Rat Femoral Head Research Articles

A novel model of traumatic femoral head necrosis in rats developed by microsurgical technique

BackgroundClinical angiography and vascular microperfusion confirmed that the femoral head retains blood supply after a collum femur fracture. However, no animal model accurately mimics this clinical situation. This study was performed to establish a rat model with retained viability of the femoral head and partial vasculature deprivation-induced traumatic caput femoris necrosis by surgery.MethodsThirty rats were randomly divided into three groups (n = 10 per group): normal group, sham-operated group (Control), and ischemic osteonecrosis group. The femoral head of the normal group of rats underwent a gross anatomy study and microangiography to identify femoral head blood supply. Microsurgical techniques were used to cauterize the anterior-superior retinacular vessels to induce osteonecrosis. Hematoxylin and Eosin (H&E) staining were used for femoral head histologic assessment. Morphologic assessments of the deformity in and trabecular bone parameters of the femoral head epiphysis were performed using micro-CT.ResultsThe blood supply of the femoral head in rats primarily came from the anterior-superior, inferior, and posterior retinacular arteries. However, anterior-superior retinacular vasculature deprivation alone was sufficient in inducing femoral head osteonecrosis. H&E showed bone cell loss in nuclear staining, disorganized marrow, and trabecular structure. The bone volume (BV) decreased by 13% and 22% in the ischemic group after 5 and 10 weeks, respectively. The mean trabecular thickness (Tb.Th) decreased from 0.09 to 0.06 mm after 10 weeks. The trabecular spacing (Tb.Sp) increased from 0.03 to 0.05 mm after 5 weeks, and the epiphyseal height-to-diameter (H/D) ratio decreased.ConclusionsWe developed an original and highly selective rat model that embodied femoral head traumatic osteonecrosis induced by surgical anterior-superior retinacular vasculature deprivation.

Bone Mesenchymal Stem Cells (BMSCs) Transplantation Can Repair Rat Femoral Head Necrosis and Inhibit the Expression of Omgp

To study the therapeutic effect of BMSCs on femoral head necrosis and whether it can inhibit the growth of OMgP. 15 healthy rats were divided into ZZ group (normal group), GT group (femoral head necrosis) and MM group (BMSCs transplantation). At 1 h, 3 h, 1 d, and 3 d, the expression of OMgP in GT group were higher than ZZ group (P <0.05) and MM group, indicating that BMSCs transplantation can decreased OMgP expression. At 1 d, 7 d, and 14 d, BBB scores of the GT group were lower than ZZ group (P < 0.05) and MM group (P < 0.05), indicating that BMSCs transplantation can improve spinal cord injury behavior. The cells in ZZ group were well stained and morphologically intact, the femoral head was not damaged, and the articular surface was smooth, GT group had cartilage necrosis with disordered inferior epiphysis, and the femoral head of the MM group had less damage and increased osteoblasts. The blood vessel counts in necrotic area in GT group were higher than ZZ group and MM group with decreased new bone area in repair area compared to ZZ and MM group (P <0.05), indicating that the area of femoral head necrosis after BMSCs transplantation was improved. The levels of ALP and BGP in GT group were lower than ZZ group (P <0.05) and MM group (P <0.05). Bone marrow mesenchymal stem cell transplantation can effectively repair new bone area, up-regulate ALP and BGP, and have a positive effect on femoral head necrosis, possibly by inhibiting OMGP activity.

Bilobalide Improves Steroid-Induced Avascular Necrosis of the Femoral Head through PI3K/AKT Pathway

Steroid-induced avascular necrosis of the femoral head is a metabolic disease that results from the use of glucocorticoid drugs, leading to impaired blood supply to the femoral head and death of bone cells and altered bone marrow composition, which in turn lead to structural changes, collapse of the femoral head, and articular dysfunction. It is a challenging disorder to manage due to frequent collapse of the femoral head and dysfunction of the hip joint often requiring joint arthroplasty surgery. Therefore, there is a critical need to develop effective strategies to prevent and treat this condition. Bilobalide, an intermediate metabolite of ginkgolide, is known to inhibit apoptosis, protects damaged cells, and protects the brain from injury. However, the role of bilobalide in steroid-induced avascular necrosis of the femoral head is poorly understood. In this study, we investigated the role of bilobalide in steroid-induced avascular necrosis of the femoral head in rats. Our data show that bilobalide reduced femoral head inflammation and oxidative stress in steroid-induced avascular necrosis of the femoral head in rats. Bilobalide reduced the apoptosis of femoral head cells in steroid-induced avascular necrosis of the femoral head in rats. In addition, bilobalide improved bone tissue injury in steroid-induced avascular necrosis of the femoral head in rats. Mechanically, bilobalide improved steroid-induced avascular necrosis of the femoral head through PI3K/AKT pathway. We therefore suggest that bilobalide may serve as a promising drug for steroid-induced avascular necrosis of the femoral head.

