Functions and Metabolism of Amino Acids in Bones and Joints of Cats and Dogs.

The bone is a large and complex organ (12-15% of body weight) consisting of specialized connective tissues (bone matrix and bone marrow), whereas joints are composed of cartilage, tendons, ligaments, synovial joint capsules and membranes, and a synovial joint cavity filled with synovial fluid. Maintaining healthy bones and joints is a dynamic and complex process, as bone deposition (formation of new bone materials) and resorption (breakdown of the bone matrix to release calcium and phosphorus) are the continuous processes to determine bone balance. Bones are required for locomotion, protection of internal organs, and have endocrine functions to maintain mineral homeostasis. Joints are responsible for resisting mechanical stress/trauma, aiding in locomotion, and supporting the overall musculoskeletal system. Amino acids have multiple regulatory, compositional, metabolic, and functional roles in maintaining the health of bones and joints. Their disorders are prevalent in mammals and significantly reduce the quality of life. These abnormalities in companion animals, specifically cats and dogs, commonly lead to elective euthanasia due to the poor quality of life. Multiple disorders of bones and joints result from genetic predisposition and are heritable, but other factors such as nutrition, growth rate, trauma, and physical activity affect how the disorder manifests. Treatments for cats and dogs are primarily to slow the progression of these disorders and assist in pain management. Therapeutic supplements such as Cosequin and formulated diets rich in amino acids are used commonly as treatments for companion animals to reduce pain and slow the progression of those diseases. Also, amino acids (e.g., taurine, arginine, glycine, proline, and 4-hydroxyproline), and glucosamine reduce inflammation and pain in animals with bone and joint disorders. Gaining insight into how amino acids function in maintaining bone and joint health can aid in developing preventative diets and therapeutic supplementations of amino acids to improve the quality of life in companion animals.

Preparation of black phosphorus@sodium alginate microspheres with bone matrix vesicle structure via electrospraying for bone regeneration.

Bone matrix vesicles are commonly acknowledged as the primary site of biomineralization in human skeletal tissue. Black phosphorus has exhibited favorable properties across various chemical and physical domains. In this investigation, a novel composite microsphere was synthesized through the amalgamation of sodium alginate (ALG) with black phosphorus nanosheets (BP) utilizing the electrospray (ES) technique. These microspheres were tailored to mimic the regulatory function of matrix vesicles (MV) upon exposure to a biomimetic mineralization fluid (SBF) during the biomineralization process. Results revealed that black phosphorus nanosheets facilitated the generation of hydroxyapatite (HA) on the microsphere surface. Live-dead assays and cell proliferation experiments showcased a cell survival rate exceeding 85 %. Moreover, wound healing assessments unveiled that M-ALG-BP microspheres exhibited superior migration capacity, with a migration rate surpassing 50 %. Furthermore, after 7 days of osteogenic induction, M-ALG-BP microspheres notably stimulated osteoblast differentiation. Particularly noteworthy, M-ALG-BP microspheres significantly enhanced osteogenic differentiation of osteoblasts and induced collagen production in vitro. Additionally, experiments involving microsphere implantation into mouse skeletal muscle demonstrated the potential for ectopic mineralization by ALG-BP microspheres. This investigation underscores the outstanding mineralization properties of ALG-BP microspheres and their promising clinical prospects in bone tissue engineering.

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.

Genistein and coumestrol reduce MCF-7 breast cancer cell viability and inhibit markers of preferential metastasis, bone matrix attachment and tumor-induced osteoclastogenesis.

