Dependence of crack shape in loaded articular cartilage on the collagenous structure.

Cartilage cracks disrupt tissue mechanics, alter cell mechanobiology, and often trigger tissue degeneration. Yet, some tissue cracks heal spontaneously. A primary factor determining the fate of tissue cracks is the compression-induced mechanics, specifically whether a crack opens or closes when loaded. Crack deformation is thought to be affected by tissue structure, which can be probed by quantitative polarized light microscopy (PLM). It is unclear how the PLM measures are related to deformed crack morphology. Here, we investigated the relationship between PLM-derived cartilage structure and mechanical behavior of tissue cracks by testing if PLM-derived structural measures correlated with crack morphology in mechanically indented cartilages. METHODS: Knee joint cartilages harvested from mature and immature animals were used for their distinct collagenous fibrous structure and composition. The cartilages were cut through thickness, indented over the cracked region, and processed histologically. Sample-specific birefringence was quantified as two-dimensional (2D) maps of azimuth and retardance, two measures related to local orientation and degree of alignment of the collagen fibers, respectively. The shape of mechanically indented tissue cracks, measured as depth-dependent crack opening, were compared with azimuth, retardance, or "PLM index," a new parameter derived by combining azimuth and retardance. RESULTS: Of the three parameters, only the PLM index consistently correlated with the crack shape in immature and mature tissues. CONCLUSION: In conclusion, we identified the relative roles of azimuth and retardance on the deformation of tissue cracks, with azimuth playing the dominant role. The applicability of the PLM index should be tested in future studies using naturally-occurring tissue cracks.

Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

IL-1Ra deficiency accelerates intervertebral disc degeneration in C57BL6J mice.

The expression of Interleukin-1ß (IL-1ß) and its antagonist and Interleukin-1 receptor antagonist (IL-1Ra) are correlated with greater human intervertebral disc (IVD) degeneration, suggesting that elevated IL-1ß activity promotes disc degeneration. Many in vitro studies support such a mechanistic relationship, whereas few in vivo investigations have been reported. The present study tests the effect of increased IL-1ß activity on intervertebral disc in mice with an IL-1Ra gene deletion. IL-1Ra-/- mice and wild-type (WT) C57Bl6J mice were examined at 3 and 12 months of age. Caudal IVD segments were evaluated for disc degeneration by histopathology, functional testing, and inflammatory gene expression relevant to IL-1ß pathways. To test differences in injury response, pinprick annular puncture was performed on IL-1Ra-/- and WT mice and evaluated similarly. IL-1Ra-/- IVDs had significantly worse histopathology at 3 months compared to WT controls, but not at 12 months. IL-1Ra-/- IVDs exhibited significantly more viscous mechanical properties than WT IVDs. qPCR revealed downregulation of inflammatory genes at 3 and 12 months in IL-1Ra-/- IVDs, with concomitant downregulation of anabolic and catabolic genes. Annular puncture yielded no appreciable differences between 2-week and 6-week post-injured WT and IL1-Ra-/- IVDs in histopathology or biomechanics, but inflammatory gene expression was sharply downregulated in IL-1Ra-/- mice at 2 weeks, returning by 6 weeks post injury. In the present study, IL-1Ra deletion resulted in increased IVD histopathology, inferior biomechanics, and transiently decreased pro-inflammatory cytokine gene expression. The histopathology of IL-1Ra-/- IVDs on a C57BL/6J background is less severe than a previous report of IL1Ra-/- on a BALB/c background, yet both strains exhibit IVD degeneration, reinforcing a mechanistic role of IL-1ß signaling in IVD pathobiology. Despite a pro-inflammatory environment, the annular puncture was no worse in IL-1Ra-/- mice, suggesting that response to injury involves pathways other than inflammation. Overall, this study supports the hypothesis that IL-1ß-driven inflammation is important in IVD degeneration.

Synovial mesenchymal progenitor derived aggrecan regulates cartilage homeostasis and endogenous repair capacity.

Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early hallmarks of osteoarthritis (OA) is the loss of aggrecan from articular cartilage followed by degeneration of the tissue. Mesenchymal progenitor cell (MPC) populations in joints, including those in the synovium, have been hypothesized to play a role in the maintenance and/or repair of cartilage, however, the mechanism by which this may occur is unknown. In the current study, we have uncovered that aggrecan is secreted by synovial MPCs from healthy joints yet accumulates inside synovial MPCs within OA joints. Using human synovial biopsies and a rat model of OA, we established that this observation in aggrecan metabolism also occurs in vivo. Moreover, the loss of the "anti-proteinase" molecule alpha-2 macroglobulin (A2M) inhibits aggrecan secretion in OA synovial MPCs, whereas overexpressing A2M rescues the normal secretion of aggrecan. Using mice models of OA and cartilage repair, we have demonstrated that intra-articular injection of aggrecan into OA joints inhibits cartilage degeneration and stimulates cartilage repair respectively. Furthermore, when synovial MPCs overexpressing aggrecan were transplanted into injured joints, increased cartilage regeneration was observed vs. wild-type MPCs or MPCs with diminished aggrecan expression. Overall, these results suggest that aggrecan secreted from joint-associated MPCs may play a role in tissue homeostasis and repair of synovial joints.

Role of Primary Afferents in Arthritis Induced Spinal Microglial Reactivity.

Increased afferent input resulting from painful injury augments the activity of central nociceptive circuits via both neuron-neuron and neuron-glia interactions. Microglia, resident immune cells of the central nervous system (CNS), play a crucial role in the pathogenesis of chronic pain. This study provides a framework for understanding how peripheral joint injury signals the CNS to engage spinal microglial responses. During the first week of monosodium iodoacetate (MIA)-induced knee joint injury in male rats, inflammatory and neuropathic pain were characterized by increased firing of peripheral joint afferents. This increased peripheral afferent activity was accompanied by increased Iba1 immunoreactivity within the spinal dorsal horn indicating microglial activation. Pharmacological silencing of C and A afferents with co-injections of QX-314 and bupivacaine, capsaicin, or flagellin prevented the development of mechanical allodynia and spinal microglial activity after MIA injection. Elevated levels of ATP in the cerebrospinal fluid (CSF) and increased expression of the ATP transporter vesicular nucleotide transporter (VNUT) in the ipsilateral spinal dorsal horn were also observed after MIA injections. Selective silencing of primary joint afferents subsequently inhibited ATP release into the CSF. Furthermore, increased spinal microglial reactivity, and alleviation of MIA-induced arthralgia with co-administration of QX-314 with bupivacaine were recapitulated in female rats. Our results demonstrate that early peripheral joint injury activates joint nociceptors, which triggers a central spinal microglial response. Elevation of ATP in the CSF, and spinal expression of VNUT suggest ATP signaling may modulate communication between sensory neurons and spinal microglia at 2 weeks of joint degeneration.

Repetitive in vivo manual loading of the spine elicits cellular responses in porcine annuli fibrosi.

Back pain and intervertebral disc degeneration are prevalent, costly, and widely treated by manual therapies, yet the underlying causes of these diseases are indeterminate as are the scientific bases for such treatments. The present studies characterize the effects of repetitive in vivo manual loads on porcine intervertebral disc cell metabolism using RNA deep sequencing. A single session of repetitive manual loading applied to the lumbar spine induced both up- and down-regulation of a variety of genes transcribed by cells in the ventral annuli fibrosi. The effect of manual therapy at the level of loading was greater than at a level distant to the applied load. Gene ontology and molecular pathway analyses categorized biological, molecular, and cellular functions influenced by repetitive manual loading, with over-representation of membrane, transmembrane, and pericellular activities. Weighted Gene Co-expression Network Analysis discerned enrichment in genes in pathways of inflammation and skeletogenesis. The present studies support previous findings of intervertebral disc cell mechanotransduction, and are the first to report comprehensively on the repertoire of gene targets influenced by mechanical loads associated with manual therapy interventions. The present study defines the cellular response of repeated, low-amplitude loads on normal healthy annuli fibrosi and lays the foundation for future work defining how healthy and diseased intervertebral discs respond to single or low-frequency manual loads typical of those applied clinically.

Contrast-enhanced x-ray microscopy of articular cartilage.

