Healthy Cartilage Research Articles

Males and Females Have Distinct Molecular Events in the Articular Cartilage during Knee Osteoarthritis.

Osteoarthritis (OA) is a major public health challenge that imposes a remarkable burden on the affected individuals and the healthcare system. Based on the clinical observation, males and females have different prevalence rates and severity levels of OA. Thus, sex-based differences may play essential roles in OA’s prognosis and treatment outcomes. To date, the comprehensive understanding of the relationship between sex and OA is still largely lacking. In the current study, we analyzed a published transcriptome dataset of knee articular cartilage (GSE114007) from 18 healthy (five females, 13 males) and 20 OA (11 females, nine males) donors to provide a slight insight into this important but complex issue. First, comparing female healthy cartilage samples with those of males revealed 36 differential expression genes (DEGs), indicating the fundamental sex-related differences at the molecular level. Meanwhile, 923 DEGs were distinguished between OA and healthy female cartilage, which can be enriched to 15 Reactome pathways. On the other hand, when comparing OA and healthy male cartilage, there are only 419 DEGs were identified, and only six pathways were enriched against the Reactome database. The different signaling response to OA in the male and female cartilage was further enforced by recognizing 50 genes with significantly different OA-responsive expression fold changes in males and females. Particularly, 14 Reactome pathways, such as “Extracellular matrix organization”, “Collagen biosynthesis and modifying enzymes”, “Dissolution of fibrin clot”, and “Platelet Aggregation (Plug formation)”, can be noted from these 50 sex-dependent OA-responsive genes. Overall, the current study explores the Sex as a Biological Variable (SABV) at the transcriptomic level in the knee articular cartilage in both healthy status and OA event, which could help predict the differential OA prognosis and treatment outcome of males and female patients.

Differentiation of Cartilage Repair Techniques Using Texture Analysis from T2 Maps.

ObjectiveThe aim of this study was to investigate texture features from T2 maps as a marker for distinguishing the maturation of repair tissue after 2 different cartilage repair procedures.DesignSeventy-nine patients, after either microfracture (MFX) or matrix-associated chondrocyte transplantation (MACT), were examined on a 3-T magnetic resonance (MR) scanner with morphological and quantitative (T2 mapping) MR sequences 2 years after surgery. Twenty-one texture features from a gray-level co-occurrence matrix (GLCM) were extracted. The texture feature difference between 2 repair types was assessed individually for the femoral condyle and trochlea/anterior condyle using linear regression models. The stability and reproducibility of texture features for focal cartilage were calculated using intra-observer variability and area under curve from receiver operating characteristics.ResultsThere was no statistical significance found between MFX and MACT for T2 values (P = 0.96). There was, however, found a statistical significance between MFX and MACT in femoral condyle in GLCM features autocorrelation (P < 0.001), sum of squares (P = 0.023), sum average (P = 0.005), sum variance (P = 0.0048), and sum entropy (P = 0.05); and in anterior condyle/trochlea homogeneity (P = 0.02) and dissimilarity (P < 0.001).ConclusionTexture analysis using GLCM provides a useful extension to T2 mapping for the characterization of cartilage repair tissue by increasing its sensitivity to tissue structure. Some texture features were able to distinguish between repair tissue after different cartilage repair procedures, as repair tissue texture (and hence, probably collagen organization) 24 months after MACT more closely resembled healthy cartilage than did MFX repair tissue.

Injectable polymeric nanoparticle hydrogel system for long-term anti-inflammatory effect to treat osteoarthritis

Treatment of osteoarthritis (OA) by administration of corticosteroids is a commonly used method in clinics using anti-inflammatory medicine. Oral administration or intra-articular injection of corticosteroids can reduce the pain and progress of cartilage degeneration, but they are usually insufficient to show local and long-term anti-inflammatory effects because of their fast clearance in the body. In this study, we suggest an injectable anti-OA drug depot system for sustained drug release that provides long-term effective therapeutic advantages. Amphiphilic poly(organophosphazene), which has temperature-dependent nanoparticle forming and sol-gel transition behaviors when dissolved in aqueous solution, was synthesized for triamcinolone acetonide (TCA) delivery. Because hydrophobic parts of the polymer can interact with hydrophobic parts of the TCA, the TCA was encapsulated into the self-assembled polymeric nanoparticles. The TCA-encapsulated polymeric nanoparticles (TePNs) were well dispersed in an aqueous solution below room temperature so that they can be easily injected as a sol state into an intra-articular region. However, the TePNs solution transforms immediately to a viscose 3D hydrogel like a synovial fluid in the intra-articular region via the conducted body temperature. An in vitro TCA release study showed sustained TCA release for six weeks. One-time injection of the TePN hydrogel system in an early stage of OA-induced rat model showed a great inhibition effect against further OA progression. The OA-induced knees completely recovered as a healthy cartilage without any abnormal symptoms.

