Mineralized Tissue Research Articles

ИЗУЧЕНИЕ ВЛИЯНИЯ РАЗЛИЧНЫХ ДОЗ ЛУЧЕВОГО ВОЗДЕЙСТВИЯ НА УРОВЕНЬ МИНЕРАЛИЗАЦИИ В РАЗНЫХ УЧАСТКАХ ТВЕРДЫХ ТКАНЕЙ ЗУБОВ МЕТОДОМ РАМАН-ФЛЮОРЕСЦЕНЦИИ

Subject. Changes in the mineralization of hard tissues of teeth under the influence of radiation factor. Objectives. To study the effect of radiation factor on the level of mineralization of hard dental tissues by Raman fluorescence. Methodology. The study was conducted at the Department of Orthopedic Dentistry and Orthodontics of the SUSMU. The study was conducted on clinically removed teeth. All teeth were divided into three groups depending on the amount of radiation exposure (2 Gy, 70 Gy, 110 Gy). The main research method was the study of raman fluorescence of tooth sections on the hardware and software complex "InSpectr" with diagnostic sensitivity for the integral concentration of aerobic-anaerobic microbial suspension up to 109 CFU/ml. Teeth were examined in three areas (neck, equator, cutting edge) before and after radiation exposure. Results. Raman fluorescence diagnostics of the tooth surface made it possible to visually see the difference in mineralization in digitized indicators. For example, according to the indicators before and after exposure to the radiation factor, it is clear that the mineralization indicators, regardless of the dose, had no significant differences. At the same time, there are significant differences in the level of mineralization in the area of different tooth settings (equator, cervical region, cutting edge). Conclusion. In different areas of the tooth surface, the level of mineralization of hard tissues differs. The smallest is observed in the area of the neck of the teeth (incisors, y = 145 ± 1.5, x = 963 cm-1, canines, y = 141 ± 1.1, x = 963 cm-1, premolars, y = 142 ± 1.8 , x = 963 cm-1, molars, y = 143 ± 1.3, x = 963 cm-1), middle, in the area of the cutting edge and occlusal surface (incisors, y = 374 ± 1.7, x = 963 cm-1, canines, y = 377 ± 1.3, x = 963 cm-1, premolars, y = 375 ± 1.2, x = 963 cm-1, molars, y = 375 ± 1.1, x = 963 cm-1), and maximum, in the equator region (incisors, y = 413 ± 1.1, x = 963 cm-1, canines, y = 414 ± 1.9, x = 963 cm-1, premolars, y = 415 ± 1.7, x = 963 cm-1, molars, y = 419 ± 1.6, x = 963 cm-1). The method of Raman-fluorescence diagnostics makes it possible to detect changes in mineralization due to the potential difference in the areas of hard tooth tissues in various areas (neck, equator, cutting edge) of all functional groups of teeth (incisors, canines, premolars, molars) both before and after direct radiation exposure, direct radiation exposure does not significantly change the level of mineralization of hard dental tissues, regardless of the dose applied in all functional groups (incisors, canines, premolars, molars), in all areas of the teeth (equator, cutting edge, cervical region).

Abstract 3077: Identifying Regulators Of Human Adventitial Fibroblast Activation Through A Genome-wide CRISPR Screen