The propensity of breast cancer to preferentially metastasize to the skeleton is well known. Once established in bone metastatic breast cancers have a poor prognosis due to their ability to promote extensive bone loss which augments tumor burden. Unfortunately, current anti-resorptive therapies for skeletal metastasis are typically prescribed after secondary tumors have formed and are palliative in nature. One group of compounds with the potential to reduce both tumor burden and osteolysis are phytoestrogens (PE), but the mechanisms mediating a beneficial effect are unclear. Therefore, the current study examined the effect of genistein and coumestrol alone or in combination on breast cancer cell number, expression of mediators of preferential skeletal metastasis, bone matrix attachment and tumor-induced osteoclast formation. Results showed that genistein and coumestrol significantly reduced viable cell number in an estrogen receptor dependent manner (p < 0.05), whereas combinations of PE had no effect. In addition, genistein and coumestrol significantly reduced expression of genes driving epithelial to mesenchymal transition (snail), bone attachment (CXCR4 and integrin αV) and osteolysis (PTHrP and TNF-α). In keeping with this genistein and coumestrol significantly suppressed attachment of breast cancer cells to bone matrix and inhibited tumor and RANKL-induced osteoclast formation. Our data suggests that phytoestrogens not only decrease breast cancer cell viability but also antagonize essential tumor bone interactions that establish and drive the progression of skeletal metastasis.

Improved osteogenesis in rat femur segmental defects treated with human allograft and zinc adjuvants.

Bone allograft is widely used to treat large bone defects or complex fractures. However, processing methods can significantly compromise allograft osteogenic activity. Adjuvants that can restore the osteogenic activity of processed allograft should improve clinical outcomes. In this study, zinc was tested as an adjuvant to increase the osteogenic activity of human allograft in a Rag2 null rat femoral defect model. Femoral defects were treated with human demineralized bone matrix (DBM) mixed with carboxy methyl cellulose containing ZnCl2 (0, 75, 150, 300 µg) or Zn stearate (347 µg). Rat femur defects treated with DBM-ZnCl2 (75 µg) and DBM-Zn stearate (347 µg) showed increased calcified tissue in the defect site compared to DBM alone. Radiograph scoring and µCT (microcomputed tomography) analysis showed an increased amount of bone formation at the defects treated with DBM-Zn stearate. Use of zinc as an adjuvant was also tested using human cancellous bone chips. The bone chips were soaked in ZnCl2 solutions before being added to defect sites. Zn adsorbed onto the chips in a time- and concentration-dependent manner. Rat femur defects treated with Zn-bound bone chips had more new bone in the defects based on µCT and histomorphometric analyses. The results indicate that zinc supplementation of human bone allograft improves allograft osteogenic activity in the rat femur defect model.

Low doses of uranium and osteoclastic bone resorption: key reciprocal effects evidenced using new in vitro biomimetic models of bone matrix.

Uranium is widely spread in the environment due to its natural and anthropogenic occurrences, hence the importance of understanding its impact on human health. The skeleton is the main site of long-term accumulation of this actinide. However, interactions of this metal with biological processes involving the mineralized extracellular matrix and bone cells are still poorly understood. To get a better insight into these interactions, we developed new biomimetic bone matrices containing low doses of natural uranium (up to 0.85 µg of uranium per cm2). These models were characterized by spectroscopic and microscopic approaches before being used as a support for the culture and differentiation of pre-osteoclastic cells. In doing so, we demonstrate that uranium can exert opposite effects on osteoclast resorption depending on its concentration in the bone microenvironment. Our results also provide evidence for the first time that resorption contributes to the remobilization of bone matrix-bound uranium. In agreement with this, we identified, by HRTEM, uranium phosphate internalized in vesicles of resorbing osteoclasts. Thanks to the biomimetic matrices we developed, this study highlights the complex mutual effects between osteoclasts and uranium. This demonstrates the relevance of these 3D models to further study the cellular mechanisms at play in response to uranium storage in bone tissue, and thus better understand the impact of environmental exposure to uranium on human bone health.

Biglycan and chondroitin sulfate play pivotal roles in bone toughness via retaining bound water in bone mineral matrix.

