Fracture Healing In Rats Research Articles

Puerarin Promotes Tibial Shaft Fracture Healing in Rats and Its Relationship with BMP-Smad Pathway

Background Fracture healing involves multiple growth factors and the interaction between multiple signaling pathways. Pue (puerarin) is a natural compound with multiple biological activities, but its effect on fracture healing and its relationship with the bone morphogenic protein (BMP)-Smad pathway is not yet clear. Purpose This study intends to assess the effect of puerarin on fracture healing. Methods Seventy rats were used for the study to establish a tibial shaft fracture rat model. The experimental rats were divided into a tibial fracture group, Pue dose group (low, medium, and high), BMP-2 group, Noggin group, Pue + SY-LB-35 group, and Pue + Noggin group through the random number method. We observed the fracture healing through micro-CT and histology, observed the tibial stump tissue with hematoxylin and eosin (HE) staining, and detected the expression of genes related to the BMP-Smad pathway through quantitative real-time polymerase chain reaction (qPCR) and Western blot. Results We found that puerarin can promote tibial shaft fracture healing in rats in a dose-dependent manner; through micro-CT and histological analysis, we observed increased trabecular bone formation during fracture healing in rats treated with puerarin. Callus formation is accelerated, and bone density increases. In addition, puerarin also significantly increased the expression of genes related to the BMP-Smad pathway, including BMP2, Smad1, Smad5, and so on. Conclusion Puerarin can promote tibial shaft fracture healing in rats, and its mechanism is related to upregulating the expression of genes related to the BMP-Smad pathway. It further improves the fracture-healing mechanism and provides candidate drugs for the treatment of tibial shaft fracture healing in clinical medicine.

Effects of Jintiange on the healing of osteoporotic fractures in aged rats

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.

Transcutaneous CO2 application combined with low-intensity pulsed ultrasound accelerates bone fracture healing in rats

BackgroundLow-intensity pulsed ultrasound (LIPUS) is a non-invasive therapy that accelerates fracture healing. As a new treatment method for fracture, we recently reported that the transcutaneous application of CO2 accelerated fracture healing in association with promoting angiogenesis, blood flow, and endochondral ossification. We hypothesized that transcutaneous CO2 application, combined with LIPUS, would promote bone fracture healing more than the single treatment with either of them.MethodsFemoral shaft fractures were produced in 12-week-old rats. Animals were randomly divided into four groups: the combination of CO2 and LIPUS, CO2, LIPUS, and control groups. As the transcutaneous CO2 application, the limb was sealed in a CO2-filled bag after applying hydrogel that promotes CO2 absorption. Transcutaneous CO2 application and LIPUS irradiation were performed for 20 min/day, 5 days/week. At weeks 1, 2, 3, and 4 after the fractures, we assessed the fracture healing process using radiography, histology, immunohistochemistry, real-time PCR, and biomechanical assessment.ResultsThe fracture healing score using radiographs in the combination group was significantly higher than that in the control group at all time points and those in both the LIPUS and CO2 groups at weeks 1, 2, and 4. The degree of bone fracture healing in the histological assessment was significantly higher in the combination group than that in the control group at weeks 2, 3, and 4. In the immunohistochemical assessment, the vascular densities of CD31- and endomucin-positive microvessels in the combination group were significantly higher than those in the control and LIPUS groups at week 2. In the gene expression assessment, significant upregulation of runt-related transcription factor 2 (Runx2) and vascular endothelial growth factor (VEGF) was detected in the combination group compared to the LIPUS and CO2 monotherapy groups. In the biomechanical assessment, the ultimate stress was significantly higher in the combination group than in the LIPUS and CO2 groups.ConclusionThe combination therapy of transcutaneous CO2 application and LIPUS had a superior effect in promoting fracture healing through the promotion of angiogenesis and osteoblast differentiation compared to monotherapy.

The Effects of Phenyramidol and Diclofenac Treatment on Fracture Healing in Rats.

