Non-collagenous Proteins Research Articles

L-histidine controls the hydroxyapatite mineralization with plate-like morphology: Effect of concentration and media

Hydroxyapatite (HA) is the main inorganic component of bone and dentin, and their non-stoichiometric compositions and plate-shaped morphology is responsible for their bioactivity and osteoconductive nature. Collagenous (CPs) and non-collagenous proteins (NCPs) facilitate mineralization and regulate structural properties of HA through their side-chains. The bioactivity of synthetic HA does not usually match with the HA found in bone and, therefore, there is a need to understand the role of biomolecules in bone mineralization in order to develop non-stoichiometric plate-shaped HA for bone grafts. Role of several amino acids has been investigated but the role of L-his has been rarely investigated under physiological conditions even though it is a part of HA inhibitor proteins, like albumin, amelogenin, and histidine-rich proteins. In this study, L-his and L-glu were used to modify the structural properties of HA in different experimental conditions and buffer systems (tris and hepes). The results showed that L-his was able to regulate the plate-shaped morphology of HA in every experimental condition, unlike the L-glu, where the crystal morphology was regulated by experimental conditions. Both amino acids behaved differently in DI water, tris, and hepes buffer, and the media used influenced the precipitation time and structural properties of HA. Hepes and tris buffers also influenced the HA precipitation process. Overall, the studies revealed that L-his may be used as an effective regulator of plate-shaped morphology of HA, instead of large NCPs/proteins, for designing biomaterials for bone regeneration applications and the choice of buffer system is important in designing and evaluating the systems for mineralization. In cell culture studies, mouse osteoblast precursor cells (MC3T3-E1) showed highest proliferation on the bone-like plate-shaped HA, among all the HA samples investigated.

Future Perspectives of Biomimetics in Restorative Dentistry

The main goal of tooth restoration aims at achieving mineralization of initial enamel and dentinal lesions in native form. Most of the restorative materials and remineralization adjuvants for enamel and dentin mineralization are evidenced in the literature. Although commercially available restorative materials exhibit superior esthetics, mechanical properties and cost effectiveness, durability of the restoration threatened by the occurrence of inadequate strength, long-term solubility, and weaker adhesion to tooth and accelerated degradation after being bonded to tooth structure. Recently, the role of biomimetic science in restorative dentistry aims at creating a restoration that can be highly compatible with the structural, functional and biologic properties of dental tissues to reproduce and emulate the original performance of the intact tooth with high durability. In order to recover the prismatic structure in mineral-depleted enamel and to achieve interfibrillar mineralization in dentin, non-collagenous protein analogues have been proposed as templates for apatite deposition. Biomimetic analogues must be necessary to achieve functional mineralization and to recover the dynamic mechanical properties of teeth. The use of these analogues associated with ion-releasing materials seems to be a promising approach for both enamel and dentin remineralization. This review enlightens the current and future perspectives of biomimetic analogues used for enamel and dentin remineralization as the clinical translation of this biomimetic research can be considered as the boon to restorative dentistry.

Functions of Osteocalcin in Bone, Pancreas, Testis, and Muscle.

Osteocalcin (Ocn), which is specifically produced by osteoblasts, and is the most abundant non-collagenous protein in bone, was demonstrated to inhibit bone formation and function as a hormone, which regulates glucose metabolism in the pancreas, testosterone synthesis in the testis, and muscle mass, based on the phenotype of Ocn−/− mice by Karsenty’s group. Recently, Ocn−/− mice were newly generated by two groups independently. Bone strength is determined by bone quantity and quality. The new Ocn−/− mice revealed that Ocn is not involved in the regulation of bone formation and bone quantity, but that Ocn regulates bone quality by aligning biological apatite (BAp) parallel to the collagen fibrils. Moreover, glucose metabolism, testosterone synthesis and spermatogenesis, and muscle mass were normal in the new Ocn−/− mice. Thus, the function of Ocn is the adjustment of growth orientation of BAp parallel to the collagen fibrils, which is important for bone strength to the loading direction of the long bone. However, Ocn does not play a role as a hormone in the pancreas, testis, and muscle. Clinically, serum Ocn is a marker for bone formation, and exercise increases bone formation and improves glucose metabolism, making a connection between Ocn and glucose metabolism.