Recent in vitro evidence shows that glycosaminoglycans (GAGs) and proteoglycans (PGs) in bone matrix may functionally be involved in the tissue-level toughness of bone. In this study, we showed the effect of biglycan (Bgn), a small leucine-rich proteoglycan enriched in extracellular matrix of bone and the associated GAG subtype, chondroitin sulfate (CS), on the toughness of bone in vivo, using wild-type (WT) and Bgn deficient mice. The amount of total GAGs and CS in the mineralized compartment of Bgn KO mouse bone matrix decreased significantly, associated with the reduction of the toughness of bone, in comparison with those of WT mice. However, such differences between WT and Bgn KO mice diminished once the bound water was removed from bone matrix. In addition, CS was identified as the major subtype in bone matrix. We then supplemented CS to both WT and Bgn KO mice to test whether supplemental GAGs could improve the tissue-level toughness of bone. After intradermal administration of CS, the toughness of WT bone was greatly improved, with the GAGs and bound water amount in the bone matrix increased, while such improvement was not observed in Bgn KO mice or with supplementation of dermatan sulfate (DS). Moreover, CS supplemented WT mice exhibited higher bone mineral density and reduced osteoclastogenesis. Interestingly, Bgn KO bone did not show such differences irrespective of the intradermal administration of CS. In summary, the results of this study suggest that Bgn and CS in bone matrix play a pivotal role in imparting the toughness to bone most likely via retaining bound water in bone matrix. Moreover, supplementation of CS improves the toughness of bone in mouse models.

Linarin and its aglycone acacetin abrogate actin ring formation and focal contact to bone matrix of bone-resorbing osteoclasts through inhibition of αvß3 integrin and core-linked CD44.

BACKGROUND: Since enhanced bone resorption due to osteoclast differentiation and activation cause skeletal diseases, there is a growing need in therapeutics for combating bone-resorbing osteoclasts. Botanical antioxidants are being increasingly investigated for their health-promoting effects on bone. Edible Cirsium setidens contains various polyphenols of linarin, pectolinarin, and apigenin with antioxidant and hepatoprotective effects. PURPOSE: This study aimed to determine whether linarin present in Cirsium setidens water extracts (CSE) and its aglycone acacetin inhibited osteoclastogenesis of RANKL-exposed RAW 264.7 murine macrophages for 5 days. METHODS: This study assessed the osteoprotective effects of CSE, linarin and acacetin on RANKL-induced differentiation and activation of osteoclasts by using MTT assay, TRAP staining, Western blot analysis, bone resorption assay actin ring staining, adhesion assay and immunocytochemical assay. This study explored the underlying mechanisms of their osteoprotection, and identified major components present in CSE by HPLC analysis. RESULTS: Linarin and pectolinarin were identified as major components of CSE. Nontoxic linarin and acacetin as well as CSE, but not pectolinarin attenuated the RANKL-induced macrophage differentiation into multinucleated osteoclasts, and curtailed osteoclastic bone resorption through reducing lacunar acidification and bone matrix degradation in the osteoclast-bone interface. Linarin and acacetin in CSE reduced the transmigration and focal contact of osteoclasts to bone matrix-mimicking RGD peptide. Such reduction was accomplished by inhibiting the induction of integrins, integrin-associated proteins of paxillin and gelsolin, cdc42 and CD44 involved in the formation of actin rings. The inhibition of integrin-mediated actin ring formation by linarin and acacetin entailed the disruption of TRAF6-c-Src-PI3K signaling of bone-resorbing osteoclasts. The functional inhibition of c-Src was involved in the loss of F-actin-enriched podosome core protein cortactin-mediated actin assembly due to linarin and acacetin. CONCLUSION: These observations demonstrate that CSE, linarin and acacetin were effective in retarding osteoclast function of focal adhesion to bone matrix and active bone resorption via inhibition of diffuse cloud-associated αvß3 integrin and core-linked CD44.

Outcomes of Radiotherapy for Osseous Echinococcosis of Meriones meridianus.

