Rat Tibia Research Articles

Stem-cell-derived ECM sheet-implant complexes for enhancing osseointegration.

Numerous treatment methods have been developed to modify the surface of dental implants to improve cell migration and proliferation, removal torque, and osseointegration. Recent studies have constructed cell sheet-implant complexes with enhanced osteogenic capabilities. However, these complexes have some limitations, such as requirements for complex preparation processes, cell vitality maintenance, strict preservation conditions, and the induction of immunogenicity. Extracellular matrix (ECM) sheets without cells may be a more desirable material. To date, the effect of ECM sheets on implant osseointegration has not been reported. In this study, we fabricated ECM sheet-implant complexes through the combination of rat bone marrow mesenchymal stem cell (BMSC)-derived ECM sheets with sandblasted, large-grit, acid-etched (SLA) implants. These complexes were characterized by light microscopy, scanning electron microscopy (SEM), and immunofluorescence (IF) assays. The adhesion, proliferation, and osteogenic differentiation of BMSCs cultured on ECM sheets were detected in vitro. Then, the ECM sheet-implant complexes were transplanted into the metaphysis of the tibias of rats to evaluate the implant osseointegration in vivo. The results showed that ECM sheets were successfully constructed and showed significantly improved adhesion and proliferation. BMSCs cultured on ECM sheets upregulated the expression levels of the osteogenic-related genes alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (Runx2) compared to controls. In vivo, ECM sheet-implant complexes demonstrated superior new bone formation. Our findings proved that the BMSC-derived ECM sheets promoted osseointegration in vitro and in vivo. The current study indicated that the ECM sheet could be an ideal tissue engineering material, and ECM sheet-implant complexes could provide a strategy with low immunogenicity and easy storage and transportation. This research provides a novel strategy for the development of implant surface modification approaches.

Orthopedic trauma or surgery does not effect on cognitive or behavioral capability of rats.

Aim: The aim of this study is to investigate the probable effects of orthopedic trauma/surgery on anxiety, memory and learning capability in rats. Material and Methods: Twenty-one Wistar albino rats grouped into three as control, sham operated, and tibia fracture. In control group, any surgery or fracture was done whereas in sham group the tibia of rats was fixed with intramedullar Kischner wire. In tibia fracture group, a closed fracture was occurred with Bonnarens and Einhorn's guillotine device then intramedullar Kischner wire sends to tibia. All rats were evaluated for memory, anxiety, and learning via behavioral tests respectively; water-maze test, elevated plus arm test and shuttle box test. Results: One month after the fractures were done and the bone healing was seen on X-ray at all rats. According to the behavioral tests results, there was not any statistically difference between groups in terms of anxiety, learning capability, locomotor activity and memory (p>0.05). Conclusion: Our results showed that, the orthopedic trauma or surgery itself does not affect the anxiety, learning capability, locomotor activity and memory in rats.

Does high fat diet effect the bone-implant connection?

Obesity induced by a high fat diet is associated with chronic up-regulation of inflammatory cytokines which stimulate osteoclast activity and bone resorption. However, the role of high-fat diet on bone-implant connectivity has not been studied in detail. In this study, we investigated whether a high-fat diet (HFD) affects bone implant connection (BIC) in periimplant bone. Twenty female Sprague Dawley rats were divided in two groups: 1) Control rats were fed with normal chow and titanium implants were integrated into tibial bones at the end of 3rd month and no treatment was applied 2) HFD group; rats were fed a high-fat diet (42 % of calories as fat), then the titanium implants were integrated into tibial bones at the end 3rd month. Following surgical integration of the implants, the rats were fed with control and HFD diets for 3 months. After the 6 months experimental period all rats were sacrificed and the implants and surrounded bone tissues were collected and the BIC was assessed histomorphometrically after the non-decalcifiing histological methods. Bone implant connection was detected with the ratio of the implant surface directly connected with the peri-implant bone tissues to the total implant surface length. Histologic analysis showed that HFD was not impaired BIC (p>0.05). In conclusion, within the limitation of this research, HFD did not effect the BIC rat tibias (Tab. 2, Fig. 2, Ref. 26). Text in PDF www.elis.sk.

