Rat Tibia Research Articles

P-1498. Contezolid is Active against Methicillin-Resistant Staphylococcus aureus in Experimental Rat Foreign Body Osteomyelitis

Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of orthopedic implant-associated infection, where S. aureus can survive intracellularly and form biofilms. Vancomycin has poor activity against staphyloccoccal biofilms. Although rifampin is effective against staphylococcoal biofilms and has intracellular activity, resistance may develop when it is used as a single agent. Contezolid is a novel oxazolidinone with reported activity against S. aureus in vitro and in vivo in both murine systemic and thigh infection models. Here, we assessed the in vivo activity of contezolid, with and without rifampin, in a rat model of foreign body osteomyelitis. Figure 1. Quantities of MRSA IDRL-6169 recovered from A. tibia B. K-wire. Circles represent values from individual animals; horizontal lines represent mean values. Compared to no treatment: *p=0.0186 **p<0.0002 Methods The left tibia of 60 male Wister rats were inoculated with arachidonic acid (sclerosing agent) and 106 cfu MRSA IDRL-6169 and a K-wire implanted. After four weeks, rats received either no treatment, contezolid (50 mg/kg orally, every 12 h), rifampin (10 mg/kg intraperitoneally every 12 h), rifampin plus contezolid, or vancomycin (75 mg/kg intraperitoneally, every 12 h) plus rifampin for 21 days. Following treatment, tibiae were harvested and cyropulverized. Tibiae and K-wires were cultured separately, and results reported as log10 cfu/gram of bone or K-wire (limit of detect 0.1 log10), respectively. Wilcoxon rank-sum test and false discovery rates were performed and p< 0.05 considered significant. Results Results are shown in Figure 1. MRSA was recovered from tibiae and K-wires of all untreated animals, at means of 5.1 log10 cfu/bone and 3.0 log10 cfu/K-wire, respectively. There was a 1.0 log10 cfu/g mean reduction in MRSA recovered from the tibiae of contezolid-treated rats compared to untreated rats (p=0.0186), and a 0.2 log10 reduction on K-wires. No MRSA was recovered from the tibiae or K-wires in animals receiving rifampin, whether alone or in combination with contezolid or vancomycin. Conclusion Contezolid was active against MRSA in a rat model of foreign body osteomyelitis, reducing bacterial loads in bone, albeit not on K-wires, unless co-administered with rifampin. Disclosures Robin Patel, MD, a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, a: See above|MicuRx Pharmaceuticals and BIOFIRE: Grant/Research Support|PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix, Oxford Nanopore Technologies and CARB-X: Advisor/Consultant|Up-to-Date and the Infectious Diseases Board Review Course.: Honoraria

Rubus Coreanus Enhances Peri-Implant Bone Healing and Biomineralization in Ovariectomized and Healthy Rats

Estrogen deficiency contributes to osteoporosis and can therefore compromise the peri-implant bone. Hence, this study evaluated peri-implant bone healing when Rubus coreanus (RC) was administered orally in ovariectomized and healthy rats. Forty 4-month-old female rats were divided into four groups: SHAM (healthy rats), SHAM/RC (healthy rats treated with RC), OVX (ovariectomized rats), and OVX/RC (ovariectomized rats treated with RC). The oral administration of RC started thirty days after ovariectomy, and implant placement into the rat tibia occurred ninety days after the ovariectomy. Euthanasia occurred sixty days after implantation. The analyses performed included removal torque, RT-PCR, confocal microscopy, and immunolabeling. A significance level of p < 0.05 was considered for all tests. The highest reverse torque values were observed in the SHAM/RC group, followed by the OVX/RC group. Confocal microscopy showed the greatest bone biomineralization in the SHAM/RC group, followed by the OVX/RC group. RT-PCR data indicated that RC decreased the RANKL/OPG ratio in both conditions. Immunohistochemistry demonstrated a balance between bone formation and resorption in all groups, especially stimulating osteoblastogenesis in both treated groups. In conclusion, RC enhanced peri-implant bone healing and biomineralization in both healthy and ovariectomized rats, with stronger effects in healthy rats, suggesting that estrogen may enhance its efficacy. These findings support RC’s potential as a prophylactic and therapeutic agent.

