Non-collagenous Proteins Research Articles

Molecular differences in collagen organization and in organic-inorganic interfacial structure of bones with and without osteocytes

Bone is a fascinating biomaterial composed mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, and water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone. In this report, we unfold some of the molecular characteristics differentiating between two load-bearing (cleithrum) bones, one from sturgeon fish, where the matrix contains osteocytes and one from pike fish where the bone tissue is devoid of these bone cells. Using enhanced solid-state NMR measurements, we underpin disparities in the collagen fibril structure and dynamics, the mineral phases, the citrate content at the organic-inorganic interface and water penetrability in the two bones. These findings suggest that different strategies are undertaken in the erection of the mineral-organic interfaces in various bones characterized by dissimilar osteogenesis or remodeling pathways and may have implications for the mechanical properties of the particular bone. Statement of significanceBone boasts unique interactions between collagen fibers and mineral phases through interfaces holding together this bio-composite structure. Over evolution, fish have gone from mineralizing their bones aided by certain bone cells called osteocytes, like tetrapod, to mineralization without these cells. Here, we report atomic level differences in collagen fiber cross linking and organization, porosity of the mineral phases and content of citrate molecules at the bio-mineral interface in bones from modern versus ancient fish. The dissimilar structural features may suggest disparate mechanical properties for the two bones. Fundamental level understanding of the organic and inorganic components in bone and the interfacial interactions holding them together is essential for successful bone repair and for treating better tissue pathologies.

Insights into the Differential Preservation of Bone Proteomes in Inhumed and Entombed Cadavers from Italian Forensic Caseworks.

Bone is a hard biological tissue and a precious reservoir of information in forensic investigations as it retains key biomolecules commonly used for identification purposes. Bone proteins have recently attracted significant interest for their potential in estimating post-mortem interval (PMI) and age at death (AAD). However, the preservation of such proteins is highly dependent on intrinsic and extrinsic factors that can hinder the potential application of molecular techniques to forensic sciences. The present study aims at investigating the effects that two commonly used types of burial practices (entombment and inhumation) have on bone protein survival. The sample consists of 14 exhumed individuals from cemeteries in Southern Italy with different AADs (29–85 years) and PMIs (1–37 years). LC-MS/MS analyses show that 16 proteins are better preserved under the entombed conditions and 4 proteins are better preserved under the inhumed conditions, whereas no clear differences are detected for post-translational protein modifications. Furthermore, several potential “stable” protein markers (i.e., proteins not affected by the burial environment) are identified for PMI and AAD estimation. Overall, these results show that the two burial environments play a role in the differential preservation of noncollagenous proteins, confirming the potential of LC-MS/MS-based proteomics in forensic sciences.

A multiscale finite element investigation on the role of intra- and extra-fibrillar mineralisation on the elastic properties of bone tissue

Lamellar bone is one of the fundamental structural units of bone tissue and it consists of mineralised collagen fibrils (MCFs) embedded within an extra-fibrillar matrix comprised of hydroxyapatite minerals distributed throughout a matrix of non-collagenous proteins (NCPs). While both intra- and extra-fibrillar phases provide a critical contribution to tissue-level behaviour, the mechanical implications of their structural arrangement, and in particular the relative distribution of HA minerals between both phases, remains poorly understood. This study presents a multiscale finite element framework to investigate the role of intra- and extra-fibrillar mineralisation on the elastic properties of bone tissue by considering two levels of structural hierarchy. At the nanoscale, representative volume elements (RVEs) of both MCFs and the extra-fibrillar matrix were developed, and a homogenisation strategy was used to determine the effective elastic properties of each phase. At the sub-micron level, an RVE of lamellar bone that accounted for newly reported patterns of mineral platelets encircling collagen fibrils was used to predict the effective response of lamellar bone tissue, with material properties established from the previous length scale. The results demonstrated that the overall mineral content in the tissue is the biggest contributor to the effective elastic properties of lamellar bone. While this is perhaps unsurprising, importantly, it was demonstrated that the extra-fibrillar matrix (and mineral therein) is the phase that makes the primary contribution to the elastic response of the tissue. The two main reasons that the extra-fibrillar matrix dominated the load-bearing response are (i) the greater proportion of mineral content compared to the intra-fibrillar regions and (ii) the highly ordered arrangement of mineral platelets that are aligned to the longitudinal axis of MCFs. Both of these features resulted in extra-fibrillar mineral strain ratios that were consistently higher than intra-fibrillar mineral strain ratios under axial loading. As a result, the predicted elastic properties of MCFs were much lower than the extra-fibrillar matrix, indicating that intra-fibrillar mineralisation only provided a modest contribution to the stiffness of bone tissue. Collectively, the predicted results of the multiscale approach compared well to the range properties measured through various experimental testing methods, highlighting its potential to provide further insight into the role of sub-tissue features of tissue biomechanics.

