mineral growth.

Using glutamine synthetase 1 to evaluate the symbionts' potential of ammonia assimilation and their responses to illumination in five organs of the giant clam, Tridacna squamosa

Nitrogen-deficient symbiotic dinoflagellates (zooxanthellae) living inside the fluted giant clam, Tridacna squamosa, need to obtain nitrogen from the host. Glutamine synthetase 1 (GS1) is a cytosolic enzyme that assimilates ammonia into glutamine. We determined the transcript levels of zooxanthellal GS1 (Zoox-GS1), which represented comprehensively GS1 transcripts of Symbiodinium, Cladocopium and Durusdinium, in five organs of T. squamosa. The outer mantle had significantly higher transcript level of Zoox-GS1 than the inner mantle, foot muscle, hepatopancreas and ctenidium, but the transcript ratios of Zoox-GS1 to zooxanthellal form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Zoox-rbcII), which represented the potential of ammonia assimilation relative to the phototrophic potential, were comparable among these five organs. Based on transcript ratios of Zoox-GS1 to zooxanthellal Urease (Zoox-URE), the outer mantle had the highest potential of urea degradation relative to ammonia assimilation among the five organs, probably because urea degradation could furnish CO2 and NH3 for photosynthesis and amino acid synthesis, respectively, in the symbionts therein. The protein abundance of Zoox-GS1 was upregulated in the outer mantle and the inner mantle during illumination. Zoox-GS1 could catalyze light-enhanced glutamine formation using ammonia absorbed from the host or ammonia released through urea degradation in the cytoplasm. The glutamine produced could be used to synthesize other nitrogenous compounds, including amino acids in the cytoplasm or in the plastid of the dinoflagellates. Some of the amino acids synthesized by the symbionts in the inner mantle and foot muscle could be donated to the host to support shell organic matrix formation and muscle production, respectively.

Fabrication of Pentoxifylline-Loaded Hydroxyapatite/Alginate Scaffold for Bone Tissue Engineering

Background: Hydroxyapatite (HAP), as a common biomaterial in bone tissue engineering, can be fabricated in combination with other osteogenic agents. Pentoxifylline (PTX) is demonstrated to have positive roles in bone defect healing. Since local administration can diminish the systemic side effects of the drug, the objectives of the current in vitro study were to find the effects of PTX on the osteoblast functions for tissue engineering applications. Methods: a HAP scaffold was fabricated by casting the HAP slurry within polyurethane foam. The scaffold was enriched with 5 mg/mL PTX. Alginate (Alg) was used as drug carrier to regulate the PTX releasing rate. MG-63 osteosarcoma cells were cultured on 3D scaffolds and 2D Alg films in the presence or absence of PTX. Results: PTX did not affect the cell viability, attachment and phenotype. Also, the ultrastructure of the scaffolds was not modified by PTX enrichment. Alizarin red S staining showed that PTX has no effect on calcium deposition. Besides, Raman confocal microscopy demonstrated an increase in the organic matrix formation including proline, valine and phenylalanine deposition (represented collagen). Although PTX increased the total protein secretion, it led to a decrease in the alkaline phosphatase activity and vascular endothelial growth factor (VEGF) content. PTX reduced the hydration and degradation rates and it was released mainly at the first 24 hours of incubation. Conclusion: Based on our in vitro study, application of engineered PTX-loaded HAP scaffold in bone regeneration can act on behalf of organic matrix production, but not angiogenesis and mineralization.

The case of oncogenic hypophosphatemic osteomalacia

Osteomalacia is a systemic bone disease, characterized by an excessive accumulation of non-mineralized osteoid and an imbalance in the organic matrix formation and mineralization. A rare cause of disease is tumor-induced osteomalacia, most often due to phosphaturic mesenchymal tumors (PMT). Usually there are benign small tumors, affecting the soft tissues and bones of any location. The basic pathogenesis of underlying oncogenic osteomalacia is a decreased renal tubular reabsorption of phosphate consequent to hyperproduction of fibroblast growth factor 23 in PMT. Clinical features are nonspecific, the average period from the symptoms onset to diagnosis reaches 3 years and at least 5 years before surgical treatment. Finding the tumour is crucial, as complete removal is curative. We present a clinical case of tumor-induced osteomalacia due to PMT required the complex differential diagnosis with other rare diseases.