BACKGROUND: Osseous cyst echinococcosis (CE) is an infectious disease that causes disability and deformity in patients, yet there is still no satisfactory treatment. Focusing on the feasibility and prognosis of radiotherapy as an adjuvant or palliative treatment for osseous CE, this study investigated the outcome of Meriones meridianus with osseous CE after radiotherapy. METHODS: The study utilized a comparison control group design with three groups of gerbils, and 240 osseous CE gerbils were randomly divided into control, 40Gy/5times, and 50Gy/5times groups. Different doses of radiotherapy were given to the gerbils, and then, the effects of radiotherapy on gerbils and lesions were observed at 3 and 6 months after radiotherapy. Statistical analysis was done using χ 2 test, unpaired t-test, and one-way ANOVA. RESULTS: Significant changes (P < 0.05) were achieved between the three groups in terms of seven parameters at 3 and 6 months, including the number of dead gerbils and lesion sites with ulceration and infection, number of dead scolices, protein content, Ca2+ concentration, the maximum diameter of lesion site, and wet weight of cysts. Except for the number of dead gerbils and lesion sites with ulceration and infection, all other parameters were observed a big difference between 3 months and 6 months in the 50Gy/5times group. CONCLUSION: Radiotherapy at a dose of 50 Gy has inhibitory and therapeutic effects on osseous CE in gerbils, and radiotherapy could probably be a treatment option for persistent or recurrent osseous CE.

Combined therapy of adipose-derived stem cells and photobiomodulation on accelerated bone healing of a critical size defect in an osteoporotic rat model.

We investigated the impact of human demineralized bone matrix (hDBM) plus adipose-derived stem cells (hADS) plus photobiomodulation (PBM) on a critical-sized femoral defect (CSFD) in ovariectomy induced osteoporosis in rats. There were 6 groups as follows. In group 1 (control, C), only CSFDs were created. Groups 2-6 were implanted with DBM into the CSFD (DBM-CSFD). In group 2 (S), only DBM was transplanted into the CSFD. In group 3 (S + PBM), the DBM-CSFDs were treated with PBM. In group 4, the DBM-CSFDs were treated with alendronate (S + ALN). In group 5, ADSs were seeded into DBM-CSFD (S + ADS). In group 6, ADSs were seeded into DBM-CSFD and the CSFDs were treated with PBM (S + PBM + ADS). At week eight (catabolic phase of bone repair), the S + ALN, S + PBM + ADS, S + PBM, and S + ADS groups all had significantly increased bone strength than the S group (ANOVA, p = 0.000). The S + PBM, S + PBM + ADS, and S + ADS groups had significantly increased Hounsfield unit than the S group (ANOVA, p = 0.000). ALN, ADS, and PBM significantly increased healed bone strength in an experimental model of DBM-treated CSFD in the catabolic phase of bone healing in osteoporotic rats. However, ALN alone and PBM plus ADS were superior to the other protocols.

Preconditioning adipose-derived stem cells with photobiomodulation significantly increased bone healing in a critical size femoral defect in rats.

We assessed the combined impacts of human demineralized bone matrix (hDBM) scaffold, adipose-derived stem cells (hADS), and photobiomodulation (PBM) on bone repair of a critical size femoral defect (CSFD) in 72 rats. The rats were divided into six groups: control (group 1); ADS (group 2 - ADS transplanted into hDBM); PBM (group 3 - PBM-treated CSFDs); ADS + PBM in vivo (group 4 - ADS transplanted into hDBM and the CSFDs were treated with PBM in vivo); ADS + PBM in vitro (group 5 - ADS were treated with PBM in vitro, then seeded into hDBM); and ADS + PBM in vitro+in vivo (group 6 - PBM-treated ADS were seeded into hDBM, and the CSFDs were treated with PBM in vivo. At the anabolic phase (2 weeks after surgery), bone strength parameters of the groups 5, 6, and 4 were statistically greater than the control, ADS, and PBM in vivo groups (all, p = 0.000). Computed tomography (CT) scans during the catabolic phase (6 weeks after surgery) of bone healing revealed that the Hounsfield unit (HU) of CSFD in the groups 2 (p = 0.000) and 5 (p = 0.019) groups were statistically greater than the control group. The groups 5, 4, and 6 had significantly increased bone strength parameters compared with the PBM in vivo, control, and ADS groups (all, p = 0.000). The group 5 was statistically better than the groups 4, and 6 (both, p = 0.000). In vitro preconditioned of hADS with PBM significantly increased bone repair in a rat model of CSFD in vivo.