Bioinspired Control of Calcium Phosphate Liesegang Patterns Using Anionic Polyelectrolytes.

The Liesegang phenomenon is a spontaneous pattern formation, which is a periodic distribution of the precipitate discovered in diffusion-limited systems. Over the past century, it has been experimentally attempted to control the periodicity of patterns and structures of precipitates by varying the concentration of the hydrogel or electrolytes, adding organic or inorganic impurities, and applying an electric or pH field. In this work, the periodic patterns of calcium phosphate were manipulated with an anionic macromolecular additive inspired by bone mineralization in which various noncollagenous proteins are involved in the formation of a polymer-induced liquid precursor. The periodic patterns were systematically controlled by adjusting the amount of poly(acrylic acid), and they were numerically simulated by adjusting the threshold concentration of nucleation. The change of the pattern is explained by improved stability and directional diffusion of the intermediate.

The role of the osteocalcin-osteopontin protein complex in bone biomechanics: a key contributor to energy dissipation at mineral-mineral interfaces

Bone is a naturally occurring composite mainly made of HydroxyApatite (HAp mineral phase and an organic matrix composed of collagenous and Non-Collagenous Proteins (NCPs). It has become apparent that NCPs role in intrinsic toughening, one of the primary contributors towards healthy tissue’s excellent fracture resistance, is considerable. Osteocalcin (OC) and Osteopontin (OPN) are the most abundant NCPs and, together, these are known to form protein complexes that mediate bonding at nanoscale interfaces in the extrafibrillar space.

The profiling and analysis of gene expression in human periodontal ligament tissue and fibroblasts.

ObjectivesThe periodontal ligament (PDL) is an important component of periodontium to support dental structure in the alveolar socket. Regeneration of PDL tissue is an effective treatment option for periodontal disease and the profiling of genes involved in this process will be informative. Therefore, our study aims to accurately delineate the profiling of gene expression for PDL tissue regeneration.Materials and MethodsWe isolated PDL tissues and PDL fibroblasts (PDLFs) from premolar teeth, which were extracted from healthy periodontal status patients undergoing orthodontic treatment. Messenger RNA (mRNA) expression in PDL tissue and PDLFs were analyzed using Cap analysis gene expression, which is a second‐generation sequencing technique to create profiling. We also determined the protein expression using Western blot.ResultsCollagens (type I, III, and VI), noncollagenous proteins (periostin and osteonectin), and proteoglycans (asporin, lumican, decorin, and osteomodulin) were highly expressed in PDL tissue. Integrin, β1 was also expressed in PDL tissue. On comparison of gene expression between PDL tissue and PDLFs, four PDL marker genes, osteopontin, asporin, periostin, and osteonectin, were decreased in PDLFs. The genes for gene regulation were also highly expressed.ConclusionsOur study demonstrated the overall profiling of mRNA expression in PDL tissue and analyzed the important genes which may be useful for providing specific information for the reconstruction of PDL. We also identified the difference in gene expression between PDL tissue and PDLFs which might provide insights towards PDL regeneration.