Fracture healing or nonunion refers to a process in which many factors interact. In this study, we aimed to evaluate the radiological, histological, and biomechanical effects of phenyramidol and diclofenac, which are frequently used to treat post-fracture ture pain worldwide, on fracture healing and nonunion in a rat femur fracture model. In this study, 72 male Wistar-Albino rats aged 2-3 months and weighing 250 ± 30 g were divided into 4 main groups. The rats were divided into 12 subgroups according to the early, middle, and late periods. A fracture model was created in rat femurs, and surgical fixation was performed. Postoperative analgesic treatment protocols included phenyramidol, diclofenac, phenyramidol + diclofenac, and the control group. The rats were sacrificed on the fifteenth, thirtieth, and forty-fifth days and were evaluated radiologically, histopathologically, and biomechanically. Scoring was conducted independently by 2 orthopedists not involved in the study. When the results were analyzed statistically, no statistically significant difference was observed between the fifteenth and thirtieth day radiology score values of the control, diclofenac, phenyramidol, and Phenyramidol + diclofenac groups (p > 0.05), but there was a statistically significant difference (p < 0.05) between the forty-fifth day radiology score values of the control, diclofenac, phenyramidol, and phenyramidol + diclofenac groups. Our study shows that the use of diclofenac or phenyramidol alone negatively affects postoperative fracture healing. However, this effect was less pronounced in the combined treatment group. Histologic examination revealed that neither treatment had a significant effect on healing. There were statistical differences in biomechanical and radiologic properties between the phenyramidol and diclofenac groups; in particular, the diclofenac group had lower biomechanical properties.

Comparison of the effects of recombinant human epidermal growth factor (rhEGF) and alendronate sodium on tibial fracture healing in rats: an experimental study

Purpose: The objective of this study is to compare the effects of recombinant human epidermal growth factor (rhEGF) on bone healing with those of alendronate, a bisphosphonate widely used in practice. Materials and Methods: An iatrogenic fracture was created in the tibial shaft of 24 Sprague-Dawley rats with osteotome and fixed with an intramedullary Kirschner wire (K-wire). After surgery, Group 1 was given 0.2 mg/kg/day of oral alendronate sodium on postoperative Day 1 to 28, Group 2 received a single dose of 0.5 mg/kg of intraosseous rhEGF on postoperative Days 1 and 14, and Group 3 was followed for a total of four weeks with oral saline. At the end of Week 4, the animals were euthanized and the lower extremities were removed by stripping the soft tissues without damaging the callus. Tissue samples of groups were prepared and and stained with hematoxylin-eosin. After staining, histological scoring was performed to evaluate the degree of union. Results: Alendronate sodium group demonstrated a mean histological score of 6.95± 1.28. The rhEGF group had a lower mean score of 4.85±1.66. The placebo group exhibited the least progress in bone healing with a mean score of 4.10±1.68. The histological score was significantly higher in the alendronate sodium group compared to both the rhEGF and placebo groups. There was also a statistically significant difference between the rhEGF and placebo groups in terms of scores. Conclusion: Alendronate sodium enhanced fracture healing processes in rats. The role of rhEGF in bone healing requires further exploration. As the understanding of bisphosphonates and growth factors in bone healing evolves, the strategies for optimizing patient care in orthopedic settings are expected to be developed.

Sclerostin Transduced Bone Marrow Mesenchymal Stem Cells Promote Fracture Healing in Rats Through the Wnt/β-Catenin Signal Pathway.

The prognosis of fracture is directly related to several factors. Due to the limitations of existing treatment strategies, there are still many fractures with poor healing. Bone marrow mesenchymal stem cells (BMSCs) have the potential to differentiate into osteoblasts and chondrocytes. Therefore, BMSC transplantation is promised as an effective method for treating bone fractures. We aim to explore whether silently expressing sclerostin gene (SOST) can promote bone formation through the SOST/Wnt/β-catenin signal pathway. We isolated rat BMSCs and the target gene (SOST shRNA) was transduced into them for osteogenic induction. The results showed that SOST significantly inhibited the proliferation and osteogenic differentiation of BMSCs during osteogenic induction, whereas silently expressing SOST not only increased the number of surviving BMSCs but also promoted the expression of osteogenesis-related proteins RUNX2, osteoprotegerin, Collagen I (COL-I), and bone morphogenetic protein-2 during osteogenic induction. The results of imaging examination in rats show that downregulating the expression of SOST can promote the formation of bony callus and the transformation of cartilage tissue into normal bone tissue, and then accelerate the healing of osteoporotic fracture. In addition, we also found that SOST silencing can activate the Wnt/β-catenin pathway to achieve these effects. In conclusion, SOST silencing can promote the proliferation and osteogenic differentiation of BMSCs in situ, and therefore may enhance the therapeutic efficiency of BMSC transplantation in OPF.

