Rat Femur Research Articles

Disulfidptosis-related Protein RPN1 may be a Novel Anti-osteoporosis Target of Kaempferol.

Osteoporosis (OP) is an age-related skeletal disease. Kaempferol can regulate bone mesenchymal stem cells (BMSCs) osteogenesis to improve OP, but its mechanism related to disulfidptosis, a newly discovered cell death mechanism, remains unclear. The study aimed to investigate the biological function and immune mechanism of disulfidptosis- related ribophorin I (RPN1) in OP and to experimentally confirm that RPN1 is the target for the treatment of OP with kaempferol. Differential expression analysis was conducted on disulfide-related genes extracted from the GSE56815 and GSE7158 datasets. Four machine learning algorithms identified disease signature genes, with RPN1 identified as a significant risk factor for OP through the nomogram. Validation of RPN1 differential expression in OP patients was performed using the GSE56116 dataset. The impact of RPN1 on immune alterations and biological processes was explored. Predictive ceRNA regulatory networks associated with RPN1 were generated via miRanda, miRDB, and TargetScan databases. Molecular docking estimated the binding model between kaempferol and RPN1. The targeting mechanism of kaempferol on RPN1 was confirmed through pathological HE staining and immunohistochemistry in ovariectomized (OVX) rats. RPN1 was abnormally overexpressed in the OP cohort, associated with TNF signaling, hematopoietic cell lineage, and NF-kappa B pathway. Immune infiltration analysis showed a positive correlation between RPN1 expression and CD8+ T cells and resting NK cells, while a negative correlation with CD4+ naive T cells, macrophage M1, T cell gamma delta, T cell follicular helper cells, activated mast cells, NK cells, and dendritic cells, was found. Four miRNAs and 17 lncRNAs associated with RPN1 were identified. Kaempferol exhibited high binding affinity (-7.2 kcal/mol) and good stability towards the RPN1. The experimental results verified that kaempferol could improve bone microstructure destruction and reverse the abnormally high expression of RPN1 in the femur of ovariectomized rats. RPN1 may be a new diagnostic biomarker in patients with OP, and may serve as a new target for kaempferol to improve OP.

Inflammatory response toward a Mg-based metallic biomaterial implanted in a rat femur fracture model

The immune system plays an important role in fracture healing, by modulating the pro-inflammatory and anti-inflammatory responses occurring instantly upon injury. An imbalance in these responses can lead to adverse outcomes, such as non-union of fractures. Implants are used to support and stabilize complex fractures. Biodegradable metallic implants offer the potential to avoid a second surgery for implant removal, unlike non-degradable implants. However, considering our dynamic immune system it is important to conduct in-depth studies on the immune response to these implants in living systems. In this study, we investigated the immune response to Mg and Mg-10Gd in vivo in a rat femur fracture model with external fixation. In vivo imaging using liposomal formulations was used to monitor the fluorescence-related inflammation over time. We combine ex vivo methods with our in vivo study to evaluate and understand the systemic and local effects of the implants on the immune response. We observed no significant local or systemic effects in the Mg-10Gd implanted group compared to the SHAM and Mg implanted groups over time. Our findings suggest that Mg-10Gd is a more compatible implant material than Mg, with no adverse effects observed in the early phase of fracture healing during our 4-week study. Statement of significanceDegradable metallic implants in form of Mg and Mg-10Gd intramedullary pins were assessed in a rat femur fracture model, alongside a non-implanted SHAM group with special respect to the potential to induce an inflammatory response. This pre-clinical study combines innovative non-invasive in vivo imaging techniques associated with multimodal, ex vivo cellular and molecular analytics. The study contributes to the development and evaluation of degradable biometals and their clinical application potential. The study results indicate that Mg-10Gd did not exhibit any significant harmful effects compared to the SHAM and Mg groups.