Lithium chloride improves bone filling around implants placed in estrogen-deficient rats

ObjectiveThis study evaluated the ability of lithium chloride (LiCl) to increase bone filling (BF) around threaded titanium implants inserted in estrogen-deficient rats and, thein-vitro effects of this drug on osteoblast-like cell viability, proliferation, mineralization and expression of bone-related markers. DesignIn vivo: Rats received sham surgery plus water (Estrogen-sufficient group), ovariectomy plus water (Estrogen-deficient group) or ovariectomy plus LiCl (150 mg/kg/every other day) (LiCl/estrogen-deficient group). On the 21st day after ovariectomy/sham surgeries, a threaded titanium implant was inserted in the rat tibia. BF and the number of TRAP + cells were assessed at 10, 20 and 30 days after implant placement. In vitro: Osteosarcoma SAOS-2 cells were exposed to 0, 0.01, 0.05, and 0.1 mM of LiCl; cell proliferation, viability, mineralization (alizarin red staining) and gene expressions of RUNX-2, OCN, OPN, BSP and ALP (Real Time PCR) were estimated in the cultures. ResultsIn vivo: The estrogen-sufficient and LiCl/estrogen-deficient groups demonstrated higher percentages of BF, within the limits of implant threads, than the estrogen-deficient group at 20 and 30 days (p < 0.05). The number of TRAP + cells was lower in LiCl/estrogen-deficient than in the estrogen-deficient group at all experimental times (p < 0.05). In vitro: Cell cultures exposed to LiCl (0.01 or 0.05 mM) exhibited larger areas of mineralized matrix than the non-exposed cultures (p < 0.05) and demonstrated the highest expressions of the genes investigated. ConclusionLiCl treatment improved BF around threaded titanium implants inserted in estrogen-deficient rats and stimulated matrix mineralization and overexpression of bone-formation markers in osteoblastic cells in culture.

Laser-structured spike surface shows great bone integrative properties despite infection in vivo.

Implant associated infections can result in devastating consequences for patients. One major cause is the formation of bacterial biofilms, which result in increased resistance against antimicrobial therapeutics. A reduction of implant associated infections can be achieved by functionalization of implant surfaces. The generation of three dimensional surface structures by femtosecond laser ablation is one method to fabricate bacterial repellent large scaled surfaces without altering the material chemical composition. The challenge is to reduce bacterial growth while improving cellular ongrowth. For this purpose, spike structures were created as small as possible by used fabrication method on cubic Ti90/Al6/V4-rods and their effectiveness against bacterial colonization was compared to unstructured Ti90/Al6/V4-rods. Rods were implanted in the rat tibia and infected intraoperatively with 103 CFU of Staphylococcus aureus. Besides clinical behaviour and lameness, the vital bacterial biomass, morphological appearance and the volume of eukaryotic cells were determined on the implant surface after 21 days. Bone alterations were examined by radiological and histological techniques. Unexpectedly, the laser-structured implants did not show a lower bacterial load on the implant surface and less severe infection related bone and tissue alterations compared to the group without structuring. Simultaneously, a better bony integration and a higher cellular colonization with eukaryotic cells was detected on the laser-structured implants. These findings don't support the previous in vitro results. Nevertheless, the strong integration into the bone is a powerful argument for further surface modifications focussing on the improvement of the antibacterial effect. Additionally, our results underline the need for in vivo testing of new materials prior to clinical use.

Early urine proteome changes in an implanted bone cancer rat model

In this study, Walker 256 cells were implanted into rat tibiae. Urine samples were then collected on days 3, 5, 7, and 13 and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). With label-free quantification, 25 proteins were found to change significantly in the urine of the tumor group rats compared with the proteins in the urine of the control group rats; this was even the case when there were no significant lesions identified in the Computed Tomography (CT) examination. Among these differentially proteins, 7 were reported to be associated with tumor bone metastasis. GO analysis shows that the differential proteins on day 3 were involved in several responses, including the acute phase response, the adaptive immune response and the innate immune response. The differentially proteins on day 7 were involved in the mineral absorption pathway. The differentially proteins on day 13 were involved in vitamin D binding and calcium ion binding. These processes may be associated with bone metastasis. Our results demonstrate that urine could sensitively reflect the changes in the early stage of implanted bone cancer; this provides valuable clues for future studies of urine biomarkers for tumor bone metastasis.