Exosomal miR-100-5p inhibits osteogenesis of hBMSCs and angiogenesis of HUVECs by suppressing the BMPR2/Smad1/5/9 signalling pathway

BackgroundNontraumatic osteonecrosis of the femoral head (NONFH) is a common, progressive, and refractory orthopaedic disease. Decreased osteogenesis and angiogenesis are considered the main factors in the pathogenesis of NONFH. We aimed to figure out whether exosomes and exosomal miRNA from necrotic bone tissues of patients with NONFH are involved in the pathogenesis of NONFH and reveal the underlying mechanisms.MethodsRT-PCR and western blotting (WB) were used to detect the expression of osteogenic, adipogenic, and angiogenic markers. ALP staining and Alizarin Red S (ARS) staining were used to evaluate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Oil Red O staining was performed to assess the adipocyte deposition. A tube formation assay was used to study angiogenesis of human umbilical vascular endothelial cells (HUVECs). H&E staining and immunohistochemistry (IHC) staining were used to detect the effect of the NONFH exosomes in vivo. MicroRNA sequencing was conducted to identify potential regulators in the NONFH exosomes. The target relationship between miR-100-5p and BMPR2 was predicted and confirmed by a dual luciferase reporter assay and WB.ResultsThe NONFH exosomes reduced the osteogenic differentiation of hBMSCs and angiogenesis of HUVECs. In addition, the injection of the NONFH exosomes caused thinning and disruption of bone trabeculae in the femoral heads of rats. MiR-100-5p expression was upregulated in the NONFH exosomes and inhibited the osteogenesis of hBMSCs and angiogenesis of HUVECs by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway. Silencing miR-100-5p expression rescued the reduction in osteogenesis and angiogenesis caused by the NONFH exosomes by activating the BMPR2/SMAD1/5/9 signalling pathway.ConclusionThe NONFH exosomal miR-100-5p can lead to NONFH-like damage by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway, which may be involved in the pathophysiological mechanisms of nontraumatic osteonecrosis of the femoral head (NONFH).

Therapeutic effect of SIRT3 on glucocorticoid-induced osteonecrosis of the femoral head via intracellular oxidative suppression

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a serious complication after long-term or excess administration of clinical glucocorticoids intervention, and the pathogenic mechanisms underlying have not been clarified yet. Oxidative stress is considered as a major cause of bone homeostasis disorder. This study is aimed to explore the potential relevance between SIRT3 and GIONFH, as well as the effect of resveratrol, which has been reported for its role in SIRT3 activation, on dexamethasone-induced oxidative stress and mitochondrial compromise in bone marrow stem cells (BMSCs). In this study, our data showed that SIRT3 level was declined in GIONFH rat femoral head, corresponding to a resultant decrease of SIRT3 expression in dexamethasone-treated BMSCs in vitro. We also found that dexamethasone could result in oxidative injury in BMSCs, and resveratrol treatment reduced this deleterious effect via a SIRT3-dependent manner. Moreover, our results demonstrated that rewarding effect of resveratrol on BMSCs osteogenic differentiation was via activation of AMPK/PGC-1α/SIRT3 axis. Meanwhile, resveratrol administration prevented the occurrence of GIONFH, enhanced SIRT3 expression and reduced oxidative level in GIONFH model rats. Therefore, our study provides basic evidence that SIRT3 may be a promising therapeutic target for GIONFH treatment and resveratrol could be an ideal agent for clinical uses.

The role of biomechanical forces and MALAT1/miR-329-5p/PRIP signalling on glucocorticoid-induced osteonecrosis of the femoral head.