Alterations of bone material properties in adult patients with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) caused by PHEX mutations results in elevated serum FGF23 levels, renal phosphate wasting and low 1,25-dihydroxyvitamin D. The glycophosphoprotein osteopontin, a potent inhibitor of mineralization normally degraded by PHEX, accumulates within the bone matrix. Conventional therapy consisting of supplementation with phosphate and vitamin D analogs is burdensome and the effects on bone material poorly characterized. We analyzed transiliac bone biopsies from four adult patients, two of them severely affected due to no diagnosis and no treatment until adulthood. We used light microscopy, qBEI and FTIRI to study histology, histomorphometry, bone mineralization density distribution, properties of the organic matrix and size of hypomineralized periosteocytic lesions. Non-treatment resulted in severe osteomalacia, twice the amount of mineralized trabecular volume, multiple osteon-like perforations, continuity of lamellae from mineralized to unmineralized areas and distinctive patches of woven bone. Periosteocytic lesions were larger than in treated patients. The latter had nearly normal osteoid thicknesses, although surface was still elevated. The median calcium content of the matrix was always within normal range, although the percentage of lowly mineralized bone areas was highly increased in non-treated patients, resulting in a marked heterogeneity in mineralization. Divalent collagen cross-links were evident independently of the mineral content of the matrix. Broad osteoid seams lacked measurable pyridinoline, a mature trivalent cross-link and exhibited considerable acidic lipid content, typically found in matrix vesicles. Based on our results, we propose a model that possibly integrates the relationship between the observed mineralization disturbances, FGF23 secretion and the known osteopontin accumulation in XLH.

The effect of hyperbaric oxygen therapy on bone macroscopy, composition and biomechanical properties after ionizing radiation injury.

BACKGROUND: Radiotherapy used in tumor treatment compromises vascularization of bone tissue. Hyperbaric oxygenation (HBO) increases oxygen availability and improves vascularization, minimizing the deleterious effects of ionizing radiation (IR). Therefore, the aim of this study was to evaluate HBO therapy effect on bone macroscopy, composition and biomechanical properties after IR damage. METHODS: Twenty male Wistar rats weighing 300 ± 20 g (10 weeks of age) were submitted to IR (30 Gy) to the left leg, where the right leg was not irradiated. After 30 days, ten animals were submitted to HBO therapy, which was performed daily for 1 week at 250 kPa for 90-min sessions. All animals were euthanized 37 days after irradiation and the tibia were separated into four groups (n = 10): from animals without HBO - right tibia Non-irradiated (noIRnoHBO) and left tibia Irradiated (IRnoHBO); and from animals with HBO - right tibiae Non-irradiated (noIRHBO) and left tibia Irradiated (IRHBO). The length (proximal-distal) and thickness (anteroposterior and mediolateral) of the tibiae were measured. Biomechanical analysis evaluated flexural strength and stiffness. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to calculate the amide I ratio, crystallinity index, and matrix to mineral ratios. RESULTS: In the macroscopic and ATR-FTIR analysis, the IRnoHBO showed lower values of length, thickness and amide I ratio, crystallinity index and matrix to mineral ratios compared to noIRnoHBO (p < 0.03). IRnoHBO showed no statistical difference compared to IRHBO for these analyses (p > 0.05). Biomechanics analysis showed that the IRnoHBO group had lower values of flexural strength and stiffness compared to noIRnoHBO and IRHBO groups (p < 0.04). In addition, the noIRHBO group showed higher value of flexural strength when compared to noIRnoHBO and IRHBO groups (p < 0.02). CONCLUSIONS: The present study concluded that IR arrests bone development, decreases the collagen maturation and mineral deposition process, thus reducing the flexural strength and stiffness bone mechanical parameters. Moreover, HBO therapy minimizes deleterious effects of irradiation on flexural strength and the bone stiffness analysis.