Background: Vascular diseases, including atherosclerosis, are the leading cause of death in the US and world-wide. A quiescent population of fibroblasts that reside in the adventitia (AdvFib) activates and expands upon different vascular insults and contribute to the progression of vascular diseases. However, what regulates AdvFib’s activation remains unknown. We designed a genome-wide CRISPRi screen on human AdvFib to identify these regulators. Methods: We generated an immortalized human coronary AdvFib line that stably expresses dCas9-KRAB, which effectively silences any genes of interest. These cells were infected with a pool of lentivirus carrying guide RNAs (gRNA) covering the entire transcriptome at 200X coverage and allowed to expand freely. Frequency of gRNAs was assessed at different cell passages to identify those that influence AdvFib activation and was followed by experimental validation. Lastly, bioinformatic analysis was performed to determine regulatory pathways and gene networks involved in AdvFib activation. Results: Comparing relative gRNA abundances from early to late passages revealed several hundred putative regulators of AdvFib activation. GO biological process analysis of the top 250 enriched gRNA targets demonstrated disproportionate representation of cyclin inhibitors, validating the effectiveness of our screening. Molecular signatures for adipogenesis and key regulators of tissue mineralization including IBSP and ENPP1 were notably enriched in our screen. Human disease association using PhenGenI revealed a significant association with atherosclerosis. Indeed, numerous coronary artery disease GWAS genes including TCF21 , ZEB2 , CDKN1A , and CDKN2A were enriched in our screen, providing additional support for AdvFib’s importance in atherosclerosis. These hits were subsequently validated individually in vitro. Conclusion: A genome-wide CRISPRi screen of AdvFib activation modulators identified numerous potential regulator genes, many of which have known genetic and observational association with human vascular diseases. This study affirms the potentially important role of AdvFib in human vascular disease and may pave the way to future therapeutics targeting this often neglected cell population.

Metabolic and Skeletal Characterization of the KK/Ay Mouse Model-A Polygenic Mutation Model of Obese Type 2 Diabetes.

Type 2 diabetes (T2D) increases fracture incidence and fracture-related mortality rates (KK.Cg-Ay/J. The Jackson Laboratory; Available from: https://www.jax.org/strain/002468 ). While numerous mouse models for T2D exist, few effectively stimulate persistent hyperglycemia in both sexes, and even fewer are suitable for bone studies. Commonly used models like db/db and ob/ob have altered leptin pathways, confounding bone-related findings since leptin regulates bone properties (Fajardo et al. in Journal of Bone and Mineral Research 29(5): 1025-1040, 2014). The Yellow Kuo Kondo (KK/Ay) mouse, a polygenic mutation model of T2D, is able to produce a consistent diabetic state in both sexes and addresses the lack of a suitable model of T2D for bone studies. The diabetic state of KK/Ay stems from a mutation in the agouti gene, responsible for coat color in mice. This mutation induces ectopic gene expression across various tissue types, resulting in diabetic mice with yellow fur coats (Moussa and Claycombe in Obesity Research 7(5): 506-514, 1999). Male and female KK/Ay mice exhibited persistent hyperglycemia, defining them as diabetic with blood glucose (BG) levels consistently exceeding 300mg/dL. Notably, male control mice in this study were also diabetic, presenting a significant limitation. Nevertheless, male and female KK/Ay mice showed significantly elevated BG levels, HbA1c, and serum insulin concentration when compared to the non-diabetic female control mice. Early stages of T2D are characterized by hyperglycemia and hyperinsulinemia resulting from cellular insulin resistance, whereas later stages may feature hypoinsulinemia due to β-cell apoptosis (Banday et al. Avicenna Journal of Medicine 10(04): 174-188, 2020 and Klein et al. Cell Metabolism 34(1): 11-20, 2022). The observed hyperglycemia, hyperinsulinemia, and the absence of differences in β-cell mass suggest that KK/Ay mice in this studyare modeling the earlier stages of T2D. While compromised bone microarchitecture was observed in this study, older KK/Ay mice, representing more advanced stages of T2D, might exhibit more pronounced skeletal manifestations. Compared to the control group, the femora of KK/Ay mice had higher cortical area and cortical thickness, and improved trabecular properties which would typically be indicative of greater bone strength. However, KK/Ay mice displayed lower cortical tissue mineral density in both sexes and increased cortical porosity in females. Fracture instability toughness of the femora was lower in KK/Ay mice overall compared to controls. These findings indicate that decreased mechanical integrity noted in the femora of KK/Ay mice was likely due to overall bone quality being compromised.

