Rat Tibia Research Articles

Optimization of Fluorapatite/Bioactive Glass Nanocomposite Foams as Bone Tissue Scaffold: An in Vivo Study.

The present study investigated the suitability of nanocomposite foams of fluorapatite and bioactive glass (FA /BG) in different weight ratios as scaffolds for bone tissue in rat tibia regeneration to determine the optimal composition. FA and BG nano powders with a weight ratio of 25% FA/75% BG (compound 1) and 75% FA/25% BG (compound 2) were used as precursors for gel casting to produce nanocomposite foams. Thirty rats were randomly divided into two equal groups. Disk-shaped samples of each compound were implanted into the tibias of 15 rats. After 15, 30, or 60 days, five rats from each group were sacrificed and subjected to radiological, histopathological, and histomorphometrical examination. Data were analyzed using SPSS software. No foreign body reaction was observed in either group at all intervals, and the bone-biomaterial junction was direct. Overall, the inflammation rate, and the number of blood vessels, osteoblasts, and osteoclasts decreased over time in both groups. However, the number of osteocytes, trabecular bone thickness, and the percentage of new bone formation increased, in contrast to the remaining biomaterial percentage. Most of the changes in the group implanted with compound 2 were significantly more significant and faster than in the other group. Although the composite with the higher percentage of FA was superior to the composite with the higher percentage of BG, considering the results of our previous similar studies, the composite with the same percentage of FA and BG is more favorable to be used as a substitute for bone tissue in the body.

Effect of thermal osteonecrosis around implants in the rat tibia: numerical and histomorphometric results in context of implant removal

The purpose of this rat study was to explore the feasibility of in vivo temperature thresholds affecting bone contact at the implant surface. Based on these data, thermal necrosis should be used for implant removal in the subsequent in vivo study. Rat tibiae of 48 animals at one site were randomly treated with heat or cold before implant insertion. Temperatures of 4 °C, 3 °C, 2 °C, 48 °C, 49 °C and 50 °C for a tempering time of 1 min were evaluated. Numerical simulations of the heat source-implant-bone system were carried out. Effects were assessed by histomorphometrical measurements. The results showed that the selected method of direct tempering using a tempering pin was suitable for maintaining a uniform layer around the pin. Starting at warm temperatures of 48 °C and rising to 50 °C, the BIC ratio revealed declining values and a significant difference was observed when comparing 50 °C to the control group (p = 0.03). However, there were no significant variations within the cold temperatures. This study pinpointed temperature discovered that could lead to the thermo-explantation and so that the number of samples used in future studies on temperature-induced bone necrosis can be reduced to a minimum. Significant BIC value reduction was seen at a temperature of 50 °C for 1 min.

Effect of Desmodium renifolium on ovariectomized rat model of osteoporosis via BMP-2/Smads signaling pathway

This study aimed to investigate the effect of Desmodium renifolium(Linn.) Schindl on ovariectomized rat model of osteoporosis and explore the underlying mechanism. Rats were randomized into a sham group, a model group, a Xianlin Gubao group, and low-, medium-, and high-dose D. renifolium groups. Bilateral oophorectomy was performed in other groups except sham group(removal of bilateral peri-ovarian adipose tissue). The sham group and model group were administrated with equal volume of 0.5% CMC-Na, Xianlin Gubao group with Xianlin Gubao, and D. renifolium groups with different concentrations of alcoholic extract of D. renifolium once a day for 14 weeks. The body weight of rats were recorded during the experiment. The levels of estradiol(E_2), 1,25-dihydroxyvitamin D_3 [1,25(OH)_2D_3], calcium(Ca), and phosphorus(P) in serum were determined after the administration. The femur microstructure was analyzed via micro-CT. RT-PCR and Western blot were employed to respectively determine the mRNA and protein levels of bone morphogenetic protein 2(BMP-2), Runt-related transcription factor 2(Runx2), and osterix(OSX) in the tibia of rats. The results indicated that D. renifolium significantly inhibited the weight growth, improved the uterus appearance, elevated the levels of E_2, Ca, P, and 1,25(OH)_2D_3 in serum, increased the number and reduced the fracture of bone trabeculae, ameliorated the bone microstructure parameters, and up-regulated the mRNA and protein levels of BMP-2, Runx2, and OSX. All the results demonstrated that D. renifolium had significant protective effect on the ovariectomized rat model of osteoporosis by regulating the BMP-2/Smads signaling pathway.