Strain Distribution Evaluation of Rat Tibia under Axial Compressive Load by Combining Strain Gauge Measurement and Finite Element Analysis.

This study is aimed at providing an effective method for determining strain-load relationship and at quantifying the strain distribution within the whole tibia under axial compressive load on rats. Rat tibial models with axial compressive load were designed. Strains in three directions (0°, 45°, and 90°) at the proximal shaft of the tibia were measured by using a strain gauge rosette, which was used to calculate the maximum and minimum principal strains. Moreover, the strain at the midshaft of the tibia was measured by a single-element strain gauge. The slopes of the strain-load curves with different peak loads were calculated to assess the stability of the strain gauge measurement. Mechanical environment in the whole tibia by the axial compressive load was quantified using finite element analysis (FEA) based on microcomputed tomography images. The von Mises elastic strain distributions of the whole tibiae were evaluated. Slopes of the strain-load curves showed no significant differences among different peak loads (ANOVA; P > 0.05), indicating that the strain-load relationship obtained from the strain gauge measurement was reasonable and stable. The FEA results corresponded to the experimental results with an error smaller than 15% (paired Student's t-test, P > 0.05), signifying that the FEA can simulate the experiment reasonably. FEA results showed that the von Mises elastic strain was the lowest in the middle and gradually increased to both sides along the lateral direction, with the maximal von Mises elastic strain being observed on the posterior side under the distal tibiofibular synostosis. The method of strain gauge measurements and FEA used in this study can provide a feasible way to obtain the mechanical environment of the tibiae under axial compressive load on the rats and serve as a reference for further exploring the mechanical response of the bone by axial compressive load.

Secondary alterations in bone mineralisation and trabecular thickening occur after long-term estrogen deficiency in ovariectomised rat tibiae, which do not coincide with initial rapid bone loss.

Recent studies have shown that osteoporosis is not simply a disease of bone loss and microarchitectural degradation but that important changes in tissue composition also occur. Such changes may be a secondary response to early bone loss, but the time sequence of changes in bone mineral distribution is not fully understood. The objective of this study was to quantify the temporal effects of estrogen deficiency on trabecular mineral distribution in the tibia of ovariectomised (OVX) rats. Weekly in vivo micro-CT scans and morphometric and bone mineral density distribution analyses of the proximal tibia were conducted for the first 4 weeks of estrogen deficiency and then at 8, 14 and 34 weeks. Here we report that although trabecular bone volume and architecture are significantly deteriorated within the first 4 weeks of estrogen deficiency, there is no change in the distribution of bone mineral within trabeculae during this initial period. The rate of bone loss in OVX animals dramatically reduced between week 4 and week 14, which coincided with the initiation of increases in trabecular thickness and mineralisation in the OVX group. Together this study reveals for the first time that alterations in bone mineralisation and trabecular thickening arise secondary to the initial rapid bone loss. We propose that these secondary mineralisation changes act to reinforce the trabecular network in an attempt to compensate for the increased loading that ensues after severe bone loss. This study provides an insight into temporal changes in bone mineral distribution in estrogen deficiency.

Evaluation of the Effect of Platelet Rich Fibrin (PRF) on Bone Regeneration in the Tibia of Diabetic Rats (Histological &amp;Immunohistochemical studies)