The potential role of bone-derived factor ucOCN in the anti-depressive effects of exercise

<p id="p00005">Depression is a major problem in the modern society. The pathogenesis of depression is a research hotspot in the field of neuroscience. The proved mechanisms of depression include monoamine neurotransmitter hypothesis, gene-environment interaction, neurogenesis, neuroplasticity, immune activation and suppression, etc. With the deepening of research, the roles of "brain-gut interaction", "muscle-brain crosstalk", and adipocytokines in the sports antidepressant field has been discovered. As an endocrine organ, the role of bone tissue in anti-depressive exercise remains to be revealed. Based on this, this research innovatively explores the biological effects and mechanisms of uncarboxylated osteocalcin (ucOCN) in exercise anti-depression from the perspective of “bone-brain crosstalk”, and further reveals the relationship between bone endocrine activity and the molecular regulation of sports antidepressant. ucOCN is a specific non-collagen protein secreted by osteoblasts (OB). After entering the blood, it acts on the hippocampus, cingulate gyrus and other brain tissues, and triggers a cascade reaction through targeting cell membrane receptors to regulate neurodevelopment, neuroplasticity and bone endocrine-nerve response system. Exercise promotes the secretion of ucOCN and has a significant antidepressant effect, but there are few studies focusing on the molecular mechanism. Based on integrated biological theory and bone endocrine function, through analysis of current research, we found several mechanisms by which ucOCN mediating exercise antidepressant. Firstly, ucOCN regulates neurotransmitters expression: exercise induces high expression of ucOCN which inhibits γ-amino groups butyric acid (GABA) expression to improve depression-like behavior. Secondly, ucOCN regulates neuroendocrine secretion: ucOCN activates the HPA axis to promote adrenocorticotropic hormone (ACTH), mineralocorticoid, cortisol and other secretions to improve depression-like behavior. In addition, ucOCN also activates G protein-coupled receptors 158 (Gpr158)/brain-derived neurotrophic factor (BDNF) pathway to promote neurotransmitter secretion and further regulate depression occurring. Thirdly, ucOCN regulates neuroimmunity: ucOCN activates the ERK and STAT pathways and down-regulates the expression of IL-6 and IL-8 in the hippocampus, and then through malondialdehyde (MDA)/super up-regulation of superoxide dismutase (SOD)/nuclear factor erythroid-derived 2-like 2(Nrf2)/heme oxygenase 1(HO1) pathway to increase expression of VGF and BDNF, which further improve depression-like behaviors. Taken together, these results provide a solid theoretical basis for the mechanism of ucOCN in sports anti-depression. We also provides new research directions and ideas for sports anti-depression, and provide a new perspective for enriching the biological mechanism network of sports brain.

Alterações das propriedades da gelatina da pele do bagre tra (Pangasianodon hypophthalmus) pela substituição alcalina por enzima no processo pré-tratado

ABSTRACT: The effect of pretreated method to remove the non-collagenous protein by using alkaline and enzyme Alcalase, as well as the temperature and time for extracting on the properties of gelatin from tra catfish skin were investigated. Yields of gelatin extracted at 70 °C for 1h from pretreated skin by enzyme method (16.2%) was significantly higher than that of the sample by alkaline method (12.14%). However, the gel strength of gelatin from skin treated via enzyme Alcalase was lower than gelatin sample pretreated by alkaline while the turbidity values was higher than gelatin from skin pretreated via alkaline. From SDS-PAGE profile, gelatin from skin pretreated by alkaline consisted of two different α- chains in protein pattern while enzymatic gelatin had low molecular weight peptides. The FT-IR spectra showed the lower wavenumber in amide I and III of enzymatic gelatin in compare to alkaline gelatin by the loss of triple helical structure during enzyme treatment. From the results, the using enzyme for pretreated material has potential to replace the alkaline method for gelatin production with purpose to reduce chemical waste caused serious ecological issues.

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Editorial introductions

Evaluation of the effect of remineralization with strontium-doped nanohydroxyapatite with noncollagenous protein analog: Chitosan on the shear bond strength of resin composite to dentin - An in vitro study.