Comparison of two subspecies of a halophytic multi-use plant Mertensia maritima in vitro and ex vitro: propagation, salinity tolerance and mineral nutrition

The aim of the present study was to compare morphological and physiological responses to increasing salinity of the two subspecies of Mertensia maritima during in vitro propagation in two different systems, and subsequent ex vitro cultivation. Plants were brought into culture using seeds and further propagated on Murashige and Skoog medium supplemented with thidiazuron and naphthaleneacetic acid either on agar-solidified or liquid medium with a temporary immersion system (Plantform bioreactors). Salinity tolerance were tested both in tissue culture and with ex vitro greenhouse-cultivated plants. Low concentration of NaCl increased explant biomass in agar-solidified medium, with no negative consequences even at high concentration. However, the effect of salinity on liquid medium was genotype-specific. Explants in Plantform bioreactors showed several-fold higher biomass in comparison to that on agar-solidified medium, but the multiplication potential was not significantly affected. Ex vitro plants were negatively influenced by increasing NaCl concentration in substrate. In contrast to in vitro plants, accumulation potential of Na⁺ in ex vitro plants was low. One of adaptations to salinity at the whole plant level was maintenance of adequate tissue mineral nutrient homeostasis, as mineral nutrient concentrations were not negatively affected by increasing salinity.

Cortical and trabecular mechanical properties in the femoral neck vary differently with changes in bone mineral density.

Graphical Abstract.

Biomechanics of the Human Osteochondral Unit: A Systematic Review.

The damping system ensured by the osteochondral (OC) unit is essential to deploy the forces generated within load-bearing joints during locomotion, allowing furthermore low-friction sliding motion between bone segments. The OC unit is a multi-layer structure including articular cartilage, as well as subchondral and trabecular bone. The interplay between the OC tissues is essential in maintaining the joint functionality; altered loading patterns can trigger biological processes that could lead to degenerative joint diseases like osteoarthritis. Currently, no effective treatments are available to avoid degeneration beyond tissues' recovery capabilities. A thorough comprehension on the mechanical behaviour of the OC unit is essential to (i) soundly elucidate its overall response to intra-articular loads for developing diagnostic tools capable of detecting non-physiological strain levels, (ii) properly evaluate the efficacy of innovative treatments in restoring physiological strain levels, and (iii) optimize regenerative medicine approaches as potential and less-invasive alternatives to arthroplasty when irreversible damage has occurred. Therefore, the leading aim of this review was to provide an overview of the state-of-the-art-up to 2022-about the mechanical behaviour of the OC unit. A systematic search is performed, according to PRISMA standards, by focusing on studies that experimentally assess the human lower-limb joints' OC tissues. A multi-criteria decision-making method is proposed to quantitatively evaluate eligible studies, in order to highlight only the insights retrieved through sound and robust approaches. This review revealed that studies on human lower limbs are focusing on the knee and articular cartilage, while hip and trabecular bone studies are declining, and the ankle and subchondral bone are poorly investigated. Compression and indentation are the most common experimental techniques studying the mechanical behaviour of the OC tissues, with indentation also being able to provide information at the micro- and nanoscales. While a certain comparability among studies was highlighted, none of the identified testing protocols are currently recognised as standard for any of the OC tissues. The fibril-network-reinforced poro-viscoelastic constitutive model has become common for describing the response of the articular cartilage, while the models describing the mechanical behaviour of mineralised tissues are usually simpler (i.e., linear elastic, elasto-plastic). Most advanced studies have tested and modelled multiple tissues of the same OC unit but have done so individually rather than through integrated approaches. Therefore, efforts should be made in simultaneously evaluating the comprehensive response of the OC unit to intra-articular loads and the interplay between the OC tissues. In this regard, a multidisciplinary approach combining complementary techniques, e.g., full-field imaging, mechanical testing, and computational approaches, should be implemented and validated. Furthermore, the next challenge entails transferring this assessment to a non-invasive approach, allowing its application in vivo, in order to increase its diagnostic and prognostic potential.

Influence of Various Strontium Formulations (Ranelate, Citrate, and Chloride) on Bone Mineral Density, Morphology, and Microarchitecture: A Comparative Study in an Ovariectomized Female Mouse Model of Osteoporosis.