Objectives: The purpose of this work was to evaluate the effect of platelet- rich fibrin (PRF) on bone defects regeneration in diabetic rats by histological studies of tibial bone and immunohistochemical examination of vascular endothelial growth factor (VEGF).Methods:The present investigation was carried out on eighty adult male albino rats. They were divided randomly into four main groups (each of 20 rats) as the following:Group (I): Normal control rats, received 0.9% saline I.P. only.Group (II): Diabetic control rats, received 0.9% saline I.P.Group (III): Diabetic experimental rats, received PRF.Group (IV): Proposed group (normal control rats received PRF).Five animals from each group were killed after 7, 14, 30 and 60 days alternatively post operatively. Diabetes was induced by injection of monohydrated alloxan. PRF was isolated from the blood of the animals in group III and IV. Bony defects were created in the tibia. The bony cavity was lifted without grafts in group I and II, while in group III and IV the bony defect received PRF. After compeletion of the experiment, the tibia was removed and the specimens were examined histologically by H&E and Masson's trichrome stains and immunohistochemically for demonstration of VEGF.Results: The histological results demonstrated that there was obvious retardation in the granulation tissue and bone formation in the diabetic control group than other groups. Moreover, there was great acceleration in the bone formation in the groups treated with PRF.Immuno-expression of the VEGF was more intense in ossifying centers, around newly formed woven bone trabeculae, in bone marrow spaces and the wall of blood vessels of diabetic and proposed groups treated with PRF.Conclusion: The best healing and bone regeneration occurred in the groups treated with PRF which was markedly increased along the intervals of the study.

Biological activity and regulatory effect of extracellular matrix expression of bone marrow mesenchymal stem cellsconditioned medium on the degenerated nucleus pulposus cells

Objective To observe the biological activities and expression of extracellular matrix of degenerative nucleus pulposus cells which were cultured in the bone marrow mesenchymal stem cells conditioned medium. Methods Bone marrow mesenchymal stem cells were extracted from the femur and tibia of adult SD rats. After subculture, the conditioned medium of bone marrow mesenchymal stem cells was collected. We destroy the annulus fibrosus of the adult rats, and the intervertebral disc was obtained 2 weeks later. Degenerative rat model was used to extract rat nucleus pulposus cells. The nucleus pulposus cells were cultured in the bone marrow mesenchymal stem cells conditioned medium for 24 hours. Results Compared with normal nucleus pulposus cells, the activity of degenerative nucleus pulposus cells [(59.43±12.33)%, t=11.647, P 0.05) and type Ⅱ collagen in cells (1.52±0.18, t=0.299, P>0.05, 1.21±0.31, t=1.543, P>0.05), proteoglycan (1.81±0.32, t=0.486, P>0.05, 0.99±0.16, t=0.094, P>0.05) and matrix metalloproteinase 3 (0.24±0.14, t=0.486, P>0.05, 0.55±0.11, t=0.593, P>0.05) Protein and mRNA expression levels were not significantly affected. Conclusion The bone marrow mesenchymal stem cell conditioned medium promoted the proliferation of extramedullary nucleus cells and the expression of extracellular matrix proteins. Key words: Intervertebral disc herniation; Nucleus pulposus cells; Extracellular matrix protein; Bone marrow mesenchymal stem cell conditioned medium; Type Ⅱ collagen; Aggrecan; Matrix metalloproteinase 3

Imaging and histomorphometric evaluation of mandible and tibia of rats treated with bisphosphonates.

To evaluate the mandible and tibia of rats treated with bisphosphonates (BPs) by imaging and histomorphometric analysis. Thirty-four rat specimens (Rattus norvegicus, Wistar strain) were distributed into 3 groups: (1) 12 rats treated with zoledronic acid; (2) 12 rats treated with clodronate; and (3) the control group, containing 10 rats that received saline. All bones were exposed to cone beam computed tomography (CBCT). The images were analyzed to determine bone density (BD), using the software OsiriX 7.0. Histological slides were prepared from the specimens and the proportion of bone volume (BV) was quantified using the software Adobe Photoshop CC. There was no statistically significant difference in BD either between the drug groups or between mandible and tibia. BV between BPs and control group did not show a significant difference. However, comparing the two bones, the mandibles in the control group displayed higher BV than did the tibiae in the same group. According to our results, we conclude that (1) BD was not altered by bone type or by type of BP administered, and (2) treatment with zoledronic acid or clodronate did not affect BV in the mandible or tibia of test groups.

