Model Of Osteonecrosis Research Articles

Inhibitory effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at early stage of steroid-induced femoral head necrosis.

Apoptosis and DNA oxidative damage serve significant roles in the pathogenesis of steroid-induced femoral head necrosis. Vitamin E demonstrates anti-apoptotic and anti-oxidant properties. Therefore, the present study investigated the effects of vitamin E on osteocyte apoptosis and DNA oxidative damage in bone marrow hemopoietic cells at an early stage of steroid-induced femoral head osteonecrosis. Japanese white rabbits were randomly divided into three groups (steroid, vitamin E-treated, and control groups), each comprising 12 rabbits. Those in the steroid group (group S) were initially injected twice with an intravenous dose of 100 µg/kg Escherichia coli endotoxin, with a 24 h interval between the two injections, and then with an intramuscular dose of 20 mg/kg methylprednisolone, three times at intervals of 24 h in order to establish a rabbit model of osteonecrosis. The vitamin E treated group (group E) received the same treatment as group S, and were administered 0.6 g/kg/d vitamin E daily from the beginning of modeling. The control group (group C) was injected with normal saline at the equivalent dosage and times as the aforementioned two groups. Two time points, weeks 4 and 6 following the completion of modeling, were selected. Osteonecrosis was verified by histopathology with hematoxylin-eosin staining. The apoptosis rate of osteonecrosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The apoptosis expression levels of caspase-3 and B-cell lymphoma 2 (Bcl-2), and DNA oxidative damage of bone marrow hematopoietic cells were analyzed by immunohistochemistry. At weeks 4 and 6 following the completion of modeling, the vacant bone lacunae rates of group E were 15.87±1.97 and 25.09±2.67%, respectively, lower than the results of 20.02±2.21 and 27.79±1.39% for group S; and the osteocyte apoptosis indexes of group E were 20.99±2.95 and 33.93±1.62%, respectively, lower than the results of 26.46±3.37 and 39.90±3.74% from group S. In addition, the Bcl-2 expression at week 4 in the femoral head tissues of group E was higher compared with group S; and the proportion of Bcl-2-positive cells of group E was 9.81±1.01%, higher compared with group S at 8.26±1.13%. The caspase-3 staining data at week 4 in femoral head tissues demonstrated that in the 12 femoral heads of group S, four were negative (32%) and eight were positive (68%); in group E, five were negative (45%) and seven were positive (55%); and in group C, 11 were negative (95%) and one was positive (5%). In addition, the DNA oxidative damage rate at week 4 in the bone marrow hemopoietic cells of group E was (7.24±1.44%), lower compared with group S (11.80±1.26%), and higher compared with group C (5.75±1.47%). Vitamin E is effective in intervening in apoptosis through decreasing caspase-3 expression and upregulating Bcl-2 expression, and by alleviating DNA oxidative damage in bone marrow hemopoietic cells at the early stage of steroid-induced femoral head necrosis in rabbit models.

Involvement of necroptosis, a newly recognized cell death type, in steroid-induced osteonecrosis in a rabbit model.

We investigated the role of programmed necrosis (necroptosis), a newly recognized form of cell necrosis that has been implicated in the development of steroid-induced osteonecrosis. We used an osteonecrosis model in which 30 Japanese white rabbits each weighing 3.5kg were injected once with methylprednisolone at 20 mg/kg body weight into the right gluteal muscle. Ten animals killed 14 days thereafter were designated as S14d groups, while another 10 animals injected with necroptosis, a specific inhibitor of necrostatin-1 i.v. at 1.65mg/kg on the same day as the steroid were also killed on the 14th day and designated as SN14d group. As a control, 10 animals injected only with physiological saline were studied as N group. After the animals were sacrificed the bilateral femoral bone was examined histopathologically and the presence of osteonecrosis determined. Furthermore, animals subjected to the same treatment and killed on the 3rd day after drug administration were set up as S3d group and SN3d group, and Western blotting of Receptor-interacting protein ( RIP ) 1 and RIP3 in femoral bone performed. The osteonecrosis rate was 70% in S14d group, and 0% in both N and SN groups. In 2 of 10 animals in SN group fatty marrow was found. On Western blotting significantly increased expression of both RIP1 and RIP3 was noted in S3d group, confirming that Nec-1 was suppressed. Necroptosis mediated by RIP1 and RIP3 expression was thought to be implicated in the development of steroid-induced osteonecrosis. Also, by suppressing expression of RIP1 and 3 with the administration of Nec-1 the osteonecrosis rate was significantly decreased. These results suggest that necroptosis may have potential as a novel target for both elucidating the mechanisms underlying steroid-induced osteonecrosis and establishing more effective prophylactic countermeasures.