Radiologic and histopathologic effects of favipiravir and hydroxychloroquine on fracture healing in rats

Fracture healing is a process in which many factors interact. In addition to many treatments, physical and biological therapy methods that affect different steps of this process, there are many biological and chemical agents that cause fracture union delay. Although the number of studies on fracture healing is increasing day by day, the mechanism of fracture healing, which is not fully understood, still attracts the attention of all researchers. In this study, we aimed to investigate the effects of favipiravir and hydroxychloroquine used in the treatment of COVID-19. In this study, 48 male Wistar rats weighing 300 ± 50 g were used. Each group was divided into eight subgroups of six rats each to be sacrificed at the 2nd and 4th weeks and evaluated radiologically and histologically. Favipiravir (group 1), hydroxychloroquine (group 2), favipiravir + hydroxychloroquine (group 3), and random control (group 4) were used. A statistically significant difference was observed between the 15th day histological scoring averages of the groups (p < 0.05). Although there was no statistically significant difference between the 15th day radiological score distributions of the groups (p > 0.05), we obtained different results in terms of complete bone union distributions and radiological images of the fracture line. Although favipiravir has a negative effect on fracture union in the early period, favipiravir may have a positive effect on fracture union in the late period. We did not find any effect of hydroxychloroquine on fracture union.

Polyene phosphatidylcholine promotes tibial fracture healing in rats by stimulating angiogenesis dominated by the VEGFA/VEGFR2 signaling pathway

One of the factors that predispose to fractures is liver damage. Interestingly, fractures are sometimes accompanied by abnormal liver function. Polyene phosphatidylcholine (PPC) is an important liver repair drug. We wondered if PPC had a role in promoting fracture healing. A rat model of tibial fracture was developed using the modified Einhorn model method. X-rays were used to detect the progression of fracture healing. Progress of ossification and angiogenesis at the fracture site were analyzed by Safranin O/fast green staining and CD31 immunohistochemistry. To investigate whether PPC has a direct angiogenesis effect, HUVECs were used. We performed MTT, wound healing, Transwell migration, and tube formation assays. Finally, RT-qPCR and Western blot analysis were used to study the underlying mechanism. The results showed that PPC significantly shortened the apparent recovery time of mobility in rats. PPC treatment significantly promoted the formation of cartilage callus, endochondral ossification, and angiogenesis at the fracture site. In vitro, PPC promoted the proliferative viability of HUVECs, their ability to heal wounds, and their ability to penetrate membranes in the Transwell apparatus and increased the tube formation of cells. The transcription of VEGFA, VEGFR2, PLCγ, RAS, ERK1/2 and MEK1/2 was significantly up regulated by PPC. Further, the protein level results demonstrated a significant increase in the expression of VEGFA, VEGFR2, MEK1/2, and ERK1/2 proteins. In conclusion, our findings suggest that PPC promotes angiogenesis by activating the VEGFA/VEGFR2 and downstream signaling pathway, thereby accelerating fracture healing.

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.

