It has been previously shown that Low-Magnitude High-Frequency Vibration (LMHFV) is able to enhance ovariectomy-induced osteoporotic fracture healing in rats. Fracture healing begins with the inflammatory stage, and all subsequent stages are regulated by the infiltration of immune cells such as macrophages and the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Therefore, the aim of this study was to investigate the effect of LMFHV treatment on the inflammatory response in osteoporotic fracture healing. In this study, ovariectomy-induced osteoporotic and sham-operated closed-femoral fracture SD-rats were randomized into three groups: sham control (SHAM), ovariectomized control (OVX-C) or ovariectomized vibration (OVX-V) (n=36, n=6 per group per time point). LMHFV (35Hz, 0.3g) was given 20 min/day and 5 days/week to OVX-V group. SHAM operation and ovariectomy were performed at 6-month and closed femoral fracture was performed at 9-month. Callus morphometry was determined by callus width from weekly radiography. Local expressions of inducible nitric oxide synthase (iNOS) (macrophage M1 marker), CD206 (macrophage M2 marker), TNF-α, IL-6 and IL-10 were detected by immunohistochemistry and quantified by colour threshold in ImageJ, assessed at weeks 1 and 2 post-fracture. Significant difference between groups was considered at p≤0.05 by one-way ANOVA. Callus formation was higher in OVX-V than that of OVX-C as shown by callus width at weeks 1 and 2 (p=0.054 and 0.028, respectively). Immunohistochemistry results showed that CD206 positive signal and the M2/M1 ratio which indicates the progression of macrophage polarization were significantly higher in OVX-V rats (p=0.053 and 0.049, respectively) when compared to OVX-C at week 1. Area fraction of TNF-α positive signal was significantly higher in SHAM and OVX-V rats at week 1 (p=0.01 and 0.033, respectively). IL-6 signal was also significantly higher in SHAM and OVX-V groups at week 1 (p=0.004 and 0.029, respectively). IL-10 expression was significantly lower in SHAM and OVX-V groups at week 1 (p=0.013 and 0.05, respectively). Here we have shown that LMHFV treatment promoted the shift from pro-inflammatory stage towards anti-inflammatory stage earlier. It has been reported that the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2 was indicative of the endochondral ossification process in the long bone fracture model. Besides, we found that LMHFV treatment enhanced pro-inflammatory markers of TNF-α and IL-6 and suppressed anti-inflammatory marker of IL-10 at week 1, showing that inflammatory response was enhanced at week 1 post-fracture. These inflammatory cytokines involved in fracture healing were shown to coordinate different fracture healing processes such as mesenchymal stem cell recruitment and angiogenesis. Our previous study has demonstrated that ovariectomized rats exhibit lower levels of inflammatory response after fracture creation. Therefore, we report that LMHFV treatment can modulate macrophage polarization from M1 to M2 at an earlier time-point and partly restore the impaired inflammatory response in OVX bones at the early stage of fracture healing that may lead to accelerated healing of osteoporotic fracture as shown by promoted callus formation

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone

Introduction:. The purpose of this study was to evaluate the basic corrosion properties of conventional CoCr (ASTM F 1537, UNS R31537), Gas Atomized Dispersion Strengthened (GADS) CoCr (ASTM 1537, UNS R31539), Ti6Al4V ELI (ASTM F 136, UNS R56401) and Ti-12Mo-6Zr-2Fe (TMZF) (ASTM F 1813) alloys that are used in joint replacement applications. Methods:. Test coupon samples made from alloys' wrought bar stocks were polished then tested. Each material had 4 groups (n. 3. 3) that were tested in 4 aqueous solutions: 1) pH 7.4, 2) pH 5, 3) pH 2, and 4) pH 2 with 1 M of H. 2. O. 2. Potential dynamic polarization was conducted using a Princeton Applied Research VMC Potentiostat, with an Ag/AgCl electrode as the reference electrode, and it resembled method in ASTM F 2129-08. The samples were evaluated with Scanning Electron Microscope (SEM). Results:. 1) Both pH and solution chemistry changes affected the corrosion behaviors and caused the polarization curves to shift; Figure 1 & 2 show the polarization curves of CoCr and Ti alloys. 2) The polarization curves for conventional CoCr and GADS alloys were identical with minor differences; Figure 1 shows the polarization curves for CoCr and GADS alloys at pH 7.4 and pH 2 conditions. 