The resin-dentin interface is less durable, which reduces the longevity of tooth-colored restorations. To encounter this shortcoming, the use of nanotechnology to mimic biomineralization proves beneficial. This study was conducted to evaluate the effect of remineralization with strontium-doped nanohydroxyapatite (Sr-nHAp) with chitosan on shear bond strength of resin composite to dentin. Sixty five extracted human premolars were divided into five groups (n = 13) based on remineralization protocol as: Group A - 20% (Sr-nHAp) with chitosan, Group B - 10% (Sr-nHAp) with chitosan, Group C - 20% (Sr-nHAp) with simulated body fluid, Group D - 10% (Sr-nHAp) with simulated body fluid, and Group E - control. Following bonding, resin composite of specified dimension was built and was subjected to shear bond strength test after 24 h and 1 week using Universal Testing Machine, and mode of failure was assessed. ANOVA and paired sample t-test were used for analyzing the data, and the level of significance was set at 5%. The highest value of shear bond strength was obtained from Group A after aging for a week, and there is a significant increase in the value of all the groups as compared to the control group after 1 week of storage than 24 h. Remineralization with Sr-nHAp and chitosan has positively improved the bond strength of resin to dentin at the end of 1 week.

Intrinsically disordered peptides enhance regenerative capacities of bone composite xenografts

Biomaterial scientists design organic bone substitutes based on the biochemical properties of the mimicked tissue to achieve near native functionality. Several non-collagenous proteins in bone are known as intrinsically disordered proteins (IDPs), as they lack detectible ordered domains and a fixed 3D structure under physiological conditions. Many IDPs perform regulatory roles in a range of cellular functions, which motivated us to design two proline-rich disordered peptides (P2 and P6) and augmented them into the SmartBone® (SBN) biohybrid substitute. Recently we reported an improved proliferation and osteogensis of human osteoblasts and mesenchymal stem cells in the composite groups containing peptides (named here as SBN + P2 and SBN + P6) in vitro. To address the effects of these composites on bone formation and biomineralization, this in vivo study investigated their functions in critical size craniotomy defects in 16 domestic pigs after 8 and 16 weeks of healing. For this purpose, we used cone beam computed tomography (CBCT), microCT (µCT), histology, immunohistochemistry, fluorescent labeling of abundant reactive entities (FLARE), synchrotron SAXS/XRD, optical photothermal IR (O-PTIR) microscopy and nanoscale atomic force microscopy-infrared (AFM-IR) analyses. Our results represent new synthetic IDPs as potential candidates for directing bone formation and biomineralization. The SBN + P6 stimulated significantly higher bone formation and biomineralization after 8 weeks of healing compared to other groups indicating its potential in stimulating early biomineralization. After 16 weeks of healing, the SBN + P2 induced significantly higher bone formation and biomineralization compared to other groups indicating its effects on later bone formation and biomineralization processes. The vigorous stretching of amide primary and secondary IR absorbance peaks at 1660 and 1546 cm−1 in the SBN + P2 group verified that this peptide experienced more conformational changes after 16 weeks of implantation with a higher phosphate intensity at 1037 cm−1 compared to peptide 6. Overall, P2 and P6 are promising candidates for bone augmentation strategies in critical clinical applications. We concluded that FLARE and O-PTIR are promising tools in evaluating and diagnosing the biochemical structure of bone tissue and the bone-biomaterial interface.

The Regulation of Collagen Processing by miRNAs in Disease and Possible Implications for Bone Turnover.

This article describes several recent examples of miRNA governing the regulation of the gene expression involved in bone matrix construction. We present the impact of miRNA on the subsequent steps in the formation of collagen type I. Collagen type I is a main factor of mechanical bone stiffness because it constitutes 90–95% of the organic components of the bone. Therefore, the precise epigenetic regulation of collagen formation may have a significant influence on bone structure. We also describe miRNA involvement in the expression of genes, the protein products of which participate in collagen maturation in various tissues and cancer cells. We show how non-collagenous proteins in the extracellular matrix are epigenetically regulated by miRNA in bone and other tissues. We also delineate collagen mineralisation in bones by factors that depend on miRNA molecules. This review reveals the tissue variability of miRNA regulation at different levels of collagen maturation and mineralisation. The functionality of collagen mRNA regulation by miRNA, as proven in other tissues, has not yet been shown in osteoblasts. Several collagen-regulating miRNAs are co-expressed with collagen in bone. We suggest that collagen mRNA regulation by miRNA could also be potentially important in bone metabolism.