Comparing biological and pathological factors affecting osteocalcin concentrations in archaeological skeletal remains

Paleoproteomics and the study of ancient proteins has become an important consideration in bioarchaeological research as we seek to understand the relationship between the physical skeleton and its underlying biochemistry. Osteocalcin is an abundant, non-collagenous protein that is accessible archaeologically due to its affinity for hydroxyapatite in bone. Manufactured by the osteoblast cells during normal bone metabolism, osteocalcin is inherently linked to the process of bone remodelling. Clinical studies have consistently shown the impact of biological (i.e., sex and age) and pathological (i.e., disease) factors on osteocalcin concentrations within the body; however, this connection has yet to be fully explored bioarchaeologically. For this study, 46 individuals from the Danish Sct. Albert’s cemetery (CE 1250–1536) were analyzed with cortical bone samples harvested from the femur. Each sample was demineralized and quantified using established paleoproteomic methods (ELISA) and tested for diagenesis using FTIR-ATR. No significant differences were found in the osteocalcin concentrations between adult males and females. However, males had a wider range of osteocalcin variability perhaps explained in part by their increased prevalence of pathological conditions characterized by bone remodelling. Similarly, non-adults had a wide range of osteocalcin variability but there were no significant differences when compared to the adult mean. No correlations were found between osteocalcin concentrations and diagenesis suggesting that the protein can survive across a range of temporal periods and variable burial environments. While no clear pattern emerged between osteocalcin concentrations and biological and/or pathological factors, this study is significant in that it further explores the use of paleoproteomics in bioarchaeological contexts and how we may unite our macroscopic and biochemical assessments of human skeletal remains.

Interfacial bonding between mineral platelets in bone and its effect on mechanical properties of bone

Bone is a composite material consisting principally of apatite mineral, collagen fibrils, non-collagenous proteins, and other organic species. Recent electron microscopy studies have shown that the mineral in bone occurs as stacks of thin polycrystalline sheets (“mineral lamellae,” MLs) which surround and lie between the collagen fibrils. We focus on the effect of the interface between these mineral lamellae on the mechanical properties of bone. Previous studies on bone treated with sodium hypochlorite (NaClO) to remove all organic material showed a greatly weakened mineral framework. Here, we treated femoral cortical bone with ethylenediamine (EDA), which only removes collagen, to study the effect of its removal on bone properties. We tested the degree of completion of the treatment by Raman spectroscopy and thermogravimetric analysis. When only collagen is removed, a continuous mineral structure remains and is less weakened than by NaClO treatment. Transmission electron microscopy study of finely ground particles of the EDA treated bone shows that stacks of MLs remain joined, whereas in NaClO treated bone, only isolated crystals are present. Thus, we infer that the MLs in bone are held together in stacks by an organic glue, which is destroyed by NaClO, but which survives the EDA treatment. We show that this glue may contribute to the stiffness, strength, and energy absorption of bone. Further studies are needed to discover the chemical nature of this glue. This study provides a starting point for such investigations.

Preparation of protein products from collagen-rich poultry tissues

Chicken stomachs are by-products obtained from the poultry processing in slaughterhouses. Their amount has been gradually increasing as a consequence of a continually rising poultry consumption. Since these animal tissues are still rich in proteins, mainly collagen, fat, and minerals, it is essential and beneficial to investigate the appropriate management and further processing. Collagen could be extracted from chicken stomachs and used as a raw material in the food, cosmetic, medical, and also pharmaceutical industry. This paper is to investigate possibilities of such extraction of collagen products, gelatines, or alternatively hydrolysates, from chicken stomachs after prior biotechnological treatment with the proteolytic enzyme Protamex. In this experiment, non-collagenous proteins were removed from stomachs using 0.03 M NaOH and 0.2 M NaCl. Subsequently, the tissue was defatted applying acetone and the enzyme Lipolase. Purified and dried collagen was then treated with the proteolytic enzyme Protamex. In the last step, gelatine was extracted from the tissue in hot water. The influence of selected processing parameters on the extraction efficiency and final product quality was monitored. The extraction conditions included the amount of the added enzyme (0.1 – 0.4%) and the extraction temperature of between 60 and 65 °C. The total gelatine yield ranged from 43.80 to 96.45% and the gel strength varied from 2 ±0 to 429 ±8 Bloom. The enzymatic treatment of the raw material is an economical and ecological alternative to traditional acid or alkaline treatments. Extracted gelatine with the gel strength of 100 – 300 Bloom would be suitable for the applications in the food industry in the production of confectionery, marshmallow, aspic or dairy products.