Glucocorticoid‐induced osteonecrosis of the femoral head (GIONFH) is a common orthopaedic disease. GIONFH primarily manifests clinically as hip pain in the early stages, followed by the collapse of the femoral head, narrowing of the hip joint space and damage to the acetabulum, resulting in severely impaired mobility. However, the pathogenesis of GIONFH is not clearly understood. Recently, biomechanical forces and non‐coding RNAs have been suggested to play important roles in the pathogenesis of GIONFH. This study aimed to evaluate the role of biomechanical forced and non‐coding RNAs in GIONFH. We utilized an in vivo, rat model of GIONFH and used MRI, μCT, GIONFH‐TST (tail suspension test), GIONFH‐treadmill, haematoxylin and eosin staining, qRT‐PCR and Western blot analysis to analyse the roles of biomechanical forces and non‐coding RNAs in GIONFH. We used RAW264.7 cells and MC3T3E1 cells to verify the role of MALAT1/miR‐329‐5p/PRIP signalling using a dual luciferase reporter assay, qRT‐PCR and Western blot analysis. The results demonstrated that MALAT1 and PRIP were up‐regulated in the femoral head tissues of GIONFH rats, RAW264.7 cells, and MC3T3E1 cells exposed to dexamethasone (Dex). Knockdown of MALAT1 decreased PRIP expression in rats and cultured cells and rescued glucocorticoid‐induced osteonecrosis of femoral head in rats. The dual luciferase reporter gene assay revealed a targeting relationship for MALAT1/miR‐329‐5p and miR‐329‐5p/PRIP in MC3T3E1 and RAW264.7 cells. In conclusion, MALAT1 played a vital role in the pathogenesis of GIONFH by binding to (‘sponging’) miR‐329‐5p to up‐regulate PRIP. Also, biomechanical forces aggravated the pathogenesis of GIONFH through MALAT1/miR‐329‐5p/PRIP signalling.

Phosphodiesterase-5 inhibition improves bone regeneration at the early stages of ischemic osteonecrosis of the femoral head in rats.

Posttraumatic osteonecrosis of the femoral head (ONFH) affects patients at different ages and may lead to functional limitation and joint replacement, with total hip arthroplasty, which is a costly procedure. Proposed methods to optimize ischemic tissue regeneration have been reported. Phosphodiesterase-5 inhibitors act by inhibiting the degradation of guanosine 3',5'-cyclic monophosphate in the nitric oxide pathway, increasing its bioavailability and promoting vascular endothelial growth factor (VEGF)-mediated neovascular recruitment and the induction of tissue regeneration in the traumatized bone. Thirty male Sprague-Dawley rats (6 months old) were subjected to an experimental model of traumatic ONFH divided into two groups, according to the administration of 5 mg/kg sildenafil or water (control group). Rats were then killed at 7, 14, and 21 days. Histological (Goldner's trichrome), histochemical (periodic acid-Schiff [PAS]), and immunohistochemical (VEGF and osteopontin [OPN]) techniques were used to quantify bone and vascular responses. Higher levels of VEGF (p < 0.01) and OPN (p < 0.01) immunostaining in the epiphysis, the greater formation of osteoid tissue (p < 0.01 on Day 7; p < 0.05 on Day 14), and higher levels of PAS staining (p < 0.01 on Day 7) were observed in the sildenafil-treated group. The present study demonstrated that sildenafil optimized bone tissue regeneration by increasing VEGF signaling and OPN expression, with increased bone formation (osteoid and carbohydrate macromolecule deposition) in the early stages following traumatic ischemic insult. Thus, sildenafil treatment may improve the prognosis of patients with osteonecrosis.

Crocin promotes osteogenesis differentiation of bone marrow mesenchymal stem cells.

Crocin has plentiful pharmacological effects, but its role in osteogenesis differentiation of bone marrow mesenchymal stem cells (BMSCs) is unexplored. This study explored the effect of crocin on osteogenesis differentiation, in order to provide evidence for its clinical application. In cell experiments, human BMSCs (hBMSCs) were induced by osteogenesis differentiation medium or crocin. In animal experiments, steroid-induced osteonecrosis of the femoral head (SANFH) rat models was established using lipopolysaccharide (LPS) plus methylprednisolone (MPS), and then treated with crocin. The osteogenesis differentiation capacity of hBMSCs was analyzed by alkaline phosphatase (ALP) and alizarin red S staining. Histopathological changes in rat femoral head tissues were observed by hematoxylin and eosin (H&E) staining. The expression levels of RUNX2, COL1A1, OCN, and GSK-3β in hBMSCs and rat femoral head tissues were measured by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot (WB) analysis. ALP and alizarin red S staining demonstrated that LAP activity and calcium nodules were increased in hBMSCs treated with crocin. From H&E staining results, femoral head tissues of SANFH models showed typical osteonecrosis, which could be ameliorated by crocin. WB and qRT-PCR assays detected that the expression levels of RUNX2, COL1A1, and OCN in hBMSCs and femoral head tissues of models were obviously increased after crocin treatment, while GSK-3β phosphorylation was reduced. In general, the action of crocin was concentration-dependent. Crocin might be beneficial to the recovery of SANFH through accelerating osteogenesis differentiation of BMSCs, which might be a novel therapy for related diseases.