Yanghe decoction attenuated pain hypersensitivity induced by michigan cancer foundation-7 injection in rats with bone metastases from breast cancer by inhibiting transient receptor potential ankyrin 1.

To study the effect and underlying mechanisms of Chinese medicine Yanghe decoction on pain relief in a rat model of bone metastasis of breast cancer induced by michigan cancer foundation-7 (MCF-7). Bone pain was induced in the tibia of rats injected with MCF-7 cells. The Chinese herbal remedy was used to decoct Yanghe decoction for the treatment of bone pain rats. The behavior study was carried out to evaluate the paw mechanical withdraw threshold and thermal withdraw latency. Liquid chromatography-mass spectrometry, Western blotting, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), immunohistochemical (IHC) staining were performed for analysis. Yanghe decoction could improve the defensive behavior similar to the transient receptor potential ankyrin 1 (TRPA1) inhibitor. In morphology study, Yanghe decoction could attenuate the cellular growth as well as inflammatory infiltration in the metastasis group. Furthermore, Yanghe decoction downregulated the TRPA1 expression on the dorsal root ganglion from the metastatic rats at both transcriptional and protein level. Yanghe decoction alleviated the inflammation in metastatic tissues by hematoxylin-eosin and IHC analysis, and Yanghe decoction also reduced the inflammatory cytokines production in the serum including tumor necrosis factor-α and interleukin-6, interleukin-1 beta by ELISA. As the cytochromec oxidase subunit II/prostaglandin E2 (PGE2) is required for cancer development, Yanghe decoction reduced the expression of PGE2 in the tissue and serum. Taken together, Yanghe decoction protected the rats from breast cancer bone metastasis through TRPA1 signaling mediated neuropathic pain and additional immune modulation in tumor microenvironment.

Effect of Flax lignans on apoptosis of growth plate chondrocytes in rats

To compare the effects of different concentrations of linolenin on inhibiting apoptosis of chondrocytes in the growth plate, and to screen the optimal concentration of linolenin, so as to provide theoretical support for delaying epiphyseal closure and promoting long bone growth in rats. Two 4-week-old male SD rats (SPF grade) with a body mass of 80 g were selected. The growth plate cartilage of rat tibia and femur was dissected and isolated in vitro to obtain growth plate chondrocytes for culture. The chondrocytes were observed and identified by inverted phase contrast microscope and typeⅡ collagen immunofluorescence test, and then 20 ng/ml IL-1β was used to induce apoptosis of growth plate chondrocytes as model group, and added with 1, 10, 20, 40 μM linolenin as the experimental group, and 5 μM letrozole as the positive control group. The cells were cultured for 24 and 48 hours respectively. The drug promoted cell proliferation was observed by MTT method, and the drug inhibited cell apoptosis was detected by flow cytometry. Contents 1, 10, 20, 40 μM could promote cell proliferation in varying degrees, and the principle was that the drug inhibits IL-1β induced chondrocyte apoptosis in the growth plate, and the optimal concentration of drugs to inhibit apoptosis was 20 μM. The appropriate concentration of linseed lignans can significantly inhibit the apoptosis of chondrocytes in the growth plate of rats, and the optimal drug concentration is 20 μM. It provides possibility for delayed bone closure and longer growth time to promote bone growth during development.

Synergistic Effect of Carbonate Apatite and Autogenous Bone on Osteogenesis.

