The novel prognostic marker SPOCK2 regulates tumour progression in melanoma.

Secreted protein acidic and cysteine rich/osteonectin, cwcv and kazal-like domain proteoglycan 2 (SPOCK2) is a protein that regulates cell differentiation and growth. Recent studies have reported that SPOCK2 plays important roles in the progression of various human cancers; however, the role of SPOCK2 in melanoma remains unknown. Therefore, this study investigated the roles of SPOCK2 and the related mechanisms in melanoma progression. To evaluate the clinical significance of SPOCK2 expression in patients with melanoma, we analysed the association between SPOCK2 expression and its prognostic value for patients with melanoma using systematic multiomic analysis. Subsequently, to investigate the roles of Spock2 in melanoma progression in vitro and in vivo, we knocked down Spock2 in the B16F10 melanoma cell line. High SPOCK2 levels were positively associated with good prognosis and long survival rate of patients with melanoma. Spock2 knockdown promoted melanoma cell proliferation by inducing the cell cycle and inhibiting apoptosis. Moreover, Spock2 downregulation significantly increased cell migration and invasion by upregulating MMP2 and MT1-MMP. The increased cell proliferation and migration were inhibited by MAPK inhibitor, and ERK phosphorylation was considerably enhanced in Spock2 knockdown cells. Therefore, Spock2 could function as a tumour suppressor gene to regulate melanoma progression by regulating the MAPK/ERK signalling pathway. Additionally, Spock2 knockdown cell injection induced considerable tumour growth and lung metastasis in C57BL6 mice compared to that in the control group. Our findings suggest that SPOCK2 plays crucial roles in malignant progression of melanoma and functions as a novel therapeutic target of melanoma.

Exploring the Role of Serum Osteonectin and Hsp27 in Pediatric MAFLD Diagnosis and Cardiometabolic Health.

BACKGROUND: Childhood obesity is one of the major challenges of public health policies. The problem of fatty liver in childhood, known as MAFLD (metabolic dysfunction-associated fatty liver disease), is of particular interest as the gold standard diagnosis technique is invasive (liver biopsy). Hence, efforts are made to discover more specific biomarkers for the MAFLD signature. Therefore, the aim of the study was to evaluate Osteonectin and Hsp27 as biomarkers for MAFLD diagnosis and to assess their links with auxological and biochemical profiles of overweight and obese pediatric subjects. METHODS: A cross-sectional study in which we (re)analyzed data from the MR PONy cohort comprising 71 pediatric subjects. Auxological data, liver ultrasonography and biochemical serum profile were recorded. Lipid-derived indices and body composition indices were calculated. Nevertheless, serum Osteonectin and Hsp27 levels were assessed using an ELISA approach. RESULTS: MAFLD prevalence was 40.8%. Higher Osteonectin levels were noted in MAFLD subjects versus non-MAFLD subjects and in dyslipidemic children regardless of their liver function status. Lipid-derived indices had good diagnostic capacity for MAFLD. CONCLUSIONS: We confirm Osteonectin as a MAFLD diagnosis biomarker in children. Also, lipid-derived indices are useful as metabolic-associated organ impairment markers in children even before the onset of obesity.

Obesity and Bone Mineral Density Protection Paradox in Chronic Kidney Disease: Secreted Protein Acidic and Rich in Cysteine as a Piece of the Puzzle?

Obesity is a health condition that represents a risk factor for numerous diseases and complications. However, obesity might also have-to some extent-some "benefits" in certain situations. This includes potential bone protection in patients suffering from chronic kidney disease. In an attempt to explain such a paradox, we highlight secreted protein acidic and rich in cysteine (SPARC) as a hypothetical mediator of this protection. Indeed, SPARC properties provide a logical rationale to describe such bone protection via its overexpression combined with its calcium-binding and collagen-binding properties. We believe that exploring such hypotheses could open new doors to elucidate unknown pathways towards developing a new generation of molecular therapies.

Osteonectin bidirectionally regulates osteoblast mineralization.