Potential Role of Lycopene in the Prevention of Postmenopausal Bone Loss: Evidence from Molecular to Clinical Studies.

Osteoporosis is a metabolic bone disease characterized by reduced bone mineral density, which affects the quality of life of the aging population. Furthermore, disruption of bone microarchitecture and the alteration of non-collagenous protein in bones lead to higher fracture risk. This is most common in postmenopausal women. Certain medications are being used for the treatment of osteoporosis; however, these may be accompanied by undesirable side effects. Phytochemicals from fruits and vegetables are a source of micronutrients for the maintenance of bone health. Among them, lycopene has recently been shown to have a potential protective effect against bone loss. Lycopene is a lipid-soluble carotenoid that exists in both all-trans and cis-configurations in nature. Tomato and tomato products are rich sources of lycopene. Several human epidemiological studies, supplemented by in vivo and in vitro studies, have shown decreased bone loss following the consumption of lycopene/tomato. However, there are still limited studies that have evaluated the effect of lycopene on the prevention of bone loss in postmenopausal women. Therefore, the aim of this review is to summarize the relevant literature on the potential impact of lycopene on postmenopausal bone loss with molecular and clinical evidence, including an overview of bone biology and the pathophysiology of osteoporosis.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.

Modeling the epithelial-mesenchymal transition process in a 3D organotypic cervical neoplasia

Here, we proposed an innovative organotypic cervical tumor model able to investigate the bi-directional crosstalk between epithelium and stroma as well as the key disease features of the epithelial-mesenchymal transition (EMT) process in vitro. By using a modular tissue assembling approach, we developed 3D cervical stromal models composed of primary human cervical fibroblasts (HCFs) or cervical cancer-associated fibroblasts (CCAFs) embedded in their own ECM to produce 3D normal cervical-instructed stroma (NCIS) or 3D cervical cancer-instructed stroma (CCIS), respectively. Then, we demonstrate the role of the tumor microenvironment (TME) in potentiating the intrinsic invasive attitude of cervical cancer derived SiHa cells and increasing their early viral gene expression by comparing the SiHa behavior when cultured on NCIS or CCIS (SiHa-NCIS or SiHa-CCIS). We proved the crucial role of the CCAFs and stromal microenvironment in the mesenchymalization of the cancer epithelial cells by analyzing several EMT markers. We further assessed the expression of the epithelial adhesion molecules, matricellular enzymes, non-collagenous proteins as well as ECM remodeling in terms of collagen fibers texture and assembly. This cervical tumor model, closely recapitulating key cervical carcinogenesis features, may provide efficient and relevant support to current approaches characterizing cancer progression and develop new anticancer therapy targeting stroma rather than cancer cells.

Spider Silk Fusion Proteins for Controlled Collagen Binding and Biomineralization.

The development of biomaterials for the interface between tendon and bone is important for realizing functional tendon replacements. Toward the development of new materials for such applications, engineered recombinant spider silk proteins were modified with peptide tag sequences derived from noncollagenous proteins in bone, so-called SIBLING proteins, such as osteopontin and sialoprotein, which are known to interact with collagen and to initiate mineralization. Materials made of these spider silk-SIBLING hybrids were analyzed concerning mineralization and interaction with cells. They showed enhanced calcium phosphate formation upon incubation in mineralization agents. In gradient films, MC3T3-E1 mouse preosteoblasts adhered preferentially along the gradient toward the variant with a collagen binding motif.

