The respective and dependent effects of scattering and bone matrix absorption on ultrasound attenuation in cortical bone.

Cortical bone is characterized by a dense solid matrix permeated by fluid-filled pores. Ultrasound scattering has potential for the non-invasive evaluation of changes in bone porosity. However, there is an incomplete understanding of the impact of ultrasonic absorption in the solid matrix on ultrasound scattering. In this study, maps were derived from scanning acoustic microscopy images of human femur cross-sections. Finite-difference time domain ultrasound scatter simulations were conducted on these maps. Pore density, diameter distribution of the pores, and nominal absorption values in the solid and fluid matrices were controlled. Ultrasound pulses with a central frequency of 8.2 MHz were propagated, both in through-transmission and backscattering configurations. From these data, the scattering, bone matrix absorption, and attenuation extinction lengths were calculated. The results demonstrated that as absorption in the solid matrix was varied, the scattering, absorption, and attenuation extinction lengths were significantly impacted. It was shown that for lower values of absorption in the solid matrix (less than 2 dB mm-1), attenuation due to scattering dominates, whereas at higher values of absorption (more than 2 dB mm-1), attenuation due to absorption dominates. This will impact how ultrasound attenuation and scattering parameters can be used to extract quantitative information on bone microstructure.

Possible involvement of HtrA1 serine protease in the onset of osteoporotic bone extracellular matrix changes.

High-temperature requirement A1 (HtrA1), a multidomain serine protease acting on Extracellular matrix (ECM) rearrangement, is also secreted by osteoblasts and osteoclasts. Recent and conflicting literature highlights HtrA1's role as a controller of bone remodeling, proposing it as a possible target for pathologies with unbalanced bone resorption, like Osteoporosis (OP). To add knowledge on this molecule function in bone physiopathology, here we compared HtrA1 distribution in the ECM of healthy (H) and OP bone tissue, also examining its localization in the sites of new bone formation. HtrA1 was homogeneously expressed in the mature bone ECM of H tissue showing a 55.6 ± 16.4% of the stained area, with a significant (p=0.0001) decrease in OP percentage stained area (21.1 ± 13.1). Moreover, HtrA1 was present in the endosteum and cells involved in osteogenesis, mainly in those "entrapped" in woven bone, whereas osteocytes in mature lamellar bone were negative. Based on our previous observation in OP tissue of a significantly increased expression of Decorin and Osteocalcin, both involved in bone mineralization and remodeling and equally substrates for HtrA1, we speculate that HtrA1 by controlling the proper amount of Decorin and Osteocalcin favors normal bone maturation and mineralization. Besides, we suggest that late-osteoblasts and pre-osteocytes secrete HtrA1 in the adjacent matrix whilst proceeding with their maturation and that HtrA1 expression is further modified during the remodeling from woven to the lamellar bone. Overall, our data suggest HtrA1 as a positive regulator of bone matrix formation and maturation: its reduced expression in mature OP bone, affecting protein content and distribution, could hamper correct bone remodeling and mineralization.

Augmented drug resistance of osteosarcoma cells within decalcified bone matrix scaffold: The role of glutamine metabolism.

Due to the lack of a precise in vitro model that can mimic the nature microenvironment in osteosarcoma, the understanding of its resistance to chemical drugs remains limited. Here, we report a novel three-dimensional model of osteosarcoma constructed by seeding tumor cells (MG-63 and MNNG/HOS Cl no. 5) within demineralized bone matrix scaffolds. Demineralized bone matrix scaffolds retain the original components of the natural bone matrix (hydroxyapatite and collagen type I), and possess good biocompatibility allowing osteosarcoma cells to proliferate and aggregate into clusters within the pores. Growing within the scaffold conferred elevated resistance to doxorubicin on MG-63 and MNNG/HOS Cl no. 5 cell lines as compared to two-dimensional cultures. Transcriptomic analysis showed an increased enrichment for drug resistance genes along with enhanced glutamine metabolism in osteosarcoma cells in demineralized bone matrix scaffolds. Inhibition of glutamine metabolism resulted in a decrease in drug resistance of osteosarcoma, which could be restored by α-ketoglutarate supplementation. Overall, our study suggests that microenvironmental cues in demineralized bone matrix scaffolds can enhance osteosarcoma drug responses and that targeting glutamine metabolism may be a strategy for treating osteosarcoma drug resistance.