In vitro and in vivo studies on exogenous polyamines and α-difluoromethylornithine to enhance bone formation and suppress osteoclast differentiation

Exogenous polyamines, including putrescine (PUT), spermidine (SPD), and spermine (SPM), and the irreversible inhibitor of the rate-limiting enzyme ornithine decarboxylase (ODC) of polyamine biosynthesis, α-difluoromethylornithine (DFMO), are implicated as stimulants for bone formation. We demonstrate in this study the osteogenic potential of exogenous polyamines and DFMO in human osteoblasts (hOBs), murine monocyte cell line RAW 264.7, and an ovariectomized rat model. The effect of polyamines and DFMO on hOBs and RAW 264.7 cells was studied by analyzing gene expression, alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) activity, and matrix mineralization. Ovariectomized rats were treated with polyamines and DFMO and analyzed by micro computed tomography (micro CT). The mRNA level of the early onset genes of osteogenic differentiation, Runt-related transcription factor 2 (Runx2) and ALP, was significantly elevated in hOBs under osteogenic conditions, while both ALP activity and matrix mineralization were enhanced by exogenous polyamines and DFMO. Under osteoclastogenic conditions, the gene expression of both receptor activator of nuclear factor-κB (RANK) and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) was reduced, and TRAP activity was suppressed by exogenous polyamines and DFMO in RAW 264.7 cells. In an osteoporotic animal model of ovariectomized rats, SPM and DFMO were found to improve bone volume in rat femurs, while trabecular thickness was increased in all treatment groups. Results from this study provide in vitro and in vivo evidence indicating that polyamines and DFMO act as stimulants for bone formation, and their osteogenic effect may be associated with the suppression of osteoclastogenesis.

Membrane Formed Around Liquid Nitrogen-treated Bone Promotes Osteogenesis and Revitalization in a Rat Model.

This study aimed to investigate the structure and functions of the membrane formed around liquid nitrogen-treated bones in the osteogenesis and revitalization of frozen bone using a rat model. Segmental defects were created in femurs of rats, and resected bones treated with liquid nitrogen [frozen bone (FB) group, n=20] or polymethylmethacrylate (PMMA group; n=20) were implanted as spacers. Histological analysis and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) of the membrane around each spacer were performed for bone morphogenetic protein 2 (BMP2), transforming growth factor (TGF)-β1, and vascular endothelial growth factor (VEGF). Furthermore, in week 2, spacers were removed from both groups (n=5 each), and autologous cancellous bone (ACB) harvested from the ilium was grafted into the defect. Radiological analysis was performed until bone union was observed. In week 2, similar two-layered membrane structures were observed in both groups; these matured into fibrous tissues over time. At each evaluation point, qRT-PCR showed higher expression of all factors in the FB than in the PMMA group. In the ACB graft model, the mean period to bone union and new bone volume were significantly shorter and greater, respectively, in the FB. Chondrocytes invaded the osteotomy site from the membrane in the FB, suggesting that endochondral ossification may occur and be related to osteogenesis. Additionally, fibroblasts and capillaries in the membrane invaded the surface of treated bone in week 2, and osteocytes were observed around them in weeks 6 and 8. Fibrous membranous tissue formed around liquid nitrogen-treated bones may be vital for osteogenesis and revitalization of frozen bones.

Morphometric analysis of rat femurs submitted to the use of dexamethasone and the consumption of curcumin and symbiotics

The aim was to analyze whether the deleterious effects of dexamethasone on bone can be prevented or mitigated by using curcumin and the symbiotic concomitantly. The rats were divided into control (CT), dexamethasone (D), dexamethasone curcumin (DC), dexamethasone symbiotic (DS) and dexamethasone symbiotic/curcumin (DCS) groups. The thickness, area, and area fraction of the bone trabeculae of the femoral head were measured. The CT group showed greater trabecular thickness when compared to the D and DS groups. The thicknesses of the DC and DCS groups were greater than those of the DS group. The area and area fraction were smaller in groups D, DC and DS compared to CT. It can be concluded that curcumin, when consumed in isolation, had a slight effect on maintaining the thickness of trabecular bone. Considering its action on the structuring of the trabecular area, it is not possible to establish a clear relationship between its benefit for bone preservation and its interference with bone cell activity.

Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women

BackgroundMechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model.MethodsBM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation.ResultsMS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model.ConclusionsData suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.

Vitamin D supplementation affects bone marrow-derived mesenchymal stem cells differentiation into insulin-producing cells.

Background this study was conducted to assess the effects of vitamin D on differentiation of bone marrow- derived mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs). Method BM-MSCs were isolated from femur and tibia of rats and incubated in low (LG) or high glucose (HG) (5mM or 25mM), or high glucose DMEM media supplemented with vitamin D (0.2nM) (HGD) for 14 days. Cells viability was analysis by MTT assay. Differentiation of SCs was confirmed using measuring genes expression level of pdx1 and insulin, and insulin secretion, glucose stimulated insulin secretion, and insulin content by ELISA method. Results Cell viability was significantly higher in HGD than LG (p < 0.05) in day 3, also, in HG and HGD than LG (p < 0.001), and HGD vs. HG (p < 0.001) in day 7. Pdx1 and insulin level was markedly higher in HGD than LG (p < 0.05 and p < 0.01). pdx1 expression was markedly higher in HGD (p < 0.05) than LG, also insulin expression the HG (p < 0.05), and HGD (p < 0.01) groups compared to the LG group. Insulin release at 5mM glucose was notably higher in the HGD group compared to LG (p < 0.05), and at 25mM glucose, both HG and HGD showed significant increases vs. LG (p < 0.05 and p < 0.01, respectively). Insulin content was significantly higher in both 5mM and 25mM glucose for HG and HGD vs. LG (p < 0.01 and p < 0.001, respectively). In conclusion, treatment BM-MSCs with vitamin D could increase their differentiation into IPCs and it can be considered as a potential supplementary agent in enhancing differentiation SCs into insulin generating cells.

MiRNA-seq analysis of high glucose induced osteoblasts provides insight into the mechanism underlying diabetic osteoporosis

The present study aims to explore the etiology of Diabetic osteoporosis (DOP), a chronic complication associated with diabetes mellitus. Specifically, the research seeks to identify potential miRNA biomarkers of DOP and investigated role in regulating osteoblasts. To achieve this, an animal model of DOP was established through the administration of a high-sugar and high-fat diet, and then injection of streptozotocin. Bone microarchitecture and histopathology analysis were analyzed. Rat calvarial osteoblasts (ROBs) were stimulated with high glucose (HG). MiRNA profiles of the stimulated osteoblasts were compared to control osteoblasts using sequencing. Proliferation and mineralization abilities were assessed using MTT assay, alkaline phosphatase, and alizarin red staining. Expression levels of OGN, Runx2, and ALP were determined through qRT-PCR and Western blot. MiRNA-sequencing results revealed increased miRNA-702-5p levels. Luciferase reporter gene was utilized to study the correlation between miR-702-5p and OGN. High glucose impaired cell proliferation and mineralization in vitro by inhibiting OGN, Runx2, and ALP expressions. Interference with miR-702-5p decreased OGN, Runx2, and ALP levels, which were restored by OGN overexpression. Additionally, downregulation of OGN and Runx2 in DOP rat femurs was confirmed. Therefore, the miRNA-702-5p/OGN/Runx2 signaling axis may play a role in DOP, and could be diagnostic biomarker and therapeutic target for not only DOP but also other forms of osteoporosis.

Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model.