Association of Neutrophil Extracellular Traps with the Development of Idiopathic Osteonecrosis of the Femoral Head

Idiopathic osteonecrosis of the femoral head (ONFH) is defined as necrosis of osteocytes due to a non-traumatic ischemia of the femoral head. Iatrogenic glucocorticoid administration and habitual alcohol intake are regarded as risk factors. It has been suggested that glucocorticoid-induced activation of platelets contributes to the local blood flow disturbance of the femoral head. Both activated platelets and alcohol can induce neutrophil extracellular traps (NETs). To determine the association of NETs with the development of idiopathic ONFH, surgically resected femoral heads of patients with idiopathic ONFH and osteoarthritis were assessed for existence of NET-forming neutrophils by immunofluorescence staining. NET-forming neutrophils were present in small vessels surrounding the femoral head of patients with idiopathic ONFH but not osteoarthritis. Moreover, Wistar-Kyoto rats were intravenously injected with NET-forming neutrophils or neutrophils without NET induction, and then the ischemic state of the tissue around the femoral head was evaluated by immunohistochemistry for hypoxia-inducible factor-1α. NET-forming neutrophils circulated into the tissue around the femoral head, and hypoxia-inducible factor-1α expression in the tissue was higher compared with that of rats intravenously administered with neutrophils without NET induction. Furthermore, ischemic change of osteocytes was observed in the femoral head of rats given an i.v. injection of NET-forming neutrophils. The collective findings suggest that NETs are possibly associated with the development of idiopathic ONFH.

Extracellular vesicles from human urine-derived stem cells inhibit glucocorticoid-induced osteonecrosis of the femoral head by transporting and releasing pro-angiogenic DMBT1 and anti-apoptotic TIMP1

Osteonecrosis of the femoral head (ONFH) frequently occurs after glucocorticoid (GC) treatment. Extracellular vesicles (EVs) are important nano-sized paracrine mediators of intercellular crosstalk. This study aimed to determine whether EVs from human urine-derived stem cells (USC-EVs) could protect against GC-induced ONFH and focused on the impacts of USC-EVs on angiogenesis and apoptosis to explore the mechanism by which USC-EVs attenuated GC-induced ONFH. The results in vivo showed that the intravenous administration of USC-EVs at the early stage of GC exposure could rescue angiogenesis impairment, reduce apoptosis of trabecular bone and marrow cells, prevent trabecular bone destruction and improve bone microarchitecture in the femoral heads of rats. In vitro, USC-EVs reversed the GC-induced suppression of endothelial angiogenesis and activation of apoptosis. Deleted in malignant brain tumors 1 (DMBT1) and tissue inhibitor of metalloproteinases 1 (TIMP1) proteins were enriched in USC-EVs and essential for the USC-EVs-induced pro-angiogenic and anti-apoptotic effects in GC-treated cells, respectively. Knockdown of TIMP1 attenuated the protective effects of USC-EVs against GC-induced ONFH. Our study suggests that USC-EVs are a promising nano-sized agent for the prevention of GC-induced ONFH by delivering pro-angiogenic DMBT1 and anti-apoptotic TIMP1. Statement of SignificanceThis study demonstrates that the intravenous injection of extracellular vesicles from human urine-derived stem cells (USC-EVs) at the early stage of glucocorticoid (GC) exposure efficiently protects the rats from the GC-induced osteonecrosis of the femoral head (ONFH). Moreover, this study identifies that the promotion of angiogenesis and inhibition of apoptosis by transferring pro-angiogenic DMBT1 and anti-apoptotic TIMP1 proteins contribute importantly to the USC-EVs-induced protective effects against GC-induced ONFH. This study suggests the promising prospect of USC-EVs as a new nano-sized agent for protecting against GC-induced ONFH, and the potential of DMBT1 and TIMP1 as the molecular targets for further augmenting the protective function of USC-EVs.