Icariin reduces high glucose-induced endothelial progenitor cell dysfunction via inhibiting the p38/CREB pathway and activating the Akt/eNOS/NO pathway.

High glucose (HG) impairs endothelial progenitor cell (EPC) function. The activation of p38 mitogen-activated protein kinase and the inhibition of the Akt/eNOS/NO pathway serve central roles in this process. Icariin has protective effects in endothelial cells. The aim of the present study was to investigate the effects of icariin on HG-induced EPC dysfunction, including proliferation, migration and tube formation. Experiments were performed with EPCs isolated from the femurs and tibias of Sprague-Dawley rats in vitro. In a dose-dependent manner, icariin reversed the inhibition of EPC proliferation induced by HG treatment, and the maximal effective concentration of icariin was 1 µM [Fold change (FC):0.90±0.07, P=0.0124 vs. HG group]. The impaired EPC migration and tube formation induced by glucose was partially restored by 1 µM icariin treatment (FC: 0.81±0.08, P=0.0148 vs. HG group for migration; 0.82±0.03, P=0.0214 vs. HG group for tube formation). Furthermore, icariin significantly suppressed HG-induced p38 and cAMP response element binding protein (CREB) phosphorylation in EPCs (FC: 1.84±0.21, P=0.0238 vs. HG group for p38; FC: 2.24±0.15, P=0.0068 vs. HG group for CREB). Increased Akt and endothelial nitric oxide (NO) synthase (eNOS) activation was also observed after icariin treatment (FC: 0.64±0.08, P=0.0047 vs. HG group for Akt; FC:0.53±0.05, P=0.0019 vs. HG group for eNOS), which was followed by increased NO production (FC: 0.69±0.06, P=0.0064 vs. HG group). In conclusion, icariin attenuated HG-induced EPC dysfunction, which may be partially attributed to the inhibition of the p38/CREB pathway and the activation of the Akt/eNOS/NO pathway. Icariin may be a therapeutic candidate for improving the function of EPC.

Effect of Giving Nano Calcium Phosphate Diet on Mineral Content and Function Groups of Ovariectomy Tibia Rats

Lack of calcium and phosphorus minerals in bones can cause decreased bone density, bone becomes brittle and at risk of osteoporosis. The purpose of this study was to analyze changes in mineral levels and functional groups in tibia osteoporosis because of ovariectomy and were treated with a nano-calcium phosphate diet. In this research, dietary formulations containing nano-calcium phosphate made. Diets given to animal models of osteoporosis rats caused by ovariectomy with the aim to balance the mineral content in the rats tibia. Dietary formulations were made of 3 types based on their calcium content, namely the first diet (D1), the second diet (D2) and the third diet (D3) each having a total calcium content of 1.0x, 1.5x and 2.0x normal body needs. Characterization and analysis of mineral content in the tibia bone using Atomic Absorption Spectroscopy (AAS) and Ultraviolet-Visible (UV−vis), while the characterization and analysis of functional groups with Fourier Transform Infrared Spectroscopy (FTIR). Based on the data obtained, ovariectomy rats showed signs of osteoporosis with decreased levels of calcium in the tibia and a decrease in phosphate ion function groups at 9 weeks since ovariectomy. The results of dietary treatment showed that postovariectomy osteoporosis rats fed a diet with a total calcium content of 1.5x normal body needs (D2) and gave more effective and efficient results compared to diets containing 1.0x (D1) and 2.0x (D3) total calcium normal body needs.

In vitro and in vivo osteogenic potential of niobium‐doped 45S5 bioactive glass: A comparative study