Modified and alternative bone cements can improve the induced membrane: Critical size bone defect model in rat femur

BackgroundAs a two-stage surgical procedure, Masquelet's technique has been used to care for critical-size bone defects (CSD). We aimed to determine the effects of modified and altered bone cement with biological or chemical enriching agents on the progression of Masquelet's induced membrane (IM) applied to a rat femur CSD model, and to compare the histopathological, biochemical, and immunohistochemical findings of these cements to enhance IM capacity. MethodsThirty-five male rats were included in five groups: plain polymethyl methacrylate (PMMA), estrogen-impregnated PMMA (E+PMMA), bone chip added PMMA (BC+PMMA), hydroxyapatite-coated PMMA (HA) and calcium phosphate cement (CPC). The levels of bone alkaline phosphatase (BALP), osteocalcin (OC), and tumor necrosis factor-alpha (TNF-α) were analyzed in intracardiac blood samples collected at the end of 4 weeks of the right femur CSD intervention. All IMs collected were fixed and prepared for histopathological scoring. The tissue levels of rat-specific Transforming Growth Factor-Beta (TGF-β), Runt-related Transcription Factor 2 (Runx2), and Vascular Endothelial Growth Factor (VEGF) were analyzed immunohistochemically. ResultsSerum levels of BALP and OC were significantly higher in E+PMMA and BC+PMMA groups than those of other groups (P = 0.0061 and 0.0019, respectively). In contrast, TNF-α levels of all groups with alternative bone cement significantly decreased compared to bare PMMA (P = 0.0116). Histopathological scores of E+PMMA, BC+PMMA, and CPC groups were 6.86 ± 1.57, 4.71 ± 0.76, and 6.57 ± 1.51, respectively, which were considerably higher than those of PMMA and HA groups (3.14 ± 0.70 and 1.86 ± 0.69, respectively) (P < 0.0001). Significant increases in TGF-β and VEGF expressions were observed in E+PMMA and CPC groups (P = 0.0001 and <0.0001, respectively) whereas Runx2 expression significantly increased only in the HA group compared to other groups (P < 0.0001). ConclusionsThe modified PMMA with E and BC, and CPC as an alternative spacer resulted in a well-differentiated IM and increased IM progression by elevating BALP and OC levels in serum and by mediating expressions of TGF-β and VEGF at the tissue level. Estrogen-supplemented cement spacer has yielded promising findings between modified and alternative bone cement.

Bifunctional Piezo‐Enhanced PLLA/ZA Coating Prevents Aseptic Loosening of Bone Implants

AbstractPrevention of aseptic loosening, the main reason for secondary surgery of bone implants, is of great crucial clinical importance. As a promising bioactive material to promote bone regeneration, the piezoelectric materials for preventing aseptic loosening has not been reported. Here, a surface coating is presented for implants based on piezoelectric poly(L‐lactic acid) (PLLA). The coating ingeniously utilizes zoledronic acid (ZA), a drug used to treat osteoporosis, as a nucleating agent to enhance the piezoelectricity of PLLA. The coating can not only provide a continuous porous piezoelectric environment to stimulate osteogenic differentiation without intervention, but also effectively inhibit the osteolytic activity of osteoclasts through the slow release of ZA, achieving long‐term effectiveness with a single implantation. The rat femur cavity implantation experiment suggests that, the PLLA/ZA coating can rapidly initiate osseointegration of the implant and effectively mitigate the risk of aseptic loosening, even in the presence of metal submicroscopic particles that induce aseptic loosening. This strategy of piezo‐enhanced PLLA drug‐loaded coating may provide a new idea for the surface modification of bone implants.

Mechanism of action of Sambucus williamsii Hance var. miquelii in the treatment of osteoporosis analyzed by UHPLC-HRMS/MS combined network pharmacology and experimental validation

