The combination of microfracture with induction of Wnt / ß- Catenin pathway, leads to enhanced cartilage regeneration.

INTRODUCTION: Microfracture does not lead to complete healing of full-thickness cartilage defects. The aim of this study was to evaluate the effect of modifying Wnt/ß-catenin signaling following microfracture, on the restoration of a full-thickness cartilage defect in a rabbit model. The modification of the canonical Wnt pathway was achieved through per os administration of lithium carbonate, which is an intracellular inhibitor of glycogen synthase kinase 3-ß (Gsk3-ß) and therefore induces Wnt/ß-catenin signaling. MATERIALS AND METHODS: Full-thickness cartilage defects of 4 mm in diameter were created in the patellar groove of the right femurs of 18 male New Zealand white rabbits. The rabbits were divided into three groups of six (n = 6) based on post-surgery treatment differences, as follows: microfracture only (group 1), microfracture plus lithium carbonate 7 mM in the drinking water for 1 week (group 2), microfracture plus lithium carbonate 7 mM in the drinking water for 4 weeks (group 3). All animals were sacrificed 9 weeks after surgery. The outcome was assessed histologically, by using the International Cartilage Repair Society (ICRS) visual histological scale. Immunohistochemistry for type II collagen was also conducted. RESULTS: Statistical analysis of the histological ICRS scores showed that group 3 was significantly superior to group 1 in four out of six ICRS categories, while group 2 was superior to 1 in only two out of six. CONCLUSION: The combination of microfracture and systematic administration of lithium carbonate 7 mM for 4 weeks shows statistically significant superiority in four out of six ICRS categories compared with microfracture only for the treatment of full-thickness cartilage defects in a rabbit experimental model.

Unique presentation of cricoid cartilage fracture causing intermittent dyspnea without preceding trauma.

Cricoid cartilage fracture is generally caused by significant neck trauma and causes continuous dyspnea, neck pain, or hoarseness developing immediately after the traumatic episode. A 69-year-old woman without any history of trauma was admitted to our hospital with intermittent dyspnea. Six months before admission she had started to complain of dyspnea occurring several times a month without warning, improving spontaneously within a few hours without treatment. Her primary care doctor diagnosed asthma and she was treated with inhaled short-acting beta agonists and glucocorticoids, without improvement. On initial evaluation at our hospital, the cause of dyspnea was unclear. Laryngoscopy was performed, which excluded vocal cord dysfunction. A further attack of dyspnea occurred on the fourth admission day. Stridor was evident during the attack, and bronchoscopy revealed subglottic narrowing of the trachea on both inspiration and expiration with no mass or foreign objects. Computed tomography (CT) of the neck revealed cricoid cartilage fracture causing airway narrowing and dyspnea. She was orally intubated, and tracheostomy was performed 2 weeks later to maintain her airway, which resolved her dyspnea. This patient's presentation was unique in two aspects. First, there was no history of trauma that may cause her cricoid cartilage fracture. Second, her symptoms of dyspnea were intermittent rather than continuous. These aspects led to suspicions of other diseases such as asthma or vocal cord dysfunction, thus delaying the diagnosis. Cricoid cartilage fracture should be considered in patients with dyspnea of unknown cause, irrespective of continuous or intermittent symptoms and preceding traumatic episodes.

Imaging of Folate Receptor Expressing Macrophages in the Rat Groove Model of Osteoarthritis: Using a New DOTA-Folate Conjugate.

Objective To evaluate the presence and localization of folate receptor expressing macrophages in the rat groove model of osteoarthritis and determine the suitability of a new folate conjugate with albumin-binding entity (cm09) for in vivo SPECT (single-photon emission computed tomography) analysis. Design In male Wistar rats, local cartilage damage was induced in addition to a standard ( n = 10) or high-fat diet ( n = 6). After 12 weeks, 111In labeled folate conjugates were administered, and SPECT/CT (computed tomography) imaging was performed after 24 hours. Subsequently, osteoarthritis severity and folate receptor expression were assessed using (immuno)-histological sections. Results In vivo SPECT/CT imaging of the new folate conjugate (cm09) was as useful as a folate conjugate without albumin-binding entity in the groove model of osteoarthritis with less renal accumulation. Induction of cartilage damage on a standard diet resulted in no effect on the amount of folate receptor expressing macrophages compared with the contralateral sham operated joints. In contrast, inducing cartilage damage in the high-fat diet group resulted in 28.4% increase of folate receptor expression as compared with the nondamaged control joints. Folate receptor expressing cells were predominantly present in the synovial lining and in subchondral bone as confirmed by immunohistochemistry. Conclusions Folate receptor expression, and thus macrophage activation, can clearly be demonstrated in vivo, in small animal models of osteoarthritis using the new 111In-folate conjugate with specific binding to the folate receptor. Increased macrophage activity only plays a role in the groove model of osteoarthritis when applied in a high-fat diet induced dysmetabolic condition, which is in line with the higher inflammatory state of that specific model.