β-ecdysone promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

β-ecdysone (βEcd) has numerous pharmacological effects, although its role in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) has not yet been explored. In cell experiments, BMSCs were induced to differentiate by osteogenic induction medium (OIM) or βEcd. In animal experiments, an osteonecrosis of the femoral head (ONFH) rat model was established using lipopolysaccharide plus methylprednisolone and treating the rats with βEcd. The osteogenic differentiation capacity of human BMSCs (hBMSCs) was analyzed by alkaline phosphatase and alizarin red S staining. Histopathological changes in rat femoral head tissues were observed by hematoxylin and eosin staining. The expression levels of RUNX2, COL1A1, OCN and phosphorylated Akt in BMSCs from rat femoral head tissues were measured by a quantitative real-time polymerase chain reaction or western blot analysis. Alkaline phosphatase activity and calcium nodules in the βEcd-treated BMSC group dose-dependently increased compared to those in the control and OIM groups. The hematoxylin and eosin staining results indicated that femoral head tissues of ONFH rats showed typical osteonecrosis, which could be ameliorated by βEcd. Western blot, quantitative real-time polymerase chain reaction and immunohistochemistry assays demonstrated that the expression levels of RUNX2, COL1A1 and OCN in hBMSCs and femoral head tissue models were obviously increased after βEcd treatment, and phosphoinositide 3-kinase and Akt phosphorylation were also increased. βEcd may be beneficial for the recovery of ONFH patients by accelerating osteogenic differentiation of BMSCs, which may be a novel therapy for related diseases.

Proper mechanical stress promotes femoral head recovery from steroid-induced osteonecrosis in rats through the OPG/RANK/RANKL system

BackgroundLong-term use of steroid may lead to osteonecrosis of the femoral head (ONFH). Mechanical stress may help bone formation and remodeling. This study aimed to probe the role of mechanical stress in the femoral head recovery in rats.MethodsRat models with ONFH were induced by steroid. Rats were subjected to different levels of mechanical stress (weight-bearing training), and then the morphology and bone density of femoral head of rats were measured. The mRNA and protein levels of the OPG/RANK/RANKL axis in rat femoral head were assessed. Gain- and loss-of function experiments of OPG were performed to identify its role in femoral head recovery following stress implement. The ex vivo cells were extracted and the effects of stress and OPG on osteogenesis in vitro were explored.ResultsSteroid-induced ONFH rats showed decreased bone density and increased bone spaces, as well as necrotic cell colonies and many cavities in the cortical bones and trabeculars. Proper mechanical stress or upregulation of OPG led to decreased RANK/RANKL expression and promoted femoral head recovery from steroid-induced osteonecrosis. However, excessive mechanical stress might impose too much load on the femurs thus leading even retard femoral head recovery process. In addition, the in vitro experimental results supported that proper stress and overexpression of OPG increased the osteogenesis of ex vivo cells of femoral head.ConclusionThis study provided evidence that proper mechanical stress promoted femoral head recovery from steroid-induced osteonecrosis through the OPG/RANK/RANKL system, while overload might inhibit the recovery process. This study may offer novel insights for ONFH treatment.

Extracellular Vesicles From Human Urine-Derived Stem Cells Inhibit Glucocorticoid-Induced Osteonecrosis of the Femoral Head by Transferring Pro-Angiogenic DMBT1 and Anti-Apoptotic TIMP1

Osteonecrosis of the femoral head (ONFH) frequently occurs after glucocorticoid (GC) treatment. Extracellular vesicles (EVs) are important nano-sized paracrine mediators of intercellular crosstalk. Herein, we found that intravenous administration of EVs from human urine-derived stem cells (USC-EVs) at the early stage of GC exposure rescues angiogenesis impairment, reduces apoptosis of bone marrow and trabecular bone cells, prevent trabecular bone destruction and improves bone microarchitecture in the femoral heads of rats. In vitro, USC-EVs reverse GC-induced suppression of endothelial angiogenesis and activation of cellular apoptosis. Deleted in malignant brain tumors 1 (DMBT1) and tissue inhibitor of metalloproteinases 1 (TIMP1) are enriched in USC-EVs and essential for USC-EVs-induced pro-angiogenic and anti-apoptotic effects in GC-treated cells, respectively. Knockdown of TIMP1 in USC-EVs attenuates their protective effects against GC-induced ONFH. Our study suggests that USC-EVs are a novel promising nano-sized agent for the prevention of GC-induced ONFH by delivering pro-angiogenic DMBT1 and anti-apoptotic TIMP1.