The Role of the Non-Collagenous Extracellular Matrix in Tendon and Ligament Mechanical Behavior: A Review.

Tendon is a connective tissue that transmits loads from muscle to bone, while ligament is a similar tissue that stabilizes joint articulation by connecting bone to bone. The 70-90% of tendon and ligament's extracellular matrix (ECM) is composed of a hierarchical collagen structure that provides resistance to deformation primarily in the fiber direction, and the remaining fraction consists of a variety of non-collagenous proteins, proteoglycans, and glycosaminoglycans (GAGs) whose mechanical roles are not well characterized. ECM constituents such as elastin, the proteoglycans decorin, biglycan, lumican, fibromodulin, lubricin, and aggrecan and their associated GAGs, and cartilage oligomeric matrix protein (COMP) have been suggested to contribute to tendon and ligament's characteristic quasi-static and viscoelastic mechanical behavior in tension, shear, and compression. The purpose of this review is to summarize existing literature regarding the contribution of the non-collagenous ECM to tendon and ligament mechanics, and to highlight key gaps in knowledge that future studies may address. Using insights from theoretical mechanics and biology, we discuss the role of the non-collagenous ECM in quasi-static and viscoelastic tensile, compressive, and shear behavior in the fiber direction and orthogonal to the fiber direction. We also address the efficacy of tools that are commonly used to assess these relationships, including enzymatic degradation, mouse knockout models, and computational models. Further work in this field will foster a better understanding of tendon and ligament damage and healing as well as inform strategies for tissue repair and regeneration.

Energy dissipation of osteopontin at a HAp mineral interface: Implications for bone biomechanics

Osteopontin (OPN) is a one of the most abundant non-collagenous proteins in the bone's organic matrix. OPN is responsible for mediating bonding at mineral interfaces in the extrafibrillar space and recent evidence shows that it is a major contributor to bone's fracture resistance. While several experimental studies have identified an important role for calcium ions in mediating energy dissipation in OPN protein networks, the underlying molecular mechanisms remain largely unknown. In the current study, the role of calcium ions on energy dissipation at OPN interface with hydroxyapatite (HAp) as the main bone mineral was investigated. For the first time, the three-dimensional structure of OPN proteins were predicted, and it was found that calcium ions greatly influenced the final protein configuration and energy dissipation performance. Under small deformation, the compact cOPN structure, resulting from calcium ions presence, facilitated greater energy dissipation through sacrificial bond breaking and mechanisms mediated by the surface-bound calcium. At larger deformation, the compact structure also enabled cOPN to dissipate higher energy. Moreover, it was found that phosphorylation of OPN played an important role in energy dissipation. While previous studies have shown that OPN dissipated energy by forming aggregate networks, this study also showed that network formation is not necessary and that individual OPN proteins can dissipate large amounts of energy at HAp interfaces.

Nanoscale Imaging and Analysis of Bone Pathologies

Understanding the properties of bone is of both fundamental and clinical relevance. The basis of bone’s quality and mechanical resilience lies in its nanoscale building blocks (i.e., mineral, collagen, non-collagenous proteins, and water) and their complex interactions across length scales. Although the structure–mechanical property relationship in healthy bone tissue is relatively well characterized, not much is known about the molecular-level origin of impaired mechanics and higher fracture risks in skeletal disorders such as osteoporosis or Paget’s disease. Alterations in the ultrastructure, chemistry, and nano-/micromechanics of bone tissue in such a diverse group of diseased states have only been briefly explored. Recent research is uncovering the effects of several non-collagenous bone matrix proteins, whose deficiencies or mutations are, to some extent, implicated in bone diseases, on bone matrix quality and mechanics. Herein, we review existing studies on ultrastructural imaging—with a focus on electron microscopy—and chemical, mechanical analysis of pathological bone tissues. The nanometric details offered by these reports, from studying knockout mice models to characterizing exact disease phenotypes, can provide key insights into various bone pathologies and facilitate the development of new treatments.