Tetramethylpyrazine promotes osteo-angiogenesis during bone fracture repair

BackgroundNonunion following a long bone fracture has gained a lot of attention due to the dreadful impact on the life quality of tremendous patients. Recent data have demonstrated the important involvement of angiogenesis in improving fracture healing. Tetramethylpyrazine (TMP) is an active component of Chinese herbal medicine with various biological activities including pro-angiogenesis property. However, the activity and mechanism of action of TMP in osteo-angiogenesis during bone fracture repair bone fracture healing remain unknown. In this study, TMP was tested for its specific activities in rat aortic endothelial cells (RAECs) and fractured rat model.MethodsThe effect of TMP on angiogenesis and migration in RAECs was detected by conducting matrigel tubulogenesis assay and transwell assay. Histopathological changes were observed in the rats from each group using H&E staining. The levels of inflammation and coagulation markers in rats were evaluated by ELISA. The expression of osteogenesis-related genes in rats was assessed by RT-qPCR and western blotting.ResultsTMP promoted angiogenesis processes and migratory ability in RAECs. TMP improved histopathological changes in fractured rat model. The concentration of inflammatory markers (IL-2, IL-6, IL-1beta) in the serum of fractured rats were suppressed by TMP treatment. TMP also had the potential to inhibit blood coagulation in rat tibia fracture model. In addition, the expression and protein levels of osteogenesis-related markers (ALP, Runx2, and OPN-1) were elevated by TMP in the tissues from the fractured rats. In mechanism, TMP significantly promoted the activation of VEGF/FLK1 pathway in vitro and in vivo.ConclusionTMP accelerated the repair of bone fracture by promoting angiogenesis and osteogenesis.

The Effect of Local Melatonin Application on Bone Fracture Healing in Rat Tibias.

Background and Objectives: This study aimed to histologically evaluate the effects of local melatonin application at different doses on bone fracture healing. Materials and Methods: Thirty rats were divided into three groups, with ten rats in each group. In the control group (n = 10), a fracture line was created in the tibial bones, and fracture osteosynthesis was performed without any additional procedure. In the local melatonin dose 1 (MLT D-1) group (n = 10), a fracture line was created in the tibial bones, and 1.2 mg of lyophilized powder melatonin was applied locally before fracture osteosynthesis. In the local melatonin dose 2 (MLT D-2) group (n = 10), a fracture line was created in the tibial bones, and 3 mg of lyophilized powder melatonin was applied locally before fracture osteosynthesis. After a 12-week healing period, all subjects were sacrificed, and tibial bones were collected for histomorphometric analysis. Results: The percentage of bone formation was significantly higher in the MLT D-1 and MLT D-2 groups than in the control group. There was no statistically significant difference between the MLT D-1 and MLT D-2 groups. Conclusions: In conclusion, the study demonstrated that local melatonin application supports bone fracture by increasing bone formation, although different doses of melatonin did not lead to significant variations in fracture healing.

Effect of Eight Months of Swimming on Bone Quality of Different Anatomical Regions: A Study on Wistar Rat Models

Swimming is a popular sport with several health benefits, but its effects on bone quality are controversial possibly due to distinct effects on different anatomical regions. Our aim was to investigate the effect of 8-month swimming on bone growth, mass, geometry, trabecular microarchitecture and osteocyte density of the lumbar vertebrae, femur and tibia of male rats. Wistar rat models were assigned to either a swimming (n = 10; 2h/d, 5 d/week) or a physically active control group (n = 10) for 8 months, after which they were sacrificed and their lumbar vertebrae, femur and tibia assessed for bone mass, cortical geometry, trabecular microarchitecture and osteocyte density through µ-CT and histology. Variables were compared between groups through independent samples t tests. Swimming animals displayed higher vertebral trabecular connectivity and lower trabecular separation compared to controls. However, femur length, trabecular and cortical bone mass and cortical thickness were lower compared to controls. At the tibia, animals from the swimming group also presented lower trabecular number and connectivity and higher trabecular separation. Osteocyte density at the femur and vertebra was similar between groups. Eight months of swimming negatively affected bone mass, cortical geometry and trabecular microarchitecture at the femur and tibia whilst having a favourable effect on vertebral trabecular microarchitecture. These results suggest that swimming has divergent effects on different anatomical regions.