Sambucus williamsii Hance var. miquelii(SWH) is a precious wild Chinese herb whose fruit, rhizome, leaves and root bark can be used as medicine. Sambucus Linn has pharmacological effects such as anti-osteoporosis, promoting fracture healing, anti-viral and anti-inflammatory. In this study, the main chemical components of the alcoholic extracts from SWH were rapidly identified by ultra-high performance liquid chromatography-quadrupole orbit trap high-resolution mass spectrometry (UHPLC- HRMS MS), and a total of 42 compounds were characterized from the alcoholic extracts of SWH. The results of network pharmacological validation showed that kaempferol, quercetin, luteolin, isorhamnetin and morroniside were the main active components, and KEGG enrichment demonstrated that SWH mainly affected the signaling pathways such as PI3K-Akt, TNF and FoxO by modulating the related targets such as AKT1, PIK3R1, EGFR, RELA SRC and PTGS2. The molecular docking results showed binding solid activity between the main active components of SWH and the targets. The network pharmacology was validated by establishing an animal model of osteoporosis (OP) in rats by gavage administration of vitamin A acid. The results of the pharmacological experiments showed that SWH could improve the degree of bone loss in the femur of osteoporotic rats, increase the number of trabeculae and decrease trabeculae porosity, up-regulate the Ca and P content in the serum of OP rats, down-regulate the scope of ALP and BGP in the serum, and promote the calcification of the bone matrix, and then exert the anti-OP efficacy. In this study, network pharmacology and pharmacological experiments verified the pharmacological mechanism of SWH in anti-OP rats. This provides a theoretical basis for the research and development of anti-OP drugs and a reference for the application of other traditional Chinese medicines in treating OP diseases.

Effects of Vancomycin/Tobramycin-Doped Ceramic Composite (Polyvinyl Alcohol Composite–Vancomycin/Tobramycin–Polymeric Dicalcium Phosphate Dihydrate) in a Rat Femur Model Implanted With Contaminated Porous Titanium Cylinders

BackgroundPeriprosthetic joint infection (PJI) remains common and problematic. We hypothesized that using a bioceramic that provided rapid release of the antibiotics (vancomycin [VAN] or VAN and tobramycin [VAN and TOB]) from a polyvinyl-alcohol-composite (PVA) combined with a delayed and sustained antibiotic release from polymeric-dicalcium-phosphate-dihydrate (PDCPD) ceramic would inhibit S. aureus-associated implant infections. MethodsA total of 50 male Sprague Dawley rats were randomly divided into 5 groups—I: negative control; II: bacteria only; III: bacteria + saline wash; IV: bacteria + PVA-VAN-PDCPD, and V: bacteria + PVA-VAN-TOB-PDCPD. A porous titanium (Ti) implant was press-fit into the rat knee. S. aureus-containing broth was added into the joint space creating a PJI. After 1 week, the joints from groups III to V were washed with saline and the fluid collected for bacterial quantification. This was followed by saline irrigation treatment (groups III to V) and application of the antibiotic-loaded PVA-PDCPD bioceramic (groups IV and V). On day 21, joint fluid was collected, and the implants harvested for bacterial quantification. ResultsNo bacteria were isolated from the negative control (group I). The positive control (group II) was positive on both days 7 and 21. Bacteria were still present on day 21 in the fluid and implant in group III. Groups (IV and V) showed a decrease in the bacterial burden in the fluid and implant on day 21. There were significant differences in bacteria levels in the collected wash fluid and on the implant at day 21 between the saline wash (group III) and treatment groups (IV and V). ConclusionsIn this animal model of acute periprosthetic infection, treatment with PVA-VAN-PDCPD and PVA-VAN/TOB-PDCPD reduced bacterial load in the infected joint and the infected Ti implant. Application of PVA-VAN-PDCPD and/or PVA-VAN/TOB-PDCPD after saline irrigation could be used as an addition to the treatment of PJI.

Feasibility Insights of the Green-Assisted Calcium-Phosphate Coating on Biodegradable Zinc Alloys for Biomedical Application: In Vitro and In Vivo Studies.