Metformin coordinates osteoblast/osteoclast differentiation associated with ischemic osteonecrosis.

In this study, we aimed to identify a candidate drug that can activate endogenous Angiopoietin 1 (Ang1) expression via drug repositioning as a pharmacological treatment for avascular osteonecrosis. After incubation with 821 drugs from the Food and Drug Administration (FDA)-approved drug library, Ang1 expression in U2OS cell culture media was examined by ELISA. Metformin, the first-line medication for treatment of type 2 diabetes, was selected as a candidate for in vitro and in vivo experimental evaluation. Ang1 was induced, and alkaline phosphatase activity was increased by metformin treatment in U2OS and MG63 cells. Wound healing and migration assay showed increased osteoblastic cell mobility by metformin treatment in U2OS and MG63 cells. Metformin upregulated expression of protein markers for osteoblastic differentiation in U2OS and MG63 cells but inhibited osteoclastic differentiation in Raw264.7 cells. Metformin (25 mg/kg) protected against ischemic necrosis in the epiphysis of the rat femoral head by maintaining osteoblast/osteocyte function and vascular density but inhibiting osteoclast activity in the necrotic femoral head. These findings provide novel insight into the specific biomarkers that are targeted and regulated by metformin in osteoblast differentiation and contribute to understanding the effects of these FDA-approved small-molecule drugs as novel therapeutics for ischemic osteonecrosis.

Evaluation of bone regeneration potential of injectable extracellular matrix (ECM) from porcine dermis loaded with biphasic calcium phosphate (BCP) powder.

Extracellular matrix (ECM) contains a wide array of complex proteins, growth factors and cytokines that regulate cell behavior and tissue development. ECM harvested from non-homologous ECM sources still provide a structural support and biochemical cues to cells for effective tissue remodeling. The aim of this study is to evaluate the effect of non-tissue specific decellularized ECM from porcine dermis loaded with biphasic calcium phosphate powder (BCP) in bone regeneration. Thermosensitive ECM hydrogels with BCP powder exhibited a porous morphology with a suitable injectability and increased mechanical stability. In-vitro studies using MC3T3-E1 pre osteoblast cells showed that the injectable ECM hydrogels were biocompatible and supported the osteogenic differentiation. The bone regeneration capacity of the injectable ECM hydrogels was evaluated in-vivo by implanting in rat femoral head for 4 and 8weeks. Micro-CT and histological staining results indicated that the injectable ECM hydrogels loaded with BCP powder showed higher and improved bone formation compared with the unfilled defect. Injectable ECM loaded with BCP powder is a good potential biomaterial for non-load bearing bone regeneration application.

Early transplantation of growth differentiation factor 11-silenced bone marrow stromal cells promotes osteogenesis in necrotic femoral head induced by steroid in rats