Histochemical analysis of osteoclast and osteoblast distributions on hydroxyapatite/collagen bone-like nanocomposite embedded in rat tibiae.

Hydroxyapatite (HAp)/collagen (Col) cylinders with laminated collagen layers were implanted into the tibial diaphysis of rats and examined histochemically to clarify how the orientation of HAp and Col bone-like nanocomposite fibers in HAp/Col blocks affects bone resorption and formation. HAp/Col fibers were synthesized and compressed into cylindrical blocks to mimic bone nanostructures. These were implanted into the cortical bone cavities of 10-week-old male Wistar rats with fiber bundles parallel to the tibial surface. The implants were histologically analyzed at 3, 5, 7, 14, and 28 days after implantation. TRAP-positive osteoclasts appeared after 3-5 days in the lateral region of the graft, where the fiber ends were exposed, but not in the bottom region, where the HAp/Col fibers were parallel to the surface. Osteoclasts were observed in both regions by day 14. PHOSPHO1-positive osteoblasts were first detected on day 5, appearing slightly away from the cylinder laterally but directly on the bottom surface. A few osteoblasts contacted the block laterally, whereas many were observed on the new bone tissue at the bottom, between days 7 and 14. Bone formation was induced earlier in the bottom region, whereas lateral resorption was dominant. This suggested the uncoupling of bone resorption and formation in the early postimplantation stages. However, bone remodeling shifted to coupling between osteoclasts and osteoblasts throughout the cylinder by day 28. The orientation of HAp/Col fibers in HAp/Col graft materials substantially affected the preferential induction of bone resorption or formation during the early stages of bone regeneration.

The intensity of subacute local biological effects after the implantation of ALBO-OS dental materialbased on hydroxyapatite and poly(lactide-co-glycolide): invivo evaluation in rats.

This study aimed to evaluate the intensity of the subacute local biological effects after implantation and osseoconductive potential of novel hydroxyapatite-based bone substitute coated with poly (lactide-co-glycolide), named ALBO-OS, in comparison to Bio-Oss®. Fifteen male Wistar rats, randomly assigned into groups: 10, 20, and 30days (n꞊5), were subcutaneously implanted with ALBO-OS and Bio-Oss®. Furthermore, artificially made bone defects on both rat's tibias were implanted with experimental materials. Unimplanted defects represented negative control. After the animals' euthanizing, tissue samples were prepared and analyzed histologically and histomorphometrically. Normal healing of the epithelial tissue was observed, with no signs of infection or necrosis. Minimal vascular congestion was noted immediately around the graft, with no signs of tissue oedema, with a minimal capsule thickness. The applied material did not cause an inflammatory response (IR) of significant intensity, and 20days after implantation, the IR was mainly assessed as minimal. Thetibial specimen showed that ALBO-OS has good osseoconductive potential, similar to Bio-Oss®, as well as low levels of acute and subacute inflammation. The tested material exhibits satisfying biocompatibility, similar to Bio-Oss®.

Creation of a Tibia Extension Model With a Perforated Ilizarov Ring System: An Experimental Study in Rats.