Exosomes Secreted from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Prevent Osteonecrosis of the Femoral Head by Promoting Angiogenesis

Background: Local ischemia is the main pathological performance in osteonecrosis of the femoral head (ONFH). There is currently no effective therapy to promote angiogenesis in the femoral head. Recent studies revealed that exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSC-Exos) have great therapeutic potential in ischemic tissues, but whether they could promote angiogenesis in ONFH has not been reported, and little is known regarding the underlying mechanism.Methods: iPS-MSC-Exos were intravenously injected to a steroid-induced rat osteonecrosis model. Samples of the femoral head were obtained 3 weeks after all the injections. The effects were assessed by measuring local angiogenesis and bone loss through histological and immunohistochemical (IHC) staining, micro-CT and three-dimensional microangiography. The effects of exosomes on endothelial cells were studied through evaluations of proliferation, migration and tube-forming analyses. The expression levels of angiogenic related PI3K/Akt signaling pathway of endothelial cells were evaluated following stimulation of iPS-MSC-Exos. The promoting effects of exosomes were re-evaluated following blockade of PI3K/Akt.Results: The in vivo study revealed that administration of iPS-MSC-Exos significantly prevented bone loss, and increased microvessel density in the femoral head compared with control group. We found that iPS-MSC-Exos significantly enhanced the proliferation, migration and tube-forming capacities of endothelial cells in vitro. iPS-MSC-Exos could activate PI3K/Akt signaling pathway in endothelial cells. Moreover, the promoting effects of iPS-MSC-Exos were abolished after blockade of PI3K/Akt on endothelial cells.Conclusions: Our findings suggest that transplantation of iPS-MSC-Exos exerts a preventative effect on ONFH by promoting local angiogenesis and preventing bone loss. The promoting effect might be attributed to activation of the PI3K/Akt signaling pathway on endothelial cells. The data provide the first evidence for the potential of iPS-MSC-Exos in treating ONFH.

Immune modulation of CD4+CD25+ regulatory T cells by zoledronic acid.

BackgroundCD4+CD25+ regulatory T (Treg) cells suppress tumor immunity by inhibiting immune cells. Manipulation of Treg cells represents a new strategy for cancer treatment. Zoledronic acid (ZA), a nitrogen-containing bisphosphonate, inhibits the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) on osteoblasts to inhibit osteoclastogenesis. In a mouse model of bisphosphonate-related osteonecrosis of the jaw, administration of ZA suppressed Treg-cell activity and activated inflammatory Th17 cells. However, the interaction between ZA and Treg cells remained unclear. This study investigated the immune modulation of Treg cells by ZA.MethodsFlow cytometry was used to analyze the phenotypic and immunosuppressive characteristics of Treg cells treated with ZA. Chemotactic migration was evaluated using transwell assays. Quantitative real-time PCR (qRT-PCR) was used to investigate the effect of ZA on the expression of suppressive molecules by Treg cells.ResultsProliferation of isolated Treg cells in culture was inhibited by ZA, although ZA did not induce apoptosis. qRT-PCR and flow cytometry showed that ZA significantly downregulated the expression of CCR4, CTLA4, PD-1 and RANKL on Treg cells. Chemotactic migration and immunosuppressive functions were also significantly attenuated in Treg cells pretreated with ZA, and these effects were dose-dependent. Co-culture with Treg cells significantly increased the migration rate of breast cancer cells, while pretreatment of Treg cells with ZA attenuated this effect.ConclusionsOur findings demonstrated that ZA acted as an immune modulator by significantly inhibiting the expansion, migration, immunosuppressive function and pro-metastatic ability of Treg cells. Immunomodulation of Treg cells by ZA represents a new strategy for cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0183-7) contains supplementary material, which is available to authorized users.