Bone augmentation using artificial bone is an important option in dental defect prostheses. A bone substitute using carbonate apatite (CO3Ap), an inorganic component of bone, was reported to have promising bone formation and bone replacement ability. However, the osteoinductivity of artificial bone is less than autogenous bone (AB). In this study, CO3Ap with AB is demonstrated as a clinically effective bone substitute. For in vitro experiments, an osteoclast-like cell (RAW-D) was cultured in the presence of AB, CO3Ap, or both (Mix), and the number of osteoclasts was evaluated. Osteoblasts were also cultured under the same conditions, and the number of adherent cells was evaluated. For in vivo experiments, a few holes were created in the rat tibia and AB, CO3Ap, or Mix were added. At 0, 14, and 21 days, the tissue morphology of the wound area was observed, and the thickness of the cortical bone was measured. In vitro, Mix did not increase the number of osteoclasts or osteoblasts. However, in vivo, the rate of bone replacement remarkably increased with Mix on dome-shape. A bone-grafting material combining osteoinductive AB with abundant artificial bone is expected to be clinically easy to use and able to form bone.

Zoledronic Acid Implant Coating Results in Local Medullary Bone Growth.

Osteoarthritis (OA) can necessitate surgical interventions to restore the function of the joint in severe cases. Joint replacement surgery is one of the procedures implemented to replace the damaged joint with prosthetic implants in severe cases of OA. However, after successful implantation, a fraction of OA patients still require revision surgery due to aseptic prosthetic loosening. Insufficient osseointegration is one of the factors that contribute to such loosening of the bone implant, which is commonly made from titanium-based materials. Zoledronic acid (ZA), a potent bisphosphonate agent, has been previously shown to enhance osseointegration of titanium implants. Herein, we fabricated ZA/Ca composites using a reverse microemulsion method and coated them with 1,2-dioleoyl-sn-glycero-3-phosphate monosodium salt (DOPA) to form ZA/Ca/DOPA composites. Titanium alloy screws were subsequently dip-coated with a suspension of the ZA/Ca/DOPA composites and poly(lactic-co-glycolic) acid (PLGA) in chloroform to yield Za/PLGA-coated screws. The coated screws exhibited a biphasic in vitro release profile with an initial burst release within 48 h, followed by a sustained release over 1 month. To assess their performance in vivo, the Za/PLGA screws were then implanted into the tibiae of Sprague-Dawley rats. After 8 weeks, microCT imaging showed new bone growth along the medullary cavity around the implant site, supporting the local release of ZA to enhance bone growth around the implant. Histological staining further confirmed the presence of new mineralized medullary bone growth resembling the cortical bone. Such local medullary growth represents an opportunity for future studies with alternative coating methods to fine-tune the local release of ZA from the coating and enhance complete osseointegration of the implant.

Angelica sinensis polysaccharides prevents hematopoietic regression in D-Galactose-Induced aging model via attenuation of oxidative stress in hematopoietic microenvironment.

Extrinsic molecular mechanisms that regulate hematopoietic stem/progenitor cell (HSPC) aging are still poorly understood, and a potential protective medication needs to be explored. The senescent parameters of hematopoietic cells and bone marrow stromal cells (BMSCs) including cell cycle analysis, senescence-associated SA-β-gal staining and signals, hematopoietic factors and cellular junction were analyzed in femur and tibia of rats. Furthermore, Sca-1+ HSPCs and BMSCs co-culture system was established to evaluate the direct effects of BMSC feeder layer to HSPCs. Oxidative DNA damage indicators in Sca-1+ HSCs and senescence-associated secretory phenotype (SASP) of BMSCs, gap junction intercellular communication between BMSCs, osteogenesis/adipogenisis differentiation balance of BMSCs were detected. In the D-gal pre-administrated rats, ASP treatment rescued senescence of hematopoietic cells and BMSCs, reserved CFU-GEMM; also, ASP treatment attenuated stromal oxidative load, ameliorated SCF, CXCL12, and GM-CSF production, increased Connexin-43 (Cx43) expression. BMSCs and Sca-1+ HSPCs co-cultivation demonstrated that ASP treatment prevented oxidative DNA damage response in co-cultured Sca-1+ HSPCs induced by D-gal pre-administration of feeder layer and the underlying mechanism may be related to ASP ameliorating feeder layer dysfunction due to D-gal induced senescence via inhibiting secretion of IL-1, IL-6, TNF-α, and RANTES, enhancing Cx43-mediated intercellular communication, improving Runx2 expression whereas decreasing PPARγ expression in BMSCs. The antioxidant property of ASP may provide a stroma-mediated potential therapeutic strategy for HSPC aging.