The water-soluble TF3 component from Eupolyphaga sinensis Walker promotes tibial fracture healing in rats by promoting osteoblast proliferation and angiogenesis

ObjectiveTo determine the active components of Eupolyphaga sinensis Walker (Tu Bie Chong) and explore the mechanisms underlying its fracture-healing ability. MethodsA modified Einhorn method was used to develop a rat tibial fracture model. Progression of bone healing was assessed using radiological methods. Safranin O/fast green and CD31 immunohistochemical staining were performed to evaluate the growth of bone cells and angiogenesis at the fracture site. Methylthiazoletetrazolium blue and wound healing assays were used to analyze cell viability and migration. The Transwell assay was used to explore the invasion capacity of the cells. Tubule formation assays were used to assess the angiogenesis capacity of human vascular endothelial cells (HUVECs). qRT-PCR was used to evaluate the changes in gene transcription levels. ResultsTu Bie Chong fraction 3 (TF3) significantly shortened the fracture healing time in model rats. X-ray results showed that on day 14, fracture healing in the TF3 treatment group was significantly better than that in the control group (P = .0086). Tissue staining showed that cartilage growth and the number of H-shaped blood vessels at the fracture site of the TF3 treatment group were better than those of the control group. In vitro, TF3 significantly promoted the proliferation and wound healing of MC3T3-E1s and HUVECs (all P < .01). Transwell assays showed that TF3 promoted the migration of HUVECs, but inhibited the migration of MC3T3-E1 cells. Tubule formation experiments confirmed that TF3 markedly promoted the ability of vascular endothelial cells to form microtubules. Gene expression analysis revealed that TF3 significantly promoted the expression of VEGFA, SPOCD1, NGF, and NGFR in HUVECs. In MC3T3-E1 cells, the transcript levels of RUNX2 and COL2A1 were significantly elevated following TF3 treatment. ConclusionTF3 promotes fracture healing by promoting bone regeneration associated with the RUNX2 pathway and angiogenesis associated with the VEGFA pathway.

Promoting a Cobalt Complex of Qingzhuan Dark Tea Polysaccharides on Fracture Healing in Rats.

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.

Silencing p75NTR regulates osteogenic differentiation and angiogenesis of BMSCs to enhance bone healing in fractured rats

BackgroundFractures heal through a process that involves angiogenesis and osteogenesis but may also lead to non-union or delayed healing. Bone marrow mesenchymal stem cells (BMSCs) have been reported to play a pivotal role in bone formation and vascular regeneration and the p75 neurotrophin receptor (p75NTR) as being an important regulator of osteogenesis. Herein, we aim to determine the potential mediation of BMSCs by p75NTR in bone healing.MethodsRat BMSCs were identified by flow cytometry (FCM) to detect cell cycle and surface markers. Then transfection of si/oe-p75NTR was performed in BMSCs, followed by Alizarin red staining to detect osteogenic differentiation of cells, immunofluorescence double staining was performed to detect the expression of p75NTR and sortilin, co-immunoprecipitation (CO-IP) was conducted to analyze the interaction between p75NTR and sortilin, and EdU staining and cell scratch assay to assess the proliferation and migration of human umbilical vein endothelial cells (HUVECs). The expression of HIF-1α, VEGF, and apoptosis-related proteins were also detected. In addition, a rat fracture healing model was constructed, and BMSCs-si-p75NTR were injected, following which the fracture condition was observed using micro-CT imaging, and the expression of platelet/endothelial cell adhesion molecule-1 (CD31) was assessed.ResultsThe results showed that BMSCs were successfully isolated, p75NTR inhibited apoptosis and the osteogenic differentiation of BMSCs, while si-p75NTR led to a decrease in sortilin expression in BMSCs, increased proliferation and migration in HUVECs, and upregulation of HIF-1α and VEGF expression. In addition, an interaction was observed between p75NTR and sortilin. The knockdown of p75NTR was found to reduce the severity of fracture in rats and increase the expression of CD31 and osteogenesis-related proteins.ConclusionSilencing p75NTR effectively modulates BMSCs to promote osteogenic differentiation and angiogenesis, offering a novel perspective for improving fracture healing.