L-Ascorbate Biosynthesis Involves Carbon Skeleton Rearrangement in the Nematode Caenorhabditis elegans

Ascorbate (AsA) is required as a cofactor and is widely distributed in plants and animals. Recently, it has been suggested that the nematode Caenorhabditis elegans also synthesizes AsA. However, its biosynthetic pathway is still unknown. To further understand AsA biosynthesis in C. elegans, we analyzed the incorporation of the 13C atom into AsA using gas chromatography-mass spectrometry (GC-MS) in worms fed with D-Glc (1-13C)-labeled Escherichia coli. GC-MS analysis revealed that AsA biosynthesis in C. elegans, similarly to that in mammalian systems, involves carbon skeleton rearrangement. The addition of L-gulono-1,4-lactone, an AsA precursor in the mammalian pathway, significantly increased AsA level in C. elegans, whereas the addition of L-galactono-1,4-lactone, an AsA precursor in the plant and Euglena pathway, did not affect AsA level. The suppression of E03H4.3 (an ortholog of gluconolactonase) or the deficiency of F54D5.12 (an ortholog of L-gulono-1,4-lactone oxidase) significantly decreased AsA level in C. elegans. Although N2- and AsA-deficient F54D5.12 knockout mutant worm (tm6671) morphologies and the ratio of collagen to non-collagen protein did not show any significant differences, the mutant worms exhibited increased malondialdehyde levels and reduced lifespan compared with the N2 worms. In conclusion, our findings indicate that the AsA biosynthetic pathway is similar in C. elegans and mammals.

The effect of total sulfur amino acid levels on growth performance and bone metabolism in pullets under heat stress

A study was conducted to investigate the effects of total sulfur amino acid (TSAA) levels on performance and bone metabolism in pullets under heat stress (HS). Hy-Line W36 day-old pullets (n = 216) were randomly distributed in 3 dietary treatments (70, 85, and 100% of TSAA), with 6 replicates of 12 birds. The treatments were defined as percentages of the TSAA level recommendation (100, 85, and 70%), and 85 and 100% of TSAA were obtained by adding L-Methionine to the basal deficient diet (70% of TSAA). The birds were raised under HS (35°C/7 h/D) from 1 to 18 wk. At 6, 12, and 18 wk, growth performance was measured. At 12 and 18 wk, bone weight, ash, collagenous (ColP), and noncollagenous proteins (NColP), tissue volume (TV), bone mineral content (BMC), and mineral density from total, cortical, and trabecular bones were evaluated. The means were subjected to ANOVA and, when significant (P ≤ 0.05), were compared by Dunnett's test. Regression analyses were performed to evaluate trends of TSAA dose response. Overall, birds fed 70% of TSAA showed poor growth and feed efficiency compared with other groups. Additionally, in at least 1 phase, birds fed 70% of TSAA showed lower bone ash, NColP, total BMC, and TV and higher ColP than the other treatments, whereas the cortical and trabecular TV and BMC were lower than 100% of TSAA (P < 0.04). Quadratic effects of TSAA levels on body weight gain (BWG) were found, and the level for maximum BWG was 95% of the TSAA recommendation (P < 0.03, R2 > 0.83). In conclusion, the use of a TSAA-deficient diet resulted in poor performance and delayed bone development. Additionally, the use of 100% of TSAA led to better initial structural bone development than 85% of TSAA. Therefore, the TSAA level recommended by the primary breeder guideline was enough to support growth and bone quality under HS, suggesting that HS does not alter TSAA requirement in pullets.

Serum Osteocalcin Level is Negatively Associated with Vascular Reactivity Index by Digital Thermal Monitoring in Kidney Transplant Recipients.