Mitochondrial dysfunction is an acute response of articular chondrocytes to mechanical injury.

Mitochondrial (MT) dysfunction is known to occur in chondrocytes isolated from end-stage osteoarthritis (OA) patients, but the role of MT dysfunction in the initiation and early pathogenesis of post-traumatic OA (PTOA) remains unclear. The objective of this study was to investigate chondrocyte MT function immediately following mechanical injury in cartilage, and to determine if the response to injury differed between a weight bearing region (medial femoral condyle; MFC) and a non-weight bearing region (distal patellofemoral groove; PFG) of the same joint. Cartilage was harvested from the MFC and PFG of 10 neonatal bovids, and subjected to injurious compression at varying magnitudes (5-17 MPa, 5-34 GPa/s) using a rapid single-impact model. Chondrocyte MT respiratory function, MT membrane polarity, chondrocyte viability, and cell membrane damage were assessed in situ. Cartilage impact resulted in MT depolarization and impaired MT respiratory function within 2 h of injury. Cartilage from a non-weight bearing region of the joint (PFG) was more sensitive to impact-induced MT dysfunction and chondrocyte death than cartilage from a weight-bearing surface (MFC). Our findings suggest that MT dysfunction is an acute response of chondrocytes to cartilage injury, and that MT may play a key mechanobiological role in the initiation and early pathogenesis of PTOA. CLINICAL SIGNIFICANCE: Direct therapeutic targeting of MT function in the early post-injury time frame may provide a strategy to block perpetuation of tissue damage and prevent the development of PTOA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:739-750, 2018.

SIRT1 alleviates senescence of degenerative human intervertebral disc cartilage endo-plate cells via the p53/p21 pathway.

Cartilage end plates (CEP) degeneration plays an integral role in intervertebral disc (IVD) degeneration resulting from nutrient diffusion disorders. Although cell senescence resulting from oxidative stress is known to contribute to degeneration, no studies concerning the role of senescence in CEP degeneration have been conducted. SIRT1 is a longevity gene that plays a pivotal role in many cellular functions, including cell senescence. Therefore, the aim of this study was to investigate whether senescence is more prominent in human degenerative CEP and whether SIRT1-regulated CEP cells senescence in degenerative IVD as well as identify the signaling pathways that control that cell fate decision. In this study, the cell senescence phenotype was found to be more prominent in the CEP cells obtained from disc degenerative disease (DDD) patients than in the CEP cells obtained from age-matched lumbar vertebral fractures (LVF) patients. In addition, the results indicated that p53/p21 pathway plays an important role in the senescence of CEP cells in vivo and vitro. Furthermore, SIRT1 was found to be capable of alleviating the oxidative stress-induced senescence of CEP cells in humans via p53/p21 pathway. Thus, the information presented in this study could be used to further investigate the underlying mechanisms of CEP.

UHMWPE-based nanocomposite as a material for damaged cartilage replacement.

In the present work dispersion-strengthened nanocomposites based on ultra-high molecular weight polyethylene (UHMWPE) after mechanical activation were studied. Mechanical activation was performed for hardening of the boundaries between the polymer particles, reducing the fusion defects and increasing of wear-resistance. Three types of samples were prepared: UHMWPE, UHMWPE/Al2O3 nanocomposite and UHMWPE/Al2O3 nanocomposite after mechanical activation. UHMWPE/Al2O3 nanocomposites prepared with mechanical activation show the best mechanical properties in compression and higher wear-resistance. UHMWPE/Al2O3 nanocomposites prepared with mechanical activation were chosen for in vivo study by orthotopical transplantation in rats. Animals' activity has been being monitored for 60days after surgery. No signs of inflammation, cellular infiltration, destruction of material or bone-cartilage defect were found. Implanted sample has not changed its position of implantation, there were no any shifts. Obtained data shows that UHMWPE-based nanocomposite is a promising material for creating bioimplants for cartilage defect replacement.

Acute mechanical injury of the human intervertebral disc: link to degeneration and pain.

Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5% (non-injured) or 30% (injured) of disc height. 30% strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured-IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROα, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.

Effects of cartilage impact with and without fracture on chondrocyte viability and the release of inflammatory markers.

Post-traumatic arthritis (PTA) frequently develops after intra-articular fracture of weight bearing joints. Loss of cartilage viability and post-injury inflammation have both been implicated as possible contributing factors to PTA progression. To further investigate chondrocyte response to impact and fracture, we developed a blunt impact model applying 70%, 80%, or 90% surface-to-surface compressive strain with or without induction of an articular fracture in a cartilage explant model. Following mechanical loading, chondrocyte viability, and apoptosis were assessed. Culture media were evaluated for the release of double-stranded DNA (dsDNA) and immunostimulatory activity via nuclear factor kappa B (NF-κB) activity in Toll-like receptor (TLR) -expressing Ramos-Blue reporter cells. High compressive strains, with or without articular fracture, resulted in significantly reduced chondrocyte viability. Blunt impact at 70% strain induced a loss in viability over time through a combination of apoptosis and necrosis, whereas blunt impact above 80% strain caused predominantly necrosis. In the fracture model, a high level of primarily necrotic chondrocyte death occurred along the fracture edges. At sites away from the fracture, viability was not significantly different than controls. Interestingly, both dsDNA release and NF-κB activity in Ramos-Blue cells increased with blunt impact, but was only significantly increased in the media from fractured cores. This study indicates that the mechanism of trauma determines the type of chondrocyte death and the potential for post-injury inflammation.

Long-term results 8 years after autologous osteochondral transplantation: 7 T gagCEST and sodium magnetic resonance imaging with morphological and clinical correlation.

OBJECTIVE: To correlate long-term clinical outcome and the results of morphological as well as advanced biochemical magnetic resonance imaging (MRI) techniques [T2-mapping, glycosaminoglycan chemical exchange saturation transfer (gagCEST), sodium-23-imaging] in patients after autologous osteochondral transplantation (AOT) in knee joints. METHOD: Nine AOT patients (two female and seven male; median age, 49) had clinical [International Knee Documentation Committee (IKDC), modified Lysholm, visual analog scale (VAS)] and radiological long-term follow-up examinations at a median of 7.9 years (inter-quartile range, 7.7-8.2). Standard morphological MRI and T2-mapping of cartilage were performed on a 3 T MR unit. Biochemical imaging further included sodium-23-imaging and chemical exchange saturation transfer (CEST) imaging at 7 T. The Magnetic resonance Observation of CArtilage Repair Tissue (MOCART) score was used for quantitative assessment of morphological MRI. RESULTS: Clinical outcome was good with a median modified Lysholm score of 90. Median VAS revealed 1.0 and median MOCART score 75 points. The difference between native and repair cartilage was statistically significant for all three biochemical imaging techniques. The strongest correlation was found between the results of the advanced biochemical imaging methods sodium-23 and CEST [ρ = 0.952, 95% confidence interval (CI): (0.753; 0.992)]. Comparing the results from morphological and biochemical imaging, a correlation was found between MOCART score and CEST ratio [ρ = -0.749, 95% CI: (-0.944; -0.169)]. Comparing the results from clinical scores with MRI, a correlation between modified Lysholm and T2-mapping [ρ = -0.667, 95% CI: (-0.992; -0.005)] was observed. CONCLUSION: Long-term clinical outcome in patients 7.9 years after AOT was good, but did not correlate with morphological and biochemical imaging results except for T2-mapping.

Protective effect of P188 in the model of acute trauma to human ankle cartilage: the mechanism of action.