What's New in Musculoskeletal Basic Science.

What's New in Musculoskeletal Basic Science.

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.

Changes of cortical bone pores structure and their effects on mechanical properties in tail-suspended rats

Cortical bone is the main mechanical bearing structure of bone, and the mechanical properties of materials are not only related to bone mineral density, but also largely depend on its pores microstructure which affected by blood vessels. However, the change of pores structure in cortical bone under microgravity was still unclear. In this study, in order to clear the changes of pore structure with cortical vascular pores and its effect on bone mechanical properties, rat tail-suspension was used to simulate microgravity and the changes of the microstructure in rat tibia cortices were investigated by high-resolution micro-CT (3 ​μm) while the bone mechanical properties were measured via three point bending test. The results showed the bone mineral density of cortical bone didn't change in tail-suspended rats. However, the pore structure of cortical bone in tail-suspended rats changed significantly, the proportion of pores greater than 15 ​μm (cortical vascular pores) increased while that less than 15 ​μm decreased. The mechanical properties of bone (such as maximum load and maximum stress) in tail-suspended rats deteriorated. And the volume ratio of pore vessels (vessel volume/tissue volume) was negatively correlated with the mechanical properties. In conclusion, the increase of cortical vascular pores in rats caused by the simulated microgravity contributes to the decrease of mechanical properties.

In vivo prevascularization strategy enhances neovascularization of β-tricalcium phosphate scaffolds in bone regeneration

BackgroundNeovascularization is critical for bone regeneration. Numerous studies have explored prevascularization preimplant strategies, ranging from calcium phosphate cement (CPC) scaffolds to co-culturing CPCs with stem cells. The aim of the present study was to evaluate an alternative in vivo prevascularization approach, using preimplant-prepared macroporous beta-tricalcium phosphate (β-TCP) scaffolds and subsequent transplantation in bone defect model.MethodsThe morphology of β-TCPs was characterized by scanning electron microscopy. After 3 weeks of prevascularization within a muscle pouch at the lateral size of rat tibia, we transplanted prevascularized macroporous β-TCPs in segmental tibia defects, using blank β-TCPs as a control. Extent of neovascularization was determined by angiography and immunohistochemical (IHC) evaluations. Tibia samples were collected at different time points for biomechanical, radiological, and histological analyses. RT-PCR and western blotting were used to evaluate angio- and osteo-specific markers.ResultsWith macroporous β-TCPs, we documented more vascular and supporting tissue invasion in the macroporous β-TCPs with prior in vivo prevascularization. Radiography, biomechanical, IHC, and histological analyses revealed considerably more vascularity and bone consolidation in β-TCP scaffolds that had undergone the prevascularization step compared to the blank β-TCP scaffolds. Moreover, the prevascularization treatment remarkably upregulated mRNA and protein expression of BMP2 and vascular endothelial growth factor (VEGF) during bone regeneration.ConclusionThis novel in vivo prevascularization strategy successfully accelerated vascular formation to bone regeneration. Our findings indicate that prevascularized tissue-engineered bone grafts have promising potential in clinical applications.The translational potential of this articleThis study indicates a novel in vivo prevascularization strategy for growing vasculature on β-TCP scaffolds to be used for repair of large segmental bone defects, might serve as a promising tissue-engineered bone grafts in the future.

Acceleration of bone formation by octacalcium phosphate composite in a rat tibia critical-sized defect.