Background and Objectives: Osteocalcin is the most abundant noncollagenous protein in bone matrix, which is considered a marker of bone formation. Previous studies indicate that circulating osteocalcin can be expressed by osteoblasts and even by osteoblast-like cells in vessel walls, and it is often associated with arterial stiffness. Our study aims to examine the potential association between osteocalcin levels and endothelial function among kidney transplant (KT) recipients. Materials and Methods: Fasting blood samples were obtained from 68 KT recipients. To measure the endothelial function and vascular reactivity index (VRI), a digital thermal monitoring test (VENDYS) was used. A commercial enzyme-linked immunosorbent assay kit was also utilized to measure serum total osteocalcin levels. In this study, a VRI of less than 1.0 indicated poor vascular reactivity; a VRI of 1.0–2.0 indicated intermediate vascular reactivity; and a VRI of 2.0 or higher indicated good vascular reactivity. Results: Our findings show that 8 KT recipients (11.8%) had poor vascular reactivity (VRI < 1.0), 26 (38.2%) had intermediate vascular reactivity (1.0 ≤ VRI < 2.0), and 34 (50%) had good vascular reactivity. Increased serum osteocalcin levels (p < 0.001) were found to be associated with poor vascular reactivity. Advanced age (r = −0.361, p = 0.002), serum alkaline phosphate level (r = −0.254, p = 0.037), and log-transformed osteocalcin levels (r = − 0.432, p < 0.001) were identified to be negatively correlated with VRI in KT recipients. Multivariable forward stepwise linear regression analysis revealed that the serum level of osteocalcin (β = −0.391, adjusted R2 change = 0.174; p < 0.001) and advanced age (β = −0.308, adjusted R2 change = 0.084; p = 0.005) were significantly and independently associated with VRI in KT recipients. Conclusions: Higher serum osteocalcin level was associated with lower VRI and poorer endothelial dysfunction among KT recipients.

Advanced Modeling Methods-Applications to Bone Fracture Mechanics.

The goal of this review is to summarize recent advances in modeling of bone fracture using fracture mechanics-based approaches at multiple length scales spanning nano- to macroscale. Despite the additional information that fracture mechanics-based models provide over strength-based ones, the application of this approach to assessing bone fracture is still somewhat limited. Macroscale fracture models of bone have demonstrated the potential of this approach in uncovering the contributions of geometry, material property variation, as well as loading mode and rate on whole bone fracture response. Cortical and cancellous microscale models of bone have advanced the understanding of individual contributions of microstructure, microarchitecture, local material properties, and material distribution on microscale fracture resistance of bone. Nano/submicroscale models have provided additional insight into the effect of specific changes in mineral, collagen, and non-collagenous proteins as well as their interaction on energy dissipation and fracture resistance at small length scales. Advanced modeling approaches based on fracture mechanics provide unique information about the underlying multiscale fracture mechanisms in bone and how these mechanisms are influenced by the structural and material constituents of bone at different length scales. Fracture mechanics-based modeling provides a powerful approach that complements experimental evaluations and advances the understanding of critical determinants of fracture risk.

Multiscale structural evolution of citrate-triggered intrafibrillar and interfibrillar mineralization in dense collagen gels

The mineralized extracellular matrix of bone is an organic–inorganic nanocomposite consisting primarily of carbonated hydroxyapatite, fibrous type I collagen, noncollagenous proteins, proteoglycans, and diverse biomolecules such as pyrophosphate and citrate. While much is now known about the mineralization-regulating role of pyrophosphate, less is known about the function of citrate. In order to assess the effect of negatively charged citrate on collagen mineralization, citrate-functionalized, bone osteoid-mimicking dense collagen gels were exposed to simulated body fluid for up to 7 days to examine the multiscale evolution of intra- and interfibrillar collagen mineralization. Here, we show by increases in methylene blue staining that the net negative charge of collagen can be substantially augmented through citrate functionalization. Structural and compositional analyses by transmission and scanning electron microscopy (including X-ray microanalysis and electron diffraction), and atomic force microscopy, all demonstrated that citrate-functionalized collagen fibrils underwent extensive intrafibrillar mineralization within 12 h in simulated body fluid. Time-resolved, high-resolution transmission electron microscopy confirmed the temporal evolution of intrafibrillar mineralization of single collagen fibrils. Longer exposure to simulated body fluid resulted in additional interfibrillar mineralization, all through an amorphous-to-crystalline transformation towards apatite (assessed by X-ray diffraction and attenuated total reflection-Fourier-transform infrared spectroscopy). Calcium deposition assays indicated a citrate concentration-dependent temporal increase in mineralization, and micro-computed tomography confirmed that >80 vol% of the collagen in the gels was mineralized by day 7. In conclusion, citrate effectively induces mesoscale intra- and interfibrillar collagen mineralization, a finding that advances our understanding of the role of citrate in mineralized tissues.