Distraction osteogenesis is a valuable clinical technique used to address length discrepancies in long bone deformities. This procedure involves performing an osteotomy at an appropriate site in the bone and correcting the deformity through an extension system. This research aims to investigate the efficacy of a newly developed device for use in rat tibias and to provide an alternative to existing devices used in animal experiments. A total of 16 male Wistar-Albino rats, each weighing approximately 300-350 grams and aged 20-28 weeks, were used in the study. On the first day, a fixator was applied to the right tibias of the rats, and Ilizarov osteotomy was performed. Distraction of the tibia commenced on the Day 7. During the distraction phase, which lasted seven days, tibia lengthening was performed twice daily at 08:00 and 16:00, with each session involving a distraction of 0.25 mm. After a 14-day waiting period, evaluations were conducted on the 28th, 35th, 42nd, and 49th days post-surgery. Following these assessments, the rats were evaluated with X-rays and subsequently sacrificed. The distraction procedures proceeded largely without issues in the rats. Union was observed during the follow-up after distraction. In the initial postoperative X-ray of one rat, no problems with the reduction of the osteotomy were detected. To verify the functionality of the system, acute distraction was tested in one rat model, and successful elongation was achieved. However, one rat experienced circulatory disturbances post-operation, with the extremity showing an ecchymotic appearance. The extremity returned to its normal state during follow-up. Infection occurred in three rats. No postoperative antibiotic therapy was administered to any of the rat models. During follow-up, the infections resolved with regular dressing changes. Due to fewer complications and improved radiological imaging with the extension models performed in the metaphyseal region of rat tibias, our system could be utilized in future fracture model applications or distraction osteogenesis studies involving rat models. We believe it could serve as an alternative to other models for creating extension models at a lower cost.

Novel Subperiosteal Device Geometry and Investigation of Efficacy on Surrounding Bone Formation and Bone-Bonding Strength.

To develop a safer bone-bonding device that promotes early osseointegration with cortical bone perforation, novel subperiosteal device geometries were proposed and evaluated for their ability to facilitate surrounding bone formation and enhance bone-bonding strength. This study used animal experiments and mechanical testing to assess the performance of these designs. The experimental device consisted of two main components: a rounded rectangular plate and a centrally positioned cylinder. To promote the recruitment of bone-marrow-derived factors, slits were incorporated into the cylinder, and a center hole was created directly above it. Four device variations, differing by the presence or absence of the slits and center hole, were fabricated and then subjected to tensile tests for mechanical property evaluation. In the animal experiments, the devices were bilaterally placed on rat tibiae, and after four weeks, bone-bonding strength tests were performed. Additionally, micro-computed tomography and histological analysis of undecalcified sections were conducted. All devices demonstrated early osseointegration, and geometric design differences, specifically the presence or absence of the slits and center hole, significantly affected the mechanical properties and bone induction. However, no significant differences in bone-bonding strength were detected. These findings suggest that the newly formed bone inside the slits and center hole contributes to the reinforcement of the device.

Strontium-containing mineralized phospholipid coatings improve osseointegration in osteoporotic rats.

Surface treatments play an important role in enhancing the osseointegration of Titanium (Ti) and its alloys. This study introduces a method employing biomimetic hydroxyapatite (Hap) deposition guided by molecularly organized phospholipids, affixed to the metal implant surface. Using the Langmuir-Blodgett technique, phospholipids were deposited onto Ti-screws by using CaCl2 or CaCl2/SrCl2 aqueous solution in the subphase of a Langmuir trough in the target proportion (i.e. 10 and 90 mol% of Sr2+ in relation of Ca2+) followed by immersion in phosphate buffer and in supersaturated simulated body fluid. Coating composition and morphology were evaluated using infrared spectroscopy and scanning electron microscopy, respectively, while contact angle measurements assessed coating wettability and surface energy. Randomized screws were then implanted into the tibias of healthy and osteoporotic female rats (G1: Control-Machined, G2: Hap, G3: HapSr10, G4: HapSr90). Osseointegration, assessed 60 days post-implantation, included reverse torque, fluorochrome area, bone tissue-screw contact area, and linear extent of bone-screw contact. Results, grouped by surface treatment (Machined, Hap, HapSr10, HapSr90), revealed that the deposition of Hap, HapSr10, and HapSr90 resulted in thin and rough coatings composed of hydroxyapatite (Hap) on the screw surface with nanoscale pores. The coatings resulted in increased wettability and surface energy of Ti surfaces. The minerals are chemically similar to natural bone apatite as revealed by FTIR analysis. In vivo analyses indicated higher torque values for strontium-containing surfaces in the osteoporotic group (p = 0.02) and, in the control group superior torque for screw removal on the Hap surface (p = 0.023). Hydroxyapatite-treated surfaces enhance morphology, composition, and reactivity, promoting screw osseointegration in healthy and osteoporotic female rats. The incorporation of strontium into the mineral phase has been proposed to not only stimulate osteoblast activity but also reduce osteoclastic resorption, which may explain the improved outcomes observed here in experimental osteoporotic conditions.