BackgroundThe osteogenic capabilities and biodegradability of octacalcium phosphate (OCP) composites make them unique. Despite the excellent characteristics of OCP, their use is limited due to handling difficulties. In this study, we aimed to evaluate and compare three types of OCPs (cemented OCP (C-OCP), C-OCP with collagen (OCP/Col), and synthetic OCP (S-OCP) with alginate (OCP/Alg)) versus commercially available β-tricalcium phosphate (β-TCP) regarding their potential to accelerate bone formation in defective rat tibias.MethodsThe specimens with OCP composite were manufactured into 5 ​mm cubes and inserted into the segmental defects of rat tibias fixed with an external fixator. In addition, 3 ​mm-hole defects in rat tibias were evaluated to compare the graft material properties in different clinical situations. Serial X-ray studies were evaluated weekly and the tibias were harvested at postoperative 6 weeks or 8 weeks for radiologic evaluation. Histological and histomorphometric analyses were performed to evaluate the acceleration of bone formation.ResultsIn the critical-defect model, OCP/Alg showed bone bridges between segmentally resected bone ends that were comparable to those of β-TCP. However, differences were observed in the residual graft materials. Most β-TCP was maintained until 8 weeks postoperatively; however, OCP/Alg was more biodegradable. In addition calcification in the β-TCP occurred at the directly contacted area between graft particles and bony ingrowth was observed in the region adjacent resected surface of tibia. In contrast, no direct bony ingrowth was observed in OCP-based materials, but osteogenesis induced from resected surface of tibia was more active. In the hole-defect model, OCP/Col accelerated bone formation. β-TCP and OCP/Alg showed similar patterns with relatively higher biodegradability. In histology, among the OCP-based materials, directly contacted new bone was formed only in OCP/Alg group. The new bone formation in the periphery area of graft materials was much more active in the OCP-based materials, and the newly formed bone showed a thicker trabecular and more mature appearance than the β-TCP group.ConclusionsIn this study, OCP/Alg was equivalent to β-TCP in the acceleration of bone formation with better biodegradability appropriate for clinical situations in different circumstances. Our OCP/Col composite showed fast degradation, which makes it unsuitable for use in mechanical stress conditions in clinical orthopedic settings.The Translational Potential of this ArticleIn our research, we compared our various manufactured OCP composites to commercially available β-TCP in critical-defect rat tibia model. OCP/Col showed acceleration in hole-defect model as previous studies in dental field but in our critical-sized defect model it resorbed fast without acceleration of bony union. OCP/Alg showed matched results compared to β-TCP and relatively fast resorption so we showed market value in special clinical indication depending on treatment strategy. This is the first OCP composite study in orthopaedics with animal critical-sized tibia bone study and further study should be considered for clinical application based on this study.

Ameliorative effect of different mesoporous bioactive glass materials in experimental tibial defects in rats.

Enhancing the bone healing procedure would resultantly improve the post-recovery life quality, as well as the speed with which the patient returns to their former life quality. Porous structures can provide a large surface area and abundant channels to facilitate mass transfer. To evaluate the application of mesoporous materials in the bone healing of surgically created defects on the tibiae of male adult Wistar rats. The defect areas were evaluated after implantation of 4 types of bioactive glass histopathologically and immunohistochemically. Fifty adult rats were divided into 5 groups including a control group without material. The used products were mesoporous bioactive glass (MBG), Cu-MBG, Zn-MBG, and Cu-Zn-MBG. Unicortical bone defects with a 3 mm diameter were performed in both tibiae of the animals and filled with 4 types of glass particles. The rats were then euthanized at 15 d and 30 d. Tibial samples were collected and the tissues forwarded for histological processing, and examined using light microscopy. Additionally, bone healing was evaluated by assessing the levels of bone morphogenetic protein BMP2, collagen 1, osteocalcin (OST), and vascular endothelial growth factor (VEGF) using immunohistochemical methods. Within the 15th day, all groups presented connective tissue septa; at the 30th day, the new bone formation was more intense in the Cu-Zn-MBG group. Additionally, BMP2, collagen 1, OST, and VEGF immune expression were more prominent in the Cu-Zn-MBG group. The study results indicated that MBG may be used for the repairing of bone defects. Cu-Zn-MBG may be the best choice for this purpose.