Osteoarthritis is a common chronic disease of joints characterized by degenerative changes of articular cartilage. An early diagnosis of osteoarthritis may be possible when imaging excised tissue for research in situ at the cellular-molecular scale. Whereas cartilage histopathology is destructive, time-consuming, and limited to 2D views, contrast-enhanced x-ray microscopy (XRM) can image articular cartilage and subchondral bone in 3D. This study evaluates articular cartilage structure ex vivo using both techniques.Osteochondral plugs were excised from non-diseased bovine knees and stained in phosphotungstic acid for 0 to 32 h. XRM imaging revealed an optimal staining time of 16 h and a saturated staining time of 24 h. Histology sections were cut and analyzed by polarized light microscopy (PLM) and second-harmonic-generation dual-photon (SHG-DP) microscopy. Histology photomicrographs were aligned with matching XRM slices and evaluated for features relevant in histopathological scoring of osteoarthritis cartilage, including the tidemark, collagen architecture and chondrocyte morphology.The cartilage collagen network and chondrocytes from the 3D contrast-enhanced XRM were correlated with the 2D histology. This technique has two distinct advantages over routine histopathology: (1) the avoidance of dehydration, demineralization, and deformation of histological sectioning, thereby preserving the intact articular cartilage and subchondral bone plate ex vivo, and (2) the ability to evaluate the entire osteochondral volume in 3D. This work explores several diagnostic features of imaging cartilage, including: visualization of the tidemark in XRM and SHG-DP microscopy, validating the morphology of chondrocytes and nuclei with XRM, SHG-DP and PLM, and correlating collagen birefringence with XRM image intensity.

Cage-lid hanging behavior as a translationally relevant measure of pain in mice.

ABSTRACT: The development of new analgesic drugs has been hampered by the inability to translate preclinical findings to humans. This failure is due in part to the weak connection between commonly used pain outcome measures in rodents and the clinical symptoms of chronic pain. Most rodent studies rely on the use of experimenter-evoked measures of pain and assess behavior under ethologically unnatural conditions, which limits the translational potential of preclinical research. Here, we addressed this problem by conducting an unbiased, prospective study of behavioral changes in mice within a natural homecage environment using conventional preclinical pain assays. Unexpectedly, we observed that cage-lid hanging, a species-specific elective behavior, was the only homecage behavior reliably impacted by pain assays. Noxious stimuli reduced hanging behavior in an intensity-dependent manner, and the reduction in hanging could be restored by analgesics. Finally, we developed an automated approach to assess hanging behavior. Collectively, our results indicate that the depression of hanging behavior is a novel, ethologically valid, and translationally relevant pain outcome measure in mice that could facilitate the study of pain and analgesic development.

Complex patterns of cell growth in the placenta in normal pregnancy and as adaptations to maternal diet restriction.

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.

Serum-Free Culture of Human Mesenchymal Stem Cell Aggregates in Suspension Bioreactors for Tissue Engineering Applications.

Mesenchymal stem cells (MSCs) have the capacity to differentiate towards bone, fat, and cartilage lineages. The most widely used culture and differentiation protocols for MSCs are currently limited by their use of serum-containing media and small-scale static culture vessels. Suspension bioreactors have multiple advantages over static culture vessels (e.g., scalability, control, and mechanical forces). This study sought to compare the formation and culture of 3D aggregates of human synovial fluid MSCs within suspension bioreactors and static microwell plates. It also sought to elucidate the benefits of these techniques in terms of productivity, cell number, and ability to generate aggregates containing extracellular matrix deposition. MSCs in serum-free medium were either (1) inoculated as single cells into suspension bioreactors, (2) aggregated using static microwell plates prior to being inoculated in the bioreactor environment, or (3) aggregated using microwell plates and kept in the static environment. Preformed aggregates that were size-controlled at inoculation had a greater tendency to form large, irregular super aggregates after a few days of suspension culture. The single MSCs inoculated into suspension bioreactors formed a more uniform population of smaller aggregates after a definite culture period of 8 days. Both techniques showed initial deposition of extracellular matrix within the aggregates. When the relationship between aggregate size and ECM deposition was investigated in static culture, midsized aggregates (100-300 cells/aggregate) were found to most consistently maximize sGAG and collagen productivity. Thus, this study presents a 3D tissue culture method, which avoids the clinical drawbacks of serum-containing medium that can easily be scaled for tissue culture applications.