OBJECTIVE: The aim of this study was to investigate whether Osteonectin/Secreted protein acidic and rich in cysteine (ON/SPARC) had a two-way dose-dependent regulatory effect on osteoblast mineralization and its molecular mechanism. METHODS: Initially, different concentrations of ON were added in osteoblasts, and the gene of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) were detected using reverse-transcription quantitative polymerase chain reaction (RT-PCR). Secondly, based on the above results, the Optima and inhibitory concentration of ON for osteoblast mineralization were determined and regrouped, the Control group was also set up, and the gene detections of Collagen 1 (Col 1), Discoidin domain receptor 2 (DDR2) and p38 mitogen­activated protein kinase were added using RT-PCR. In the third stage of the experiment, osteoblasts were pretreated with 0.4Mm ethyl-3,4-dihydroxybenzoate (DHB) (a specific inhibitor of collagen synthesis) for 3 h before adding the optima SPARC, the gene and protein expressions of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 were detected by RT­qPCR and western blot analysis, and the mineralized nodules were observed by alizarin red staining. RESULTS: The results showed that the expression of OCN, OPN, BSP, ALP, DDR2, ALP, Col 1, DDR2 and P38 genes and proteins in osteoblasts were significantly enhanced by 1 ug/ml ON, 100 ug/ml ON or 1 ug/ml ON added with 3,4 DHB significantly inhibited the expressions of DDR2, P38 and the above-mentioned mineralization indexes, and significantly reduced the formation of mineralized nodules. CONCLUSION: This study suggested that ON had a bidirectional dose-dependent regulatory effect on osteoblast mineralization, and the activation of P38 pathway by collagen binding to DDR2 was also an important molecular mechanism.

Effect of a 3-month L-carnitine supplementation and resistance training program on circulating markers and bone mineral density in postmenopausal women: a randomized controlled trial.

BACKGROUND: Higher circulating levels of trimethylamine N-oxide (TMAO), which is a metabolite that can be produced by the gut microbiota from L-carnitine (LC), have been associated with bone mineral density (BMD). Because LC supplementation can improve bone density and microstructural properties in animal models, this study aimed to examine the effects of 12 weeks of LC supplementation on BMD and selected blood markers involved in bone metabolism of postmenopausal women participating in a resistance training (RT) program. METHODS: Twenty-seven postmenopausal women, who had not been treated for osteoporosis, with a total T-score above - 3.0 and no diet differences completed 12 weeks of RT. The participants' diets were supplemented with either 1 g of LC-L-tartrate and 3 g of leucine per day (LC group) or 4 g of leucine per day as a placebo (PLA group), in a double-blind fashion. RESULTS: After the intervention in the LC group, plasma total carnitine and serum decorin levels were higher than the corresponding preintervention values (p = 0.040 and p = 0.042, respectively). Moreover, plasma TMAO and serum SPARC levels were higher in the LC group than the corresponding postintervention values in the PLA group (p < 0.001 and p = 0.030, respectively). No changes in the BMD were observed after 3 months of the intervention. CONCLUSIONS: Twelve weeks of LC supplementation during RT program increased plasma TMAO levels and appeared to affect signaling molecules, as indicated by the increase in the resting SPARC and decorin levels, with no significant modification in the BMD. TRIAL REGISTRATION: Retrospectively registered at the ClinicalTrials.gov (NCT05120011).

Polymeric and Composite Carriers of Protein and Non-Protein Biomolecules for Application in Bone Tissue Engineering.

Conventional intake of drugs and active substances is most often based on oral intake of an appropriate dose to achieve the desired effect in the affected area or source of pain. In this case, controlling their distribution in the body is difficult, as the substance also reaches other tissues. This phenomenon results in the occurrence of side effects and the need to increase the concentration of the therapeutic substance to ensure it has the desired effect. The scientific field of tissue engineering proposes a solution to this problem, which creates the possibility of designing intelligent systems for delivering active substances precisely to the site of disease conversion. The following review discusses significant current research strategies as well as examples of polymeric and composite carriers for protein and non-protein biomolecules designed for bone tissue regeneration.

SPARC in cancer-associated fibroblasts is an independent poor prognostic factor in non-metastatic triple-negative breast cancer and exhibits pro-tumor activity.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). We also quantified PD-L1 and PD-1 expression. We detected SPARC expression in tumor cells (42.4%), cancer-associated fibroblasts (CAFs; 88.1%), tumor-associated macrophages (77.1%), endothelial cells (75.2%) and tumor-infiltrating lymphocytes (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient-derived xenografts and cell lines. Furthermore, we analyzed publicly available single-cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor-related processes. We then showed that fibroblast-secreted SPARC had a tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC targeted therapy.