In vitro and in vivo experiments were undertaken to evaluate the solubility, apatite-forming ability, cytocompatibility, osteostimulation, and osteoinduction for a series of Nb-containing bioactive glass (BGNb) derived from composition of 45S5 Bioglass. Inductively coupled plasma optical emission spectrometry (ICP-OES) revealed that the rate at which Na, Ca, Si, P, and Nb species are leached from the glass decrease with the increasing concentration of the niobium oxide. The formation of apatite as a function of time in simulated body fluid was monitored by 31P Magic Angle Spinning (MAS) Nuclear magnetic resonance spectroscopy. Results showed that the bioactive glasses: Bioglass 45S5 (BG45S5) and 1 mol%-Nb-containing-bioactive glass (BGSN1) were able to grow apatite layer on their surfaces within 3 h, while glasses with higher concentrations of Nb2 O5 (2.5 and 5 mol%) took at least 12 h. Nb-substituted glasses were shown to be compatible with bone marrow-derived mesenchymal stem cells (BMMSCs). Moreover, the bioactive glass with 1 mol% Nb2 O5 significantly enhanced cell proliferation after 4 days of treatment. Concentrations of 1 and 2.5 mol% Nb2 O5 stimulated osteogenic differentiation of BMMSCs after 21 days of treatment. For the in vivo experiments, trial glass rods were implanted into circular defects in rat tibia in order to evaluate their osteoconductivity and osteostimulation. Two morphometric parameters were analyzed: (a) thickness of new-formed bone layer and (b) area of new-formed subperiostal bone. Results showed that BGNb bioactive glass is osteoconductive and osteostimulative. Therefore, these results indicate that Nb-substituted glass is suitable for biomedical applications.

Growth Factor Delivery for the Repair of a Critical Size Tibia Defect Using an Acellular, Biodegradable Polyethylene Glycol-Albumin Hydrogel Implant.

Growth factor delivery using acellular matrices presents a promising alternative to current treatment options for bone repair in critical-size injuries. However, supra-physiological doses of the factors can introduce safety concerns that must be alleviated, mainly by sustaining delivery of smaller doses using the matrix as a depot. We developed an acellular, biodegradable hydrogel implant composed of poly(ethylene glycol) (PEG) and denatured albumin to be used for sustained delivery of bone morphogenic protein-2 (BMP2). In this study, poly(ethylene glycol)-albumin (PEG-Alb) hydrogels were produced and loaded with 7.7 μg/mL of recombinant human BMP2 (rhBMP2) to be tested for safety and performance in a critical-size long-bone defect, using a rodent model. The hydrogels were formed ex situ in a 5 mm long cylindrical mold of 3 mm diameter, implanted into defects made in the tibia of Sprague-Dawley rats and compared to non-rhBMP2 control hydrogels at 13 weeks following surgery. The hydrogels were also compared to the more established PEG-fibrinogen (PEG-Fib) hydrogels we have tested previously. Comprehensive in vitro characterization as well as in vivo assessments that include: histological analyses, including safety parameters (i.e., local tolerance and toxicity), assessment of implant degradation, bone formation, as well as repair tissue density using quantitative microCT analysis were performed. The in vitro assessments demonstrated similarities between the mechanical and release properties of the PEG-Alb hydrogels to those of the PEG-Fib hydrogels. Safety analysis presented good local tolerance in the bone defects and no signs of toxicity. A significantly larger amount of bone was detected at 13 weeks in the rhBMP2-treated defects as compared to non-rhBMP2 defects. However, no significant differences were noted in bone formation at 13 weeks when comparing the PEG-Alb-treated defects to PEG-Fib-treated defects (with or without BMP2). The study concludes that hydrogel scaffolds made from PEG-Alb containing 7.7 μg/mL of rhBMP2 are effective in accelerating the bridging of boney defects in the tibia.

The effects of transplanted mesenchymal stem cells treated with 17-b estradiol on experimental autoimmune encephalomyelitis.