Porcelain veneer for a simple and esthetic treatment on anterior teeth with enamel hypoplasia: a case report

Introduction: the esthetic factor should be considered for every patient who seeks a comprehensive dental treatment, especially for women. The anterior teeth deformities will diminish the levels of confidence and decrease self-esteem of the patients. The tooth development disturbances, such as enamel hypoplasia, may present an esthetic problem. Enamel hypoplasia is an alteration of organic matrix formation which characterized by the defect or porosity on the enamel surface. It can be restored with a variety of restorative treatment options including indirect porcelain veneers. Case: a 30 years old female patient was referred with chief complaint of anterior teeth discoloration. Clinical examination on the maxillary left central and lateral incisors revealed the defects on nearly half of the labial surface. Case management: the incisal lapping technique were chosen to prepared teeth #21 and #22 for indirect porcelain veneer fabrications. Conclusion: indirect porcelain veneers established an excellent esthetic result and restored the size, shape and color of anterior teeth with long term stability.

Metabolomic and transcriptomic profiling reveals the alteration of energy metabolism in oyster larvae during initial shell formation and under experimental ocean acidification

Marine bivalves secrete calcified shells to protect their soft bodies from predation and damages, which is of great importance for their survival, and for the safety of the coastal ecosystem. In recent years, larval shell formation of marine bivalves has been severely affected by ocean acidification (OA), and previous study indicated that OA might affect such process by disrupting endogenous energy metabolism. Developmental stages from trochophore to D-shape larvae are extremely important for initial shell formation in oyster since a calcified shell was formed to cover the chitin one. In the present study, metabolomic and transcriptomic approaches were employed to investigate the energy metabolism of oyster larvae during initial shell (prodissoconch I, PDI shell) formation and under experimental OA treatment. Totally 230 chemical compounds were identified from the present dataset, most of which were highly expressed in the “middle” stage (early D-shape larvae) which was critical for PDI shell formation since a calcified shell was formed to cover the chitin one. Several compounds such as glucose, glutarylcarnitine (C5), β-hydroxyisovaleroylcarnitine, 5-methylthioadenosine (MTA), myristoleate (14:1n5) and palmitoleate (16:1n7) were identified, which were involved in energy metabolic processes including amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. In addition, mRNA expressions of genes related to protein metabolism, glycolysis, lipid degradation, calcium transport and organic matrix formation activities were significantly down-regulated upon experimental OA. These results collectively suggested that formation of the initial shell in oyster larvae required endogenous energy coming from amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. These metabolic activities could be severely inhibited by experimental OA, which might alter the allocation of endogenous energy. Insufficient endogenous energy supply then suppressed the mobilization of calcium and resulted in a failure or delay in PDI shell formation.

Impact of 3-year vitamin D and calcium supplementation on mineral and organic matrix formation of trabecular bone in postmenopausal osteoporosis

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Impact of 3-year vitamin D and calcium supplementation on mineral and organic matrix formation of trabecular bone in postmenopausal osteoporosis

Impact of 3-year vitamin D and calcium supplementation on mineral and organic matrix formation of trabecular bone in postmenopausal osteoporosis

Tibial dysplasia. A clue to the diagnosis of NF1.

The subjects with NF1 present some characteristic bone abnormalities. Among the latter there is tibial dysplasia, which is usually located between the middle third and the distal third of the tibia (2). In this site there is thinning of the bone, due to narrowing of the medullary, whereas the cortical can be normal or thickened. The weakening of the bone is responsible for an anterolateral bending of the tibia -tibia procurvata et vara- (2). The latter can be complicated by pathological fractures and mainly by defective formation of bone callus and non rejoining of the two fragments (pseudoarthrosis). The bone fragility is partly due to an increased osteocyte-related micro-porosity with persistence of ectopic blood vessels, hypomineralization, and generalized defect of organic matrix formation (1).

Computational prediction of candidate miRNAs and their potential functions in biomineralization in pearl oyster Pinctada martensii

MicroRNAs (miRNAs) are a class of non-coding RNA molecules with presumed post-transcriptional regulatory activity in various biological processes, such as development and biomineralization. Pinctada martensii is one of the main species cultured for marine pearl production in China and Japan. In our previous research, 258 pm-miRNAs had been identified by solexa deep sequencing in P. martensii, while it is far from the number of miRNAs found in other species. In this study, based on the transcriptome database of pearl sac, we identified 30 candidate pm-miRNAs by computational prediction. Among the obtained 30 pm-miRNAs, 13 pm-miRNAs were generated from the complementary strand of protein-coding mRNAs, and 17 pm-miRNAs could not be annotated using blastx and tblastn analysis. Notably, 10 of the 30 pm-miRNAs, such as pm-miR-1b, pm-miR-205b and pm-miR-375b, were homologous with the reported pm-miRNAs, respectively. To validate the existence of the identified pm-miRNAs, eight randomly selected pm-miRNAs were tested by stem loop quantitative RT-PCR analyses using 5.8S as the internal reference gene. Target prediction between the obtained pm-miRNAs and biomineralization-related genes by microTar, miRanda and RNA22 indicated pm-miR-2386 and pm-miR-13b may be the key factors in the regulation network by regulating the formation of organic matrix or the differentiation of mineralogenic cell during shell formation. Thus, this study enriched miRNA databases of pearl oyster and provided a new way to understand biomineralization.

