Effects of Vitamin C Supplementation on Histology of Callus Diameter and Osteoblast Number in Male Wistar Rats With Complete Femur Bone Fracture

Our previous study documented that melatonin (MLT) induced the osteogenic differentiation of mesenchymal stem cells (MSCs) and promoted the healing of femoral fractures in rats via the neuropeptide Y (NPY)/neuropeptide Y1 receptor (NPY1R) signaling pathway. MLT treatment upregulated the expression of the zinc uptake transporter zinc transporter 1 (Zip1) in nerve cells. Prior research demonstrated that oral zinc upregulated NPY expression. MSCs were isolated from rat bone marrow and identified using flow cytometry in our study. The results showed that MLT treatment upregulated NPY and NPY1R levels in MSCs with osteogenic differentiation, which was accompanied by upregulated Zip1 expression. However, the MLT-induced osteogenic differentiation of MSCs was reversed after interference of Zip1 expression. It was confirmed by the decreased alkaline phosphatase (ALP) level; downregulated activities of type I collagen α1 chain (COL1A1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2) and ALP; and reduced mineralized nodule formation. MLT promoted fracture healing in rats with femoral fracture, which was accompanied by increased expression of NPY and NPY1R and significantly increased expression of Zip1. In contrast, the silencing of Zip1 expression reversed MLT-mediated fracture healing. In summary, Zip1 participated in the regulation of the NPY/NPY1R signaling pathway via MLT to promote the osteogenic differentiation of MSCs and fracture healing.

Effects of social stress and fluoxetine treatment on fracture healing in a rat femur fracture model

Effects of botulinum toxin A on fracture healing in rats: an experimental study

Reply to “Comment on Aydin et al. entitled ‘Effects of botulinum toxin A on fracture healing in rats: an experimental study’”

Bone fractures are the most common form of musculoskeletal trauma worldwide. Numerous microRNAs (miRNAs) have been suggested to be participants in regulating bone-related diseases. Recent studies revealed the regulatory role of miR-22-3p in osteogenic differentiation, but its role in fracture healing has not been investigated previously. Here, a rat femoral fracture model was established, Bone marrow mesenchymal stem cells (BMSCs) were isolated to detect the specific function and underlying mechanisms of miR-22-3p. MiR-22-3p and sclerostin domain-containing 1 (SOSTDC1) expression was determined by RT-qPCR and immunohistochemistry staining. The levels of proteins associated with osteogenic differentiation were assessed by western blotting. Flow cytometry was conducted to identify the isolated rat BMSCs. Alizarin red staining, alkaline phosphatase staining and Oil Red O staining were used to evaluate the osteogenic and adipogenic differentiation of rat BMSCs. The interaction between miR-22-3p and SOSTDC1 was verified using a luciferase reporter assay. Haematoxylin and Eosin (H&E) staining of the bone tissues was performed to analyse the effect of miR-22-3p on histopathological changes invivo. MiR-22-3p was downregulated in the callus tissues of rat femoral fracture, while the expression of SOSTDC1 was upregulated. The isolated rat BMSCs had the capacity for both osteogenic and adipogenic differentiation. The differentiation capacity of BMSCs into osteoblasts was increased by miR-22-3p overexpression. MiR-22-3p activated the PI3K/AKT pathway by targeting SOSTDC1. SOSTDC1 overexpression and PI3K/AKT signalling inhibitor LY294002 abolished the enhancing effect of miR-22-3p overexpression on the osteogenesis of BMSCs. Thus MiR-22-3p facilitated the femoral fracture healing in rats. MiR-22-3p overexpression promoted fracture healing via the activation of PI3K/AKT pathway by targeting SOSTDC1.

Chitosan-containing compounds have been shown to be suitable for bone replacement, but few studies demonstrate the impact of the chitosan as a free drug on the fracture.In this study, we aimed to evaluate possible effects of free chitosan on fracture healing. Thirty adult male Sprague-Dawley rats with a mean body weight of 205 g (range from 200g to 210g) were randomly and equally divided into two groups. Standardized femur fractures were created in all rats. Treatments were administered intraperitoneally twice weekly at 1 mg chitosan per injection and the controls were administered physiological saline. The site of the fracture was compared with the control group at 1, 2 and 4 weeks after surgery (n=5 in each group). The weight, activity and reaction of the rats were observed at all the timepoints. Anterior-posterior radiographs and micro-CT scans of all fractures were taken after surgery, and the parameters included: the volume of callus that was calculated using the Perkins volume formula, BV/TV, BV, BMD of cortical bone, cortical thickness, and cortical number at the fracture sites. After sacrifice, fractured femurs from rats were dissected and carefully cleaned of muscle around the fracture callus to preserve callus integrity. Sections were stained with haematoxylin and eosin for histological evaluation of healing. Radiological (X-ray and micro-CT) evaluation showed that fracture healing of the experimental group was better than control group at the second week and fourth week. Histological evaluation revealed fracture healing of the experimental group was better than control group at the same time. There was no statistically significant difference in fracture healing between the two groups at the first week. Systemic delivery of free chitosan can accelerate the bone healing process in rat femur fracture at the early-middle stage.