3) The polarization curves for Ti6Al4V ELI and TMZF alloys were also similar at most conditions with some minor differences; Figure 2 shows the polarization curves of Ti6Al4V ELI and TMZF alloys at pH 2. It should also be noted that at pH 2 plus H. 2. O. 2. condition, Ti6Al4V ELI showed increased current at voltages near 1V as shown in Figure 2. This might suggest an onset of accelerated corrosion or breakdown that is not normally observed with Ti alloys. TMZF showed constant current in the same region, suggesting no breakdown or accelerated corrosion at this condition. SEM revealed corrosion of TI6Al4V ELI alloy similar to etching as shown in Figure 2. Discussion:. The fact that GADS and conventional CoCr alloys behaved identically suggests similar corrosion properties and resistance in normal conditions; the same applies to TI6Al4V ELI and TMZF alloys. It was unexpected to see the seemingly “breakdown” or accelerated corrosion for Ti6Al4V ELI in pH 2 solution with added H. 2. O. 2. at voltages near 1 V. Note that the test condition was very aggressive and the proper amount or concentration of H. 2. O. 2. in a test solution has not been well documented in the literature. However, it should be kept in mind that alloys' corrosion properties under extreme test conditions should not be overlooked as similar conditions could happen clinically in crevice environments for example. Regardless, it is interesting to observe the difference between Ti6Al4V ELI and TMZF at this particular condition, and it warrants further investigation

Bone is a connective tissue that undergoes constant remodeling. Any disturbances during this process may result in undesired pathological conditions. A single nucleotide substitution (596T-A) in exon eight which leads to a M199K mutation in human RANKL was found to cause osteoclast-poor autosomal recessive osteopetrosis (ARO). Patients with ARO cannot be cured by hematopoietic stem cell transplantation and, without proper treatments, will die in their early age. To date, how this mutation alters RANKL function has not been characterized. We thus hypothesized that hRANKL M199 residue is a structural determinant for normal RANKL-RANK interaction and osteoclast differentiation. By sharing our findings, we aim to achieve an improved clinical outcome in treating bone-related diseases such as osteoporosis, ARO and osteoarthritis. Site-directed mutagenesis was employed to create three rat RANKL mutants, replacing the methionine 200 (human M199 equivalent residue) with either lysine (M200K), alanine (M200A) or glutamic acid (M200E). Recombinant proteins were subsequently purified through affinity chromatography and visualized by Coomassie blue staining and western blot. MTS was carried out before osteoclastogenesis assay in vitro to measure the cellular toxicity. Bone resorption pit assay, immuno-fluorescent staining, luciferase reporter assay, RT-PCR, western blot and calcium oscillation detection were also conducted to explore the biological effect of rRANKL mutants. Computational modeling, thermal Shift Assay, western blot and protein binding affinity experiments were later carried out for structural analyses. rRANKL mutants M200K/A/E showed a drastically reduced ability to induce osteoclast formation and did not demonstrate features of competitive inhibition against wild-type rRANKL. These mutants are all incapable of supporting osteoclastic polarization and bone resorption or activating RANKL-induced osteoclast marker gene transcription. Consistently, they were unable to induce calcium flux, and also showed a diminished induction of IκBa degradation and activation of NF-kB and NFATc1 transcriptional activity. Furthermore, the transcriptional activation of the antioxidant response element (ARE) crucial in modulating oxidative stress and providing cytoprotection was also unresponsive to stimulation with rM200s. Structural analyses showed that rM200 is located in a hydrophobic pocket critical for protein folding. Thermal shift and western blot assays suggested that rM200 mutants formed unstructured proteins, with disturbed trimerisation and the loss of affinity to its intrinsic receptors RANK and OPG. Taken together, we first demonstrates the underlying cause of M199-meidated ARO in a cellular and molecular level by establishing a phenotype in BMMs similar to observed in human samples. Further investigation hints the structural significance of a hydrophobic pocket within the TNF-like region. Combined with pharmaceutical studies on small-molecule drugs, this finding may represent a therapeutic target motif for future development of anti-resorptive treatments