Multiscale, Converging Defects of Macro-Porosity, Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.

Developmental structure of the vertebral column, fins, scutes and scales in bester sturgeon, a hybrid of beluga Huso huso and sterlet Acipenser ruthenus

In the larval bester, a hybrid sturgeon of beluga Huso huso and sterlet Acipenser ruthenus, development of cartilage around the notochord began 7 days post hatch (dph) (14.0 mm, total length, L(T)). The vertebral cartilage develops in the following sequence: basidorsals and basiventrals, neural canals, neural spines and ribs. The development of ribs remained incomplete in the largest specimen (181 dph, 179 mm L(T) ) that was examined. Endoskeletal development of the fins began 4 dph for the dorsal and anal fins, 6 dph for the pectoral fin and 10 dph for the caudal and pelvic fins. Complete elements of all fins were observed by 91 dph and complete ossification of fin rays was observed by 122 dph in the double-stained specimens. Observation of the histological sections, however, suggested that ossification occurred soon after the formation of the organic matrix in the fin rays. Dorsal scutes were first visible by 25 dph, followed by the lateral and ventral scutes, which were visible by 37 and 44 dph, respectively. The number of scutes was fixed at 44, 59 and 91 dph and ossification was complete by 59 (dorsal) and 91 dph (lateral and ventral scutes) in the double-stained specimens. Ossification occurred soon after the formation of the scute organic matrix in the histological sections. Four types of scales were observed in the H. huso×A. ruthenus hybrid. Median predorsal, preanal and small scales on the anterior section of the head were visible by 59 dph. Scales on the caudal fin were visible by 91 dph and a variable assemblage of scales anterior to the anal fin was visible by 122 dph. Both the scutes and scales developed in a process that is similar to that of intramembranous ossification.

Lactic acid bacterial extract as a biogenic mineral growth modifier

The formation of minerals and mechanisms by which bacteria could control their formation in natural habitats is now of current interest for material scientists to have an insight of the mechanism of in vivo mineralization, as well as to seek industrial and technological applications. Crystalline uniform structures of calcium and barium minerals formed micron-sized building blocks when synthesized in the presence of an organic matrix consisting of secreted protein extracts from three different lactic acid bacteria (LAB) viz.: Lactobacillus plantarum MTCC 1325, Lactobacillus acidophilus NRRL B4495 and Pediococcus acidilactici CFR K7. LABs are not known to form organic matrix in biological materialization processes. The influence of these bacterial extracts on the crystallization behavior was investigated in details to test the basic coordination behavior of the acidic protein. In this report, varied architecture of the mineral crystals obtained in presence of high molecular weight protein extracts of three different LAB strains has been discussed. The role of native form of high molecular weight bacterial protein extracts in the generation of nucleation centers for crystal growth was clearly established. A model for the formation of organic matrix–cation complex and the subsequent events leading to crystal growth is proposed.

Biochemical markers of bone metabolism: an overview.

An overview of biochemical markers of bone metabolism is presented along with indications for their clinical utilization. The structure, cyclical metabolism, and hormone regulation of bone is reflected by markers of resorption, formation and/or turnover. Markers of resorption representing degradation of type 1 collagen, include N-telopeptides, C-telopeptides, hydroxyproline, and the collagen crosslinks pyridinoline and deoxypyridinoline; acid phosphatase, a marker of osteoclast activity, and urinary calcium are also indicators of bone resorption. Bone formation markers indicate osteoblast activity; bone-specific alkaline phosphatase and the N-terminal and C-terminal extension peptides of procollagen reflect formation of organic matrix in bone. Osteocalcin, produced by osteoblasts but also released during osteoclastic degradation, may indicate either formation when resorption and formation are coupled or turnover when they are uncoupled. Bone markers respond to intervention more rapidly than techniques such bone mineral density. Resorption markers respond approximately 1 to 3 months after intervention; markers of formation respond later, after 6 to 9 months. Bone markers may add useful information for assessing fracture risk and for monitoring osteoporosis, Paget's disease of bone, cancer metastasis, and metabolic disease. Various therapeutic interventions may affect release of some bone markers. Bone disease has high prevalence in adults so bone markers will become even more important for assessing fracture risk and monitoring therapy as populations age. Characteristics of bone markers are dependent on biology and the assay used. Substantial work remains in characterizing existing assays, identifying better markers and performing the clinical studies to define which bone markers should be measured and when.