Anatomic study on the blood supply to the femoral head following hip resurfacing using the posterior approach.

OBJECTIVES: The aim of this study was to investigate femoral head perfusion following cadaveric hip resurfacing using the posterior approach. METHODS: This cadaveric study involved injecting Higgins India ink into the common iliac arteries and evaluating the distribution of ink in the resurfaced heads using the modified Spalteholz technique. The study consisted of 2 parts. The 1st part involved utilisation of 22 cadaveric hips for establishing the injection and histological technique. The 2nd part of the study included 4 control cadaveric hips and 12 cadaveric hips with posterior approach hip resurfacing. Each specimen was divided into 15 zones (12 head zones and 3 neck zones) to evaluate detailed geographic distribution of dye-containing blood vessels. RESULTS: All 4 controls had good flow of ink to all head zones and the neck region. In all the resurfaced heads, there was good flow to all the neck zones. 6 resurfaced specimens had no dye flow to any of the head zones. In the remaining 6, dye-stained vessels were seen variably in the anterior and middle zones but were consistently absent in the posterior zones of the head. Zones representing the antero-inferior parts of femoral head had the maximum flow of ink, followed by zones representing middle-inferior parts. CONCLUSIONS: Posterior approach for hip resurfacing arthroplasty results in vascular insult to the femoral head, with posterior zones more affected than the anterior zones. The persistence of the dye in the intraosseous blood vessels of the neck and in anteroinferior head may be a source of revascularisation of the femoral head after posterior approach hip resurfacing.

Microglial pannexin-1 channel activation is a spinal determinant of joint pain.

Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5'-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1ß (IL-1ß) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide 10panx suppressed the aberrant discharge of spinal laminae I-II neurons evoked by innocuous mechanical hindpaw stimulation in arthritic rats. Furthermore, mice with a microglia-specific genetic deletion of Panx1 were protected from developing mechanical allodynia. Treatment with probenecid, a clinically used broad-spectrum Panx1 blocker, resulted in a striking attenuation of MIA-induced mechanical allodynia and normalized responses in the dynamic weight-bearing test, without affecting acute nociception. Probenecid reversal of mechanical allodynia was also observed in rats 13 weeks after anterior cruciate ligament transection, a model of posttraumatic osteoarthritis. Thus, Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.

Correction to: LyP-1 Conjugated Nanoparticles for Magnetic Resonance Imaging of Triple Negative Breast Cancer.

This article was updated to correct the spelling of B. Gino Fallone's name; it is correct as displayed above. Correction to: Mol Imaging Biol (2017). DOI: https://doi.org/10.1007/s11307-017-1140-4.

LyP-1 Conjugated Nanoparticles for Magnetic Resonance Imaging of Triple Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, or Her2/neu. Both diagnosis and treatment of TNBC remain a clinical challenge. LyP-1 is a cyclic 9 amino acid peptide that can bind to breast cancer cells. The goal of this study was to design and characterize LyP-1 conjugated to fluorescent iron oxide nanoparticles (LyP-1-Fe3O4-Cy5.5) as a contrast agent for improved and specific magnetic resonance imaging (MRI) in a preclinical model of TNBC. PROCEDURES: The binding of LyP-1-Fe3O4-Cy5.5 to MDA-MB-231 TNBC cells was evaluated and compared to scrambled peptide bio-conjugated to iron oxide nanoparticles (Ctlpep-Fe3O4-Cy5.5) as a negative control. Following the in vitro study, the MDA-MB-231 cells were injected into mammary glands of nude mice. Mice were divided into two groups: control group received Ctlpep- Fe3O4-Cy5.5 and LyP-1 group received LyP-1-Fe3O4-Cy5.5 (tail vein injection at 2 mg/kg of Fe3O4). Mice were imaged with an in vivo fluorescence imager and a 9.4 T MRI system at various time points after contrast agent injection. The T2 relaxation time was measured to observe accumulation of the contrast agent in breast tumor and muscle for both targeted and non-targeted contrast agents. RESULTS: Immunofluorescence revealed dense binding of the LyP-1-Fe3O4-Cy5.5 contrast agent to MDA-MB-231 cells; while little appreciable binding was observed to the scrambled negative control (Ctlpep-Fe3O4-Cy5.5). Optical imaging performed in tumor-bearing mice showed increased fluorescent signal in mammary gland of animals injected by LyP-1-Fe3O4-Cy5.5 but not Ctlpep- Fe3O4-Cy5.5. The results were confirmed ex vivo by the 2.6-fold increase of fluorescent signal from LyP-1-Fe3O4-Cy5.5 in extracted tumors when compared to the negative control. In MR imaging studies, there was a statistically significant (P < 0.01) difference in normalized T2 between healthy breast and tumor tissue at 1, 2, and 24 h post injection of the LyP-1-Fe3O4-Cy5.5. In animals injected with LyP-1-Fe3O4, distinct ring-like structures were observed with clear contrast between the tumor core and rim. CONCLUSION: The results demonstrate that LyP-1-Fe3O4 significantly improves MRI contrast of TNBC, hence has the potential to be exploited for the specific delivery of cancer therapeutics.