Demineralized bone matrix paste formulated with biomimetic PLGA microcarriers for the vancomycin hydrochloride controlled delivery: Release profile, citotoxicity and efficacy against S. aureus.

Infection and resulting bone defects caused by Staphylococcus aureus is one of the major issues in orthopaedic surgeries. Vancomycin hydrochloride (VaH) is largely used to manage these events. Here, a human derived bone paste supplemented with biopolymer microcarriers for VaH sustained delivery to merge osteoinductive and antimicrobial actions is described. In detail, different emulsion formulations were tested to fabricate micro-carriers of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) by a proprietary technology (named Supercritical Emulsion Extraction). These carriers (mean size 827 ± 68 µm; loading 47 mgVaH/gPLGA) were assembled with human demineralized bone matrix (DBM) to obtain an antimicrobial bone paste system (250 mg/0.5 cm3 w/v, carrier/DBM). Release profiles in PBS indicated a daily drug average release of about 4 µg/mL over two weeks. This concentration was close to the minimum inhibitory concentration and able to effectively inhibit the S. aureus growth in our experimental sets. Carriers cytotoxicity tests showed absence of adverse effects on cell viability at the concentrations used for paste assembly. This approach points toward the potential of the DBM-carrier-antibiotic system in hampering the bacterial growth with accurately controlled antibiotic release and opens perspectives on functional bone paste with PLGA carriers for the controlled release of bioactive molecules.

Photobiomodulation Therapy Associated with Heterologous Fibrin Biopolymer and Bovine Bone Matrix Helps to Reconstruct Long Bones.

Bone defects cause aesthetic and functional changes that affect the social, economic and especially the emotional life of human beings. This complication stimulates the scientific community to investigate strategies aimed at improving bone reconstruction processes using complementary therapies. Photobiomodulation therapy (PBMT) and the use of new biomaterials, including heterologous fibrin biopolymer (HFB), are included in this challenge. The objective of the present study was to evaluate the influence of photobiomodulation therapy on bone tibial reconstruction of rats with biomaterial consisting of lyophilized bovine bone matrix (BM) associated or not with heterologous fibrin biopolymer. Thirty male rats were randomly separated into three groups of 10 animals. In all animals, after the anesthetic procedure, a noncritical tibial defect of 2 mm was performed. The groups received the following treatments: Group 1: BM + PBMT, Group 2: BM + HFB and Group 3: BM + HFB + PBMT. The animals from Groups 1 and 3 were submitted to PBMT in the immediate postoperative period and every 48 h until the day of euthanasia that occurred at 14 and 42 days. Analyses by computed microtomography (µCT) and histomorphometry showed statistical difference in the percentage of bone formation between Groups 3 (BM + HB + PBMT) and 2 (BM + HFB) (26.4% ± 1.03% and 20.0% ± 1.87%, respectively) at 14 days and at 42 days (38.2% ± 1.59% and 31.6% ± 1.33%, respectively), and at 42 days there was presence of bone with mature characteristics and organized connective tissue. The µCT demonstrated BM particles filling the defect and the deposition of new bone in the superficial region, especially in the ruptured cortical. It was concluded that the association of PBMT with HFB and BM has the potential to assist in the process of reconstructing bone defects in the tibia of rats.

Multimodal X-ray imaging of nanocontainer-treated macrophages and calcium distribution in the perilacunar bone matrix.