ABSTRACTBACKGROUND:The aim of this study was to investigate the effects of acrylamide (AA) on fracture healing histologically, biochemically, and radiologically in a rat femur fracture model.METHODS:Scanning electron microscopy imaging, Fourier transform infrared spectroscopy, and ultraviolet-visible spectrophotometer examination were performed for AA characterization. In this study, after the femur fracture model was created, the groups were formed to include eight rats in each group (G) as follows: G1: 15th-day control, G2: 15th-day AA, G3: 30th-day control, and G4: 30th-day AA. In G2 and G4, 5 mg/kg AA was administered 3 times a week by gastric gavage. The fracture was evaluated radiologically according to Lane-Sandhu scoring and histologically according to Huo scoring. The weight changes of the rats were recorded. Albumin, total protein, cholesterol, high-density lipoprotein, low-density lipoprotein, triglyceride, alkaline phosphatase, lactate dehydrogenase, vit. D, parathormone, calcium, phosphorus (P), white blood cell (WBC), hemoglobin (Hb), and Platelet values were examined in the blood samples.RESULTS:The characterization properties of AA were confirmed. No significant weight change was observed in the rats during the study. When blood values were compared, a statistically significant difference was determined between albumin, total protein, P, WBC, and Hb groups (p=0.41, p=0.00, p=0.003, p=0.019, and p=0.017, respectively). According to the histological score comparisons, G3 was significantly different from G1, G2, and G4 (p<0.05), and G4 was significantly different from G1 and G2 (p<0.05). According to Lane-Sandhu scoring, there was a significant difference between G2 and G3 and G4 (p=0.0, p=0.034), G1 and G3 (p=0.001), respectively.CONCLUSION:AA adversely affects fracture healing even at low doses, as in the present study. According to the results of this study, the authors recommend a diet poor in AA during fracture treatment. Therefore, further human studies are required to find out the complex effect of AA on bone healing and the body.

Fractures occur commonly with multiple injuries, and their incidence has increased in recent years. Trace amounts of cobalt are necessary for many living organisms as it stimulates hematopoiesis and improves bone health. However, cobalt is also toxic, as it might cause allergic reactions and tissue destruction. These factors limit the application of cobalt in some medical fields. We studied the tea polysaccode-cobalt complex (TPS-Co) prepared from Qingzhuan Dark Tea polysaccharides. We used 6-week-old Sprague-Dawley rats to establish a femoral fracture model and evaluated the effects of CoCl2 and TPS-Co on the healing of femoral fractures. In this study, treatment with TPS-Co for the same content of cobalt intake decreased the side effects associated with CoCl2 treatment and accelerated the healing of femoral fractures in rats. This treatment method promoted angiogenesis by upregulating the expression of vascular endothelial growth factor and hypoxia-inducible factor. Bone formation was promoted via the upregulation of the expression of bone morphogenetic protein 2 and serum bone alkaline phosphatase. TPS-Co was found to actively regulate bone and vascular systems, resulting in significant bone regeneration effects. Therefore, the Qingzhuan Dark Tea polysaccharide cobalt complex might be used as an additive or drug to promote fracture healing, and thus, it might have a huge market value.

PurposeFractures in patients with type 2 diabetes mellitus are at a high risk of delayed union or non-union. Previous studies have shown a protective effect of liraglutide on bone. In the present study, we aimed to investigate the effects of a combination of liraglutide and insulin on fracture healing in a rat model of diabetes and the mechanisms involved.Materials and MethodsClosed femoral mid-shaft fractures were established in male Sprague-Dawley rats with or without diabetes mellitus, and the diabetic rats were administered insulin and/or liraglutide. Six weeks after femoral fracture, the femoral callus was evaluated by immunohistochemistry, histology, and micro-computed tomography. Additionally, the effects of liraglutide on high-glucose-stimulated MC3T3-E1 cells were analyzed by Western blotting.ResultsMicro-computed tomography and safranin O/fast green staining showed that fracture healing was delayed in the diabetic rats, and this was accompanied by much lower expression of osteogenic markers and greater osteoclast activity. However, treatment with insulin and/or liraglutide prevented these changes. Liraglutide in combination with insulin treatment resulted in lower blood glucose concentrations and significantly higher osteocalcin (OCN) and collagen I (Col I) expression six weeks following fracture. Western blot analysis showed that liraglutide prevented the low expression of the bone morphogenetic protein-2, osterix/SP7, OCN, Col I, and β-catenin in high-glucose-stimulated MC3T3-E1 cells.ConclusionThese results demonstrate that insulin and/or liraglutide promotes bone fracture healing in the DF model. The combination was more effective than either single treatment, which may be because of the two drugs’ additive effects on the osteogenic ability of osteoblast precursors.