Preparation, identification and screening of anti-osteoporosis milk-derived peptides: Intervention effects in osteoporosis rats

To identify milk-derived peptides with both antioxidant and calcium absorption activities in combating osteoporosis, we employed a comprehensive screening approach that included virtual enzymatic hydrolysis, molecular docking, and cellular experiments using osteoblasts. Under the optimal conditions for dual-enzyme hydrolysis, the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging rate and soluble calcium binding capacity of the milk-derived peptides were 19.69% and 0.6965 μg/mL, respectively. Six peptide segments, namely KEDVPSER, HKEMPFPK, YPSYG, EDVPSE, VPQLE, and IPAVF, were identified through UPLC-Q-Exactive Orbitrap MS and molecular docking for further validation. Among the peptides, YPSYG significantly promoted the proliferation of MC3T3-E1 cells both with and without CaCl2 (P < 0.05), increasing proliferation by 38.27% and 20.67%, respectively, compared to the control group. Additionally, YPSYG significantly improved proliferation after H2O2-induced oxidative damage (P < 0.05), with a 38.23% higher rate than the model group. Compared with rats in the osteoporosis model group, YPSYG significantly enhanced serum alkaline phosphatase (ALP) and N-terminal propeptide of type I procollagen in rats (s-PINP) levels and decreased tartrate-resistant acid phosphatase (TRAP) levels (P < 0.05). Furthermore, milk-derived peptides and YPSYG significantly increased the bone weight index, maximum load, and bending energy of the femur and tibia in osteoporotic rats (P < 0.05). Additionally, these peptides significantly reduced the number of osteoclasts in the metaphysis of the femur and tibia in osteoporotic rats and alleviated microstructural damage. This study confirmed that milk-derived peptides, including YPSYG, effectively promoted bone formation and improved bone microstructure in osteoporotic rats. These findings provided a foundation for developing functional foods for elderly bone health.

The effects of endurance trainability phenotype, sex, and interval running training on bone collagen synthesis in adult rats

Bone is influenced by many factors such as genetics and mechanical loading, but the short-term physiological effects of these factors on bone (re)modelling are not well characterised. This study investigated the effects of endurance trainability phenotype, sex, and interval running training (7-week intervention) on bone collagen formation in rats using a deuterium oxide stable isotope tracer method. Bone samples of the femur diaphysis, proximal tibia, mid-shaft tibia, and distal tibia were collected after necropsy from forty-six 9 ± 3-month male and female rats selectively bred for yielding low (LRT) or high (HRT) responses to endurance training. Bone collagen proteins were isolated and hydrolysed, and fractional synthetic rates (FSRs) were determined by the incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. There was a significant large main effect of phenotype at the femur site (p < 0.001; η2g = 0.473) with HRT rats showing greater bone collagen FSRs than LRT rats. There was a significant large main effect of phenotype (p = 0.008; η2g = 0.178) and a significant large main effect of sex (p = 0.005; η2g = 0.196) at the proximal site of the tibia with HRT rats showing greater bone collagen FSRs than LRT rats, and male rats showing greater bone collagen FSRs compared to female rats. There was a significant large main effect of training at the mid-shaft site of the tibia (p = 0.012; η2g = 0.159), with rats that underwent interval running training having greater bone collagen FSRs than control rats. Similarly, there was a significant large main effect of training at the distal site of the tibia (p = 0.050; η2g = 0.156), with rats in the interval running training group having greater bone collagen FSRs compared to rats in the control group. Collectively, this evidence highlights that bone responses to physiological effects are site-specific, indicating that interval running training has positive effects on bone collagen synthesis at the tibial mid-shaft and distal sites, whilst genetic factors affect bone collagen synthesis at the femur diaphysis (phenotype) and proximal tibia (phenotype and sex) in rats.