Protocol for Recovery of a Rat Tibia Fracture through 3D-Printed PLA-Scaffold Implantation

One of the more recent fracture-healing methods comprises applying patient-specific 3D printing. An optimal fracture-healing method would accurately print and embed a customized implant at the defect site of the fracture. In this study, to provide a standardized 3D printing process, the entire process of making a fracture model, manufacturing the customized 3D printing, and implanting the result was conducted through animal experiments. A fracture treatment 3D printing process was established and a fracture model was produced. X-ray and micro-computed tomography (CT) images were acquired and the portions with the fractured bones were extracted by segmentation and reconstruction using image preprocessing software. Based on this, 3D modeling of the fracture site scaffold was performed, and the 3D printing protocol was experimentally verified through polylactic acid (PLA)-based 3D printing and implantation at the fracture site. The entire process of making the fracture model, manufacturing the customized 3D-printed bone fragment, and implanting it in the defect site was performed based on 3D printing. There is a need for further animal clinical validation and histological studies on the fracture recovery process according to 3D printing protocols. Nevertheless, this approach (performing the implantation process of a customizable 3D printing scaffold for replacing atypical fractures using X-ray and micro-CT images) is expected to play a new role in fracture treatment.

Immunohistochemical evaluation for integrin binding sialoprotein on healing process of intrabony defect treated by bone sialoprotein

Background: Bone defect healing is a multidimensional procedure with an overlapping timeline that involves the regeneration of bone tissue. Due to bone's ability to regenerate, the vast majority of bone abnormalities can be restored intuitively under the right physiological conditions. The goal of this study is to examine the immunohistochemistry of bone sialoprotein in order to determine the effect of local application of bone sialoprotein on the healing of a rat tibia generated bone defect. Materials and Methods: In this experiment, 48 albino male rats weighing 300-400 grams and aged 6-8 months will be employed under controlled temperature, drinking, and food consumption settings. The animals will be subjected to a surgical procedure on the medial side of the tibiae bone, with the bone defect repaired with absorbable hemostatic material in the control group (12 rats). The experimental group (12 rats) will be treated with local administration of 30 μl bone sialoprotein fixed by absorbable hemostatic sponge. After surgery, the rats will be slaughtered at 7, 14, and 28 days (four rats for each period). Results: Immunohistochemical analysis of bone sialoprotein by stromal cells reveal a substantial difference between the bone sialoprotein group and the control group. Conclusion: The study concludes that local application of bone sialoprotein could be a successful therapeutic treatment for bone injuries; these findings are encouraging for future clinical use.

Overexpression of GDNF in Spinal Cord Attenuates Morphine Analgesic Tolerance in Rats with Bone Cancer Pain

Bone cancer pain (BCP) is one of the typical and distressing symptoms in cancer patients. Morphine is a widely used analgesic drug for BCP; however, long-term morphine administration will lead to analgesic tolerance. Our previous study indicated that spinal glial cell line-derived neurotrophic factor (GDNF) exerts analgesic effects in rats with BCP. In this study, BCP was established by inoculated Walker 256 carcinoma cells into rat tibias, while morphine tolerance (MT) was induced by intrathecally injecting morphine twice daily from the 9th to 15th postoperative day (POD) in BCP rats. The BCP rats developed mechanical and thermal hyperalgesia on POD 5 and it lasted to POD 15. The analgesic effect of morphine was decreased after repeat administration. Western blots and immunochemistry tests showed that GDNF was gradually decreased in the spinal cord after the development of MT in rats with BCP, and GDNF was colocalized with the μ opioid receptor (MOR) in the superficial laminate of the spinal cords. The overexpression of GDNF by lentivirus significantly attenuated MT, and restored the expression of MOR in the spinal cord. In summary, our results suggest that the reduction of GDNF expression participated in the development of MT in rats with BCP and could be a promising therapeutic option for BCP.

Surgical technique and comparison of autologous cancellous bone grafts from various donor sites in rats.

