Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee.

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

Reduced Capsaicin-Induced Mechanical Allodynia and Neuronal Responses in the DRG in the Presence of Ptpn6 Overexpression.

Transient Receptor Potential Vanilloid 1 (TRPV1) is a nonselective cation channel expressed by pain-sensing neurons and has been an attractive target for the development of drugs to treat pain. Recently, Src homology region 2 domain-containing phosphatase-1 (SHP-1, encoded by Ptpn6) was shown to dephosphorylate TRPV1 in dorsal root ganglia (DRG) neurons, which was linked with alleviating different pain phenotypes. These previous studies were performed in male rodents only and did not directly investigate the role of SHP-1 in TRPV-1 mediated sensitization. Therefore, our goal was to determine the impact of Ptpn6 overexpression on TRPV1-mediated neuronal responses and capsaicin-induced pain behavior in mice of both sexes. Twelve-week-old male and female mice overexpressing Ptpn6 (Shp1-Tg) and their wild type (WT) littermates were used. Ptpn6 overexpression was confirmed in the DRG of Shp1-Tg mice by RNA in situ hybridization and RT-qPCR. Trpv1 and Ptpn6 were found to be co-expressed in DRG sensory neurons in both genotypes. Functionally, this overexpression resulted in lower magnitude intracellular calcium responses to 200 nM capsaicin stimulation in DRG cultures from Shp1-Tg mice compared to WTs. In vivo, we tested the effects of Ptpn6 overexpression on capsaicin-induced pain through a model of capsaicin footpad injection. While capsaicin injection evoked nocifensive behavior (paw licking) and paw swelling in both genotypes and sexes, only WT mice developed mechanical allodynia after capsaicin injection. We observed similar level of TRPV1 protein expression in the DRG of both genotypes, however, a higher amount of tyrosine phosphorylated TRPV1 was detected in WT DRG. These experiments suggest that, while SHP-1 does not mediate the acute swelling and nocifensive behavior induced by capsaicin, it does mediate a protective effect against capsaicin-induced mechanical allodynia in both sexes. The protective effect of SHP-1 might be mediated by TRPV1 dephosphorylation in capsaicin-sensitive sensory neurons of the DRG.

The relative contribution of bone microarchitecture and matrix composition to implant fixation strength in rats.

Bone microarchitectural parameters significantly contribute to implant fixation strength but the role of bone matrix composition is not well understood. To determine the relative contribution of microarchitecture and bone matrix composition to implant fixation strength, we placed titanium implants in 12-week-old intact Sprague-Dawley rats, ovariectomized-Sprague-Dawley rats, and Zucker diabetic fatty rats. We assessed bone microarchitecture by microcomputed tomography, bone matrix composition by Raman spectroscopy, and implant fixation strength at 2, 6, and 10 weeks postimplantation. A stepwise linear regression model accounted for 83.3% of the variance in implant fixation strength with osteointegration volume/total volume (50.4%), peri-implant trabecular bone volume fraction (14.2%), cortical thickness (9.3%), peri-implant trabecular crystallinity (6.7%), and cortical area (2.8%) as the independent variables. Group comparisons indicated that osseointegration volume/total volume was significantly reduced in the ovariectomy group at Week 2 (~28%) and Week 10 (~21%) as well as in the diabetic group at Week 10 (~34%) as compared with the age matched Sprague-Dawley group. The crystallinity of the trabecular bone was significantly elevated in the ovariectomy group at Week 2 (~4%) but decreased in the diabetic group at Week 10 (~3%) with respect to the Sprague-Dawley group. Our study is the first to show that bone microarchitecture explains most of the variance in implant fixation strength, but that matrix composition is also a contributing factor. Therefore, treatment strategies aimed at improving bone-implant contact and peri-implant bone volume without compromising matrix quality should be prioritized.

Competitive Binding of Bilirubin and Fatty Acid on Serum Albumin Affects Wear of UHMWPE.