Modelling stromal compartments to recapitulate the ameloblastoma tumour microenvironment.

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling.

Histological and Immunohistochemical Analysis of Peri-Implant Soft and Hard Tissues in Patients with Peri-Implantitis.

Currently, researchers are focused on the study of cytokines as predictive biomarkers of peri-implantitis (PI) in order to obtain an early diagnosis and prognosis, and for treatment of the disease. The aim of the study was to characterize the peri-implant soft and hard tissues in patients with a peri-implantitis diagnosis. A descriptive observational study was conducted. Fifteen soft tissue (ST) samples and six peri-implant bone tissue (BT) samples were obtained from 13 patients who were diagnosed with peri-implantitis. All the samples were processed and embedded in paraffin for histological and immunohistochemical analyses. A descriptive and quantitative analysis of mast cells and osteocytes, A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), osteonectin (ON), and ∝-smooth muscle actin (∝-SMA) was performed. We observed the presence of mast cells in peri-implant soft tissue in all samples (mean 9.21 number of mast cells) and osteocytes in peri-implant hard tissue in all samples (mean 37.17 number of osteocytes). The expression of APRIL-ST was 32.17% ± 6.39%, and that of APRIL-BT was 7.09% ± 5.94%. The BAFF-ST expression was 17.26 ± 12.90%, and the BAFF-BT was 12.16% ± 6.30%. The mean percentage of ON was 7.93% ± 3.79%, and ∝-SMA was 1.78% ± 3.79%. It was concluded that the expression of APRIL and BAFF suggests their involvement in the bone resorption observed in peri-implantitis. The lower expression of osteonectin in the peri-implant bone tissue can also be associated with a deficiency in the regulation of bone remodeling and the consequent peri-implant bone loss.

Activity of acute pancreatitis is modified by secreted protein acidic and rich in cysteine ablation.

BACKGROUND: Acute pancreatitis (AP) is a frequent cause for hospitalization. However, molecular determinants that modulate severity of experimental pancreatitis are only partially understood. OBJECTIVE: To investigate the role of secreted protein acidic and rich in cysteine (SPARC) during cerulein-induced AP in mice. METHODS: AP was induced by repeated cerulein injections in SPARC knock-out mice (SPARC-/- ) and control littermates (SPARC+/+ ). Secreted protein acidic and rich in cysteine expression and severity of AP were determined by histopathological scoring, immunohistochemistry, and biochemical assays. For functional analysis, primary murine acinar cell cultures with subsequent amylase release assays were employed. Proteome profiler assay and ELISA were conducted from pancreatic tissue lysates, and co-immunofluorescence was performed. RESULTS: Upon cerulein induction, SPARC expression was robustly induced in pancreatic stellate cells (PSCs) but not in acinar cells. Genetic SPARC ablation resulted in attenuated severity of AP with significantly reduced levels of pancreatic necrosis, apoptosis, immune cell infiltration, and reduced fibrosis upon chronic stimulation. However, the release of amylase upon cerulein stimulation in primary acinar cell culture from SPARC+/+ and SPARC-/- was indistinguishable. Notably, immune cell derived C-C Motif Chemokine Ligand 2 (CCL2) was highly elevated in SPARC+/+ pancreatic tissue potentially linking PSC derived SPARC with CCL2 induction in AP. CONCLUSION: SPARC mediates the severity of AP. The potential link between SPARC and the CCL2 axis could open new avenues for tailored therapeutic interventions in AP patients and warrants further investigations.

Implantation of bovine hydroxyapatite and secretome with different oxygen concentration may improve massive bone defect regeneration: An experimental study on animal model.