Osteoporosis stands out as a prevalent skeletal ailment, prompting exploration into potential treatments, including dietary strontium ion supplements. This study assessed the efficacy of supplementation of three strontium forms-strontium citrate (SrC), strontium ranelate (SrR), and strontium chloride (SrCl)-for enhancing bone structure in 50 female SWISS mice, aged seven weeks. In total, 40 mice underwent ovariectomy, while 10 underwent sham ovariectomy. Ovariectomized (OVX) mice were randomly assigned to the following groups: OVX (no supplementation), OVX + SrR, OVX + SrC, and OVX + SrCl, at concentrations equivalent to the molar amount of strontium. After 16 weeks, micro-CT examined trabeculae and cortical bones, and whole-bone strontium content was determined. Results confirm strontium administration increased bone tissue mineral density (TMD) and Sr content, with SrC exhibiting the weakest effect. Femur morphometry showed limited Sr impact, especially in the OVX + SrC group. This research highlights strontium's potential in bone health, emphasizing variations in efficacy among its forms.

Optimal dual calcium and cholecalciderol dosages for osteoporotic fracture risk patients

Background: Vitamin D is crucial for musculoskeletal health, promoting calcium absorption, osteoid tissue mineralization, and muscle function. Insufficient levels can lead to bone dystrophy, muscle weakness, and osteoporotic fractures. Aims: This research investigates the correlation between bone mineral density and osteoporotic fracture risk, including positive and negative influences, and aims to determine the optimal vitamin D level. Methods: This retrospective observational study examined 206 Jordanian rehabilitation and rheumatology clinic patients from September to November 2021. The participants were post-menopausal women and men over 60. The Age-adjusted Charlson Co-Morbidity Index and Functionality Grade system was used to assess participants' co-morbidity burden and functionality. DEXA scans assessed participants' proximal femoral hip and anteroposterior spine. Participants were divided into two Vit D groups: those below 30 ng/ml and those above 30. Results were compared using a Chi Square test. The study examined correlations, total variations, and Vit D prediction quality using logistic regression analyses. SPSS 23.0 was used for statistical analysis with a 5% significance level. Results: A binary logistic regression model was employed to simulate the correlation between the vitamin D levels of patients and their bone mineral density. The model indicated a 61.39% likelihood of having a fH_BMD (femoral head bone mineral density) equal to or greater than 0.755 g/cm2 when the vitamin D level is at its optimal value of 27.25 ng/ml. The model indicated a 27.25% likelihood of a fHOPF risk-free tool with a value of ≥3% when the optimal vitamin D level is 27.25 ng/ml. The model indicated a 17.74% likelihood of experiencing a significant osteoporotic fracture within the next 10 years. Conclusion: The findings of our study demonstrated a direct correlation between elevated levels of vitamin D and improved bone mineral quality indices that were examined. The serum 25-OH Cholecalciferol levels are more likely to have a beneficial effect on bone health status.

Morphological peculiarities of regeneration phases and dynamics of growth factor levels at using 2-ethyl-6-methyl-3-hydroxypyridinium n-acetyl-6-aminohexanoate for rat skin burns repairation

BACKGROUND: It was found that new derivatives of acexamic acid — N-acetyl-6-aminohexanoate of silver and 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate stimulate regeneration and mineralization of bone tissues in osteoporosis. However, to date, the effect of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate on the features of reparative histogenesis and the relationship of its stages with the levels of growth factors in regenerating tissues during skin restoration after thermal burn has not been studied.
 AIM: To estimate the morphology dynamics of skin burn healing and its relationship with the levels of vascular endothelial growth factor and basic fibroblast growth factor growth factors in rat regenerating tissues to characterize the reparative properties of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate.
 MATERIALS AND METHODS: 63 nonlinear rats were used to establish thermal burn wound model according to standard methodics. Burn wounds of the experimental group animals were treated with 2% ointment of 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate daily, control group 1 — the burns were not treated, control group 2 — ointment base was applied daily. Morphological examinations of regenerating tissues from the burn area were carried out on days 7, 14 and 21 of the experiment after standard sample preparation. The concentrations of growth factors in homogenates of regenerating tissues were determined by Enzyme Immunoassay.
 RESULTS: The inflammation phase is characterized by vascular endothelial growth factor active production and release from cells and morphologically accompanied by early neovasculogenesis in granulation tissue and significant differences in its thickness in rats of the experimental group. During the proliferation phase, activation of fibroblast reproduction and differentiation was associated with an increase in basic fibroblast growth factor levels and the maintenance of elevated vascular endothelial growth factor levels in all groups, especially high in experimental group rats. The epithelialization phase was accompanied by a statistically significant decrease in the content of basic fibroblast growth factor and vascular endothelial growth factor in regenerating skin tissues by 1.9 and 1.8 times, respectively, in comparison with the control groups.
 CONCLUSIONS: The obtained experimental data indicate 2% ointment with 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate has a reparative potential, as evidenced by the accelerated reduction both the area of burn defects and the average time for their healing in the experimental group compared to the control groups.