Subcutaneously Engineered Decalcified Bone Matrix Xenografts Promote Bone Repair by Regulating the Immune Microenvironment, Prevascularization, and Stem Cell Homing.

Immunoregulatory and vascularized microenvironments play an important role in bone regeneration; however, the precise regulation for vascularization and inflammatory reactions remains elusive during bone repair. In this study, by means of subcutaneous preimplantation, we successfully constructed demineralized bone matrix (DBM) grafts with immunoregulatory and vascularized microenvironments. According to the current results, at the early time points (days 1 and 3), subcutaneously implanted DBM grafts recruited a large number of pro-inflammatory M1 macrophages with positive expression of CD68 and iNOS, while at the later time points (days 7 and 14), these inflammatory cells gradually subsided, accompanying increased presence of anti-inflammatory M2 macrophages with positive expression of CD206 and Arg-1, indicating a gradually enhanced anti-inflammatory microenvironment. At the same time, the gradually increased angiogenesis was observed in the DBM grafts with implantation time. In addition, the positive cells of CD105, CD73, and CD90 were observed in the inner region of the DBM grafts, implying the homing of mesenchymal stem cells. The repair results of cranial bone defects in a rat model further confirmed that the subcutaneous DBM xenografts at 7 days significantly improved bone regeneration. In summary, we developed a simple and novel strategy for bone regeneration mediated by anti-inflammatory microenvironment, prevascularization, and endogenous stem cell homing.

Annexin A5 derived from matrix vesicles protects against osteoporotic bone loss via mineralization.

Matrix vesicles (MVs) have shown strong effects in diseases such as vascular ectopic calcification and pathological calcified osteoarthritis and in wound repair of the skeletal system due to their membranous vesicle characteristics and abundant calcium and phosphorus content. However, the role of MVs in the progression of osteoporosis is poorly understood. Here, we report that annexin A5, an important component of the matrix vesicle membrane, plays a vital role in bone matrix homeostasis in the deterioration of osteoporosis. We first identified annexin A5 from adherent MVs but not dissociative MVs of osteoblasts and found that it could be sharply decreased in the bone matrix during the occurrence of osteoporosis based on ovariectomized mice. We then confirmed its potential in mediating the mineralization of the precursor osteoblast lineage via its initial binding with collagen type I to achieve MV adhesion and the subsequent activation of cellular autophagy. Finally, we proved its protective role in resisting bone loss by applying it to osteoporotic mice. Taken together, these data revealed the importance of annexin A5, originating from adherent MVs of osteoblasts, in bone matrix remodeling of osteoporosis and provided a new strategy for the treatment and intervention of bone loss.

Centrosome clustering control in osteoclasts through CCR5-mediated signaling.

Osteoclasts uniquely resorb calcified bone matrices. To exert their function, mature osteoclasts maintain the cellular polarity and directional vesicle trafficking to and from the resorbing bone surface. However, the regulatory mechanisms and pathophysiological relevance of these processes remain largely unexplored. Bone histomorphometric analyses in Ccr5-deficient mice showed abnormalities in the morphology and functional phenotype of their osteoclasts, compared to wild type mice. We observed disorganized clustering of nuclei, as well as centrosomes that organize the microtubule network, which was concomitant with impaired cathepsin K secretion in cultured Ccr5-deficient osteoclasts. Intriguingly, forced expression of constitutively active Rho or Rac restored these cytoskeletal phenotypes with recovery of cathepsin K secretion. Furthermore, a gene-disease enrichment analysis identified that PLEKHM1, a responsible gene for osteopetrosis, which regulates lysosomal trafficking in osteoclasts, was regulated by CCR5. These experimental results highlighted that CCR5-mediated signaling served as an intracellular organizer for centrosome clustering in osteoclasts, which was involved in the pathophysiology of bone metabolism.

Bone Material Properties in Bone Diseases Affecting Children.

PURPOSE OF REVIEW: Metabolic and genetic bone disorders affect not only bone mass but often also the bone material, including degree of mineralization, matrix organization, and lacunar porosity. The quality of juvenile bone is moreover highly influenced by skeletal growth. This review aims to provide a compact summary of the present knowledge on the complex interplay between bone modeling and remodeling during skeletal growth and to alert the reader to the complexity of bone tissue characteristics in children with bone disorders. RECENT FINDINGS: We describe cellular events together with the characteristics of the different tissues and organic matrix organization (cartilage, woven and lamellar bone) occurring during linear growth. Subsequently, we present typical alterations thereof in disorders leading to over-mineralized bone matrix compared to those associated with low or normal mineral content based on bone biopsy studies. Growth spurts or growth retardation might amplify or mask disease-related alterations in bone material, which makes the interpretation of bone tissue findings in children complex and challenging.