A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies.

The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4×104 MSCs/cm2 at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd.

Experimental model of osteonecrosis of the jaw in rats treated with zoledronic acid

We have examined the development of medication-related osteonecrosis of the jaws (MRONJ) in rats with no previous accumulation of zoledronic acid in the mandible. Ten male Wistar rats (weight 350–400g) were anaesthetised with chloral hydrate 450mg/kg intraperitoneally and the first and second mandibular molars on the left side were extracted. The five experimental rats were given six injections of zoledronic acid 0.18mg/kg over the next four weeks (total dose 1.08mg/kg). Two injections were given at once as an intravenous bolus injection (0.36mg/kg). Then rats were given 4 injections (0.18mg/kg) with 1 week interval over the next four weeks, after which they observed for a further four weeks. The five control rats were injected with saline. At the end of the eighth week, the animals were killed by asphyxiation in a carbon dioxide chamber, and their bone structure was visualised using cone-beam computed tomography (CT) and Galaxis software. We then studied the mandibles histopathologically to investigate the incidence of necrosis and infiltration of inflammatory cells. The cone-beam CT images in the experimental group showed deficiencies in the bone structure in the extracted molar area of the lower alveolar ridge. The histological findings in the mandibles of the group given zoledronic acid showed necrosis and infiltration of inflammatory cells, which were not present in the control group. We conclude that the immediate effect of zoledronic acid on the bone tissue during regeneration is an important factor in the development of MRONJ, in addition to the previously reported effects of the duration of treatment with zoledronic acid.

NEL-like type 1 gene transfection for prevention of traumatic femoral head necrosis in rats

Objective To determine the effect of NEL-like type 1 gene (NELL-1) transfection in vivo in the repair of traumatic femoral head necrosis. Methods Twenty-four SD rats were randomly divided into three groups (8 rats per group) according to the lottery method, ie, sham group (served as normal control), NELL-1 treatment group (injected NELL-1 gene by recombinant adenovirus vectors around the hip one week after osteonecrosis model induced surgically) and placebo group (given an equal volume of saline solution at the same time after the induction of osteonecrosis). Femurs were taken from the animals 5 weeks after surgery. Gross observation was performed for morphology changes, X-ray assessment for femoral head height and length ratio (H/L), Micro-CT measure for bone parameters of femoral head including total volume (TV), bone volume (BV), total mineralized content (TMC), trabecular thickness (Tb.Th) and trabecular space (Tb.SP), and histological study for osteocytes, osteoblasts and osteoclasts. Results Preserved femoral head shape was noted in NELL-1 treatment group compared to the obvious flattening of the femoral head in placebo group. No heterotopic osteogenesis was observed in any group. Femoral head H/L ratio for 0.753 2±0.040 2 in NELL-1 treatment group was higher than 0.598 4±0.037 0 in placebo group (P 0.05), but all were increased compared to placebo group (P 0.05). Most osteocytes were alive in NELL-1 treatment group. More active osteoblasts and osteoclasts were noted in NELL-1 treatment group than those in placebo group. Conclusion NELL-1 gene transfection can preserve femoral head shape and bone content, promote osteoblast activity and neovascularization and hence is an effective treatment for rat traumatic osteonecrosis. However, the activity of osteoclasts is stimulated simultaneously. Key words: Osteonecrosis; Femoral head; Genes; Transfection

Severe compromise of preosteoblasts in a surgical mouse model of bisphosphonate-associated osteonecrosis of the jaw.