Total Joint Replacement (TJR) devices undergo standardized wear testing in mechanical simulators while submerged in a proteinaceous testing solution to mimic the environmental conditions of artificial joints in the human body. Typically, bovine calf serum is used to provide the required protein content. However, due to lot-to-lot variability, an undesirable variance in testing outcome is observed. Based on an earlier finding that yellowish-orange serum color saturation is associated with wear rate, we examined potential sources of this variability, by running a comparative wear test with bilirubin; hemin; and a fatty acid, oleic acid, in the lubricant. All these compounds readily bind to albumin, the most abundant protein in bovine serum. Ultrahigh molecular weight polyethylene (UHMWPE) pins were articulated against CoCrMo discs in a pin-on-disc tribometer, and the UHMWPE wear rates were compared between lubricants. We found that the addition of bilirubin increased wear by 121%, while hemin had a much weaker, insignificant effect. When added at the same molar ratio as bilirubin, the fatty acid tended to reduce wear. Additionally, there was a significant interaction with respect to bilirubin and hemin in that UHMWPE wear rate decreased with increasing fatty acid concentration. We believe the conformational change in albumin by binding bilirubin makes it more likely to form molecular bridges between UHMWPE and the metal counterface, thus increasing adhesive wear. However, fatty acids compete for binding sites on albumin, and can prevent this conformational change. Hence, the protein is stabilized, and the chance for albumin to form bridges is lowered. Ultimately, UHMWPE wear rate is driven by the competitive binding of bilirubin and fatty acid to albumin.

Wear Characteristics of Conventional Ultrahigh-Molecular-Weight Polyethylene Versus Highly Cross-Linked Polyethylene in Total Ankle Arthroplasty.

BACKGROUND: The aim of this study was to compare the polyethylene wear rate, particle size, and particle shape of primary semiconstrained, fixed-bearing, bone-sparing total ankle arthroplasty using conventional ultrahigh-molecular-weight polyethylene (CPE) versus highly cross-linked polyethylene (HXLPE) by applying a level walking input using a joint simulator. METHODS: Two fixed-bearing total ankle replacement systems with different types of polyethylene liners were tested: (1) CPE sterilized in ethylene oxide, and (2) HXLPE sterilized with gas plasma after electron beam irradiation. Three implants for each design underwent wear testing using gravimetric analysis over 5 million simulated walking cycles. A fourth implant was used as a load soak control. Equivalent circle diameter (ECD) and equivalent shape ratio (ESR) were computed to determine particle size and particle shape, respectively. RESULTS: The mean wear rate from 1.5 to 5 million cycles (MC) was 2.0 ± 0.3 mg/MC for HXLPE and 16.7 ± 1.3 mg/MC for CPE ( P < .001). The total number of particles per cycle generated for HXLPE and CPE were 0.17 × 106 particles/cycle and 0.53 × 106 particles/cycle, respectively ( P < .001). The mean ECD of HXLPE particles (0.22 ± 0.11 µm) was significantly smaller than the mean ECD of CPE particles (0.32 ± 0.14 µm) ( P < .001). HXLPE particles were significantly more round than CPE particles ( P < .001). CONCLUSIONS: HXLPE liners had a significantly lower wear rate and produced significantly fewer and rounder particles than CPE liners. The results of this study suggest that HXLPE has more favorable wear characteristics for total ankle arthroplasty. CLINICAL RELEVANCE: Polyethylene wear particles have been linked to osteolysis after total ankle arthroplasty. There is no consensus on the importance of highly cross-linked polyethylene in total ankle arthroplasty with regard to implant wear. This is the first nonindustry study to compare the polyethylene wear rate, particle size, and particle shape of fixed-bearing total ankle arthroplasty conventional polyethylene versus highly cross-linked polyethylene. The lower wear rate and different particle size/morphology of highly cross-linked polyethylene could be beneficial in vivo to decrease osteolysis.

Finite element evaluation of the newest ISO testing standard for polyethylene total knee replacement liners.

Current treatment for end-stage osteoarthritis is total knee replacement. Given that the number of total knee replacement surgeries is expected to approach 3.48 million by 2030, understanding long-term failure is important. One of the preclinical tests for total knee replacements is carried out using mechanical wear testing under generic walking conditions. Used for this purpose is the International Standards Organization's generic walking profile. Recently this standard was updated by reversing the direction of anterior/posterior translation and internal/external rotation. The effects of this change have not been investigated, and therefore, it is unknown if comparisons between wear tests utilizing the old and new version of the standard are valid. In this study, we used a finite element model along with a frictional energy-based wear model to compare the kinematic inputs, contact conditions, and wear from the older and newer versions of the ISO standard. Simulator-tested components were used to validate the computational model. We found that there were no visible similarities in the contact conditions between the old and new versions of the standard. The new version of the standard had a lower wear rate but covered a larger portion of the articular surface. Locations of wear also varied considerably. The results of the study suggest that major differences between the old and new standard exist, and therefore, historical wear results should be compared with caution to newly obtained results.