A cooperative study assessing the effects of a second iron injection administered before weaning on growth performance, hematological status, and tissue mineral concentrations of nursery pigs*

A cooperative study assessing the effects of a second iron injection administered before weaning on growth performance, hematological status, and tissue mineral concentrations of nursery pigs*

Knocking out FAM20C in pre-osteoblasts leads to up-regulation of osteoclast differentiation to affect long bone development

Family with sequence similarity 20 member C (FAM20C) is a Golgi casein kinase that phosphorylates extracellularly-secreted regulatory proteins involved in bone development and mineralization, but its specific role in bone development is still largely unknown. In this study, to examine the specific mechanisms that FAM20C influences bone development, we cross-bred Osx-Cre with FAM20Cflox/flox mice to establish a Osx-Cre; FAM20Cflox/flox knockout (oKO) mouse model; FAM20C was KO in pre-osteoblasts. oKO development was examined at 1–10 weeks, in which compared to control FAM20Cflox/flox, they had lower body weights and bone tissue mineralization. Furthermore, oKO had lower bone volume fractions, thickness, and trabecular numbers, along with higher degrees of trabecular separation. These mice also had decreased femoral metaphyseal cartilage proliferation layer, along with thickened hypertrophic layer and increased apoptotic cell counts. Transcriptomic analysis found that differentially-expressed genes in oKO were concentrated in the osteoclast differentiation pathway, in line with increased osteoclast presence. Additionally, up-regulation of osteoclast-related, and down-regulation of osteogenesis-related genes, were identified, in which the most up-regulated genes were signal regulatory protein β-1 family (Sirpb1a-c) and mitogen-activated protein kinase 13. Overall, FAM20C KO in pre-osteoblasts leads to abnormal long bone development, likely due to subsequent up-regulation of osteoclast differentiation-associated genes.

The use of raman-fluorescent medical technologies to assess the effect of physical and chemical factors on the mineralization of hard tooth tissues in normal and noncarious lesions

BACKGROUND: Assessment of the rapid state of mineralization of hard tooth tissues in normal and in various carious and noncarious lesions based on objective in situ methods is an unsolved and urgent issue in dental science and practice.
 AIM: To substantiate the need for the clinical application in situ of express digital raman-fluorescent technology to increase the objectivity and effectiveness of assessing the degree of mineralization of hard tooth tissues in carious (initial form) and noncarious (wedge-shaped defect, pathological erasability) lesions under the influence of various physical and chemical factors on tissues.
 MATERIALS AND METHODS: A survey of 80 patients aged 25–50 years was conducted; the patients were divided into three groups: comparison and with carious and with noncarious (wedge-shaped defect, pathological erasability) lesions of hard dental tissues. In each group, changes in hygienic conditions and mineralization parameters of hard tooth tissues were assessed by the difference in the spectral intensity of raman-fluorescent measurements before and after controlled and/or uncontrolled hygienic treatment. Previously, under experimental conditions, the effects of various physical (mechanical processing, X-ray irradiation on the LNK-268 installation) and chemical (citric acid, hydroxylapatite (GAP) dry and moistened) factors on the hard tissues of the tooth were studied. Simultaneously, “locally” in real time, the mineralization parameters of the hard tissues of the tooth were recorded before and after hygienic treatment. In this study, we used an installation of the “InSpectrM” type in our modification.
 RESULTS: Experimental studies have shown the effectiveness and objectivity of the use of raman-fluorescence diagnostics to assess the hygienic condition and mineralization of hard dental tissues. The objectivity of the assessment of mineralization in a moistened state was investigated (dry GAP, 702 rel. units; wet GAP, 1520 rel. units), and the absence of the effect of radiation on the mineralization of hard tissues of the teeth (p 0.95) was revealed.
 Clinical observations showed that in patients with a wedge-shaped defect and increased erasability of hard tooth tissues, the hygienic condition is 5–10% worse than that in patients with intact tooth tissues and initial formation of caries. The main difference in the condition of hard tissues of teeth with noncarious lesions is a statistically significantly reduced (p 0.95) mineralization of hard tissues of the tooth, requiring remineralizing therapy immediately after hygienic treatment.
 CONCLUSION: In experimental and clinical conditions, the objectivity and effectiveness of the use of express raman-fluorescent technologies to assess the mineralization of hard tooth tissues when exposed to various physical and chemical factors in normal conditions, with carious (initial form) and noncarious (wedge-shaped defect, pathological erasability) lesions, are substantiated.