Hyperbranched Poly-l-Lysine Modified Titanium Surface With Enhanced Osseointegration, Bacteriostasis, and Anti-Inflammatory Properties for Implant Application: AnExperimental InVivo Study.

This study aimed to explore multiple effects of hyperbranched poly-l-lysine (HBPL) titanium (Ti) surfaces on osseointegration, bacteriostasis, and anti-inflammation across three different animal models. Ti surfaces were covalently modified with HBPL, with uncoated surfaces as controls. Characterization included scanning electron microscopy (SEM) and surface chemistry and elemental analysis (EDX). Ti and Ti-HBPL implants were placed in conventional canine edentulous sites, post-operative infection canine edentulous sites, and diabetic rat tibias. Implants from canine edentulous models were analyzed using micro-CT and histomorphometry to assess osseointegration at 8 weeks. Post-operative infection beagles were used to evaluate antibacterial efficacy through clinical parameters and bacterial cultures at 1 week. In diabetic rats, micro-CT and histomorphometry were performed at 8 weeks. HBPL was uniformly grafted on Ti-HBPL surfaces. Ti-HBPL surfaces showed higher bone volume/total volume (BV/TV, p < 0.001), bone-implant contact (BIC%, p < 0.001), and trabecular number (Tb.N, p < 0.01) in beagles. Besides, it displayed higher BIC% (p < 0.001) and bone area fraction occupancy (BAFO%, p < 0.01) in hard tissue sections. In an infected model, Ti-HBPL surfaces exhibited lower bleeding on probing (BOP, p < 0.001), and plaque index (DI, p < 0.01), with reduced bacterial colony formation (p < 0.001) compared to the control group. In diabetic rats, Ti-HBPL surfaces showed an increase in BV/TV (p < 0.01) and Tb.N (p < 0.001), downregulated TNF-α and IL-1β (p < 0.01), and upregulated IL-10 (p < 0.01) and osteocalcin (OCN) expression (p < 0.01). HBPL-Ti surfaces demonstrated enhanced osseointegration, bacteriostasis, and anti-inflammatory effects invivo.

A novel Osteoarthritis scoring system to separate typical OA joint degeneration from non-typical lesions in male Sprague Dawley rats

ObjectiveTo develop a novel scoring system to characterize osteoarthritis-related degeneration distinct from spontaneous subchondral bone lesions observed in the tibia and femur of male Sprague Dawley rats. MethodKnee joints from male rats following 12 weeks of a diet-induced obesity model of osteoarthritis (OA) were assessed. OA histopathological changes (OAHC) were assessed in the knee joints. All scores were evaluated using a modified Mankin score and a modified Osteoarthritis Research Society International histological score. OAHC were divided into 3 categories: (I) Typical OA score evaluating the changes in cartilage structure, cellularity, proteoglycan depletion, and tidemark integrity, (II) A novel Non-typical OA score evaluating cartilage integrity, and the size of local thickening, fragmentation and degeneration along the tidemark and the size and severity of the subchondral bone lesion, and (III) Total OA score comprised of both, the Typical and the Non-typical scores. ResultsRats exposed to a high fat/high sucrose diet had higher Typical OA score compared to a control group (Chow). Non-typical and Total OA scores revealed no differences in the severity of the lesions between the HFS and the Chow group animals. All scoring systems had excellent intra- and inter-examiner reliability. ConclusionThe spontaneous bone lesions observed in male Sprague Dawley rats can obscure the effect of the diet-induced obesity if the classical scoring system is used to assess joint degeneration. The newly proposed scoring method provides a reliable method to distinguish classical OA joint degeneration from spontaneous Non-typical lesions occurring in these rats.