Enhancing the effectiveness of Alkaline Phosphatase and bone matrix proteins by tunable metal-organic composite for accelerated mineralization.

The irregular expression of bone matrix proteins occurring during the mineralization of bone regeneration results in various deformities which poses a major concern of orthopedic reconstruction. The limitations of the existing reconstruction practice paved a way for the development of a metal-organic composite [TQ-Sr-Fe] with Metal ions strontium [Sr] and iron [Fe] and a biomolecule Thymoquinone [TQ] in an attempt to enhance the bone mineralization due to their positive significance in osteoblast differentiation, proliferation and maturation. TQ-Sr-Fe was synthesized by in-situ coprecipitation and subjected to various characterization to determine their nature, compatibility and osteogenic efficiency. The crystallographic and electron microscopy analysis reveals sheet like structure of the composite. The negative cytotoxicity of TQ-Sr-Fe in the MG 63 cell line signified their biocompatibility. Cell adhesion and proliferation rate affirmed osteoconductive and osteoinductive nature of the composites and it was further supported by the gene expression of osteoblastic differentiation. The sequential expression of bone matrix proteins such as OCN, SPARC, COL 1, and Alkaline Phosphatase elevate the calcium deposition of MG-63 osteoblast like cells and initiates mineralization compared to control. Thus, the metal-organic composite TQ-Sr-Fe would make a suitable composite for accelerating mineralization process which would leads to faster bone regeneration.

Age-related decline in bone mineral transport and bone matrix proteins in osteoblasts from stromal stem cells.

We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3 or 4 mo) and old (18 or 19 mo) mice, age correlated with reduced mRNA of mineral transport proteins: alkaline phosphatase (ALP), ankylosis (ANK), the Cl-/H+ exchanger ClC3, and matrix proteins collagen 1 (Col1) and osteocalcin (BGLAP). Some proteins, including the neutral phosphate transporter2 (NPT2), were not reduced. These are predominately osteoblast proteins, but in mixed cell populations. Remarkably, in osteoblasts differentiated from preparations of stromal stem cells (SSCs) made from bone marrow cells in young and old mice, differentiated in vitro on perforated polyethylene terephthalate membranes, mRNA confirmed decreased expression with age for most transport-related and bone matrix proteins. Additional mRNAs in osteoblasts in vitro included ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), unchanged, and ENPP2, reduced with age. Decrease with age in ALP activity and protein by Western blot was also significant. Transport protein findings correlated with micro-computed tomography of lumbar vertebra, showing that trabecular bone of old mice is osteopenic relative to young mice, consistent with other studies. Pathway analysis of osteoblasts differentiated in vitro showed that cells from old animals had reduced Erk1/2 phosphorylation and decreased suppressor of mothers against decapentaplegic 2 (Smad2) mRNA, consistent with TGFß pathway, and reduced ß-catenin mRNA, consistent with WNT pathway regulation. Our results show that decline in bone density with age reflects selective changes, resulting effectively in a phenotype modification. Reduction of matrix and mineral transport protein expression with age is regulated by multiple signaling pathways.NEW & NOTEWORTHY This work for the first time showed that specific enzymes in bone mineral transport, and matrix synthesis proteins, in the epithelial-like bone-forming cell layer are downregulated with aging. Results were compared using cells extracted from long bones of young and old mice, or in essentially uniform osteoblasts differentiated from stromal stem cells in vitro. The age effect showed memory in the stromal stem cells, a remarkable finding.

Germ-Free C57BL/6 Mice Have Increased Bone Mass and Altered Matrix Properties but Not Decreased Bone Fracture Resistance.