Targeted cell therapy for partial-thickness cartilage defects using membrane modified mesenchymal stem cells by transglutaminase 2

Unlike full-thickness cartilage defects (FCD), partial-thickness cartilage defects (PCD) may still have residual healthy cartilage tissue, and therefore, the conventional clinical treatments such as microfracture and autologous chondrocyte implantation (ACI) are so traumatic that they may not be the suitable therapies for PCD. Although intra-articular injection of mesenchymal stem cells (MSCs) is a minimally invasive treatment, its therapeutic efficacy is markedly limited due to anoikis caused by failure of cell colonization in the injured area. By modifying a functional polypeptide on the MSC plasma membrane and exploiting the high expression of transglutaminase 2 (TGase2) in the regions of injured cartilage, we achieved targeted recognition and capture of modified MSCs by injured articular chondrocytes (ACs). In the in vitro co-culture model, MSCs improved the function of injured ACs and enhanced the chondrogenic differentiation potential of MSCs. Results of in vitro study also revealed that the activation of the AKT/mTOR signaling pathway may play an important role in the treatment of injured ACs by MSCs. Further, membrane-modified MSCs exhibited a better therapeutic effect than wide-type MSCs in a rabbit model of PCD. Thus, this unique cell membrane modification strategy provides a new cell-based therapeutic approach for the early treatment of articular cartilage defects and other joint diseases.

Bevacizumab Arrests Osteoarthritis Progression in a Rabbit Model: A Dose-Escalation Study.

Cartilage neoangiogenesis holds a prominent role in osteoarthritis (OA) pathogenesis. This study aimed to assess the efficacy bevacizumab, an antibody against vascular endothelial growth factor and inhibitor of angiogenesis, in a rabbit OA model. Animals were divided into four groups: one receiving a sham intra-articular knee injection and three groups undergoing 5, 10, and 20 mg intra-articular bevacizumab injections. The effect of the antibody on articular cartilage and synovium was assessed through histology and quantified with the Osteoarthritis Research Society International (OARSI) scores. Immunohistochemistry was performed to investigate type 2 collagen, aggrecan, and matrix metalloproteinase 13 (MMP-13) expression. Bevacizumab treatment led to a significant reduction of cartilage degeneration and synovial OA changes. Immunohistochemistry revealed significantly lower cartilage MMP-13 expression levels in all experimental groups, with the one receiving 20 mg bevacizumab showing the lowest. The antibody also resulted in increased production of aggrecan and type 2 collagen after administration of 5, 10, and 20 mg. The group treated with 20 mg showed the highest levels of type 2 collagen, while aggrecan content was even higher than in the healthy cartilage. Intra-articular bevacizumab has been demonstrated to effectively arrest OA progression in our model, with 20 mg being the most efficacious dose.

Primary Osteoarthritis Early Joint Degeneration Induced by Endoplasmic Reticulum Stress Is Mitigated by Resveratrol

Increasing numbers of people are living with osteoarthritis (OA) due to aging and obesity, creating an urgent need for effective treatment and preventions. Two top risk factors for OA, age and obesity, are associated with endoplasmic reticulum (ER) stress. The I-ERS mouse, an ER stress-driven model of primary OA, was developed to study the role of ER stress in primary OA susceptibility. The I-ERS mouse has the unique ability to induce ER stress in healthy adult articular chondrocytes and cartilage, driving joint degeneration that mimics early primary OA. In this study, ER stress-induced damage occurred gradually and stimulated joint degeneration with OA characteristics including increased matrix metalloproteinase activity, inflammation, senescence, chondrocyte death, decreased proteoglycans, autophagy block, and gait dysfunction. Consistent with human OA, intense exercise hastened and increased the level of ER stress-induced joint damage. Notably, loss of a critical ER stress response protein (CHOP) largely ameliorated ER stress-stimulated OA outcomes including preserving proteoglycan content, reducing inflammation, and relieving autophagy block. Resveratrol diminished ER stress-induced joint degeneration by decreasing CHOP, TNFα, IL-1β, MMP-13, pS6, number of TUNEL-positive chondrocytes, and senescence marker p16 INK4a. The finding, that a dietary supplement can prevent ER stressed-induced joint degeneration in mice, provides a preclinical foundation to potentially develop a prevention strategy for those at high risk to develop OA.

Automatic knee cartilage and bone segmentation using multi-stage convolutional neural networks: data from the osteoarthritis initiative.