EFFECT OF POLYMER-INDUCED LIQUID PRECURSOR PROCESS CONTAINING POLYASPARTIC ACID ON INTRAFIBRILLAR DENTIN REMINERALIZATION (MICRO-COMPUTED TOMOGRAPHY ANALYSIS)

Objective: Caries treatment can be performed by minimal intervention, i.e., by removing the infected dentin and leaving the affected dentin and theninducing remineralization in the affected dentin. The affected dentin still consists of collagen cross bonds. Polymer-induced liquid precursor processis a guided tissue remineralization method that aims to remineralize intrafibrillar and extrafibrillar dentin by adding polymers that are similar tonon-collagen protein. One of the non-collagen protein analog materials is polyaspartic acid. The aim of this study was to evaluate the remineralizationof dentin on the demineralized dentin surface after immersed in remineralization solutions containing polyaspartic acid as a non-collagen proteinanalog.Methods: Human premolar teeth extracted for orthodontic purposes were divided into four groups, namely, one control and three treatment groups.Teeth in the control group were immersed only in the demineralization solution containing acetate buffer (pH 5.0, 66 h). Teeth in the three treatmentgroups were immersed in acetate buffer (pH 5.0, 66 h) and then continue to immersed in the remineralization solution containing polyaspartic acidfor 3, 7, and 14 days. Remineralization was evaluated by micro-CT.Results: Remineralization appeared on the demineralized dentin surface, characterized by an increase in the grayscale index, after immersion inthe remineralization solution containing polyaspartic acid. Significant differences were observed in the mean grayscale index values among the fourgroups.Conclusion: Polyaspartic acid has the potential to induce dentin remineralization.

CONCENTRATION DEPENDENT EFFECTS OF CARBOXYMETHYL CHITOSAN ON DENTIN REMINERALIZATION WITH AMORPHOUS CALCIUM PHOSPHATE

Objective: Carboxymethyl chitosan (CMC) is a non-collagenous protein analog which has a similar role as dentin matrix protein 1. CMC stabilizes amorphous calcium phosphate (ACP); hence forming nanocomplexes of CMC-ACP. The purpose of this study was to evaluate the effect of CMC concentration in CMC-ACP on dentin remineralization.Methods: Cavities were formed on the occlusal surfaces of freshly extracted premolar teeth. All samples were demineralized and immersed in phosphate-buffered saline and stored in a shaking incubator at 37°C. The teeth were randomly divided into five groups. Group 1 was control group (no treatment), whereas Groups 2, 3, 4, and 5 were treated with CMC-ACP containing 1%, 2.5%, 5%, and 10% CMC. The remineralized layer on the dentin surface was evaluated using scanning electron microscopy and energy-dispersive X-ray analysis.Results: The highest dentin remineralization capacity was achieved in Group 5 (10% CMC), whereas diminishing effects were observed in Group 4 (5% CMC), Group 3 (2.5% CMC), and Group 2 (1% CMC). Although no significant differences in calcium levels were observed between 2.5%, 5%, and 10% CMC groups, phosphate levels differed significantly in all treatment groups.Conclusion: Optimal dentin remineralization was achieved by the application of CMC–ACP containing 2.5% CMC.