The gut microbiome impacts bone mass, which implies a disruption to bone homeostasis. However, it is not yet clear how the gut microbiome affects the regulation of bone mass and bone quality. We hypothesized that germ-free (GF) mice have increased bone mass and decreased bone toughness compared with conventionally housed mice. We tested this hypothesis using adult (20- to 21-week-old) C57BL/6J GF and conventionally raised female and male mice (n = 6-10/group). Trabecular microarchitecture and cortical geometry were measured from micro-CT of the femur distal metaphysis and cortical midshaft. Whole-femur strength and estimated material properties were measured using three-point bending and notched fracture toughness. Bone matrix properties were measured for the cortical femur by quantitative back-scattered electron imaging and nanoindentation, and, for the humerus, by Raman spectroscopy and fluorescent advanced glycation end product (fAGE) assay. Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue strength and decreased whole-bone strength that was not explained by differences in bone size, increased tissue mineralization and fAGEs, and altered collagen structure that did not decrease fracture toughness. We observed several sex differences in GF mice, most notably for bone tissue metabolism. Male GF mice had a greater signature of amino acid metabolism, and female GF mice had a greater signature of lipid metabolism, exceeding the metabolic sex differences of the conventional mice. Together, these data demonstrate that the GF state in C57BL/6J mice alters bone mass and matrix properties but does not decrease bone fracture resistance. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Mimicking bone matrix through coaxial electrospinning of core-shell nanofibrous scaffold for improving neurogenesis bone regeneration.

There is a significant clinical demand for bone repair materials with high efficacy. This study was designed to fabricate nanofibrous scaffolds to promote bone defect regeneration using magnesium doped mesoporous bioactive glass (MBG), a fusion protein Osteocalcin-Osteopontin-Biglycan (OOB), silk fibroin (SF) and nerve growth factor (NGF) for facilitating accelerated bone formation. We found that MBG adsorbed with OOB (OOB@MBG) as core, and SF adsorbed with NGF (SF@NGF) as shell to fabricate the nanofibrous scaffolds (OOB@MBG/NGF@SF) through coaxial electrospinning. OOB@MBG/NGF@SF scaffolds could effectively mimic the component and structure of bone matrix. Interestingly, we observed that OOB@MBG/NGF@SF scaffolds could substantially promote bone mesenchymal stem cells (BMSCs) osteogenesis through stimulating Erk1/2 activated Runx2 and mTOR pathway, and it could also activate the expression level of various osteogenic marker genes. Intriguingly, OOB@MBG/NGF@SF scaffolds could also enhance BMSCs induced neural differentiation cells differentiated into neuron, and activate the expression of the different neuron specific marker genes. Moreover, it was found that OOB@MBG/NGF@SF scaffolds accelerated bone regeneration with neurogenesis, and new neurons were formed in Haversian canal in vivo. Consistent with these observations, we found that Erk1/2 and mTOR signaling pathways also regulated osteogenesis with the neurogenesis process from RNA sequencing result. Overall, our findings provided novel evidence suggesting that OOB@MBG/NGF@SF scaffolds could function as a potential biomaterial in accelerating bone defect regeneration with neurogenesis, as well as in recovering the motor ability and improving the quality of life of patients.

Osteocyte Remodeling of the Lacunar-Canalicular System: What's in a Name?

PURPOSE OF REVIEW: Osteocytes directly modify the bone surrounding the expansive lacunar-canalicular system (LCS) through both resorption and deposition. The existence of this phenomenon is now widely accepted, but is referred to as "osteocyte osteolysis," "LCS remodeling," and "perilacunar remodeling," among other names. The uncertainty in naming this physiological process reflects the many persistent questions about why and how osteocytes interact with local bone matrix. The goal of this review is to examine the purpose and nature of LCS remodeling and its impacts on multiscale bone quality. RECENT FINDINGS: While LCS remodeling is clearly important for systemic calcium mobilization, this process may have additional potential drivers and may impact the ability of bone to resist fracture. There is abundant evidence that the osteocyte can resorb and replace bone mineral and does so outside of extreme challenges to mineral homeostasis. The impacts of the osteocyte on organic matrix are less certain, especially regarding whether osteocytes produce osteoid. Though multiple lines of evidence point towards osteocyte production of organic matrix, definitive work is needed. Recent high-resolution imaging studies demonstrate that LCS remodeling influences local material properties. The role of LCS remodeling in the maintenance and deterioration of bone matrix quality in aging and disease are active areas of research. In this review, we highlight current progress in understanding why and how the osteocyte removes and replaces bone tissue and the consequences of these activities to bone quality. We posit that answering these questions is essential for evaluating whether, how, when, and why LCS remodeling may be manipulated for therapeutic benefit in managing bone fragility.

Bone matrix quality in a developing high-fat diet mouse model is altered by RAGE deletion.