OBJECTIVE: Because P188 poloxamer is effective in promoting cell survival in models of acute trauma, the objectives were to understand the mechanism of its action focusing on glycogen synthase kinase-3 (GSK3) activation, interleukin-6 (IL-6), and p38 signaling. DESIGN: Sixteen normal human tali were impacted using a 4-mm diameter indenter with an impulse of 1 Ns. Eight-millimeter cartilage plugs containing the 4-mm impacted core and 4-mm adjacent nonimpacted ring were removed and cultured with or without P188. Cell lysates were analyzed using Western blots with antibodies against total and phosphorylated extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, ATF-2, GSK3, Stat1, and Stat3. Additional tests were performed with the p38 inhibitor (p38i) SB203580. RESULTS: Studied pathways were activated after impaction with the peak of activity at 1 hour. P188 completely attenuated phosphorylation of Stat1 and ATF-2 and inhibited p38, Stat3, JNK, ERK, and GSK3. The p38i partially offset phosphorylation of Stat3, GSK3, and ERK suggesting a role of p38 in these three pathways. Additionally, the p38i improved cell survival (P = 0.053) and reduced apoptosis (by approximately 20%, P = 0.046, versus almost 40% by P188), thus confirming that P188 acts (at least in part) through the p38 pathway. CONCLUSION: Our results report a novel mechanism through which P188 exerts its protective effects on cartilage in the model of acute injury. In addition to its effect on cellular membrane, P188 affects stress-related p38 signaling, apoptosis-related GSK3, and inflammation-related IL-6 signaling. Taken together, these findings suggest that P188 alone or in combination with proanabolic agents may have a therapeutic potential in preventing progressive cartilage degeneration and the development of posttraumatic osteoarthritis.

[Glycosaminoglycanes of cartilage tissue of rats after the mechanical trauma: the influence of low-molecular fraction from the cord blood and blood of dairy calves].

Influence of low-molecular fraction (to 5 kDa) from cord blood (FCB) compared with "Actovegin" derived from the blood of veal calves on the content of the matrix polysaccharide components from and proteins of the cartilage connecting tissue after the mechanical trauma was investigated. It is established, that intramuscular administration of FCB essentially stimulates the accumulation of the basic components of the matrix: hexosamine, uronic acid, hyaluronic acid, chondroitinsulfate and heparin, and also the most important components of structural proteins in the cartilage regenerate of the rat knee joint.

Longitudinal evaluation of cartilage composition of matrix-associated autologous chondrocyte transplants with 3-T delayed gadolinium-enhanced MRI of cartilage.

OBJECTIVE: The purposes of this study were to use delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) to evaluate the zonal distribution of glycosaminoglycans (GAGs) in normal cartilage and repair tissue and to use 3-T MRI to monitor the GAG content in matrix-associated autologous chondrocyte transplants. SUBJECTS AND METHODS: Fifteen patients who underwent matrix-associated autologous chondrocyte transplantation in the knee joint underwent MRI at baseline and 3-T follow-up MRI 1 year later. Total and zonal changes in longitudinal relaxivity (deltaR1) and relative deltaR1 were calculated for repair tissue and normal hyaline cartilage and compared by use of analysis of variance. RESULTS: There was a significant difference between the mean deltaR1 of repair tissue and that of reference cartilage at baseline and follow-up (p < 0.001). There was a significant increase in deltaR1 value and a decrease in GAG content from the deep layer to the superficial layer in the reference cartilage and almost no variation and significantly higher values for the repair tissue at both examinations. At 1-year follow-up imaging, there was a 22.7% decrease in deltaR1 value in the deep zone of the transplant. CONCLUSION: T1 mapping with dGEMRIC at 3 T shows the zonal structure of normal hyaline cartilage, highly reduced zonal variations in repair tissue, and a tendency toward an increase in global and zonal GAG content 1 year after transplantation.

Influence of exogenous hyaluronan on synthesis of hyaluronan and collagenase by equine synoviocytes.

OBJECTIVE: To evaluate the influence of exogenous hyaluronan (HA) on in vitro synthesis of HA and collagenase by equine synoviocytes from normal and inflamed joints. ANIMALS: 9 adult horses. PROCEDURE: Synoviocytes for culture were taken from the middle carpal joint of 3 horses with normal joints (control) and 6 horses with osteochondral fractures (principal). Synoviocytes were propagated in monolayer cultures and were incubated with 3 commercial HA products at concentrations of 0, 200, 400, and 1,500 micrograms/ml. Newly synthesized HA was radiolabeled with [3H]glucosamine and quantified by cetylpyridinium chloride precipitation and liquid scintillation counting. The hydrodynamic size of radioactive HA was determined by high-performance liquid chromatography, and collagenase activity was evaluated by use of a quantitative radioactive collagen film assay. RESULTS: Exogenous HA influenced neither the rate of synthesis nor the hydrodynamic size of the newly produced HA by control or principal cell cultures. Culture supernatants from abnormal synovium, exposed to 400 and 1,500 micrograms of exogenous HA/ml, contained significantly more collagenase activity than did those exposed to lower concentrations. CONCLUSION: Although HA is thought to have beneficial effects in equine arthropathies, the principal mechanisms of action of HA do not appear to be stimulation of synthesis of HA of augmented molecular weight or marked inhibition of collagenase synthesis.