THE EFFECTS OF MIXTURES OF VARIOUS CONCENTRATIONS OF CARBOXYMETHYL CHITOSAN/AMORPHOUS CALCIUM PHOSPHATE WITH GYPSUM ON DENTIN REMINERALIZATION

Objective: Carboxymethyl chitosan/amorphous calcium phosphate (CMC/ACP) is a non-collagen protein analog with the ability of dentinremineralization, and gypsum is a compound material than can facilitate application of CMC/ACP. CMC/ACP concentration is claimed to increasecalcium and phosphate concentration, which indicates dentin remineralization. The aim of this study is to observe the effects of different concentrationsof CMC/ACP mixed with gypsum on dentin remineralization.Methods: Occlusal cavities were created in 18 freshly extracted teeth, and the teeth were divided into three groups. The first group consisted ofuntreated demineralized dentin, and the second and third groups were treated with mixtures of 2.5% and 5% CMC/ACP and gypsum. All groupswere checked and analyzed on the 14th day using scanning electron microscopy/energy-dispersive X-ray spectroscopy and one-way ANOVA (p&lt;0.05).Results: Significant differences (p=0.001) of calcium and phosphate concentrations were found between Groups I, II, and III (p=0.001), whereas nodifferences were found between Groups II and III.Conclusion: Gypsum and CMC/ACP work synergistically to induce dentin remineralization. The highest dentin remineralization was shown by 5%CMC/ACP-gypsum mixture, but the level was not statistically different from that of 2.5% CMC/ACP-gypsum mixture.

Free of salt high-pressure deliming of animal hides

The wastewater pollution of tanneries is of high concern. The investigation of technologies to minimize the consumption of chemicals in the leather production process can reduce the environmental burden. We focus on the reduction of ammonium salts in the leather production process. Salt-free deliming of animal hides with compressed carbon dioxide as deliming agent is performed for the first time in a technical scale 20-L drum. As a result, CO2-deliming at 30 bar and 30 °C is two times faster than conventional deliming. In addition, the deliming efficiency is slightly improved. The initial calcium (Ca) content of the hides of 8 g/kg reaches the lowest value of 2 g/kg after a process time of 3 h. However, a process time of 60 min is sufficient to reach an elimination of 50 wt% of the initial lime. The resulting Ca-content of 4 g/kg after 60 min CO2-deliming at 30 bar is comparable with the Ca-content of conventional delimed hide. We clarify that the ampholytic character of the collagen itself enables a buffering of the pH-value at pH-7. The stable pH-value supports the selection of specific bating enzymes that decompose non-collagen proteins. No buffering salts contaminate the wastewater. The high-pressure CO2-deliming process has high potential to reduce wastewater emissions, save costs for chemicals, and process time in industrial beamhouse applications.

FAM20C-Mediated Phosphorylation of MEPE and Its Acidic Serine- and Aspartate-Rich Motif.

ABSTRACTMatrix extracellular phosphoglycoprotein (MEPE) is expressed in bone and teeth where it has multiple functions. The C‐terminus of MEPE contains a mineral‐binding, acidic serine‐ and aspartate‐rich motif (ASARM) that is also present in other noncollagenous proteins of mineralized tissues. MEPE‐derived ASARM peptides function in phosphate homeostasis and direct inhibition of bone mineralization in a phosphorylation‐dependent manner. MEPE is phosphorylated by family with sequence similarity 20, member C (FAM20C), which is the main kinase phosphorylating secreted phosphoprotein. Although the functional importance of protein phosphorylation status in mineralization processes has now been well‐established for secreted bone and tooth proteins (particularly for osteopontin), the phosphorylation pattern of MEPE has not been previously determined. Here we provide evidence for a very high phosphorylation level of this protein, reporting on the localization of 31 phosphoresidues in human MEPE after coexpression with FAM20C in HEK293T cells. This includes the finding that all serine residues located in the canonical target sequence of FAM20C (Ser‐x‐Glu) were phosphorylated, thus establishing the major target sites for this kinase. We also show that MEPE has numerous other phosphorylation sites, these not being positioned in the canonical phosphorylation sequence. Of note, and underscoring a possible important function in mineralization biology, all nine serine residues in the ASARM were phosphorylated, even though only two of these were positioned in the Ser‐x‐Glu sequence. The presence of many phosphorylated amino acids in MEPE, and particularly their high density in the ASARM motif, provides an important basis for the understanding of structural and functional interdependencies in mineralization and phosphate homeostasis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.