The effect of amino-bisphosphonates on osteoblastic lineage and its potential contribution to the pathogenesis of bisphosphonate-associated osteonecrosis of the jaw (BONJ) remain controversial. We assessed the effects of zoledronic acid (ZOL) on bone and vascular cells of the alveolar socket using a mouse model of BONJ. Thirty-two mice were treated twice a week with either 100μg/kg of ZOL or saline for 12 weeks. The first left maxillary molar was extracted at the third week. Alveolar sockets were assessed at both 3 weeks (intermediate) and 9 weeks (long-term) after molar extraction by semi-quantitative histomorphometry for empty lacunae, preosteoblasts (Osterix), osteoclasts (TRAP), and pericyte-like cells (CD146). Also, the bone microarchitecture was assessed by micro-CT. Osteonecrotic-like lesions were observed in 21% of mice. Moreover, a decreased number of preosteoblasts contrasted with the increased number of osteoclasts at both time points. In addition, osteoclasts display multinucleation and detachment from the endosteal surface. Furthermore, the number of pericyte-like cells increased at the intermediate time point. The alveolar bone mass increased exclusively with long-term ZOL treatment. The severe imbalance between bone-forming cells and bone-resorbing cells shown in this study could contribute to the pathogenesis of BONJ.

Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw.

Characterize the cell profile and immunostaining of proinflammatory markers in an experimental model of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Male Wistar rats (n=6-7) were treated chronically with saline solution or zoledronic acid (ZA) at 0.04, 0.20, and 1.00mgkg(-1) (1.4×10(-7) , 6.9×10(-6) , and 3.4×10(-5) molkg(-1) ), and subsequently, the first left inferior molar was extracted. Were performed counting of viable and empty osteocyte lacunae, viable and apoptotic osteoclasts, polymorphonuclear neutrophil, mast cells (toluidine blue), and the positive presence cells for CD68, tumor necrosis factor-alpha (TNF-α), IL (interleukin)-1β, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-kB) and IL-18 binding protein (IL-18bp). BRONJ was showed in ZA treated with 0.20 and 1.00mgkg(-1) . There is a dose dependent increase in percentage of empty osteocyte lacunae (P<0.001) and apoptotic osteoclasts (P<0.001), counting of total osteoclasts (P=0.003), polymorphonuclear neutrophil cells (P=0.009), cytoplasmic-positive cells of CD68 (P<0.001), TNF-α (P=0.001), IL-1β (P=0.001), iNOS (P<0.001), NF-kB (P=0.006), and nuclear-positive cells of NF-kB (P=0.011). Consequently, there is no difference in mast cells (P=0.957), and IL-18bp immunostaining decreases dose dependently (P=0.005). BRONJ is characterized by increases in immunostaining for proinflammatory markers and NF-kB and inversely associated with cells exhibiting IL-18bp.

Experimental model of osteonecrosis of the jaw associated to bisphosphonates (ONJBPs) in ewes chronically treated with high doses of zoledronic acid

Experimental model of osteonecrosis of the jaw associated to bisphosphonates (ONJBPs) in ewes chronically treated with high doses of zoledronic acid

Search for a reliable model for bisphosphonate-related osteonecrosis of the jaw: establishment of a model in pigs and description of its histomorphometric characteristics

The pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ) remains unknown, and the development of a reliable experimental model would help to improve our understanding of it. We used 12 domestic pigs of which 6 made up the experimental group and were treated with zoledronate 4mg intravenously weekly for 5 weeks, while the control group (n=6) were given no drugs. On day 60 the right second maxillary and mandibular third molars were extracted. Thirty days later 3 animals in each group were killed; the rest were killed 90 days later. Histopathological specimens from the extraction sites were analysed for bone density, collagen architecture, density of osteons, and the amount of non-mineralised bone. Bone density, amount of non-mineralised bone, and density of osteons differed significantly between the 2 groups (p<0.001 in each case), but the chromatic pattern dictated by the collagen architecture did not. Our results correspond to the observations that have been made in patients with BRONJ, which means that the histomorphometric conditions seen in patients can be reproduced in this experimental setting.

Medication-related osteonecrosis of the jaw. Introduction of a new modified experimental model.