Abstract 1965: 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors

Abstract FDA-approved pan-FGFR inhibitors bring encouraging results in solid tumors. However, resistance mechanisms and side effect profiles may limit their clinical utility. We designed 3HP-2827, a highly selective and potent small molecule inhibitor of FGFR2, to treat solid tumors harboring FGFR2 alterations, including amplifications, mutations, and fusions to overcome these limitations. 3HP-2827 inhibits FGFR2 enzyme activity with more than 1623-fold selectivity over FGFR1 and FGFR3. 3HP-2827 inhibits cell proliferation with IC50 <10 nM and 30 nM in FGFR2 amplified and FGFR2-mutant cancer cell lines, respectively. 3HP-2827 demonstrates high kinome selectivity for FGFR2 against a panel of 485 human kinases, resulting in 90.1% inhibition of FGFR2, and no other kinases showed greater than 75% inhibition. 3HP-2827 leads only modest activity at 10 µM against PDE4D2 and LCK in 44 distinct common adverse drug reaction targets, supporting its high degree of safety. 3HP-2827 also demonstrates a dose-dependent inhibition of phosphorylation of FGFR2 and FGFR2 signaling pathway nodes, including ERK and AKT, in the FGFR2-dependent cancer cell line. In vivo, 3HP-2827 demonstrates potent antitumor activity in cell- and patient-derived xenograft mouse models, including FGFR2-amplified gastric cancer, FGFR2-mutant (N549K) endometrial cancer, FGFR2 fusion ICC and gastric cancers. Strikingly, 3HP-2827 demonstrates superior activity in patient-derived ICC and gastric cancer, cell-derived endometrial cancer (CDX) in tumor and pFGFR2 inhibitions. In the ICC PDX model, 3HP-2827 also induces the regression of pemigatinib-resistant tumors. 3HP-2827 is well tolerated in the toxicology studies, and no hyperphosphatemia or tissue mineralization is observed at 150 mg/kg/kg/day in rats and 120 mg/kg/day in dogs. The data support that 3HP-2827, a potential to be a best-in-class selective FGFR2 inhibitor, will be studying clinically at the beginning of 2024. Citation Format: Susan S. Liu-Chen, Jufang Lin, Jingyu Hu, Yanchao Liu, Weibo Zhang, Zhixiong Zhang, Yan Ding, Jinqiu Liu, Kevin Chen, Shaojing Hu. 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1965.

Dietary zinc requirement of juvenile stinging catfish Heteropneustes fossilis based on growth performance, haematology, and tissue mineral composition

This investigation was done to determine how much zinc (Zn) the stinging catfish, Heteropneustes fossilis, needs in its diet. Five isonitrogenous (34.5% protein) and isolipidic (6.0% lipid) diets were prepared to contain graded levels of Zn (0, 10, 20, 30, and 40 mg kg−1), supplied as zinc sulfate (ZnSO4·7H2O), and referred to as Zn0, Zn10, Zn20, Zn30, and Zn40, respectively. A total of 600 fish (initial body weight: 1.41 ± 0.02 g) were stocked in 15 glass aquaria (40 fish/aquarium), each with 180 L water capacity. For ten weeks, each diet was hand fed to three groups of fish twice daily until they appeared satisfied. The highest weight gain and specific growth rate, and lowest feed conversion ratio were recorded in fish fed with a 30 mg Zn kg−1 diet. Zn contents in bone and muscle linearly increased up to 30 mg kg−1 Zn and then remained stable, while iron (Fe) and copper (Cu) contents in bone and muscle had an inverse pattern with the inclusion level of dietary Zn. Increasing dietary Zn levels up to 30 mg kg−1 was found to improve values of hematological parameters such as red blood cell (RBC), white blood cell (WBC), haemoglobin (Hb), and haematocrit (HCT). These values, however, decreased when the dietary Zn level was further increased. The serum alkaline phosphatase level was the highest in fish fed a diet containing 30 mg kg-1 of Zn. Regression analyses based on weight gain, specific growth rate, and bone and muscle Zn concentrations indicated that the optimum dietary Zn requirement for stinging catfish was in a range of 27.4–36.5 mg kg−1.