The most widely used biomaterials in the treatment of massive bone defects are allograft bone or metal implants. The current problem is that the availability of allographs is limited and metal implants are very expensive. Mass production of secretome can make bone reconstruction of massive bone defects using a scaffold more effective and efficient. This study aims to prove bone regeneration in massive bone defects using bovine hydroxyapatite reconstruction with normoxic and hypoxic secretome conditions using collagen type 1 (COL1), alkaline phosphate (ALP), osteonectin (ON), and osteopontin (OPN) parameters. This is an in vivo study using male New Zealand white rabbits aged 6-9 months. The research was carried out at the Biomaterials Center-Tissue Bank, Dr. Soetomo Hospital for the manufacturer of bovine hydroxyapatite (BHA) and secretome BM-MSC culture under normoxic and hypoxic conditions, and UNAIR Tropical Disease Institute for implantation in experimental animals. Data analysis was carried out with the one-way ANOVA statistical test and continued with the Post Hoc test LSD statistical test to determine whether or not there were significant differences between groups. There were significant differences between hypoxic to normoxic group and hypoxic to BHA group at day-30 observation using ALP, COL 1, ON, and OPN parameters. Meanwhile, there is only osteonectin parameter has significant difference at day-30 observation. At day-60 observation, only OPN parameter has significant differences between hypoxic to normoxic and hypoxic to BHA group. Between day-30 and day-60 observation, BHA and normoxic groups have a significant difference at all parameters, but in hypoxic group, there are only difference at ALP, COL 1, and ON parameters. Hypoxic condition BM-MSC secretome with BHA composite is superior and could be an option for treating bone defect.

Effects of teriparatide on bone formation in rats with experimentally induced premaxillary expansion

ABSTRACT Objective: This study aimed to evaluate the effects of systemic teriparatide on sutural bone formation after premaxillary suture expansion in rats. Material and Methods: Twenty Wistar male rats (8-10 weeks old) were randomly divided into two groups, namely, control (C, n=10) and teriparatide (T, n=10). An expansion force was applied to the maxillary incisors using helical spring for a seven-day expansion period, for both groups. On the eighth day, the rats were kept for a seven-day consolidation period, and then 60 µg/kg teriparatide (once a day) was administered to group T subcutaneously for seven days. Then, all the rats were sacrificed, and histological sections were stained with hemotoxylin-eosin for examination. Anti-osteonectin, anti-osteocalcin, anti-Vascular endothelial growth factor (VEGF) and anti-transforming growth factor beta (TGF-β) were evaluated by immunohistochemical analysis in the midpalatal suture area. Results: Histologically, the newly formed bone tissue was observed to be larger in group T than in group C. The number of immunoreactive osteoblasts for osteonectin, osteocalcin and VEGF antibodies was significantly higher in group T than in group C (p = 0.0001). The TGF-β antibody showed a mild reaction in group T, but did not reach significance in comparison with group C (p ˃ 0.05). Conclusion: Systemic teriparatide application following the premaxillary expansion of the suture area may stimulate bone formation and add to the consolidation of the expansion in rats by regulating osteonectin, osteocalcin and VEGF.

RESUMO Objetivo: O presente estudo teve como objetivo avaliar os efeitos do uso sistêmico da teriparatida na formação óssea sutural após a expansão da pré-maxila em ratos. Material e Métodos: Vinte ratos machos da raça Wistar (com oito a dez semanas de vida) foram divididos aleatoriamente em dois grupos: controle (C, n=10) e teriparatida (T, n=10). Uma força de expansão foi aplicada aos incisivos superiores, usando uma mola helicoidal, por um período de expansão de sete dias em ambos os grupos. No oitavo dia, os ratos iniciaram um período de sete dias de consolidação, nos quais 60 µg/kg de teriparatida foram administrados (uma vez ao dia), por via subcutânea, para o grupo T. Posteriormente, todos os ratos foram sacrificados e cortes histológicos corados com hemotolixina-eosina foram examinados. Por meio de análise imuno-histoquímica da região da sutura palatina mediana, avaliou-se a presença de anti-ostenectina, anti-osteocalcina, anti-fator de crescimento endotelial vascular (VEGF) e anti- fator transformador de crescimento (TGF-β). Resultados: Histologicamente, observou-se que o tecido ósseo recém-formado foi maior no grupo T do que no grupo C. O número de osteoblastos imunorreativos para anticorpos de osteonectina, osteocalcina e VEGF foi significativamente maior no grupo T do que no grupo C (p = 0,0001). O anticorpo TGF-β mostrou uma pequena reação no grupo T; porém, sem diferença significativa para o grupo C (p ˃ 0,05). Conclusão: O uso sistêmico de teriparatida após a expansão da sutura na região da pré-maxila pode estimular a formação óssea e melhorar a consolidação da expansão em ratos, por meio da regulação de osteonectina, osteocalcina e VEGF.