Extraction Of Collagen From Fish Orientai Sole Brachirus orientalis Skin And Studying Of Some Chemical Charactristics And Amino Acid Composition

This study includes the extraction of acid soluble collagen (Acetic acid 0.5 M) from fish Oriental sole skin . After the removal of fat and non-collagenous proteins and dialyzed against distilled water, some the chemical properties were studied, that includes chemical composition and the percentage of yield were 12.15 % on the basis of wet weight. According to the electrophoretic patterns, the collagen consisted of two different chains (α1 and α 2), were characterized to be type I, β chain components as well as the observed was larger molecular weight , When estimating amino acid collagen observed that there is variation in the percentage of amino acid major such as glycine , proline , hydroxyproline and hydroxy lysine, When measuring the absorption spectrum of ultraviolet (UV) there was a single peak absorption at 232 nm for collagen fish skin, FTIR showed regions of amides A, B, I, II and III were 3408.57, 2934.89, 1651.73, 1540.85 and 1243.38 cm-1 respectively.

Bone matrix quality in paired iliac bone biopsies from postmenopausal women treated for 12months with strontium ranelate or alendronate.

Bone quality is altered mainly by osteoporosis, which is treated with modulators of bone quality. Knowledge of their mechanisms of action is crucial to understand their effects on bone quality. The goal of our study was to compare the action of alendronate (ALN) and strontium ranelate (SrRan) on the determinants of bone quality. The investigation was performed on over 60 paired human iliac biopsies. Paired samples correspond to biopsies obtained from the same patient, one before treatment (baseline) and one after 12months of treatment, in postmenopausal women with osteoporosis. Vibrational spectroscopy (Raman and FTIRM) and nanoindentation were used to evaluate the effect of both drugs on bone quality at the ultrastructural level. Outcomes measured by vibrational spectroscopy and nanoindentation are sensitive to bone age. New bone packets are distinguished from old bone packets. Thus, the effect of bone age is distinguished from the treatment effect. Both drugs modify the mineral and organic composition in new and old bone in different fashions after 12months of administration. The new bone formed during ALN administration is characterized by an increased mineral content, carbonation and apatite crystal size/perfection compared to baseline. Post-translational modifications of collagen are observed through an increase in the hydroxyproline/proline ratio in new bone. The proteoglycan content is also increased in new bone. SrRan directly modulates bone quality through its physicochemical actions, independent of an effect on bone remodeling. Strontium cations are captured by the hydrated layer of the mineral matrix. The mineral matrix formed during SrRan administration has a lower carbonate content and crystallinity after 12months than at baseline. Strontium might create bonds (crosslinks) with collagen and noncollagenous proteins in new and old bone. The nanomechanical properties of bone were not modified with either ALN or SrRan, probably due to the short duration of administration. Our results show that ALN and SrRan have differential effects on bone quality in relation to their mechanism of action.

Acellular Porcine Cornea Produced by Supercritical Carbon Dioxide Extraction: A Potential Substitute for Human Corneal Regeneration.

The aim of this study was to develop a non-cytotoxic, biocompatible innovative acellular porcine cornea (APC) for corneal wound healing and corneal blindness treatment. APC was produced by using supercritical carbon dioxide (SCCO2) to decellularize the porcine cornea. Decellularization of the porcine cornea was examined by hematoxylin and eosin staining and 4,6-diamidino-2-phenylindole, dihydrochloride staining. The residual DNA content of APC was analyzed in comparison with the native porcine cornea. Virus inactivation up to at least 6 log10 was confirmed for the stepwise process of APC for 4 different model viruses. In addition, a series of in vitro and in vivo tests in accordance with ISO-10993 biocompatibility assay and animal performance tests were performed. APC produced by the SCCO2 process revealed complete decellularization, without any residual non-collagenous proteins. The scanning electron microscopy structural features of the decellularized cornea were similar to those of human. APC was found to be nontoxic and exhibited excellent biocompatibility in both in vitro and in vivo studies. The animal performance test proved that APC exerted excellent adaptability on the cornea and no sign of irritation and good compatibility in lamellar corneal transplantation. APC manufactured by SCCO2 technology revealed complete cells and non-collagenous protein removal compared with the Triton-sodium dodecyl sulfate decellularization process. APC showed excellent biocompatibility in rabbit lamellar corneal transplantation with a follow-up to 1 year. APC can be a potential substitute for human-donated cornea for corneal transplantation in the near future.