Lovastatin Combination Therapy Increases the Survival and Proliferation of Rat Bone Marrow-Derived Mesenchymal Stem Cells Against the Inflammatory Activity of Lipopolysaccharide.

Oxidative stress hurts the survival of transplanted mesenchymal stem cells (MSCs). Lipopolysaccharide (LPS) preconditioning inhibits apoptotic death in MSCs. Also, Lovastatin's protective effect was reported on MSCs. Here, we investigated the potential of LPS and Lovastatin combination therapy on the survival and proliferation of MSCs. MSCs harvested from adult rats (240-260 g) femur and tibia bone marrow. Third passage MSCs were divided into 6 groups control group, LPS, LPS + Lovastatin (10 and 15 µM), and Lovastatin (10 and 15 µM). Cell survival and proliferation were assessed using an MTT assay 24 h after LPS, Lovastatin, or LPS + Lovastatin treatment. Also, Malondialdehyde (MDA) as a lipid peroxidation marker and antioxidant enzymes such as Glutathione peroxidase (GPX) and Superoxide dismutase (SOD) activity levels evaluated. Finally, the expression level of tumor protein P53 (P53) and octamer-binding transcription factor 4 (OCT4) genes were measured by qRT-PCR test. Lovastatin 10 μM potentiated proliferation and survival of MSCs. It can increase the activity of GPX and SOD. 10 µM Lovastatin could not affect MDA amounts but decreased the expression levels of P53 and Oct4 significantly. Nevertheless, treatment with LPS reduced the survival and proliferation of MSCs, along with a significant reduction in GPX activity. LPS + Lovastatin could increase SOD activity, however, GPX enzyme activity and MSCs proliferation did not change so, and it was not effective. We propose Lovastatin at the dose of 10 µM as a suitable combination agent to increase the survival and proliferation of MSCs in oxidative stress conditions.

Optimization of the formulation of Tubiechong gel plaster and its effect on fracture healing by promoting angiogenesis in a rat model

Background and aimTransdermal drug delivery has been used in traditional Chinese medicine for thousands of years and is worth further developing. The purpose of this study was to investigate the preparation method of Tubiechong gel plaster (TGP) and evaluate its activity to promote fracture healing. Experimental procedureUsing rat skin as a barrier, and the human umbilical vein endothelial cells (HUVECs) proliferation promotion rate of the receiving solution as the evaluation index, the in vitro permeability of the plaster was studied by Franz diffusion cell method. A modified Einhorn method was used to model rat tibia fractures. The bone healing process was monitored by radiography. Bone development and angiogenesis were assessed by Safranin O Fast Green staining and CD31 immunohistochemistry, respectively. Serum bone metabolites and VEGF levels were determined by ELISA method. Results and conclusionThe optimal dose of azone for TGP is 2 %. The transdermal penetration equation for this plaster is consistent with the Ritger-Peppas equation. The prepared plaster was stable for at least 3 months. X-ray images showed that the fracture healing was promoted by TGP in a dose-related form at all periods. Serum BALP, OC, CTX-I, and VEGF levels also indicated better fracture healing after treatment. TGP significantly stimulated angiogenesis and bone growth at the fracture site on days 7,14 and 21, and the effect of promoting bone growth was shown on the third day. These data prove that the prepared Tubiechong gel plasters can promote fracture healing by promoting angiogenesis.

Initial bone tissue reactions of hydroxyapatite/collagen-(3-glycidoxypropyl)trimethoxysilane injectable bone paste.