The dynamics of spicule calcification in whole colonies of the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

The dynamics of calcification was examined in fractionated segments throughout entire colonies of the gorgonian Leptogorgia virgulata (Lamarck). The uptake of 45Ca, 3H-aspartic acid and 3H-thymidine was evaluated in isolated fractions including tissues, spicules and axes. Polyp number and distribution, axis weight and spicule type and size were also examined. The highest rates of uptake for all labeled materials were in the branch tips, indicating that the greatest degree of organic matrix formation, subsequent spicule formation and cell division occur in this apical region of the colony. Ca uptake decreased from the branch tips to the mid-portions of colonies, followed by an increase toward the bases. This latter rise in uptake is due to the increase in size and density of spicules which causes an increase in isotopic exchange. At branch junctions, the predominant cellular activity appears to be spicule formation vs. cell division. Axes of apical regions of branches displayed high rates of Ca uptake supporting its active role in the calcification process. The number and distribution of polyps, as well as spicule types, was variable among colonies indicating individual variability and/or the impact of varying environmental conditions. Data indicate that, although spicule formation occurs at its highest rate at branch tips, it occurs throughout the remainder of the colony as well.

Physical and Biochemical Changes of the Mandible and Long Bone in Protein-Energy Malnourished Newborn Rats

The activities of alkaline and acid phosphatases, the linear dimensions and volumes of mandibles and long bones, and the level of calcitonin in serum of rat pups suckled on dams fed 25 or 6% protein diets were determined. In both mandibles and long bones, alkaline phosphatase activity corresponded to the calcification pattern, and acid phosphatase activity paralleled organic matrix formation. The calcitonin level in blood serum of the malnourished pups differed from that of controls only on day 5. The linear dimensions and volume were consistently more affected in long bones than in mandibles. These results may be explained in terms of the critical growth periods of these bones. In the mandible, the critical growth period appears to occur prenatally, whereas in the long bone it occurs postnatally, when the nutritional stress of this experiment was applied. Long bones were thus generally more affected than mandibles.

On the daily incremental lines in human dentine

Assuming that the cross-striations on enamel rods represent daily increments, the various incremental lines in dentine between selected pairs of tetracycline markers were identified and correlated with Retzius lines in the enamel, in order to obtain comparative information on the rate of dentine formation. The incremental lines described by Andresen (1898) and von Ebner (1906) are the same and are separated by approx. 20 μm. However, a regular series of lines approx. 4 μm apart were also found and considered to be due to a 24-h rhythm in organic matrix formation. A separate 12-h rhythm influencing mineralization was deduced by comparing the number of calcospherite striae with the 4 μm rhythmic change in fibre orientation of the matrix.

Ribonucleic acid within the extracellular matrix during embryonic tooth formation.

AbstractDuring embryonic tooth formation, interactions between epithelial and mesenchymal cells results in the formation of a metachromatic interface or extracellular matrix. The cervical or germinative region of this epidermal organ system is populated by an increasing gradient of cellular differentiation and an extracellular matrix which is the progenitor for subsequent dentine organic matrix formation. Embryonic rabbit tooth primordia can be maintained in culture enabling kinetic studies of labeled precursor incorporation. Autoradiographs of tooth organ cultures continusly incubated with labeled uridine for periods up to eight hours, demonstrated initial cellular incorporation with subsequent transfer of 2% of the grain density to the extracellular matrix by four hours. The grain density was removed by ribonuclease treatment. No incorporation of tritiated thymidine into the matrix was observed. The incorporation of C14‐uridine during organ culture was inhibited by actinomycin D. Micrurgy was employed to isolate the extracellular matrix free of adherent cells. Electron microscopy demonstrated membrane‐bound, electron dense bodies within the matrix, presumably cytoplasmic extensions. No cells per se were observed on the isolated matrix. Several experimental criteria suggested that uridine incorporation into the extracellular matrix was regulated by epithelial and mesenchymal cells. Phenol extraction procedures of labeled cervical matrices demonstrated an ultraviolet absorption maximum at 260 μU. Both spectrophotometric determinations and orcinol assays found RNA to be 0.4–0.5% of the cervical extracellular matrix.These results are interpreted to indicate that RNA is a component of the metachromatic extracellular matrix during epithelio‐mensenchymal interactions associated with tooth formation. The functional significance of these observations is premature at this time.