Enumeration and Localization of Mesenchymal Progenitor Cells and Macrophages in Synovium from Normal Individuals and Patients with Pre-Osteoarthritis or Clinically Diagnosed Osteoarthritis.

Osteoarthritis (OA) is a degenerative disorder characterized by chondrocyte apoptosis and degeneration of articular cartilage resulting in loss of mobility and pain. Inflammation plays a key role in the development and progression of OA both on the side of apoptosis and repair, while its exact role in pathogenesis has yet to be fully elucidated. Few studies have examined the cellular composition (inflammatory cells and/or progenitor cells) in the synovium of patients with pre-OA (asymptomatic with cartilage damage). Therefore, in the current study, mesenchymal progenitor cells (MPCs) and macrophages were enumerated within normal, pre-OA and OA synovium. No differences were observed between MPCs in normal vs. pre-OA, however, fewer macrophages were observed in pre-OA vs. normal synovium. Osteoarthritic synovium contained greater numbers of both MPCs and macrophages. Interestingly, the localization of MPCs and macrophages was affected by disease severity. In normal and pre-OA synovium, MPCs and macrophages co-localized, while in OA synovium, MPCs and macrophage populations were spatially distinct. Examining the cellular interactions between MPCs and macrophages in synovium may be essential for understanding the role of these cells in the onset and/or pathogenesis of the disease. This study has provided a first step by examining these cell types both spatially and temporally (e.g., disease severity). Further cellular and molecular studies will be needed to determine the functions of these cells in the context of disease and in relation to each other and the joint as a whole.

Topographical variations in zonal properties of canine tibial articular cartilage due to early osteoarthritis: a study using 7-T magnetic resonance imaging at microscopic resolution.

OBJECTIVE: Our aim was to determine topographical variations in zonal properties of articular cartilage over the medial tibia in an experimental osteoarthritis (OA) model using 7-T magnetic resonance imaging (MRI). MATERIALS AND METHODS: An anterior cruciate ligament (ACL)-transection canine model was subjected to study at 8 (six) and 12 (seven) weeks after the surgery. Each medial tibia was divided into five topographical locations. For each specimen, T2 relaxation (at 0° and 55°) was quantified at microscopic resolution. The imaging data grouped the five locations into two topographical areas (meniscus-covered and -uncovered). RESULTS: The T2 (55°) bulk values from the meniscus-covered area were significantly lower than those from the uncovered area. The total cartilage thicknesses on the meniscus-covered area were significantly thinner than those on the meniscus-uncovered area. Significant differences in the T2 (0°) values were observed in most thicknesses of the four subtissue zones and whole-tissue from the uncovered area, while the same significant changes were detected in the superficial zone from the meniscus-covered area. CONCLUSION: By quantifying high-resolution imaging data both topographically and depth-dependently (zonal-wise), this study demonstrates that the rate of disease progression varies topographically over the medial tibia. Future correlation with OA pathology could lead to better detection of early OA.

Effect of disulfide bonding and multimerization on proteoglycan 4's cartilage boundary lubricating ability and adsorption.