In the field of bone tissue engineering, recently developed Zn alloy scaffolds are considered potential candidates for biodegradable implants for bone regeneration and defect reconstruction. However, the clinical success of these alloys is limited due to their insufficient surface bioactivities. Further, the higher concentration of Zn2+ produced during degradation promotes antibacterial activity, but deteriorates osteogenic properties. This study fabricated an Azadirachta indica (neem)-assisted brushite-hydroxyapatite (HAp) coating on the recently developed Zn-2Cu-0.5Mg alloy to tackle the above dilemma. The microstructure, degradation behavior, antibacterial activity, and hemocompatibility, along with in vitro and in vivo cytocompatibility of the coated alloys, are systematically investigated. Microstructural analysis reveals flower-like morphology with uniformly grown flakes for neem-assisted deposition. The neem-assisted deposition significantly improves the adhesion strength from 12.7 to 18.8 MPa, enhancing the mechanical integrity. The potentiodynamic polarization study shows that the neem-assisted deposition decreases the degradation rate, with the lowest degradation rate of 0.027 mm/yr for the ZHN2 sample. In addition, the biomineralization process shows the apatite formation on the deposited coating after 21 days of immersion. In vitro cytotoxicity assay exhibits the maximum cell viability of 117% for neem-assisted coated alloy in 30% extract after 5d and the improved cytocompatibility which is due to the controlled release of Zn2+ ions. Meanwhile, neem-assisted coated alloy increases the ZOI by 32 and 24% for Gram-positive and Gram-negative bacteria, respectively. Acceptable hemolysis (<5%) and anticoagulation parameters demonstrate a promising hemocompatibility of the coated alloy. In vivo implantation illustrates a slight inflammatory response and vascularization after 2 weeks of subcutaneous implantation, and neo-bone formation in the defect areas of the rat femur. Micro-CT and histology studies demonstrate better osseointegration with satisfactory biosafety response for the neem-assisted coated alloy as compared to that without neem-assisted deposition. Hence, this neem-assisted brushite-Hap coating strategy elucidates a new perspective on the surface modification of biodegradable implants for the treatment of bone defects.

Ex vivo organotypic bone slice culture reveals preferential chondrogenesis after sustained growth plate injury

Postnatal bone growth primarily relies on chondrocyte proliferation and osteogenic differentiation within the growth plate (GP) via endochondral ossification. Despite its importance, the GP is vulnerable to injuries, affecting 15–30 % of bone fractures. These injuries may lead to growth discrepancies, influence bone length and shape, and negatively affecting the patient's quality of life. This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained growth plate injury (GPI) in an ex vivo rat femur organotypic culture (OTC) model. Specifically, focusing on postnatal endochondral ossification process. 300 μm thick ex vivo bone cultures with a 2 mm long horizontal GPI was utilized. After 15 days of cultivation, gene expression analysis, histological and immunohistochemistry staining's were conducted to analyze key markers of endochondral ossification. In our OTCs we observed a significant increase in Sox9 expression due to GPI at day 15. The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation. Ihh levels increased significantly on day 7 and day 15, while PTHrP was downregulated on day 7. GPI had no impact on osteoclast number and activity, but gene expression analysis indicated OTCs' efforts to inhibit osteoclast differentiation and activation, thereby reducing bone resorption.In conclusion, our study provides novel insights into the molecular and cellular mechanisms underlying postnatal bone growth and regeneration following growth plate injury (GPI). We demonstrate that chondrocyte proliferation and differentiation play pivotal roles in the regeneration process, with the Ihh-PTHrP feedback loop modulating these processes. Importantly, our ex vivo rat femur organotypic culture model allows for the detailed investigation of these processes, providing a valuable tool for future research in the field of skeletal biology and regenerative medicine.

The Effect of Cell Culture Passage on the Efficacy of Mesenchymal Stromal Cells as a Cell Therapy Treatment.