To evaluate a modified experimental model for medication-related osteonecrosis of the jaw (MRONJ) through the upper right central incisor extraction followed by intravenous bisphosphonate administration. Forty five rats underwent the upper right central incisor tooth extraction were divided in 2 groups: Group I - experimental group, 30 rats received an intravenous administration protocol of zoledronic acid 35μg/kg into the tail vein every two weeks, totalizing four administrations, during eight weeks of administration, previously the extraction, and Group II - control group, 15 rats didn't received any medication before extraction. The groups were subdivided in postoperative periods: 14/28/42 days. Clinical analysis and microtomography were performed to verify the presence of osteonecrosis. In addition, descritive histological analysis of hematoxylin-eosin stained sections was performed to evaluate the presence of osteonecrosis or necrotic foci. Twelve (40%) rats, from experimental group, showed clinical signs of MRONJ (p=0.005), however, all samples showed imaginologic findings like osteolysis and loss of integrity of the cellular walls (p≤0.001). Microscopic evaluation revealed osteonecrosis areas with microbial colonies and inflammatory infiltrate (p≤0.001). In the control group, all animals presented the chronology of a normal wound healing. The presence of medication-related osteonecrosis of the jaw after maxillary central incisor extraction in rats. This new experimental model may be considered an option for the study of MRONJ.

Fate of bone marrow mesenchymal stromal cells following autologous transplantation in a rabbit model of osteonecrosis

Background aimsInternalizing quantum dots (i-QDs) are a useful tool for tracking cells in vivo in models of tissue regeneration. We previously synthesized i-QDs by conjugating QDs with a unique internalizing antibody against a heat shock protein 70 family stress chaperone. In the present study, i-QDs were used to label rabbit mesenchymal stromal cells (MSCs) that were then transplanted into rabbits to assess differentiation potential in an osteonecrosis model. MethodsThe i-QDs were taken up by bone marrow-derived MSCs collected from the iliac of 12-week-old Japanese white rabbits that were positive for cluster of differentiation (CD)81 and negative for CD34 and human leukocyte antigen DR. The average rate of i-QD internalization was 93.3%. At 4, 8, 12, and 24 weeks after transplantation, tissue repair was evaluated histologically and by epifluorescence and electron microscopy. ResultsThe i-QDs were detected at the margins of the drill holes and in the necrotized bone trabecular. There was significant colocalization of the i-QD signal in transplanted cells and markers of osteoblast and mineralization at 4, 8, and 12 weeks post-transplantation, while i-QDs were detected in areas of mineralization at 12 and 24 weeks post-transplantation. Moreover, i-QDs were observed in osteoblasts in regenerated tissue by electron microscopy, demonstrating that the tissue was derived from transplanted cells. ConclusionThese results indicate that transplanted MSCs can differentiate into osteoblasts and induce tissue repair in an osteonecrosis model and can be tracked over the long term by i-QD labeling.

Functional investigation of bone implant viability using radiotracers in a new model of osteonecrosis

OBJECTIVES:Conventional imaging methods are excellent for the morphological characterization of the consequences of osteonecrosis; however, only specialized techniques have been considered useful for obtaining functional information. To explore the affinity of radiotracers for severely devascularized bone, a new mouse model of isolated femur implanted in a subcutaneous abdominal pocket was devised. To maintain animal mobility and longevity, the femur was harvested from syngeneic donors. Two technetium-99m-labeled tracers targeting angiogenesis and bone matrix were selected.METHODS:Medronic acid and a homodimer peptide conjugated with RGDfK were radiolabeled with technetium-99m, and biodistribution was evaluated in Swiss mice. The grafted and control femurs were evaluated after 15, 30 and 60 days, including computed tomography (CT) and histological analysis.RESULTS:Radiolabeling achieved high (>95%) radiochemical purity. The biodistribution confirmed good blood clearance 1 hour after administration. For 99mTc-hydrazinonicotinic acid (HYNIC)-E-[c(RGDfK)2, remarkable renal excretion was observed compared to 99mTc-methylene diphosphonate (MDP), but the latter, as expected, revealed higher bone uptake. The results obtained in the control femur were equal at all time points. In the implanted femur, 99mTc-HYNIC-E-[c(RGDfK)2 uptake was highest after 15 days, consistent with early angiogenesis. Regarding 99mTc-MDP in the implant, similar uptake was documented at all time points, consistent with sustained bone viability; however, the uptake was lower than that detected in the control femur, as confirmed by histology.CONCLUSIONS:1) Graft viability was successfully diagnosed using radiotracers in severely ischemic bone at all time points. 2) Analogously, indirect information about angiogenesis could be gathered using 999mTc-HYNIC-E-[c(RGDfK)2. 3) These techniques appear promising and warrant further studies to determine their potential clinical applications.