Accurate and efficient knee cartilage and bone segmentation are necessary for basic science, clinical trial, and clinical applications. This work tested a multi-stage convolutional neural network framework for MRI image segmentation. Stage 1 of the framework coarsely segments images outputting probabilities of each voxel belonging to the classes of interest: 4 cartilage tissues, 3 bones, 1 background. Stage 2 segments overlapping sub-volumes that include Stage 1 probability maps concatenated to raw image data. Using sixfold cross-validation, this framework was tested on two datasets comprising 176 images [88 individuals in the Osteoarthritis Initiative (OAI)] and 60 images (15 healthy young men), respectively. On the OAI segmentation dataset, the framework produces cartilage segmentation accuracies (Dice similarity coefficient) of 0.907 (femoral), 0.876 (medial tibial), 0.913 (lateral tibial), and 0.840 (patellar). Healthy cartilage accuracies are excellent (femoral = 0.938, medial tibial = 0.911, lateral tibial = 0.930, patellar = 0.955). Average surface distances are less than in-plane resolution. Segmentations take 91 ± 11s per knee. The framework learns to automatically segment knee cartilage tissues and bones from MR images acquired with two sequences, producing efficient, accurate quantifications at varying disease severities.

Intra-operative Raman spectroscopy and ex vivo Raman mapping for assessment of cartilage degradation

The development of a label-free, non-destructive and safe analytical method such as Raman spectroscopy for assessing cartilage degradation is highly desirable. Compared to non-optical imaging modalities, Raman mapping offers a more sensitive means of directly assessing the chemical composition of cartilage in three-dimensional space and the potential to monitor cartilage degeneration to inform intervention and treatment strategies. Herein, we report the application of Raman spectroscopic methods ex vivo and at arthroscopy to identify molecular alterations in cartilage specimens containing minor focal lesions characteristic of the early disease phase. Our initial ex vivo analysis, obtained by single-point Raman spectroscopy of cartilage samples, supports previous findings based on S-O stretching vibration bands associated with sulphated glycosaminoglycans (sGAGs). We extended the analyses to the high-wavenumber region where we observed that vibrational bands assigned to C-H and O-H stretching modes discriminated early cartilage alterations from healthy cartilage samples. Furthermore, we performed a proof-of-concept in-clinic study using a custom-built optical probe to acquire Raman spectral measurements for the first time in patients undergoing arthroscopy of knee joints. Spectra were obtained with adequate signal-to-noise ratios that similarly discriminated between lesion and adjacent cartilage sites and identified reductions in sGAGs in apparently healthy cartilage. Building on this, we present initial results from Raman mapping to spatially resolve the molecular constituents of cartilage through its depth and across a lesion. Mapping revealed a non-uniform and reduced sGAG distribution within the lesion and peripheral cartilage that was otherwise visually normal, similar to the in-clinic observations, showing that the degradative influence of the lesion extended beyond its border. This was accompanied by a decreased fluorescence signal intensity, which suggests that fluorescence may provide valuable information as an adjunct to the Raman signal in discriminating normal and degenerating cartilage. This work demonstrates the value of Raman mapping over single-point Raman measurements for the analysis of the anisotropy of articular cartilage and highlights the potential of the technology for in vivo articular joint arthroscopy applications.

Maturation process of regenerated tissues after single-stage simultaneous autologous particulated cartilage implantation and open wedge high tibial osteotomy for articular cartilage defects with medial osteoarthritis of bilateral knees: a case report

BackgroundOpen wedge high tibial osteotomy (OWHTO) is an effective treatment option for young and middle-aged active patients with medial unicompartmental knee osteoarthritis (OA). In addition, particulated cartilage implantation has been developed as a simple procedure for cartilage regeneration. Thus, to improve the OWHTO outcomes, a single-stage, simultaneous bilateral knee arthroscopic particulated cartilage implantation with OWHTO was performed.Case presentationA 60-year-old male patient presented with severe bilateral knee pain, with grade 2 varus knee OA of the Kellgren–Lawrence classification. Primary arthroscopic evaluations based on the International Cartilage Repair Society grading system showed grade 3c articular cartilage defects of 1.5 cm in diameter at the center of the bilateral medial femoral condyles. Following bilateral OWHTO, the healthy cartilage tissue was harvested from the lateral wall of the unilateral femoral intercondylar notch and minced with the cartilage processor. Then, subchondral drillings and cartilage fragment implantations into the bilateral defects were performed arthroscopically. One year postsurgery, second-look arthroscopy findings revealed that the defects were filled with cartilage-like tissues. The maturation process of the regenerated tissues was confirmed with T2 mapping magnetic resonance imaging during the 3-year follow-up period. The patient could walk without a cane, and all Knee Injury and Osteoarthritis Outcome Score parameters were improved without any correction loss in 3 years.ConclusionsThis is the first report to evaluate the maturation process of the implanted particulated cartilage tissue with T2 mapping magnetic resonance imaging for 3 years. The effect of chondral resurfacing procedure with OWHTO remains unclear; however, the implantation of arthroscopic particulated cartilage fragments is a single-stage and less-invasive procedure. This treatment could regenerate cartilage-like tissue in the present case. Therefore, this additional procedure could potentially improve the long-term outcomes of OWHTO.