Background/Objective: Mesenchymal Stromal Cells (MSCs) have been considered a promising treatment for several diseases, such as cardiac injuries. Many studies have analyzed their functional properties; however, few studies have characterized MSCs through successive culture passages. The main objective of this work was to analyze the phenotype and functionality of MSCs isolated from two different sources in five culture passages to determine if the culture passage might influence the efficacy of MSCs as a cell therapy treatment. Methods: Bone Marrow (BM)-MSCs were harvested from the femur of Wistar rats (n = 17) and Adipose Tissue(AT)-MSCs were isolated from inguinal fat (n = 17). MSCs were cultured for five culture passages, and the immunophenotype was analyzed by flow cytometry, the functionality was characterized by adipogenic, osteogenic, and chondrogenic differentiation assays, and cytokine secretion capacity was determined through the quantification of the Vascular Endothelial Growth-Factor, Fibroblast Growth-Factor2, and Transforming Growth-Factorβ1 in the cell supernatant. The ultrastructure of MSCs was analyzed by transmission electron microscopy. Results: BM-MSCs exhibited typical phenotypes in culture passages two, four, and five, and their differentiation capacity showed an irregular profile throughout the five culture passages analyzed. AT-MSCs showed a normal phenotype and differentiation capacity in all the culture passages. BM- and AT-MSCs did not modify their secretion ability or ultrastructural morphology. Conclusions: Throughout the culture passages, BM-MSCs, but not AT-MSCs, exhibited changes in their functional and phenotypic characteristic that might affect their efficacy as a cell therapy treatment. Therefore, the culture passage selected should be considered for the application of MSCs as a cell therapy treatment.

The state of the liver, reproductive and musculoskeletal systems in female rats with pro-longed exposure to α-cypermethrin

Pyrethroid insecticides are currently a widely used class of pesticides. It is therefore important to determine the mechanism of disorders in some mammalian systems due to prolonged exposure to these pesticides and to justify means to prevent poisoning complications. The study was conducted on 30 female rats aged 3 months for 120 days. Intoxication was modeled using α-cypermethrin at a dose of 10 mg/kg. The prevention of intoxication complications was carried out with an adaptogenic complex consisting of the flavonoid quercetin, vitamins C and D3, and minerals (Ca, Mn, Zn, Cu, Se, Mg) at a dose of 500 mg/kg. Assessment of intoxication and preventive efficacy of the adaptogen was performed by determining the duration of the estrous cycle, ovarian organ index, degree of atrophy of the alveolar process in the jaws, as well as density, content of mineral and organic components in the bones. Hepatotoxicity markers were determined in the blood serum, osteoresorption markers in the alveolar bone homogenate, inflammation indicators in the liver, and antioxidant system status in all tissues. Prolonged exposure to α-cypermethrin was associated with a prolongation of the estrous cycle due to a reduction in the diestrus stage and a decrease in the ovarian organ index along with a significant increase in blood estradiol levels. In intoxicated animals, the degree of atrophy of the alveolar process increased and the density of femurs and vertebrae decreased due to a decrease in the weight fraction of the mineral component in the bones. In the bone tissue of the alveolar process, an increase in the activity of elastase and markers of oxidative stress (an increase in the content of malondialdehyde and a decrease in catalase activity), as well as a compensatory increase in the alkaline phosphatase activity were found. In the rat liver, an increase in the acid phosphatase activity and the inhibition of antioxidant defense were observed. An increase in the elastase activity and a decrease in the catalase activity with an increase in the malondialdehyde content were found in the serum of the animals. The use of an adaptogen under conditions of α-cypermethrin intoxication contributed to the normalization of the estrous cycle, ovarian organ index and blood estradiol levels. In the tissue of the femur and vertebrae of rats, the treatment by the prophylactic complex led to an increase in bone density due to an increase in the content of the mineral component, and a decrease in the degree of atrophy of the jaws, in the tissue of which the activation of the enzymatic link of antioxidant defense and a decrease in the activity of destructive elastase too were found. The developed adaptogen prevented the development of oxidative stress and inflammation in the blood serum and liver in the animals. The results of the research indicate a negative effect of α-cypermethrin on the sexual function in female rats, on the state of bone tissue along with the hepatotoxic effect of the pesticide. The proposed agent for the prevention of α-cypermethrin intoxication effectively prevented endocrine disruption in the ovaries, bone destruction and inflammation in the liver.

PPARγ inhibition promotes osteogenic differentiation of bone marrow mesenchymal stem cells and fracture healing.