Three-dimensional cell-laden collagen scaffolds: From biochemistry to bone bioengineering.

The bones can be viewed as both an organ and a material. As an organ, the bones give structure to the body, facilitate skeletal movement, and provide protection to internal organs. As a material, the bones consist of a hybrid organic/inorganic three-dimensional (3D) matrix, composed mainly of collagen, noncollagenous proteins, and a calcium phosphate mineral phase, which is formed and regulated by the orchestrated action of a complex array of cells including chondrocytes, osteoblasts, osteocytes, and osteoclasts. The interactions between cells, proteins, and minerals are essential for the bone functions under physiological loading conditions, trauma, and fractures. The organization of the bone's organic and inorganic phases stands out for its mechanical and biological properties and has inspired materials research. The objective of this review is to fill the gaps between the physical and biological characteristics that must be achieved to fabricate scaffolds for bone tissue engineering with enhanced performance. We describe the organization of bone tissue highlighting the characteristics that have inspired the development of 3D cell-laden collagenous scaffolds aimed at replicating the mechanical and biological properties of bone after implantation. The role of noncollagenous macromolecules in the organization of the collagenous matrix and mineralization ability of entrapped cells has also been reviewed. Understanding the modulation of cell activity by the extracellular matrix will ultimately help to improve the biological performance of 3D cell-laden collagenous scaffolds used for bone regeneration and repair as well as for in vitro studies aimed at unravelling physiological and pathological processes occurring in the bone.

Aesculetin Accelerates Osteoblast Differentiation and Matrix-Vesicle-Mediated Mineralization.

The imbalance between bone resorption and bone formation in favor of resorption results in bone loss and deterioration of bone architecture. Osteoblast differentiation is a sequential event accompanying biogenesis of matrix vesicles and mineralization of collagen matrix with hydroxyapatite crystals. Considerable efforts have been made in developing naturally-occurring plant compounds, preventing bone pathologies, or enhancing bone regeneration. Coumarin aesculetin inhibits osteoporosis through hampering the ruffled border formation of mature osteoclasts. However, little is known regarding the effects of aesculetin on the impairment of matrix vesicle biogenesis. MC3T3-E1 cells were cultured in differentiation media with 1–10 μM aesculetin for up to 21 days. Aesculetin boosted the bone morphogenetic protein-2 expression, and alkaline phosphatase activation of differentiating MC3T3-E1 cells. The presence of aesculetin strengthened the expression of collagen type 1 and osteoprotegerin and transcription of Runt-related transcription factor 2 in differentiating osteoblasts for 9 days. When ≥1–5 μM aesculetin was added to differentiating cells for 15–18 days, the induction of non-collagenous proteins of bone sialoprotein II, osteopontin, osteocalcin, and osteonectin was markedly enhanced, facilitating the formation of hydroxyapatite crystals and mineralized collagen matrix. The induction of annexin V and PHOSPHO 1 was further augmented in ≥5 μM aesculetin-treated differentiating osteoblasts for 21 days. In addition, the levels of tissue-nonspecific alkaline phosphatase and collagen type 1 were further enhanced within the extracellular space and on matrix vesicles of mature osteoblasts treated with aesculetin, indicating matrix vesicle-mediated bone mineralization. Finally, aesculetin markedly accelerated the production of thrombospondin-1 and tenascin C in mature osteoblasts, leading to their adhesion to preformed collagen matrix. Therefore, aesculetin enhanced osteoblast differentiation, and matrix vesicle biogenesis and mineralization. These findings suggest that aesculetin may be a potential osteo-inductive agent preventing bone pathologies or enhancing bone regeneration.