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Combined X-ray fluorescence and ptychography offer excellent elemental and structural imaging contrasts at the nanoscale. They enable a robust correlation of elemental distributions with respect to the cellular morphology. Here we extend the applicability of the two modalities to higher X-ray excitation energies, permitting iron mapping. Using a long-range scanning setup, we applied the method to two vital biomedical cases. We quantified the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers. Our work allowed to visualize the internalization of the nanocontainer agglomerates in the cytosol. From the iron areal mass maps, we obtained a distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates. In the second application we mapped the calcium content in a human bone matrix in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was used to remove the mass-thickness effect from the raw calcium map. The resulting ptychography-enhanced calcium distribution allowed then to observe a locally lower degree of mineralization of the perilacunar matrix.

Betulin Promotes Differentiation of Human Osteoblasts In Vitro and Exerts an Osteoinductive Effect on the hFOB 1.19 Cell Line Through Activation of JNK, ERK1/2, and mTOR Kinases.

Although betulin (BET), a naturally occurring pentacyclic triterpene, has a variety of biological activities, its osteogenic potential has not been investigated so far. The aim of this study was to assess the effect of BET on differentiation of human osteoblasts (hFOB 1.19 and Saos-2 cells) in vitro in osteogenic (with ascorbic acid as an osteogenic supplement) and osteoinductive (without an additional osteogenic supplement) conditions. Osteoblast differentiation was evaluated based on the mRNA expression (RT-qPCR) of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), type I collagen-α1 (COL1A1), and osteopontin (OPN). Additionally, ALP activity and production of COL1A1 (western blot analysis) and OPN (ELISA) were evaluated. The level of mineralization (calcium accumulation) was determined with Alizarin red S staining. BET upregulated the mRNA level of RUNX2 and the expression of other osteoblast differentiation markers in both cell lines (except the influence of BET on ALP expression/activity in the Saos-2 cells). Moreover, it increased mineralization in both cell lines in the osteogenic conditions. BET also increased the mRNA level of osteoblast differentiation markers in both cell lines (except for ALP in the Saos-2 cells) in the osteoinductive conditions, which was accompanied with increased matrix mineralization. The osteoinductive activity of BET in the hFOB 1.19 cells was probably mediated via activation of MAPKs (JNK and ERK1/2) and mTOR, as the specific inhibitors of these kinases abolished the BET-induced osteoblast differentiation. Our results suggest that BET has the potential to enhance osteogenesis.

L-Carnitine Reduces Oxidative Stress and Promotes Cells Differentiation and Bone Matrix Proteins Expression in Human Osteoblast-Like Cells.

Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance.

Development of a 3D Collagen Model for the In Vitro Evaluation of Magnetic-assisted Osteogenesis.

Magnetic stimulation has been applied to bone regeneration, however, the cellular and molecular mechanisms of repair still require a better understanding. A three-dimensional (3D) collagen model was developed using plastic compression, which produces dense, cellular, mechanically strong native collagen structures. Osteoblast cells (MG-63) and magnetic iron oxide nanoparticles (IONPs) were incorporated into collagen gels to produce a range of cell-laden models. A magnetic bio-reactor to support cell growth under static magnetic fields (SMFs) was designed and fabricated by 3D printing. The influences of SMFs on cell proliferation, differentiation, extracellular matrix production, mineralisation and gene expression were evaluated. Polymerase chain reaction (PCR) further determined the effects of SMFs on the expression of runt-related transcription factor 2 (Runx2), osteonectin (ON), and bone morphogenic proteins 2 and 4 (BMP-2 and BMP-4). Results demonstrate that SMFs, IONPs and the collagen matrix can stimulate the proliferation, alkaline phosphatase production and mineralisation of MG-63 cells, by influencing matrix/cell interactions and encouraging the expression of Runx2, ON, BMP-2 and BMP-4. Therefore, the collagen model developed here not only offers a novel 3D bone model to better understand the effect of magnetic stimulation on osteogenesis, but also paves the way for further applications in tissue engineering and regenerative medicine.