Objective To study the effect of growth differentiation factor (GDF) 11-silenced bone marrow stromal cells (BMSCs) on bone regeneration in early steroid-induced osteonecrosis of the femoral head in rats. Methods After GDF11 expression in BMSCs was inhibited by siRNA, the knockdown efficiency and transfection cytotoxicity were detected. The further experiments both in vitro (n=3) and in vivo (n=8) were divided into 4 groups respectively: blank control group (without any intervention), model group (glucocorticoid treatment), experimental group (siRNA transfection and glucocorticoid treatment) and negative control group (negative control transfection and glucocorticoid treatment). The BMSCs were induced into osteogenic differentiation. Alkaline phosphatase (ALP) staining and alizarin red staining were applied to evaluate the osteogenic differentiation ability of the cells while reverse transcription polymerase chain reaction (qRT-PCR) was employed to detect the relative expression levels of osteogenic markers. The osteogenesis in the necrotic femoral head was evaluated by microCT, H&E staining, immunohistochemistry staining and biomechanical test. Results No transfection cytotoxicity was found (P>0.05). The ALP staining and alizarin red staining showed that the osteogenic differentiation of BMSCs in the experimental group was better than that in the model group. At the level of mRNA, the relative expression of ALP, runt-related transcription factor (Runx) 2, osteocalcin (OCN) and type Ⅰ collagen (α1) (COL1A1) in the blank control group (1.00±0.09, 1.02±0.23, 1.03±0.30 and 1.02±0.25, respectively) were significantly higher than those in the model group (0.46±0.11, 0.50±0.11, 0.35±0.01 and 0.57±0.02, respectively) but significantly lower than those in the experimental group (1.97±0.30, 0.94±0.19, 1.50±0.18 and 1.28±0.37) (all P<0.05). MicroCT images and quantitative analysis showed that the bone mass in the experimental group was significantly increased compared with the model group (P<0.05). Histological examination showed better bone regeneration and higher expression of Runx2 and COL1 in the necrotic femoral head in the experimental group than in the model group. Improved biomechanical properties were shown in the experimental group compared with the model group (P<0.05). Conclusions Silence of GDF11 expression may alleviate the inhibitory effect of glucocorticoid on osteogenic differentiation of BMSCs. Early transplantation of GDF11-silenced BMSCs may promote osteogenesis in the necrotic femoral head in rats. Key words: Femoral head necrosis; Bone marrow cells; Growth differentiation factor 11; Glucocorticoids; Osteogenic differentiation

Puerarin alleviates streptozotocin (STZ)-induced osteoporosis in rats through suppressing inflammation and apoptosis via HDAC1/HDAC3 signaling

Diabetic osteoporosis is a severe public health concern in the world. Puerarin (PU) is extensively attractive due to its superior bioactivities. In the study, we found that PU protected against streptozotocin (STZ)-induced osteoporotic changes in rats. PU treatment improved STZ-induced diabetes in rats, as evidenced by the reduced serum glucose and insulin levels. PU administration markedly attenuated bone loss and tartarate-resistant acid phosphatase (TRAP) activity in STZ-induced rats. Bone mineral density (BMD) was significantly decreased in diabetic rats, while being prevented by PU. STZ-induced impairments in microarchitecture of femoral tissues were markedly alleviated by PU treatment. In addition, bone-specific alkaline phosphatase (BALP), osteoprotegerin (OPG) and Runt-related transcription factor 2 (Runx2) levels in serum or tibia were improved by PU in STZ-injected rats; however, TRACP isoform 5b (TRACP-5b), carboxy-terminal collagen cross-links (β-CTX) and receptor activator of nuclear factor-κB ligand (RANKL) levels were decreased. Further, PU treatment inhibited inflammation and apoptosis in STZ-treated rats. Additionally, STZ injection increased histone deacetylase (HDAC)-1 and -3 expressions in femoral heads of rats, which were relieved by PU treatment. Notably, both HDAC1 and HDAC3 could enhance osteoporosis in vitro, as proved by the decreased ALP and Runx2 levels and the increased TRAP expression. Inflammation and apoptosis were exacerbated by HDAC1/3 over-expression, which were markedly diminished by PU treatment. In contrast, suppressing HDAC1/3 significantly abrogated fructose (Fru)-elicited inflammation and apoptosis in cells. Collectively, our data illustrated that PU is a potential therapeutic option to prevent diabetic osteoporosis by inhibiting HDAC1/HDAC3 signaling.

Intermittent Parathyroid Hormone Injection Can Decrease Femoral Head Collapse in the Vascular Deprivation of Rat Femoral Head Model.

Backgrounds:Intermittent parathyroid hormone (intermittent PTH) injection has been shown to improve osteogenesis. We hypothesized that intermittent PTH injection could stimulate osteogenesis during the early phase of vascular deprivation-induced femoral neck osteonecrosis in a rat model.Materials and Methods:Eighteen Sprague-Dawley rats were divided into three groups (normal saline [CON], PTH 10 μg/kg [PTH-H], and PTH 1 μg/kg [PTH-L]) for 8 weeks by subcutaneous injection. All rats were sacrificed at postoperative 8 weeks, and all underwent a micro-computed tomography (μ-CT) examination for bone quality and quantity evaluation and histomorphometric analysis for microscopic histologic differences.Results:Under μ-CT examination, both the PTH-H and PTH-L groups revealed less bone resorption than the control group. The PTH-H group had a better bone protective effect than the PTH-L group. Bone mineral density was increased in the PTH-H and PTH-L groups compared to the control group. The uninjured left femoral head was enlarged in both PTH groups. The histologic examination showed that both PTH groups had new bone and cartilage formation. The control group had only dead bone without any osteogenesis.Conclusion:Intermittent PTH injection could decrease bone resorption and improve bone density, compared to the control group, in vascular deprivation of the femoral head in a rat model. High-level intermittent PTH injection had a better effect than low-level intermittent PTH injection.