Superior Retinacular Artery Did Not Occlude in a Rat Model of the Non-Traumatic Osteonecrosis of the Femoral Head

Non-traumatic osteonecrosis of the femoral head (ONFH) is believed to be avascular necrosis. Certainly, a disruption of a supplying artery induces necrosis in the supplying artery-dominant region. However, when an abnormality such as cell apoptosis, or another reason, is developed in the supplying artery-dominant region, it induces a disruption of the supplying artery. Therefore, to prove that ONFH is avascular necrosis, it is necessary to demonstrate the disruption of the supplying artery prior to histological osteonecrosis development. Here we investigate histologically, using a rat model, whether disruption of the supplying artery occurs before the initial development of ONFH following corticosteroid treatment. Rats were given imiquimod and methylprednisolone, and were sacrificed 1, 2, 3, 7 or 14 days after the last injection. At the sacrifice, the rat was perfused with 20 ml black Indian ink through the left ventricle. ONFH was observed in the Imiquimod + Methylprednisolone group at 7 and 14 days. The osteonecrotic area was not stained with perfused black Indian ink. However, the lateral portion of the femoral head near the superior retinacular artery was stained with the ink. In conclusion, the present study shows that the superior retinacular artery did not occlude before the initial development of ONFH histologically in rats.

Efficacy of a new porous titanium compression screw using an ovine osseointegration model