Discoidin domain receptor 2 activation of p38 mitogen-activated protein kinase as an important pathway for osteonectin-regulating osteoblast mineralization.

OBJECTIVE: The present study aimed to determine the role of the discoidin domain receptor 2 (DDR2) in the osteonectin (ON) regulation of osteoblast mineralization through the activation of p38 mitogen-activated protein kinase (MAPK). METHODS: Four groups were established: the ON group, the inhibitor group, the Ddr2-small interfering ribonucleic acid (siRNA) group, and the control group. Osteoblasts from the parietal bones of neonatal Sprague-Dawley rats were isolated and cultured. In the ON group, 1 µg/mL ON was added to the osteoblasts. The gene expressions of collagen 1 (Col 1) and Ddr2 were detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the inhibitor group, the osteoblasts were added to WRG-28 (a specific DDR2 inhibitor), and in the Ddr2-siRNA group, the osteoblasts were transfected with Ddr2-siRNA. The gene and protein expressions of DDR2, bone sialoprotein, osteocalcin, osteopontin, and p38 MAPK were determined using RT-qPCR and western blot analysis. Alizarin red staining and transmission electron microscopy were used to detect mineralization. RESULTS: The results showed that ON enhanced the osteoblast Col 1 and Ddr2 gene expressions, while the use of a Ddr2-siRNA/DDR2-blocker decreased the OPN, BSP, OCN, and P38 gene and protein expressions and reduced osteoblast cellular activity and mineralized nodules. CONCLUSION: The present study demonstrated that DDR2 activation of p38 MAPK is an important approach to ON-regulating osteoblast mineralization.

Osteosarcoma: A case report and evaluation.

Osteosarcoma (OS) comprises 2.1% of all malignant oral and maxillofacial tumors. OS arising from the jaw OS differs from OS of the long bones in its biological behavior, presenting a lower incidence of metastasis and a better prognosis. The morphologic and behavioral heterogeneity observed in OS and the perplexity of the varied histological features mimicking other primary and metastatic bone tumors has emphasized the need of advanced molecular techniques in its diagnosis. Hereby, we present a case of OS which was diagnosed by immunohistochemical analysis, aiding in establishing its histogenetic origin.

AT1 and AT2 Receptor Knockout Changed Osteonectin and Bone Density in Mice in Periodontal Inflammation Experimental Model.

BACKGROUND: The aim of this study was to evaluate the role of AT1 and AT2 receptors in a periodontal inflammation experimental model. METHODS: Periodontal inflammation was induced by LPS/Porphyromonas gingivalis. Maxillae, femur, and vertebra were scanned using Micro-CT. Maxillae were analyzed histopathologically, immunohistochemically, and by RT-PCR. RESULTS: The vertebra showed decreased BMD in AT1 H compared with WT H (p < 0.05). The femur showed increased Tb.Sp for AT1 H and AT2 H, p < 0.01 and p < 0.05, respectively. The Tb.N was decreased in the vertebra (WT H-AT1 H: p < 0.05; WT H-AT2 H: p < 0.05) and in the femur (WT H-AT1 H: p < 0.01; WT H-AT2 H: p < 0.05). AT1 PD increased linear bone loss (p < 0.05) and decreased osteoblast cells (p < 0.05). RANKL immunostaining was intense for AT1 PD and WT PD (p < 0.001). OPG was intense in the WT H, WT PD, and AT2 PD when compared to AT1 PD (p < 0.001). AT1 PD showed weak immunostaining for osteocalcin compared with WT H, WT PD, and AT2 PD (p < 0.001). AT1 H showed significantly stronger immunostaining for osteonectin in fibroblasts compared to AT2 H (p < 0.01). CONCLUSION: AT1 receptor knockout changed bone density, the quality and number of bone trabeculae, decreased the number of osteoblast cells, and increased osteonectin in fibroblasts.

Supplementation of Calcium and Fluoride-Free Water Mitigates Skeletal Fluorosis in Fluoride-Intoxicated Rats.