Glucocorticoid induces osteonecrosis of the femoral head in rats through GSK3β-mediated osteoblast apoptosis

ObjectiveOne of the important causes of glucocorticoids (GCs)-induced osteonecrosis of the femoral head (ONFH) is osteoblast apoptosis. Glycogen synthase kinase 3β (GSK3β) has been reported to be related to dexamethasone (Dex)-induced osteoblast apoptosis. This study aimed to determine whether GSK3β plays role in GC-induced ONFH and investigate the underlying mechanism. Methods18 male Sprague-Dawley rats were divided into 3 groups. Rats from ONFH group underwent lipopolysaccharide and methylprednisolone injection. Lithium chloride (LiCl, a GSK3β inhibitor) group were fed with LiCl solution. The control group were untreated. Osteonecrosis, apoptosis and bone loss were evaluated by HE staining, TUNEL staining and micro-CT respectively. Protein expressions were examined by western blotting. In addition, primary osteoblast cells were transfected by GSK3β-siRNA and related signaling pathway and proteins were examined. ResultsONFH group showed a relative high percentage of empty lacunae and apoptotic cells, whilst LiCl treatment markedly decreased the percentage. LiCl treatment decreased GC-induced bone loss. Immunoblot analysis for GSK3β showed decreased level of Ser9-phosphorylated GSK3β in ONFH group compared with control group. Knockdown of GSK3β by siRNA in primary osteoblast cells attenuated DEX-induced apoptosis and loss of mitochondrial transmembrane potential (Δψm). GSK3β knockdown also reversed the release of cytochrome C (Cyt C) from mitochondria to the cytosol. GSK3β decreased apoptosis-related protein expression both in vitro and in vivo. ConclusionOur findings suggest that GC induces ONFH in rats through GSK3β-mediated osteoblast apoptosis, with involvement of mitochondrial apoptotic pathway.

Repair of osteonecrosis of the femoral head : 3D printed Cervi cornus Colla deproteinized bone scaffolds.

BackgroundOsteonecrosis of the femoral head (ONFH) is a common joint disease and a major cause of morbidity.ObjectiveIn this study Cervi cornus Colla (CCC) deproteinized bone scaffolds were designed and three dimensional (3D)-printed for the repair of ONFH in rats.Material and methodsThe CCC-deproteinized bone scaffolds were 3D-printed using polycaprolactone mixed with the CCC-deproteinized bone powder. The scaffolds were viewed under a scanning electron microscope and subjected to compression analysis. Osteoblasts were isolated from rats and coated onto the scaffolds. Cell proliferation assays were performed with the MTT (3‑[4,5-dimethylthiazole‑2]-2,5-diphenyltetrazolium bromide) kit from Promega. An ONFH was induced in rats and a CCC-deproteinized bone scaffold was implanted into the necrotic femoral head. General observations, X‑ray imaging, and pathological examination of the femoral head were performed to evaluate the treatment of ONFH in the rats.ResultsThe scaffolds were porous with a mean pore diameter of 315.70 ± 41.52 nm and a porosity of 72.86 ± 5.45% and exhibited favorable mechanical properties and degradation. In vitro assays showed that osteoblasts accumulated in the pores and adhered to the scaffolds. The CCC-deproteinized bone scaffolds enhanced the proliferation of osteoblasts. The in vivo experiments revealed that the general observation score of rats in the CCC-scaffold implanted group was significantly higher than that in the control group. The X‑ray images showed significant alleviation of ONFH in the CCC-deproteinized bone scaffold implanted rats. The femoral heads of rats in the treatment group showed less destruction or ossification of cartilage cells, few bone cement lines, very little necrosis or irregularities on the cartilage surface and only a small amount of inflammatory cell infiltration in the medullary cavity.ConclusionThese results suggest that CCC-deproteinized bone scaffold implants facilitated the repair of ONFH in rats. This research provides a new therapeutic approach for the repair of early and mid-term ONFH.