Event Abstract Back to Event Efficacy of a new porous titanium compression screw using an ovine osseointegration model Robert A. Poggie1, Louis-Philippe Lefebvre2, Stephanie Grenier2, Madeleine Chagnon3 and Michel Assad3 1 BioVera Inc., Canada 2 National Research Council Canada, Medical Devices, Canada 3 AccelLAB Inc., Orthopedics and Biomaterials, Canada Introduction: Fracture fixation of small bones remains challenging for pathologies relating to tenuous retrograde blood supply. For example, acute scaphoid fractures are among the most common traumatic injuries and account for 50-80% of carpal injuries[1]. Solid screws without bone grafting are often ineffective in cases of osteonecrosis. The objectives of this study were to evaluate bone healing and strength of fixation of a new porous titanium (Ti) compression screw compared to a conventional titanium compression screw in cortical bone and in challenging models of gap-healing and osteonecrosis. Materials and Methods: Six (6) female Dorset sheep (>18mo.; 75-90kg) were prepared for bilateral hind limb surgeries using either porous Ti (NRC) or conventional solid Ti (Acutrak) headless cannulated compression screws (Ø=3.3-3.8mm; L=15mm). Porous titanium screws presented a 40% porosity with a mean pore size of 344µm, and compressive, shear, and tensile strengths of 157 MPa, 113 MPa and 77 MPa, respectively. The 1st model evaluated non-union following removal of a bone core (Ø=8mm; L=8mm) from the proximal tibia and screw placement in its middle, resulting in a 2-mm peripheral gap. The 2nd model simulated osteonecrosis with a 2nd core removal (Ø=14mm; L=8mm) from the distal femur, submersion in liquid N2, and screw fixation of the devitalized core. The 3rd model quantified bone ingrowth and torsional strength at mid-diaphyseal tibial cortical locations. Sheep were then euthanized at 6 and 12 weeks post-implantation. Bone ingrowth was reconstructed by Micro-CT (SkyScan 1172) and quantified with histomorphometry. Explanted screws were sectioned longitudinally, processed for undecalcified histology, infiltrated with PMMA, microground (Exakt 400 CS), and stained with Goldner’s Trichrome. Digital images were analyzed for bone-implant contact and volumetric bone ingrowth. Torsional strength was recorded with a continuous digital torque meter at necropsy. Results: Following 6 weeks of implantation in the non-union model, 75% of new bone formed adjacent to porous Ti as opposed to 38% with solid Ti screws. At 12 weeks, porous Ti screws exhibited 89% new bone formation vs. 79% for controls. Below the actual gap, bone filled 99% of the area within threads in porous Ti implants vs. 83% for controls at 12 weeks. Following bone devitalization, porous Ti showed up to 51% of new bone formation within its cannulated center as opposed to ≤3% for conventional screws. For the bone ingrowth model (Figure 1), linear bone apposition reached 55% for porous Ti vs. 36% (controls). Limited by failure of the Allen driver, the maximum torque for porous screws was 0.78Nm vs. 0.42Nm for controls. Histopathology revealed excellent safety and biocompatibility as evidenced by a mild tissue reaction. Discussion: Superior bone apposition, volumetric bone ingrowth and torsional strength were observed using the porous titanium screw, likely attributed to its interconnected porosity and higher surface area. A beneficial bone conduction effect was pronounced within the screw cannula. The clinical implication is that porous Ti does not occlude bone ingrowth and vascularization is observed within the compression device itself, translating into bone fixation and healing. Conclusion: Internal fixation with porous titanium compression screws increases the fixation strength and the potential for bone ingrowth in clinically challenging conditions of non-union and osteonecrosis. The authors would like to sincerely thank Dr. Paul A. Martineau and Dr. Edward J. Harvey (McGill University Health Centre), who co-developed this porous implant with the National Research Council of Canada; Special thanks to Dr. Cathy Tkaczyk and Mr. Yannick Trudel for their respective scientific and highly-skilled surgical support

Establishment of an Animal Model of Bisphosphonate-Related Osteonecrosis of the Jaws in Spontaneously Diabetic Torii Rats.

BackgroundWe evaluated the side effects of bisphosphonate (BP) on tooth extraction socket healing in spontaneously diabetic Torii (SDT) rats, an established model of non-obese type 2 diabetes mellitus, to develop an animal model of BP-related osteonecrosis of the jaws (BRONJ).Materials and MethodsMale Sprague-Dawley (SD) rats and SDT rats were randomly assigned to the zoledronic acid (ZOL)-treated groups (SD/ZOL or SDT/ZOL) or to the control groups (SD/control or SDT/control). Rats in the SD/ZOL or SDT/ZOL groups received an intravenous bolus injection of ZOL (35 μg/kg) every 2 weeks. Each group consisted of 6 rats each. Twenty-one weeks after ZOL treatment began, the left maxillary molars were extracted. The rats were euthanized at 2, 4, or 8 weeks after tooth extraction, and the total maxillae were harvested for histological and histochemical studies.ResultsIn the oral cavity, bone exposure persisted at the tooth extraction site in all rats of the SDT/ZOL group until 8 weeks after tooth extraction. In contrast, there was no bone exposure in SD/control or SDT/control groups, and only 1 of 6 rats in the SD/ZOL group showed bone exposure. Histologically, necrotic bone areas with empty lacunae, microbial colonies, and less invasion by inflammatory cells were observed. The number of tartrate-resistant acid phosphatase-positive osteoclasts was lower in the SDT/ZOL group than in the SD/control group. The mineral apposition rate was significantly lower in the SDT/ZOL group compared with the SD/control group.ConclusionsThis study demonstrated the development of BRONJ-like lesions in rats and suggested that low bone turnover with less inflammatory cell infiltration plays an important role in the development of BRONJ.

Synergistic local drug delivery in a piglet model of ischemic osteonecrosis: a preliminary study.