Osteoinductive Dental Pulp Stem Cell-Derived Extracellular Vesicle-Loaded Multifunctional Hydrogel for Bone Regeneration.

Stem cell-derived extracellular vesicles (EVs) show great potential for promoting bone tissue regeneration. However, normal EVs (Nor-EVs) have a limited ability to direct tissue-specific regeneration. Therefore, it is necessary to optimize the osteogenic capacity of EV-based systems for repairing extensive bone defects. Herein, we show that hydrogels loaded with osteoinductive dental pulp stem cell-derived EVs (Ost-EVs) enhanced bone tissue remodeling, resulting in a 2.23 ± 0.25-fold increase in the expression of bone morphogenetic protein 2 (BMP2) compared to the hydrogel control group. Moreover, Ost-EVs led to a higher expression of alkaline phosphatase (ALP) (1.88 ± 0.16 of Ost-EVs relative to Nor-EVs) and the formation of orange-red calcium nodules (1.38 ± 0.10 of Ost-EVs relative to Nor-EVs) in vitro. RNA sequencing revealed that Ost-EVs showed significantly high miR-1246 expression. An ideal hydrogel implant should also adhere to surrounding moist tissues. In this study, we were drawn to mussel-inspired adhesive modification, where the hydrogel carrier was crafted from hyaluronic acid (HA) and polyethylene glycol derivatives, showcasing impressive tissue adhesion, self-healing capabilities, and the ability to promote bone growth. The modified HA (mHA) hydrogel was also responsive to environmental stimuli, making it an effective carrier for delivering EVs. In an ectopic osteogenesis animal model, the Ost-EV/hydrogel system effectively alleviated inflammation, accelerated revascularization, and promoted tissue mineralization. We further used a rat femoral condyle defect model to evaluate the in situ osteogenic ability of the Ost-EVs/hydrogel system. Collectively, our results suggest that Ost-EVs combined with biomaterial-based hydrogels hold promising potential for treating bone defects.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey’s fibres and not by its mineral phase

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey’s fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Citrate Improves Biomimetic Mineralization Induced by Polyelectrolyte-Cation Complexes Using PAsp-Ca&Mg Complexes.

Magnesium ions are highly enriched in early stage of biological mineralization of hard tissues. Paradoxically, hydroxyapatite (HAp) crystallization is inhibited significantly by high concentration of magnesium ions. The mechanism to regulate magnesium-doped biomimetic mineralization of collagen fibrils has never been fully elucidated. Herein, it is revealed that citrate can bioinspire the magnesium-stabilized mineral precursors to generate magnesium-doped biomimetic mineralization as follows: Citrate can enhance the electronegativity of collagen fibrils by its absorption to fibrils via hydrogen bonds. Afterward, electronegative collagen fibrils can attract highly concentrated electropositive polyaspartic acid-Ca&Mg (PAsp-Ca&Mg) complexes followed by phosphate solution via strong electrostatic attraction. Meanwhile, citrate adsorbed in/on fibrils can eliminate mineralization inhibitory effects of magnesium ions by breaking hydration layer surrounding magnesium ions and thus reduce dehydration energy barrier for rapid fulfillment of biomimetic mineralization. The remineralized demineralized dentin with magnesium-doped HAp possesses antibacterial ability, and the mineralization mediums possess excellent biocompatibility via cytotoxicity and oral mucosa irritation tests. This strategy shall shed light on cationic ions-doped biomimetic mineralization with antibacterial ability via modifying collagen fibrils and eliminating mineralization inhibitory effects of some cationic ions, as well as can excite attention to the neglected multiple regulations of small biomolecules, such as citrate, during biomineralization process.