Introduction. Corrosion at modular junctions of total hip replacements has been identified as a potential threat to implant longevity, resulting in efforts to determine appropriate countermeasures. Visual scoring and volumetric material loss measurements have been useful tools to elucidate various clinical and design factors associated with corrosion damage. However, corrosion involves electron exchange that results in chemical changes to biomedical alloys, and electrochemical assessment may therefore be a more appropriate approach to understand the phenomenon. The purpose of this pilot study was to electrochemically distinguish the severity of corrosion in retrieved femoral heads. A secondary goal was to identify the potential of electrochemical impedance spectroscopy (EIS) as a method to identify different forms of corrosion damage. Methods. Twenty femoral heads were identified from a larger study of total hip replacements, obtained as part of an ongoing multi- center IRB-approved retrieval program. Using a previously established 4-point scoring method, components were binned by taper damage: 10 components were identified as having severe damage, 7 with moderate damage and 2 with mild damage. One (1) unimplanted control was included to represent minimal corrosion damage. All components were then characterized using electrochemical impedance spectroscopy under the frequency domain: a 10 mV sinusoidal voltage, ranging from 20 kHz to 2 mHz, was applied to the taper of a femoral head (working electrode) filled with a 1M solution of PBS, a platinum counter electrode and a chlorided silver reference electrode. Absolute impedance at 2 mHz (|Z. 0.002. |), and max phase angle (θ) were assessed relative to taper damage severity. After least-squares fitting of the EIS data to a Randles circuit with a constant phase element, circuit elements: polarization resistance (Rp), CPE-capacitance, and CPE-exponent were also evaluated. The seven (7) most severely corroded components were further examined with scanning electron microscopy to identify corrosion modes. For all statistical analyses, significance was determined at alpha=0.05. Results. Taper damage was strongly correlated with both |Z. 0.002. | (ρ = −0.857, p<0.001) and CPE-capacitance (ρ=0.913, p<0.001). Taper damage was moderately associated with max phase angle (ρ= −0.483, p=0.031), CPE-exponent (ρ= −0.653, p=0.002) and Rp (ρ=0.556, p=0.011). Log-log plots of the strongest predictors of taper damage (|Z. 0.002. | and CPE- capacitance) identified some clustering among severely corroded components. SEM analysis identified evidence of grain/phase boundary corrosion on four components, all with log CPE-capacitance ≥ −4.4. Discussion. The results of this pilot study highlight that electrochemical impedance spectroscopy is useful in determining corrosion severity in retrieved femoral heads, and may also identify intergranular corrosion attack. For an undamaged taper, the self- passivating behavior of CoCrMo creates a surface that opposes charge transfer, but greater corrosion appears to compromise this barrier. The observed trend of low impedance but high capacitance for severely corroded components with intergranular corrosion may signal charge storage at the boundaries of individual grains. Additional work is underway to characterize this behavior

Introduction. Fretting crevice-corrosion (tribocorrosion) of metallic biomaterials is a major concern in orthopedic, spinal, dental and cardiovascular devices. 1. Stainless steel (i.e., 316L SS) is one alloy that sees extensive use in applications where fretting, crevices and corrosion may be present. While fretting-corrosion of this alloy has been somewhat studied, the concept of fretting-initiating crevice corrosion (FICC), where an initial fretting corrosion process leads to ongoing crevice-corrosion without continued fretting, is less understood. This study investigated the susceptibility of 316L SS to FICC and the role of applied potential on the process. The hypothesis is crevice-corrosion can be induced in 316L SS at potentials well below the pitting potential. Materials and Methods. A pin-on-disk fretting test system similar to that of Swaminathan et al. 2. was employed. Disks were ∼35 mm in diameter and the pin area was ∼500 mm. Samples were polished to 600 mm finish, cleaned with ethanol and distilled water. An Ag/AgCl wire as the reference, a carbon counter electrode and phosphate buffered saline (PBS, pH 7.4, Room T) were used for electrochemical testing. Load was controlled with a dead-weight system, monitored with a six-axis load cell (ATI Inc.). Interfacial motion was captured with a non-contact eddy current sensor (0.5 mm accuracy). Motion and load data acquisition was performed with Labview (National Instruments). Samples were loaded to ∼2 N. The potential per tests was increased from −250 to 250 mV (50 mV increments) with new locations and pins used in each repeat (n=3). Testing incorporated a 1 min rest before fretting (5 min, 1.25 Hz, 60 mm displacement saw tooth pattern). Fretting ceased and the load was held while currents were captured for another 5 min to assess ongoing crevice corrosion. Results. Testing showed that crevice corrosion can be initiated