Studies in animal models for alcohol abuse have suggested that ethanol inhibits bone growth, decreases bone formation, and increases fracture risk. Experimental tibia fracture healing in rats fed a liquid diet containing 7.2% ethanol for 8 weeks was investigated with histological and osteodensitometric studies with respect to the control group. After 4 weeks of vitamin A- and sucrose-enriched milk containing 7.2% ethanol feeding, we created closed tibia fractures, which were then fixed with intramedullary nails, in 10 rats. After a follow-up time of 4 weeks, the rats were killed for examination. The same procedure was performed in another 10 rats, which were fed on the same diet (isocaloric modified liquid diet) but without ethanol and used as the control group. A histological scoring system was developed for fracture healing. Histological evaluation of fracture region revealed an average fracture healing score of 1.9 in the ethanol-fed group versus 2.6 in the control group (p = 0.014). In the test group, dual-energy x-ray absorptiometry measurements in the fracture region showed a mean bone mineral density of 0.11 +/- 0.03 g/cm(2), whereas it was 0.130 +/- 0.051 g/cm(2) in the control group (p = 0.000). The mean bone mineral content in the fracture region was 0.103 +/- 0.08 g/cm(3) in the test group versus 0.128 +/- 0.06 g/cm(3) in the control group (p = 0.000). A significant correlation was found among histological scores, bone mineral density (r = 0.64, p = 0.04), and bone mineral content (r = 0.63, p = 0.04). This study showed that rats fed on a diet mixed with ethanol have a histologically delayed fracture healing associated with decreased bone density and mineral content. Besides the negative effects of ethanol on bone metabolism, it also interferes with the fracture-healing process.

Objective To observe the effects of transplantation of bone marrow stem cells(BMSCs)transfected with bone morphogenetic protein-2(BMP-2)on fracture healing in rats with diabetes so as to provide a new therapy for diabetic fractures.Methods Fifty male adult Wistar rats aged six weeks randomized to the control group and experimental group were all employed to establish models with diabetic fractures.Under high glucose condition,BMSCs were transfected with BMP-2 by adenovirus vector in vitro.BMSCs transfected by BMP-2 were transplanted into the fracture area of rats in the experimental group,while non-transfected BMSCs into the corresponding area of rats in the control group.X-ray examination was performed at 1,2,3,4 and 6 weeks after transplantation.Bony calluses were collected for HE staining and gray scales of BMP-2 in calluses were determined by immunohistochemical method.Meanwhile,serum levels of BMP-2 were measured by ELtSA.Results The gray scales of BMP-2 in the calluses were 83±3 in the experimental group and 118±4 in the control group at four weeks(P＜0.01).The serum concentrations of BMP-2 were(203.80±8.96)ng/L in the gxperimental group and(139.15±4.19)ng/L in the control group at four weeks(P＜0.01).Conclusion Key words: Myeloid progenitor cells; Bone morphogenetic proteins; Diabetes mellitus; Gene transfer techniques

ObjectiveTo examine the effects of Jintiange on enhancing the healing of osteoporotic fractures in aged rats.MethodsAn osteoporotic fracture model of femur was established using 70 SD rats (aged > 12 months), which were randomly numbered and divided into an experimental group and a control group, each with an equal sample size (n = 35). The experimental group received Jintiange capsule ingredients via intragastric administration, while the control group received an equal volume of saline via the same method. X-ray examinations were conducted at the 4th and 12th weeks to evaluate fracture healing in the rats. After 12 weeks, micro-CT scanning was employed to assess the microstructure of the healthy femurs, and the parameters of the experimental and control groups were compared.ResultsAfter 4 weeks, the mean fracture healing scores were 0.78 ± 0.43 in the experimental group and 0.56 ± 0.51 in the control group. After 12 weeks, the mean scores were 1.50 ± 0.71 in the experimental group and 0.96 ± 0.68 in the control group. The scores in the experimental group were higher than those in the control group (P < 0.05). Micro-CT examination of the femurs showed an improvement trend in bone microarchitecture in the experimental group, indicated by increased bone volume fraction (BV/TV), trabecular number (Tb. N), and trabecular thickness (Tb. Th). However, these improvements were not statistically significant. A negative correlation with statistical significance was observed between trabecular separation (Tb. Sp) and fracture healing scores.Conclusions Jintiange may promote osteoporotic fracture healing in aged rats by enhancing bone microstructure.