This study aimed to explore the effects of peroxisome proliferator-activated receptor γ (PPARγ) inhibition on fracture healing of nonunion and the underlying mechanisms. Bone marrow mesenchymal stem cells (BMSCs) were treated with PPARγ antagonist GW9662 (5 μM, 10 μM). Alkaline phosphatase (ALP) staining and Alizarin Red S was used to assess early stage of osteogenesis and osteogenic differentiation. GW9662 (1 mg/kg/day) were administered intraperitoneally into the rats with bone fracture. Bone healing processes in the rat femur fracture model were recorded and assessed by radiographic methods on Weeks 8, 14, and 20 postoperation. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative Week 20. GW9662 treatment increased ALP activity and Alp mRNA expression in rat BMSCs. Moreover, GW9662 administration increased matrix mineralization and mRNA and protein levels of Bmp2 and Runx2 in the BMSCs. In addition, GW9662 treatment improved radiographic score in the fracture rats and increased osteogenesis-related proteins, including type I collagen, osteopontin, and osteoglycin, in the bone tissues of the fracture sites. In conclusion, PPARγ inhibition promotes osteogenic differentiation of rat BMSCs, as well as improves the fracture healing of rats through Bmp2/Runx2 signaling pathway in the rat model of bone fracture.

Zinc-energized dynamic hydrogel accelerates bone regeneration via potentiating the coupling of angiogenesis and osteogenesis.

Insufficient initial vascularization plays a pivotal role in the ineffectiveness of bone biomaterials for treating bone defects. Consequently, enhancing the angiogenic properties of bone repair biomaterials holds immense importance in augmenting the efficacy of bone regeneration. In this context, we have successfully engineered a composite hydrogel capable of promoting vascularization in the process of bone regeneration. To achieve this, the researchers first prepared an aminated bioactive glass containing zinc ions (AZnBg), and hyaluronic acid contains aldehyde groups (HA-CHO). The composite hydrogel was formed by combining AZnBg with gelatin methacryloyl (GelMA) and HA-CHO through Schiff base bonding. This composite hydrogel has good biocompatibility. In addition, the composite hydrogel exhibited significant osteoinductive activity, promoting the activity of ALP, the formation of calcium nodules, and the expression of osteogenic genes. Notably, the hydrogel also promoted umbilical vein endothelial cell migration as well as tube formation by releasing zinc ions. The results of in vivo study demonstrated that implantation of the composite hydrogel in the bone defect of the distal femur of rats could effectively stimulate bone generation and the development of new blood vessels, thus accelerating the bone healing process. In conclusion, the combining zinc-containing bioactive glass with hydrogels can effectively promote bone growth and angiogenesis, making it a viable option for the repair of critical-sized bone defects.

Ginsenoside Rc alleviates osteoporosis by the TGF-β/Smad signaling pathway.

Osteoporosis is a common chronic bone disorder in postmenopausal women. Ginsenosides are primary active components in ginseng and the effects of various ginsenoside variants in osteoporosis treatment have been widely revealed. We planned to explore the impact of ginsenoside Rc on bone resorption in an osteoporosis rat model. We used ovariectomized rats to assess the potential impact of ginsenoside Rc on osteoporosis. μ-CT was implemented for analyzing the microstructure of the distal left femur in rats. H&E staining together with Masson staining were applied for bone histomorphometry evaluation. ELISA kits were implemented to detect serum concentrations of TRACP-5b, OCN, CTX, as well as PINP. Ginsenoside Rc treatment lessened the serum levels of TRACP-5b as well as CTX, while increasing serum levels of OCN, and PINP of OVX rats. Moreover, we found that ginsenoside Rc contributed to the synthesis of type I collagen via increasing Col1a1 and Col1a2 levels in femur tissues of ovariectomized rats. Our findings also revealed that ginsenoside Rc activated the TGF-β/Smad pathway by increasing TGF-β as well as phosphorylated Smad2/3 protein levels. Ginsenoside Rc alleviates osteoporosis in rats through promoting the TGF-β/Smad pathway.