Efectos de la aplicación local de líquido amniótico bovino en la curación de fracturas en ratas (Rattus norvegicus)

In this study, it was aim to examine the local application of bovine amniotic fluid on bone fracture healing in rats. Twenty female sprague dawley rats included in the study were divided into 2 groups of 10. The sham group (n=10): Bone fractures were created in the right tibia bones of the rats and fixed with kirschner wire. After a four–week recovery period, the subjects were sacrificed. Local bovine amniotic fluid group (n=10): Bone fractures were created in the right tibia bones of the rats and local bovine amniotic fuid was applied during fixation with kirschner wire. After a four–week recovery period, the subjects were sacrificed. Samples from all subjects were decalcified, stained with hematoxylin and eosin, and new bone formation and fibrosis were analyzed. When the groups were evaluated in terms of new bone regeneration, it was determined that the new bone regeneration in the subjects treated with local bovine amniotic fluid were statistically significantly higher than sham group (P&lt;0.05). When the groups were evaluated in terms of fibrosis, the fibrosis value in the sham group was found to be statistically significantly higher when compared with the local bovine amniotic fluid group (P&lt;0.05). It can be stated that local bovine amniotic fluid application may positively affect the healing of bone fractures.

The Influence of Vitamin B12 on the Progression of Fracture Healing in Rats

The Influence of Vitamin B12 on the Progression of Fracture Healing in Rats

MiR-22-3p facilitates bone marrow mesenchymal stem cell osteogenesis and fracture healing through the SOSTDC1-PI3K/AKT pathway.

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.

Hydroxychloroquine induces oxidative stress and impairs fracture healing in rats.

The aim of this study was to investigate whether hydroxychloroquine sulfate (HCQS) induced oxidative stress and how it affected the union of bone fractures in an experimental rat model. A total of 48 Wistar albino male rats were used. The rats were divided into six groups. To investigate the effects of oral administration of HCQS at varying doses between the third and sixth weeks, fracture healing processes were evaluated using radiography, histopathology, biochemistry, and dual-energy X-ray absorptiometry. The activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) were measured to analyze the relationship between HCQS and oxidative stress. Radiographic scores, alkaline phosphatase levels, callus/diaphysis ratio, callus development, and bone mineral density were significantly lower in rats given HCQS at three and six weeks compared to the control group (p<0.005). When oxidative stress parameters were compared among the groups, all antioxidant parameters were statistically significant, indicating that antioxidant systems played a role in peripheral blood, when HCQS was used (p<0.005). Oral HCQS intake impairs the fracture healing process by causing oxidative stress in rats. However, further biomolecular researches are needed to understand the underlying mechanism of these effects.

Liraglutide in Combination with Insulin Has a Superior Therapeutic Effect to Either Alone on Fracture Healing in Diabetic Rats.

Fractures 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. Closed 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. Micro-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. These 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.

The Influence of Zoledronic Acid on the Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells and Tibial Fracture in Rats via Phosphatidylinositol-3-Kinase/AKT Pathway

Fracture healing is an extremely complex physiological process, involving a sequence of crucial mechanisms. Whether zoledronic acid (ZA) affects proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and tibial fractures still remains unknown. We performed construction of the tibial fracture model in rats and analyzed the impact of ZA and ZA+LY294002 on fracture healing in rats. Test of the influence of ZA and ZA+LY294002 on crucial osteogenic genes alkaline phosphatase (ALP), RUNX2, OCN and OPN was performed. Examination of the influence of suppressing the PI3K/AKT pathway on the proliferation with bone differentiation of ZA. Results showed ZA distinctly accelerated the proliferation and ALP activity of BMSC cells, BMP2, RUNX2, OCN, OPN and the activation of the PI3K/AKT pathway. Repression of PI3K/AKT pathway suppressed the proliferation and osteogenic differentiation action of ZA. ZA boosted tibial fracture healing in rats via activating the PI3K/AKT pathway. ZA facilitates the proliferation with osteogenic differentiation of BMSC cells and tibial fracture healing in rats via activating the PI3K/AKT pathway.