Overweightness and obesity in adolescents are epidemics linked to chronic low-grade inflammation and elevated fracture risk. The increased fracture risk observed in overweight/obese adolescence contrasts the traditional concept that high body mass is protective against fracture, and thus highlights the need to determine why weight gain becomes detrimental to fracture during growth and maturity. The Receptor for Advanced Glycation End products (RAGE) is a central inflammatory regulator that can influence bone metabolism. It remains unknown how RAGE removal impacts skeletal fragility in overweightness/obesity, and whether increased fracture risk in adolescents could result from low-grade inflammation deteriorating bone quality. We characterized the multiscale structural, mechanical, and chemical properties of tibiae extracted from adolescent C57BL/6J (WT) and RAGE null (KO) mice fed either low-fat (LF) or high-fat (HF) diet for 12 weeks starting at 6 weeks of age using micro-computed tomography, strength, Raman spectroscopy, and nanoindentation. Overweight/obese WT HF mice possessed degraded mineral-crystal quality and increased matrix glycoxidation in the form of pentosidine and carboxymethyl-lysine, with HF diet in females only showing reduced cortical surface expansion and TMD independently of RAGE ablation. Furthermore, in contrast to males, HF diet in females led to more material damage and plastic deformation. RAGE KO mitigated glycoxidative matrix accumulation, preserved mineral quantity, and led to increased E/H ratio in females. Taken together, these results highlight the complex, multi-scale and sex-dependent relationships between bone quality and function under overweightness, and identifies RAGE-controlled glycoxidation as a target to potentially preserve matrix quality and mechanical integrity.

New Bone Formation in the Whole Decellularized Cortical Bone Scaffold Using the Model of Revitalizing a Haversian System.

ABSTRACT: Decellularized allogeneic bone chips act as scaffolds for bone tissue regeneration. Owing to their lack of osteogenic potentials compared to autologous bone graft, decellularized bone scaffolds (DBSs) have applied only to small partial bone defects in clinical settings. Furthermore, only decellularized cancellous bone chips have been limitedly used for the purpose of bone regeneration. The cortical bone has less porosity and less osteogenic materials such as bone morphogenetic proteins in comparison with cancellous bone. In this study, we tried to accelerate new bone formation within the decellularized cortical bone scaffold using a vascular pedicle as an in vivo bioreactor.Forty DBSs were divided into 4 groups with different conditionings (DBS+ demineralized bone matrix [DBM], DBS+DBM+me+mesenchymal stem cells, DBS+DBM+vascular pedicle, and DBS+DBM+vascular pedicle+mesenchymal stem cells) and implanted into the back of 5 rabbits. Half of the DBSs were examined at 8 weeks and the other half at 16 weeks to determine vascularization level and osteogenesis within each group. New bone formation and bone-forming cells related to osteogenesis were observed via histological staining. Inclusion of the vascular pedicle resulted in larger areas of bone regeneration. With time, osteon structures became more prominent in groups containing the vascular pedicle.In summary, vascularized DBSs combined with a vascular pedicle have shown promising results for bone regeneration, thereby representing potential therapeutic alternatives for autologous bone grafts or bone tissue free transfer in large or segmental bone defects. In addition, demineralized whole cortical bone matrix along with vascular pedicle and various bone inductive materials, such as DBM and recombinant human bone morphogenetic protein-2, may be an additional new option of an ideal osteoinductive system.

Demineralized bone matrix combined with cytotoxic T-lymphocyte-associated protein 4 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells and suppresses the activation of T lymphocytes in vitro.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) can promote osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs), and CTLA4-modified bone marrow mesenchymal stem cells possess immunoregulatory effects. In the present study, we aimed to construct a new tissue engineering bone using demineralized bone matrix and CTLA4 protein, designated as DBM-CTLA4 (+). The effects of DBM-CTLA4 (+) on the osteogenic differentiation of hBMMSCs and T lymphocyte activation were evaluated through in vitro experiments. The cumulative release of CTLA4 from DBM-CTLA4 (+) was determined using enzyme-linked immunosorbent assay. DBM-CTLA4 (+) was co-cultured in a Transwell chamber with either phytohemagglutinin-treated hBMMSCs or human peripheral blood mononuclear cells (hPBMCs). Osteogenic differentiation of hBMMSCs was assessed by calcium deposition, ALP activity, and the protein levels of COL1A1, RUNX2, BMP2, and OPN. T lymphocyte activity was assessed by measuring the protein levels of IL-2, L-17, HLA-DRA1, IFN-γ, and RANKL. Our results showed that the cumulative release rates of CTLA4 at 7, 14, 21, and 28 days were 12.6% ± 1.4%, 30.2% ± 2.3%, 49.8% ± 3.8%, and 60.5% ± 2.7%, respectively. Compared to the negative control, DBM-CTLA4 (+) promoted the proliferation of hBMMSCs, and enhanced calcium deposition, ALP activity, and protein levels of COL1A1, RUNX2, BMP2, and OPN. Moreover, DBM-CTLA4 (+) decreased the levels of IL-2, IL-17, HLA-DR, IFN-γ, and RANKL in hPBMCs treated with phytohemagglutinin. In conclusion, DBM-CTLA4 (+) promoted proliferation and osteogenic differentiation of hBMMSCs and suppressed T lymphocyte activation.