We have previously reported that a novel bioresorbable self-setting injectable bone paste composed of hydroxyapatite/collagen bone-like nanocomposite (HAp/Col) and (3-glycidoxypropyl)trimethoxysilane (GPTMS) was successfully prepared and was replaced with new bone within 3 months of implantation in defects created in porcine tibia. In this study, the HAp/Col-GPTMS paste was implanted into bone defects in rat tibiae to investigate the initial kinetics and bone tissue response. Even though more than 35% of GPTMS molecules should be eluted rapidly from directly injected pastes according to previously reported cell culture tests, in this study, energy-dispersive X-ray spectrometry did not detect Si (GPTMS) deposition in tissues surrounding the paste at 1 day postimplantation. Further, no abnormal inflammatory responses were observed in the surrounding tissues over the test period for both directly injected and prehardened pastes. Companying these observations with the results of the previous animal test (in which the paste was fully resorbed and was substituted with new bone), the eluted GPTMS resolved in no harm in vivo from the initial to final (completely resorbed) stages. Material resorption rates calculated from X-ray microcomputed tomography (μ-CT) images decreased with increasing in GPTMS concentration. Histological observations indicated that tartrate-resistant acid phosphatase (TRAP) active cells, (assumed to be osteoclasts), exist on the periphery of pastes. This result suggested that the paste was resorbed by osteoclasts in the same way as the HAp/Col. Since a good correlation was observed between TRAP active areas in histological sections and material resorption rate calculated from μ-CT, the TRAP activity coverage ratio offers the possibility to estimate the osteoclastic resorption ratio of materials, which are replaced with bone via bone remodeling process.

Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis.

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials and bone tissue. The rat's tibia is frequently chosen due to its easy surgical access through thin tissue layers (skin and muscle) and the flattened shape of its medial face, facilitating the surgical insertion of intraosseous devices. Additionally, this model enables the induction of specific diseases, mimicking various clinical conditions to assess biological responses to different implant conditions like geometry, surface texture, or biological cues. However, despite its robust cortical structure, certain intraosseous devices may require adaptations in design and size for successful implantation. Therefore, establishing standardized surgical methods for manipulating both soft and hard tissues in the implantation region is essential for ensuring proper implant or screw device placement, particularly in fields like implant dentistry and orthopedics. This study included eighty Sprague Dawley rats divided into two groups based on their respective diseases: Group 1 with osteoporosis and Group 2 with Type 2 Diabetes. Implantations were performed at 4 weeks and 12 weeks, with the same surgeon following a consistent surgical technique. A positive biological response was observed, indicating complete osseointegration of all implants placed. These results validate the success of the surgical protocol, which can be replicated for other studies and serve as a benchmark for the biomaterials community. Notably, osseointegration values remained stable at both 4 weeks and 12 weeks for both disease models, demonstrating a durable integration of the implant over time and emphasizing the establishment of an intimate bone connection as early as 4 weeks.

Surface bioactivation of Polyetheretherketone (PEEK) by magnesium chondroitin sulfate (MgCS) as orthopedic implants for reconstruction of skeletal defects

Polyether-ether-ketone (PEEK) is clinically used as a bio-implant for the healing of skeletal defects. However, the osseointegration of clinical-sized bone grafts remains limited. In this study, surface-porous PEEK was created by using a sulfonation method and a metal-polysaccharide complex MgCS was introduced on the surface of sulfonated PEEK to form MgCS@SPEEK. The as-prepared MgCS@SPEEK was found to have a porous surface with good hydrophilicity and bioactivity. This was followed by an investigation into whether MgCS loaded onto sulfonated PEEK surfaces could promote osseointegration and angiogenesis. The in vitro results showed that MgCS@SPEEK had a positive effect on reducing the expression levels of inflammatory genes and promoting osteogenesis and angiogenesis-related genes expression levels. Furthermore, porous MgCS@SPEEK was implanted in critical-sized rat tibial defects for in vivo evaluation of osseointegration. The micro-computed tomography evaluation results revealed substantial bone formation at 4 and 8 weeks. Collectively, these findings indicate that MgCS@SPEEK could provide improved osseointegration and an attractive strategy for orthopaedic applications.

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.