PURPOSE: The objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface. MATERIALS AND METHODS: PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi-), using size exclusion chromatography (SEC) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi- was compared at a physiological concentration (450 µg/mL) and assessed over a range of concentrations (45, 150, and 450 µg/mL). R/A and NR PRG4Multi- were evaluated at 450 µg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualize the adsorption of PRG4 preparations to the surface of articular cartilage explants. RESULTS: Separation into enriched populations of PRG4Multi+ and PRG4Multi- was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi- both functioned as effective friction-reducing cartilage boundary lubricants at 450 µg/mL, with PRG4Multi+ being more effective than PRG4Multi-. PRG4Multi+ lubricated in a dose-dependent manner, however, PRG4Multi- did not. R/A PRG4Multi- lubricated similar to NR PRG4Multi-. PRG4-containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi- appeared to have less intensity. CONCLUSIONS: These results demonstrate that the intermolecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure-lubrication function relationship will further contribute to the understanding of PRG4's role in diarthrodial joint homeostasis and disease.

Meniscus Induced Cartilaginous Damage and Non-linear Gross Anatomical Progression of Early-stage Osteoarthritis in a Canine Model.

BACKGROUND: The predictable outcome of the anterior cruciate ligament transection (ACLT) canine model, and the similarity to naturally occurring osteoarthritis (OA) in humans, provide a translatable method for studying OA. Still, evidence of direct meniscus-induced cartilaginous damage has not been identified, and gross-anatomical blinded scoring of early-stage OA has not been performed. OBJECTIVE: A gross anatomical observation and statistical analysis of OA progression to determine meniscus induced cartilaginous damage, to measure the macroscopic progression of OA, and to address matters involving arthroscopic and surgical procedures of the knee. METHOD: Unblinded assessment and blinded scoring of meniscal, tibial, femoral, and patellar damage were performed for control and at four time points following unilateral ACLT: 3-week (N=4), 8-week (N=4), 12-week (N=5), and 25-week (N=4). Mixed-model statistics illustrates damage (score) progression; Wilcoxon rank-sum tests compared time-point scores; and Wilcoxon signed-rank tests compared ACLT and contralateral scores, and meniscus and tibia scores. RESULT: Damage was manifest first on the posterior aspect of the medial meniscus and subsequently on the tibia and femur, implying meniscal damage can precede, coincide with, and aggravate cartilage damage. Damage extent varied chronologically and was dependent upon the joint component. Meniscal damage was evident at 3 weeks and progressed through 25-weeks. Meniscal loose bodies corresponded to tibial cartilage damage location and extent through 12 weeks, followed by cartilage repair activity after complete meniscal degeneration. CONCLUSION: This study provides additional information for understanding OA progression, identifying OA biomarkers, and arthroscopic and meniscectomy procedures.

Full-Length Recombinant Human Proteoglycan 4 Interacts with Hyaluronan to Provide Cartilage Boundary Lubrication.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid and at the surface of articular cartilage. The objectives of this study were to (1) assess the articular cartilage surface adsorption and in vitro cartilage boundary lubricating ability of full-length recombinant human PRG4 (rhPRG4), and (2) cartilage boundary lubricating ability of purified rhPRG4, both alone and in combination with hyaluronan (HA). rhPRG4 adsorption onto articular cartilage explants was assessed by immunohistochemistry and dot blot. An in vitro cartilage-cartilage friction test was used to assess rhPRG4's cartilage boundary lubricating ability compared to bovine PRG4, and that of purified rhPRG4 both alone and in combination with HA. rhPRG4 was able to adsorb to the articular surface, as well as the cut surface, of cartilage explants. The kinetic coefficient of friction of rhPRG4 was similar to that of PRG4 (p = 0.16) and lower than phosphate-buffered saline (p < 0.05), while that of purified rhPRG4 + HA was significantly lower than rhPRG4 alone (p < 0.05). This study demonstrates that rhPRG4 can adsorb to an intact articular cartilage surface and functions as an effective boundary lubricant, both alone and with HA, and provides the foundation for in vivo evaluation of this clinically relevant full-length rhPRG4 for treatment of osteoarthritis.