Fluorosis is a public health concern in 25 countries around the globe. The present study is about the mitigation of fluoride (F) toxicity by giving F-free water (FFW) and calcium (Ca). A study was conducted by taking 76 Wistar rats in two phases, phase I (6 months), where rats were randomly divided into four groups: normal-Ca diet (NCD) 0.5%; low-Ca diet (LCD) 0.25%; NCD + 100 ppm F and LCD + 100 ppm F in groups 1, 2, 3 and 4, respectively. F and Ca were given through water and diet respectively. Phase II is the reversal of fluorosis for 3 months, where LCD group 2 was treated with NCD. Groups 3 and 4 were divided into two subgroups each: 3X and 3Y, and 4X and 4Y, respectively. Groups 3X and 4X received FFW with NCD. Group 3Y continued as phase I and 4Y NCD and F. The biochemical expression, gene expression, biomechanical properties and DXA were studied by standard methods. The results revealed that in phase I, bone turnover was significantly increased whereas bone mineral content and biomechanical properties of group 4 were significantly decreased (p ≤ 0.05) as compared with that of all other groups. Trabecular separation and total porosity increased in groups 2 and 4. Expression of osteocalcin, osteonectin and osteopontin genes was significantly downregulated in group 4. Bone turnover in group 4X was normalised. Expressions of osteocalcin, osteonectin and osteopontin were upregulated after providing NCD and FFW. In conclusion, low calcium aggravates skeletal fluorosis which could be mitigated on supplementation of Ca and FFW.

The Influence of Calcification Factors and Endothelial-Dysfunction Factors on the Development of Unstable Atherosclerotic Plaques.

BACKGROUND: This study aimed to evaluate changes in markers of calcification and of endothelial dysfunction during the development of calcification and instability of atherosclerotic plaques and to identify associations of calcification factors with the formation of unstable plaques. METHODS: We analyzed 44 male patients with coronary atherosclerosis who underwent endarterectomy in coronary arteries during coronary bypass surgery. The endarterectomy material (intima/media) was examined using histological and biochemical methods, and the stability and calcification degree of atherosclerotic plaques were assessed. In homogenates of the tissue samples and in blood, concentrations of osteoprotegerin, osteocalcin, osteopontin, osteonectin, monocyte-chemoattractant protein type 1 (MCP-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), and E-selectin were determined by enzyme immunoassays. RESULTS: Unstable atherosclerotic plaques proved to be calcified more frequently (80.4% of plaques) than stable ones (45.0%). Osteonectin, E-selectin, and sVCAM-1 levels were lower in unstable plaques and plaques with large calcification deposits. Osteocalcin content increased with the increasing size of the calcification deposits in plaque. Blood osteocalcin concentration directly correlated with osteocalcin concentration in atherosclerotic plaques and was higher in the blood of patients with calcified plaques in coronary arteries. CONCLUSIONS: The results provide the basis for further research on the suitability of osteocalcin as a potential biomarker of an unstable calcified atherosclerotic plaque in a coronary artery.

Graft application and dexamethasone treatment influences new bone formation in rat tibial bone defects / Aplicación de injerto y tratamiento con dexametasona influyen en la formación de hueso nuevo en los defectos en la tibia de rata

SUMMARY: The objective of this study were bone defect complications that occur due to traumas or infections. Bone grafts are required to provide support, fill gaps and improve biological repair in skeletal damage. Dexamethasone plays role in calcium signaling modulation and used in diseases. Aim of this study was to evaluate osteonectin and osteopontin expressions in new bone development after dexamethasone application on tibial bone defects. Rats were divided into defect, defect+graft and defect+graft+dexamethasone treated groups. Tibial bone defect created, and rats were kept immobile for 28 days. Alloplastic material was placed in defect area in second and group third groups. 2.5 mg/kg Dex and normal saline were injected to dexamethasone and defect groups twice a week for 56 days. Inflammation and congestion were increased in defect and defect+graft groups. Defect+graft+dexamethasone group; increased number of osteoblast and osteocyte cells, dense bone matrix, formation of new bone trabeculae was observed. Defect+graft group; osteonectin expression in graft regions, osteoblast cells, some connective tissue cells and fibers were seen whereas in defect+graft+dexamethasone group; osteopontin expression in osteoblast and osteocyte cells of new bone trabeculae were observed. Dexamethasone may lead to formation of new bone trabeculae into the graft material resulting in increased osteoconduction and osteoinductive effect for differentiation of osteon.