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.

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

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

Plasma Membrane Receptors Involved in the Binding and Response of Osteoclasts to Noncellular Components of the Bone.

Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various signals, some of which are received directly from noncellular elements of the bone. In vitro, adherence to the bone triggers the reduction of cell-cell fusion events between osteoclasts and the activation of osteoclasts to form unusual dynamic cytoskeletal and membrane structures that are required for degrading the bone. Integrins on the surface of osteoclasts are known to receive regulatory signals from the bone matrix. Regulation of the availability of these signals is accomplished by enzymatic alterations of the bone matrix by protease activity and phosphorylation/dephosphorylation events. Other membrane receptors are present in osteoclasts and may interact with as yet unidentified signals in the bone. Bone mineral has been shown to have regulatory effects on osteoclasts, and osteoclast activity is also directly modulated by mechanical stress. As understanding of how osteoclasts and other bone cells interact with the bone has emerged, increasingly sophisticated efforts have been made to create bone biomimetics that reproduce both the structural properties of the bone and the bone's ability to regulate osteoclasts and other bone cells. A more complete understanding of the interactions between osteoclasts and the bone may lead to new strategies for the treatment of bone diseases and the production of bone biomimetics to repair defects.

Low concentrations of caffeic acid phenethyl ester stimulate osteogenesis in vitro.

OBJECTIVE: The aim of this study was to evaluate the effects of caffeic acid phenethyl ester (CAPE) on osteoblast-like cell cultures (SAOS-2). METHODS: SAOS-2 were exposed to CAPE at 1 nM, 10 nM, 100 nM, 1 µM, and 10 µM. Non-exposed cultures were used as control. The following parameters were assayed: 1) cell viability at 1, 3, and 7 days; 2) alkaline phosphatase (ALP) activity at 5 and 10 days; 3) matrix mineralization at 14 days; and 4) Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (SPP1), and osteocalcin (BGLAP) gene expression at 5 and 10 days. The data were analyzed by ANOVA two-way or Kruskal-Wallis (α = 5%). RESULTS: At day 1, cell viability was similar among all groups (p > 0.05). At days 3 and 7, cultures exposed to CAPE at 10 µM exhibited a significant reduction in cell viability compared with the others groups (p < 0.05). At day 5, ALP activity was similar among all experimental groups; at day 10, however, the stain intensity was higher in cultures exposed to CAPE at 100 nM and 10 nM in comparison with the other groups (p < 0.05). At days 5 and 10, RUNX2, ALP, SPP1, and BGLAP gene expression was greater in cultures exposed to CAPE in comparison with the control (p < 0.05). At day 14, matrix mineralization was similar in cultures exposed to CAPE at 1 nM and 10 nM (p > 0.05), but superior to those ones observed in the other experimental groups (p < 0.05). CONCLUSION: CAPE at low concentrations can positively module the osteogenesis in vitro.

Poor bone matrix quality: What can be done about it?

PURPOSE OF THE REVIEW: Bone's ability to withstand load resisting fracture and adapting to it highly depends on the quality of its matrix and its regulators. This review focuses on the contribution of bone quality to fracture resistance and possible therapeutic targets for skeletal fragility in aging and disease. RECENT FINDINGS: The highly organized, hierarchical composite structure of bone extracellular matrix together with its (re)modeling mechanisms and microdamage dynamics determines its stiffness, strength, and toughness. Aging and disease affect the biological processes regulating bone quality, thus resulting in defective extracellular matrix and bone fragility. Targeted therapies are being developed to restore bone's mechanical integrity. However, their current limitations include low tissue selectivity and adverse side effects. Biological and mechanical insights into the mechanisms controlling bone quality, together with advances in drug delivery and studies in animal models, will accelerate the development and translation to clinical application of effective targeted-therapeutics for bone fragility.