The present study was conducted aimed at exploring the modulatory effects of 17-b estradiol (17-bED) on mesenchymal stem cells (MSCs) in the EAE (experimental autoimmune encephalomyelitis) animal model of multiple sclerosis (MS). Following the isolation of bone marrow-derived MSCs from the bilateral femurs and tibias of the male Wistar rats, the cells were harvested and cultured in the presence of 100nM 17-bED for 24h. EAE was induced in male Wistar rats (8-12weeks old) using guinea pig spinal cord homogenate, in combination with the complete Freund's adjuvant. The MSC therapy was triggered when all of the animals obtained a disability score. The symptoms were monitored on a daily basis throughout the study until the rats were euthanized. The mRNA expression of cytokines, including IL-17, IFN-γ, TNF-α, IL-10, IL-4, and TGF-β together with MMP8 and MMP9 as the family members of matrix metalloproteinases(MMPs) in the brain and spinal cord tissues were examined using real-time PCR. The levels of splenocytes-originated IL-10 and IFN-γ cytokines were also measured by ELISA. The MTT-based research findings showed that the infiltration of lymphocytes into the spleen decreased considerably. It was also observed that the mRNA expression of proinflammatory cytokines decreased significantly, while the mRNA levels of anti-inflammatory cytokines increased remarkably. It was also found that the mRNA levels of the examined matrix metalloproteinases(MMP8 and MMP9) were downregulated significantly. The findings of the present study indicated that the administration of 17-bED enhanced the efficacy of MSCs transplantation and modulated immune responses relatively in the EAE model, via the regulation of either pro- or anti-inflammatory cytokines and matrix metalloproteinases.

Guhong Injection promotes fracture healing by activating Wnt/beta-catenin signaling pathway in vivo and in vitro

Guhong Injection (GHI), composed of aceglutamide and safflower aqueous extract, has been applied to the clinical treatment of orthopedic diseases, but the relevant mechanism by which GHI exerts effects on bone remodeling has not been reported. In the present study, we investigated the effects of various concentrations of GHI (2.5, 5 and 10 ml/kg) in accelerating rat tibia healing progress by observing haematoxylin and eosin (HE) stained sections, detecting the activity of bone metabolism biochemical markers such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), osteocalcin (OC) and C-terminal crosslinking telopeptide of type Ⅰ collagen (CTX-1) in rat serum, as well as measuring the expressions of collagen I (COL-1) and collagen II (COL-2) in rat tibia. Also, we investigated the effects of different concentrations of GHI (30, 60 and 90 μl/ml) on the proliferation and differentiation of osteoblasts (OBs) through proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP) and type I collagen (COL-1). At the same time, the expression of important factors of Wnt/beta-catenin signaling pathway including Wnt-3a, beta-catenin, disheveled-1 (Dvl-1), glycogen synthase kinases-3beta (GSK-3beta), lymphoid enhancing factor-1 (LEF-1) and axis inhibition protein-2 (Axin-2) after GHI intervention was detected by quantitative real-time PCR (q-PCR), immunohistochemistry and Western blotting. In vivo, rats of tibia fracture model treated with intraperitoneal injection (ip) of GHI had more mature fibroblasts, as well as shorter period formation of new bone. The levels of BMP-2, TGF-beta and OC in rat serum were significantly up-regulated, while the level of CTX was down-regulated. After 4 weeks of drug treatment, the level of COL-1 in the rat tibia increased, but there was no significant change in the level of COL-2. In vitro, after drug intervention, the number of OBs increased significantly, the activities of PCNA, ALP and COL-1 were enhanced. Treatment with GHI increased the mRNA and protein expression of Wnt-3a, beta-catenin, Dvl-1 and LEF-1, and decreased the expression of mRNA of Axin-2 and GSK-3beta. All results demonstrate that GHI accelerates the proliferation of OBs and shortens the recovery time of bone structure, and the Wnt/beta-catenin signaling pathway is involved in the regulation process.

Phosphoproteomic profiling of oxycodone‑treated spinal cord of rats with cancer‑induced bone pain.

Treatment of cancer‑induced bone pain (CIBP) is challenging in clinical settings. Oxycodone (OXY) is used to treat CIBP; however, a lack of understanding of the mechanisms underlying CIBP limits the application of OXY. In the present study, all rats were randomly divided into three groups: The sham group, the CIBP group, and the OXY group. Then, a rat model of CIBP was established by inoculation of Walker 256 tumor cells from rat tibia. Phosphoproteomic profiling of the OXY‑treated spinal dorsal cords of rats with CIBP was performed, and 1,679 phosphorylated proteins were identified, of which 160 proteins were significantly different between the CIBP and sham groups, and 113 proteins were significantly different between the CIBP and OXY groups. Gene Ontology analysis revealed that these proteins mainly clustered as synaptic‑associated cellular components; among these, disks large homolog 3 expression was markedly increased in rats with CIBP and was reversed by OXY treatment. Subsequent domain analysis of the differential proteins revealed several significant synaptic‑associated domains. In conclusion, synaptic‑associated cellular components may be critical in OXY‑induced analgesia in rats with CIBP.