Autologous cancellous bone graft is the gold standard in large bone defect repair. However, studies using autologous bone grafting in rats are rare. To determine the feasibility of autologous cancellous bone graft harvest from different anatomical donor sites (humerus, ilium, femur, tibia, and tail vertebrae) in rats and compare their suitability as donor sites, a total of 13 freshly euthanized rats were used to describe the surgical technique, determine the cancellous bone volume and microstructure, and compare the cancellous bone collected quantitatively and qualitatively. It was feasible to harvest cancellous bone grafts from all five anatomical sites with the humerus and tail being more surgically challenging. The microstructural analysis using micro-computed tomographyshowed a significantly lower bone volume fraction, bone mineral density, and trabecular thickness of the humerus and iliac crest compared to the femur, tibia, and tail vertebrae. The harvested weight and volume did not differ between the donor sites. All donor sites apart from the femur yielded primary osteogenic cells confirmed by the presence of alkaline phosphatase and Alizarin Red S stain. Bone samples from the iliac crest showed the most consistent outgrowth of osteoprogenitor cells. In conclusion, the tibia and iliac crest may be the most favorable donor sites considering the surgical approach. However, due to the differences in microstructure of the cancellous bone and the consistency of outgrowth of osteoprogenitor cells, the donor sites may have different healing properties, that need further investigation in an in vivo study.

In Vivo Effects of Nanotechnologically Synthesized and Characterized Fluoridated Strontium Apatite Nanoparticles in the Surgical Treatment of Endodontic Bone Lesions

In this study, fluoridated strontium apatite (SAP) nanoparticles with different mole percentages (5%, 10%, 30%, and 50%) synthesized using a hydrothermal method were used as biomaterials. The in vivo biocompatibility of the synthesized nanoparticles was investigated by embedding them as biomaterials in bone defects created in rat tibiae. Through the hematoxylin-eosin staining method, a histopathological analysis was performed for new bone formation, osteoblast density, and fibrotic tissue formation. Fluorine (F) addition affected the structural and morphological properties of the nanoparticles. With the F doping, the shapes of the nanoparticles changed from nano-rods to almost spherical. The Sr/P ratios, with a stoichiometric value of 1.67, were 1.76, 1.53, 1.54, 1.68, and 1.79 in pure, 5%, 10%, 30%, and 50% F-doped nanoparticles, respectively. The F/Sr ratios of 5%, 10%, 30%, and 50% F-doped nanoparticles were 0.05, 0.13, 0.16, and 0.20, respectively. The highest values in terms of fibrotic tissue formation were obtained in the group containing pure SAP. The best results in terms of new bone formation and osteoblast density in bone defects were observed in the groups with higher F ratios (30% and 50% F-doped). Pure and F-doped strontium apatite nanoparticles showed good results for new bone formation and osteoblast levels compared to the control group. It was observed that an increase in the fluorine ratio resulted in better bone healing. The results showed that pure and F-doped SAP nanoparticles synthesized by a hydrothermal method can be used as biomaterials in orthopedics and dentistry, especially in the surgical treatment of endodontic lesions.

Collagen membrane from bovine pericardium for treatment of long bone defect.

The treatment of bone regeneration failures has been constantly improved with the study of new biomaterials. Techgraft® is a collagen membrane derived from bovine pericardium, which has been shown to have biocompatibility and effectiveness in tissue repair. However, its use in orthopedics has not yet been evaluated. Therefore, the purpose of this study was to characterize a bovine pericardium collagen membrane and evaluate the effects of its use in the regeneration of a bone defect in rat tibia. Scanning electron microscopy, atomic force microscopy, weight lost and water uptake tests, and mechanical test were performed. Afterwards, the membrane was tested in an experimental study, using 12 male Sprague Dawley rats. A bone defect was surgically made in tibiae of animals, which were assigned to two groups (n=6): bone defect treated with collagen membrane (TG) and bone defect without treatment (CONT). Then, tibiae were submitted to micro-CT. The membranes preserved their natural collagen characteristics, presenting great strength, high water absorption, hydrophilicity, and almost complete dissolution in 30 days. In the experimental study, the membrane enhanced the growth of bone tissue in contact with its surface. A higher bone volume and trabeculae number and less trabecular space was observed in bone defects of the membrane group compared to the control group at 21 days. In conclusion, the Techgraft membrane seems to have favorable characteristics for treatment of long bone repair.