An experimental study on the effects of splenectomy on bone fracture healing

Bone fracture healing is a complex process involving multiple mechanisms where immune system has vital role. The spleen is a part of this process and its effect on bone metabolism is not completely understood. Herein, the effects of simultaneous bone fractures and splenectomy were evaluated regarding early and late periods of fracture healing in rats. The closed fracture model was applied in sixty-four healthy male Wistar-albino rats. Rats were randomly selected and divided into four groups. The splenectomy was carried out in two groups. The early period was declared as three weeks and late period as five weeks for evaluating the fracture healing in rats,(Groups A: splenectomized 3rd week, Groups B: non-splenectomized 3rd week, Groups C: splenectomized 5th week, and Groups D: non-splenectomized 5th week). The groups were compared regarding radiological, biomechanical, and histopathological aspects at 3rd and 5th weeks of fracture healing. The callus/bone volume ratios of Groups C and D were lower than those of Groups A and B (A–C, p ≤ 0.001; B–D, p ≤ 0.001). Radiographic Union Score for Tibial Fracture scores of Groups C and D were higher than those for Groups A and B (A–C, p ≤ 0.001; B–D, p ≤ 0.001). Bone mineral density measurements of Groups C and D were higher than those for Groups A and B (A–C, p ≤ 0.001; A–D, p ≤ 0.001; B–C, p ≤ 0.001 and B–D, p ≤ 0.001). The histological scoring of Groups A was lower than Groups C and D (A–C, p = 0.004; A–D, p = 0.001). Splenectomy may thus adversely affect the early phase fracture healing.

Tubiechong patching promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway

Ethnopharmacological relevanceThe external use of traditional Chinese medicine (TCM) to treat fractures has a long history of clinical application and theoretical basis, and is also one of the characteristic treatment methods of TCM with significant efficacy and many advantages. Among the commonly used external Chinese medicines, Tubiechong is noteworthy. Aim of the studyTo elucidate whether local patching of Tubiechong can promote fracture healing and explore its mechanism of action. Materials and methodsA rat tibia fracture model was constructed by the modified Einhorn modeling method. X-ray films were taken to evaluate the progress of fracture healing. Serum bone alkaline phosphatase (BALP), osteocalcin (BGP) and the C-terminal content of collagen type I (CTX-I) were analyzed by ELISA. CD31 immunohistochemistry was used to evaluate angiogenesis in the tibia segment. The effects of Tubiechong decoction (TD) on HUVEC proliferation, migration and invasion were detected by MTT assay, wound healing assay and Transwell migration assay, respectively. RNA-seq was performed to identify differentially expressed genes (DEGs). Enrichment of functions and signaling pathway analysis were performed based on the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Quantitative real time polymerase chain reaction (qRT-PCR) was used to study gene expression levels. Western blotting (WB) was used to detect the expression of relevant regulatory proteins. ResultsThe healing time of rat tibia fractures in the three TD dose groups was shortened. The serum levels of BALP, BGP and CTX- I in the TD-treated group were higher than those in the NC group. The X-ray results showed that on the 7th day after surgery, the fracture healing degree of the high-dose TD group was significantly better than that of the NC group, and the fracture healing degrees of each TD treatment group were significantly higher than those of the NC group on the 14th, 17th, and 21st days after the operation. The CD31 immunohistochemistry results showed that the number of blood vessels and the vascular area in the TD treatment group were higher than those in the NC group. In vitro, TD promoted the proliferation, wound healing and migration of HUVECs. GO analysis of transcriptome sequencing results showed that TD significantly altered the expression of genes related to cell growth, metabolism, and motility. According to KEGG annotations, VEGFA was upregulated. Eight DEGs were enriched in the VEGFA-VEGFR2 signaling pathway, of which six were upregulated. KEGG signaling pathway analysis showed that the most abundant DEGs were in mitogen-activated protein kinase (MAPK) signaling pathway. qRT-PCR showed that VEGFA gene expression in HUVECs was 7.8 times that of the control group after 1 mg/mL TD treatment for 24 h, and WB experiments showed that its protein expression was 3 times that of the control group. WB results showed that the phosphorylated ERK gene was highly expressed, while the expression levels of phosphorylated P38 and phosphorylated JNK protein remained unchanged. ConclusionTubechong patching therapy promotes tibia fracture healing in rats by regulating angiogenesis through the VEGF/ERK1/2 signaling pathway.