Exploring the mechanism behind improved osteointegration of phosphorylated titanium implants with hierarchically structured topography

Titanium (Ti) and its alloys have been frequently used in dental and orthopedic implants, but the undesired oxide layer easily formed on the surface tends to be the cause of implant failure for Ti-based implants. To address this problem, we herein prepared a phosphorylated Ti coating (TiP-Ti) with a micro/nano hierarchically structured topography on commercially pure Ti implants by a hydrothermal method to improve its osteointegration capacity. The surface morphology, chemical composition, and biological activity were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), contact-angle measurement, and protein adsorption assay. Osteointegration of TiP-Ti implants in rat tibia was investigated by biomechanical testing, micro-CT and histological analyses. We further explored the proposed mechanism which improves osteointegration of TiP-Ti implants by proliferation, adhesion, and differentiation assays of rat bone marrow mesenchymal stem cells (BMSCs). Our results demonstrated that the improved osteointegration mainly benefited from the better spread and adhesion of BMSCs on the micro/nano hierarchically structured TiP-Ti surfaces compared to hydroxyapatite coated Ti (HA-Ti), the positive control, and untreated Ti (untreated-Ti), the negative control. In conclusion, TiP-Ti surface is a promising candidate implant surface design to accelerate the osteointegration of Ti-based implants in biomedical applications.

Co-culture of the bone and bone marrow: a novel way to obtain mesenchymal stem cells with enhanced osteogenic ability for fracture healing in SD rats

BackgroundMesenchymal stem cells (MSCs) have great potential for the repair and regeneration of bone fracture, but their optimal origins remain controversial.MethodsBone marrow-MSCs (BM-MSCs) and bone-bone marrow-MSCs (B-BM-MSCs) were isolated from 12 SD rats, and the morphology, MSC-associated markers, and proliferative capacity of these cells were compared using an inverted microscope, flow cytometry, and CCK-8 assays, respectively. After 14 days of osteoblastic induction, osteoblast phenotypes were detected by ALP and calcium nodule staining, and the expression of BMP-2 and TGF-β1 was observed by western blotting. Then, the rat tibia fracture model was established with 3 groups (n = 6 per group), the control, BM-MSC, and B-BM-MSC groups. Computed tomography (CT) imaging was performed to evaluate fracture healing at weeks 2, 4, and 6. Finally, the fractured bones were removed at weeks 4 and 6, and HE staining was performed to evaluate fracture healing.ResultsAlthough the 2 types of MSCs shared the same cellular morphology and MSC-associated markers, B-BM-MSCs had a higher proliferative rate than BM-MSCs from day 9 to day 12 (p < 0.05), and the expression levels of ALP and calcium were obviously higher in B-BM-MSCs than in BM-MSCs after osteogenic induction (p < 0.01 and p < 0.001, respectively). Western blot results showed that the expression levels of BMP-2 and TGF-β1 in B-BM-MSCs were higher than in BM-MSCs before and after osteogenic induction (p < 0.01). In the animal experiments, CT imaging and gross observation showed that B-BM-MSCs had a greater capacity than BM-MSCs to promote fracture healing, as the Lane-Sandhu scores of B-BM-MSCs at weeks 4 and 6 after operation (3.00 ± 0.81 and 9.67 ± 0.94, respectively) were higher than those of BM-MSCs (1.33 ± 0.47 and 6.67 ± 1.25, respectively; both p < 0.05). The HE staining results further supported this conclusion.ConclusionsTaken together, our study results proved that MSCs obtained by co-culturing the bone and bone marrow from SD rats had better proliferative, osteogenic differentiation, and fracture healing capacities than BM-MSCs, perhaps suggesting a novel way to obtain MSCs for bone tissue repair.