Osteogenic growth peptide normally stimulated by blood loss and marrow ablation has local and systemic effects on fracture healing in rats.

Osteogenic growth peptide, a histone H4-related, 14-amino-acid peptide, is an active mediator of local, as well as systemic, osteogenic activity in response to marrow ablation, trauma, and blood loss. In this study, the effect of exogenous osteogenic growth peptide on the healing of femoral fractures in rats was investigated. A fracture at the midshaft of the femur was created in 50 rats. Half of the rats were injected subcutaneously with 25 ng of osteogenic growth peptide per rat per day for the first 7 days after fracture. Radiographs were taken each week, and the diameter of the callus was measured. The femurs of four animals from each group were harvested 1, 2, 3, and 4 weeks after fracture. Two femurs from each group were sectioned for histologic examination, and two were sectioned for measurement of density and mineral content. Marrow was aspirated from the contralateral femurs to establish adhering cell cultures, which were examined for osteogenicity. At 2 weeks, a large increase in mitogenicity and osteogenicity was seen in the marrow-derived cultures from the rats treated with osteogenic growth peptide; this increase was sustained through 4 weeks. Extraction of RNA from the contralateral marrow (systemic expression) and callus (local expression) for amplification with reverse transcription-polymerase chain reaction revealed greater systemic expression of transforming growth factors beta1, beta2, and beta3, fibroblast growth factor-2, insulin-like growth factor-1, and aggrecan throughout the 4 weeks after fracture, whereas types IIA and IIB collagen, link protein, and fibroblast growth factor receptor-3 had a greater local expression. The specimens treated with osteogenic growth peptide had a stronger expression of transforming growth factor-beta1, both locally and systemically. The average diameter of the callus was greater for the treated rats at all time intervals, and peak diameters were 7.58 mm at 3 weeks for the treated rats and 6.64 mm at 2 weeks and 6.63 mm at 3 weeks for the controls. Histological study revealed an earlier organization and faster healing of the treated fractures, as evidenced by the larger, earlier appearance of cartilaginous soft callus and the more rapid organization of bridging trabecular bone. No statistical significance was obtained when these comparisons were made between the groups. These results suggest that osteogenic growth peptide can be used to promote earlier proliferation and differentiation of osteogenic cells in marrow and bone-repair callus, possibly through its effect on the transforming growth factor-beta family.

BackgroundDiabetes mellitus (DM) significantly impairs fracture healing, necessitating reliable animal models to study diabetic fracture repair mechanisms and therapeutic interventions. This study aimed to develop and comprehensively validate a standardized three-point bending system for inducing precise, reproducible mid-shaft transverse femoral fractures in rats, addressing existing methodological gaps of insufficient reproducibility and detailed calibration in previous models.MethodsA custom-designed three-point bending fracture induction system was developed using AutoCAD software based on the original principle introduced by Einhorn et al. (1984). After manufacturing and calibration, the system was validated first using 22 cadaveric rat femurs and subsequently applied to live rats (n = 44), including diabetic (streptozotocin-induced, n = 22) and non-diabetic animals (n = 22). Fracture induction reproducibility was assessed through radiographic, histologic and mechanic analysis. Additionally, statistical analysis was conducted using GraphPad Prism 9 software. Coefficients of variation (CV) for fracture-healing parameters (callus diameter, calcification ratio, maximum bending force at re-fracture) were calculated and compared statistically with similar parameters from previously published rat femoral fracture studies.ResultsCadaveric validation confirmed that the optimal blade travel distance, set as half the femoral diameter, consistently produced standardized transverse fractures without comminution. A significant correlation between body weight and femoral diameter (Femoral diameter = 1.0276 ln [Body weight] − 1.349) allowed accurate preoperative determination of optimal blade travel distance. Live animal testing demonstrated consistent fracture patterns, stable intramedullary pin fixation, and no complications during surgical procedures. Statistical analysis revealed significantly lower coefficients of variation for healing parameters in this study compared to previously published models (p < 0.05). Histological analysis indicated the fracture type was transverse. Callus was found around fracture site.ConclusionOur validated three-point bending system significantly enhances reproducibility, consistency, and methodological rigor in animal fracture research. This standardized model provides an ideal foundation for future preclinical studies investigating diabetic fracture healing mechanisms and potential therapeutic interventions.