within minutes of fretting (or in a few cycles depending on potential; Fig. 1). Potentials as low as −100 mV showed evidence of corrosion, while sustained crevice corrosion was seen at −50 mV. As the potential increased above −50 mV, susceptibility to FICC increased. Fig. 2 is a typical cyclic polarization curve for 316L SS in PBS without fretting. Pitting starts at 400 mV vs Ag/AgCl, and the protection potential in this case is around potentials where FICC can be induced. Discussion. This study showed that 316L SS is prone to FICC starting at −100 mV and the severity of the crevice-corrosion damage depends on the applied potential (Fig. 3). Current after cessation of fretting takes longer to return to baseline or does not return indicating ongoing corrosion without fretting (Fig. 1). If the pin and disk are separated, the crevice-corrosion process stops immediately. The region immediately outside the fretting contact was crevice-like with a very small separation distance between the pin and disk surface which allowed crevice corrosion to develop (Fig. 3). Conclusion. 316L SS can undergo FICC at potentials close to normal physiological electrode potential conditions. Few fretting cycles are required to develop conditions for continued crevice-corrosion. Higher potentials increased the susceptibility of FICC in 316L SS

Introduction. There are increasing reports of total hip replacement (THR) failure due to corrosion within modular taper junctions, and subsequent adverse local tissue reactions (ALTRs) to corrosion products. Modular junction corrosion is a multifactorial problem that depends on material, design, patient and surgical factors. However, the influence of alloy microstructure on corrosion has not been studied sufficiently. Especially for cast CoCrMo, there are concerns regarding microstructure variability with respect to grain size and hard-phase volume fraction. Therefore, it was the goal of this study to (1) identify different types of microstructures in contemporary implants, and (2) determine implications of alloy microstructure on the occurring corrosion modes. Methods. Fifteen surgically retrieved femoral stems made from cast CoCrMo alloy were analyzed for this study. Damage on the taper surfaces was investigated by scanning electron microscopy (SEM) and damage was assessed with the Goldberg Score. The alloy microstructure was evaluated by standard metallographic techniques. Alloy samples were sectioned off the femoral stem, and microstructural features were visualized by chemical etching. Cyclic potentio-dynamic polarization tests were carried out with alloy samples from two implants with different commonly occurring types of microstructures. Both had a similar grain size, but type 1 had no hard-phases, where as type 2 exhibited hard-phases along the grain boundaries, as well as intra-granular hard-phase clusters. Tests were performed in bovine serum at 37°C with a saturate calomel reference electrode and a graphite counter electrode. In vitro generated corrosion damage was then compared to in vivo generated damage features on the taper surfaces of the corresponding implants. Results. Tapers with high damage scores exhibited varying degrees of grain and phase boundary corrosion, along with fretting and pitting corrosion. In several cases thick chromium oxide films were observed. The metallographic analysis showed that nominally identical alloys (ASTM F75) exhibited a broad variability in grain size (250 micrometers to several millimeters), hard-phase volume fraction (0–6%), and hard-phase type (carbides and intermetallic phases). The corrosion tests revealed that the alloy without hard-phases (type 1) had a significantly higher pitting potential (p=0.001) than type 2 alloy without hard-phases. After testing, both alloys exhibited grain boundary corrosion. However, type 2 had a higher degree of material loss due to hard-phase detachment. Additionally, type 2 exhibited pitting within the grains around hard-phases, along with the formation of thick oxide films which was consistent with the lower pitting potential. The results also corresponded with the damage features on the corresponding tapers, where type 1 exhibited only mild damage features, and type 2 underwent severe grain and phase boundary corrosion along with thick oxide films (Figure 3). Discussion. It appears that the alloy microstructure drives local modes of corrosion. Additional phase boundaries due to hard-phase content promote corrosion. The fact that the same alloy can differ broadly even within the same design shows that material standards are currently not sufficient. Optimizing implant alloys will help to reduce in vivo corrosion processes, and subsequently the risk of implant failure due to ALTRs. For figures/tables, please contact authors directly.