A locally injectable system sequentially delivering an antiresorptive drug (clodronate) followed by an osteogenic agent (simvastatin) was hypothesized to improve femoral head microarchitecture, size, and shape compared with untreated or partial treatment groups in an established piglet osteonecrosis model. After 6 weeks, the clodronate+simvastatin treatment resulted in no collapse, microCT measurements and epiphyseal quotients within 10% of control, normal microstructure, and healthy histology. All other groups exhibited collapse, lower epiphyseal quotients and total femoral head volumes (P<0.05), and abnormal histology. This pilot study provides evidence of synergistic antiresorptive and osteogenic activities, which may prevent femoral head collapse in Perthes disease.

An osteonecrosis model induced by oral bisphosphonate in ovariectomised rats.

To develop a model of osteonecrosis using oral bisphosphonate in ovariectomy-induced osteoporotic rats. Thirty-six rats were subjected to ovariectomy or sham surgery. After 8 weeks, rats received oral alendronate (1.0 mg kg(-1) ) or saline once weekly for 4 weeks; then, serum C-telopeptide cross-linked collagen type I levels were measured to evaluate bone metabolism. Twelve rats from each group were injected with either lipopolysaccharide or saline into the bone marrow of the mandibles and femurs, and the areas of osteonecrosis were evaluated by histomorphometry. Serum C-telopeptide cross-linked collagen type I levels were significantly increased in the ovariectomy group (105.1 ± 2.1 ng ml(-1) ) compared with the sham group (78.9 ± 12.5 ng ml(-1) ); they were significantly reduced following oral alendronate administration in the ovariectomy group (91.0 ± 4.4 ng ml(-1) ). Following alendronate and lipopolysaccharide administration, extensive osteonecrosis was observed in the mandibles and femurs of ovariectomy (0.45 ± 0.08 mm(2) , 1.69 ± 0.72 mm(2) , respectively) and sham (1.12 ± 0.45 mm(2) , 1.84 ± 0.66 mm(2) , respectively) groups. Significantly wider osteonecrosis occurred in the mandibles of sham-operated rats than ovariectomy rats following alendronate or lipopolysaccharide treatment. We successfully developed a model of osteonecrosis in ovariectomised rats following oral bisphosphonate administration.

MicroRNA expression in bone marrow mesenchymal stem cells from mice with steroid-induced osteonecrosis of the femoral head.

The present study aimed to identify microRNAs (miRNAs) from bone marrow mesenchymal stem cells (BMSCs) in a rat model of steroid-induced osteonecrosis of the femoral head (ONFH) using Affymetrix GeneChip®. Following identification of miRNAs, the present study aimed to elucidate the molecular mechanisms underlying steroid‑induced ONFH. A total of six C57BL/6J mice were randomly divided into two groups, control and experimental groups (n=3 per group). The mice in the experimental group were subcutaneously injected with 21 mg/kg methylprednisolone for 4 weeks, while the mice in the control group were injected with the identical dose of normal saline. The femoral head was subsequently removed and sectioned. Following sectioning, hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling were performed to confirm the establishment of the model. To replicate the animal model ex vivo, the bone marrow was isolated. Next, different miRNAs were screened for using GeneChip®, and the key miRNAs were assessed by bioinformatics analysis and their functions were confirmed. Compared with the control, 23 miRNAs in the experimental group were identified, with seven upregulated and 16 downregulated. Of these miRNAs, putative target miRNAs were predicted by bioinformatics analysis, with two being upregulated (miR‑21‑3p and miR‑652‑5p) and five downregulated (miR‑206‑3p, miR‑196a‑5p, miR‑34b‑3p, miR‑34c‑5p and miR‑148a‑3p). The results of reverse transcription‑quantitative polymerase chain reaction were consistent with the gene‑chip results. Steroid‑induced ONFH may cause miRNA changes in BMSCs. Numerous miRNAs regulate osteogenic differentiation and the decrease in miRNA‑196a‑5p may be important in steroid-induced ONFH.