RESUMEN: Los defectos óseos son complicaciones que ocurren debido a traumas o infecciones. Se requieren injertos óseos para proporcionar apoyo, llenar los espacios y mejorar la reparación biológica en el hueso dañado. La dexametasona desempeña un papel importante en la modulación de la señalización del calcio y se usa en enfermedades. El objetivo de este estudio fue evaluar las expresiones de osteonectina y osteopontina en el desarrollo óseo después de la aplicación de dexametasona en defectos óseos tibiales. Las ratas se dividieron en grupos: defecto, defecto + injerto y defecto + injerto + grupos tratados con dexametasona. Se creó un defecto óseo tibial, y las ratas se mantuvieron inmóviles durante 28 días. El material aloplástico se colocó en el área del defecto en el segundo y tercer grupo. Se inyectaron 2,5 mg / kg de dexametasona y solución salina normal a grupos de defectos dos veces por semana durante 56 días. La inflamación y la congestión aumentaron en los grupos de defectos y defectos + injerto; En el grupo defecto + injerto + grupo tratado con dexametasona se observó un aumento en el número de osteoblastos y osteocitos, de matriz ósea densa y en la formación de nuevas trabéculas óseas. En el grupo defecto + grupo de injerto se observó la expresión de osteonectina en las áreas de injerto, osteoblastos, algunas células y fibras de tejido conectivo, mientras que en el grupo defecto + injerto + dexametasona se observó la expresión de osteopontina en osteoblastos y osteocitos y formación de nuevas trabéculas óseas . En conclusión la dexametasona puede conducir a la formación de nuevas trabéculas óseas en el material de injerto, lo que resulta en un aumento de la osteoconducción y un efecto osteoinductivo para la diferenciación del osteón.

A Unique Population of Regulatory T Cells in Heart Potentiates Cardiac Protection From Myocardial Infarction.

BACKGROUND: Regulatory T cells (Tregs), traditionally recognized as potent suppressors of immune response, are increasingly attracting attention because of a second major function: residing in parenchymal tissues and maintaining local homeostasis. However, the existence, unique phenotype, and function of so-called tissue Tregs in the heart remain unclear. METHODS: In mouse models of myocardial infarction (MI), myocardial ischemia/reperfusion injury, or cardiac cryoinjury, the dynamic accumulation of Tregs in the injured myocardium was monitored. The bulk RNA sequencing was performed to analyze the transcriptomic characteristics of Tregs from the injured myocardium after MI or ischemia/reperfusion injury. Photoconversion, parabiosis, single-cell T-cell receptor sequencing, and adoptive transfer were applied to determine the source of heart Tregs. The involvement of the interleukin-33/suppression of tumorigenicity 2 axis and Sparc (secreted acidic cysteine-rich glycoprotein), a molecule upregulated in heart Tregs, was further evaluated in functional assays. RESULTS: We showed that Tregs were highly enriched in the myocardium of MI, ischemia/reperfusion injury, and cryoinjury mice. Transcriptomic data revealed that Tregs isolated from the injured hearts had plenty of differentially expressed transcripts in comparison with their lymphoid counterparts, including heart-draining lymphoid nodes, with a phenotype of promoting infarct repair, indicating a unique characteristic. The heart Tregs were accumulated mainly because of recruitment from the circulating Treg pool, whereas local proliferation also contributed to their expansion. Moreover, a remarkable case of repeatedly detected T-cell receptor of heart Tregs, more than that of spleen Tregs, suggests a model of clonal expansion. Besides, HelioshighNrp-1high phenotype proved the mainly thymic origin of heart Tregs, with a small contribution of phenotypic conversion of conventional CD4+ T cells, proved by the analysis of T-cell receptor repertoires and conventional CD4+ T cells adoptive transfer experiments. The interleukin-33/suppression of tumorigenicity 2 axis was essential for sustaining heart Treg populations. Last, we demonstrated that Sparc, which was highly expressed by heart Tregs, acted as a critical factor to protect the heart against MI by increasing collagen content and boosting maturation in the infarct zone. CONCLUSIONS: We identified and characterized a phenotypically and functionally unique population of heart Tregs that may lay the foundation to harness Tregs for cardioprotection in MI and other cardiac diseases.