Direct coupled electrical stimulation towards improved osteogenic differentiation of human mesenchymal stem/stromal cells: a comparative study of different protocols

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols.

Lanthanum Hydroxide and Chronic Kidney Disease Mineral and Bone Disorder: A Rat Model.

To investigate the pharmacological effects and molecular mechanisms of lanthanum hydroxide(LH) on ectopic mineralization of soft tissue and abnormal bone in rats with acute kidney injury(AKI). Wistar rats were modeled by 5/6 nephrectomy. After the operation, the rats were divided into different groups, the biochemical indexes of serum collected at different times. LH was administered by intragastric tube at doses of 0.4, 0.2, and 0.1g/kg, respectively. Rats were sacrificed in the 16th week after LH treatment. Observation of pathological changes in tissues were made by specific staining. Western Blot, Real-Time Quantitative PCR, and immunohistochemistry techniques were used to detect the impact on pathway-related proteins. Compared with the control group (no LH administered), the serum phosphate level of the LH group was significantly reduced (p<0.01), calcification of the thoracic aorta was reduced (p<0.05, p<0.01) (Serum biochemical tests before dosing and during drug treatment cycles), renal fibrosis was improved (p<0.01), nuclear entry of nuclear factor kappa-B (NF-κB) was reduced (p<0.01), and the expression of the smooth muscle protein 22α (SM22α) was significantly increased (p<0.01). The expression of osteogenic marker genes was decreased. In addition, compared with the controls, the receptor activator for nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio of the femur in the model group was increased (p<0.05). LH can inhibit the occurrence and development of vascular calcification and bone abnormalities in AKI rats by inhibiting the NF-κB and RANKL/OPG signaling pathways.

Micromechanical modelling of transverse fracture behaviour of lamellar bone using a phase-field damage model: The role of non-collagenous proteins and mineralised collagen fibrils

At the tissue-scale and above, there are now well-established structure-property relationships that provide good approximations of the biomechanical performance of bone through, for example, power-law relationships that relate tissue mineral density to elastic properties. However, below the tissue-level, the individual role of the constituents becomes prominent and these simple relationships tend to break down, with more detailed theoretical and computational models are required to describe the mechanical response. In this study, a two-dimensional micromechanics damage-based representative volume element (RVE) of lamellar bone was developed, which included a novel implementation of a phase-field damage model to describe the behaviour of non-collagenous proteins at mineral-mineral and mineral-fibril interface regions. It was found that, while the stiffness of the tissue was governed by the relative proportion of extra-fibrillar mineral and mineralised collagen fibrils, the strength and toughness of the tissue in transverse direction relied on the interactions occurring at mineral-mineral and mineral-fibril interfaces, highlighting the prominence of non-collagenous proteins in determine fracture-based processes at this scale. While fractures tended to initiate in mineral rich areas of the extra-fibrillar mineral matrix, it was found that the presence of mineralised collagen fibrils at low density did not provide a substantial contribution to crack propagation behaviour under transverse loading. However, at physiological volume fraction (VfMCF=50%), different scenarios could arise depending on the relative strength value of the interphase around the MCFs (σfInterphaseMCF) to the interphase between individual minerals (σfInterphaseHA): (i) When γ=σfInterphaseMCFσfInterphaseHA<1, MCFs appear to facilitate crack propagation with MCF-mineral debonding being the dominant failure mode; (ii) once γ>1, the MCFs hinder the microcracks, leading to inhibition of crack propagation, which can be regarded as an energy dissipation mechanism. The effective fracture properties of the tissue also experience a sudden increase in fracture work density (J-integral) once the crack is arrested by MCFs or severely deflected. Collectively, the predicted behaviour of the model compared well to those reported through experimental and computational methods, highlighting its potential to provide further understanding into the mechanistic response of bone ultrastructure alterations related to the structural and compositional changes resulting from disease and aging.