Quantitative MRI T2 Mapping is Able to Assess Tissue Quality After Reparative and Regenerative Treatments of Osteochondral Lesions of the Talus.

BackgroundQuantitative MRI has potential for tissue characterization after reparative and regenerative surgical treatment of osteochondral lesions of the talus (OCLTs). However available data is inconclusive and quantitative sequences can be difficult to implement in real‐time clinical application.PurposeTo assess the potential of T2 mapping in discriminating articular tissue characteristics after reparative and regenerative surgery of OCLTs in real‐world clinical settings.Study TypeObservational and prospective cohort study.Population15 OCLT patients who had received either reparative treatment with arthroscopic microfracture surgery (MFS) for a grade I lesion or regenerative treatment with bone marrow derived cell transplantation (BMDCT) for a grade II lesion.Field Strength/Sequence1.5 T, proton density weighted TSE, T2‐weighted true fast imaging with steady‐state‐free precession and multi‐echo T2 mapping sequences.AssessmentPatients were evaluated at a minimum postoperative follow‐up of 24 months. T2 maps of the ankle were generated and the distribution of T2 values was analyzed in manually identified volumes of interest (VOIs) for both treated lesions (TX) and healthy cartilage (CTRL). The amount of fibrocartilage, hyaline‐like and remodeling tissue in TX VOIs was obtained, based on T2 thresholds from CTRL VOIs.Statistical TestsFisher's exact test for categorical data, nonparametric Mann–Whitney U test for continuous data. The statistical significance level was P < 0.05.ResultsFrom CTRL VOI analysis, T2 < 25 msec, 25 msec ≤ T2 ≤ 45 msec, and T2 > 45 msec were considered as representative for fibrocartilage, hyaline‐like and remodeling tissue, respectively. Tissue composition of the two treatment groups was different, with significantly more fibrocartilage (+28%) and less hyaline‐like tissue (−15%) in MFS than in BMDCT treated lesions. No difference in healthy tissue composition was found between the two groups (P = 0.75).Data ConclusionsT2 mapping of surgically treated OCLTs can provide quantitative information about the type and amount of newly formed tissue at the lesion site, thereby facilitating surgical follow‐up in a real‐word clinical setting.Level of Evidence2Technical EfficacyStage 3

Chondrocyte morphology as an indicator of collagen network integrity

ABSTRACT Osteochondral allograft (OCA) transplantation offers an attractive treatment option as it can be used to repair large cartilage defects that otherwise would not heal. The currently accepted criterion for OCA selection for joint reconstruction is the percentage of viable chondrocytes, but this criterion alone may not be sufficient to ensure structural integrity and functional performance of allografts following transplantation. We sought to determine an additional parameter that indicates matrix integrity. We used multi-photon microscopy to quantitatively assess chondrocyte viability, chondrocyte shape, and collagen structure of articular cartilage of OCAs. Chondrocyte shape varied considerably in otherwise macroscopically healthy-looking OCAs with good (>90%) cell viability. Shape varied from the expected ellipsoidal form found in healthy cartilage, to excessively elongated and flattened cells that often contained multiple cytoplasmic processes reminiscent of those observed in fibroblasts. Chondrocytes with abnormal morphology were associated with degradation of their pericellular matrix and disruption of the collagen fiber orientation, reflected by an increase in heterogeneity of second harmonic signal intensity. Cell shape may be an important marker for collagen network integrity in articular cartilage in general and OCAs specifically. We propose that, aside from cell viability, cell shape may be used as an additional criterion measure for the selection of OCAs. OCAs selected for transplantation based on these criteria showed good graft-host integration post-operation. In view of the rapid and nondestructive nature of the current approach, it may be suitable for clinical application in the future.

POS0376 MONOMERIC C REACTIVE PROTEIN (mCRP) REGULATES INFLAMMATORY RESPONSES IN HUMAN AND MOUSE CHONDROCYTES

Background:C-reactive protein (CRP) is an acute-phase protein that is used as an established biomarker to follow disease severity and progression in a plethora of inflammatory diseases. However, its pathophysiologic mechanisms of action are still poorly defined and remain elusive. CRP, in its pentameric form, exhibits weak anti-inflammatory activity. On the contrary, the monomeric isoform (mCRP) exhibits potent pro-inflammatory properties in endothelial cells, leukocytes, and platelets. So far, no data exists regarding mCRP effects in human or mouse chondrocytesObjectives:This work aimed to verify the pathophysiological relevance of mCRP in the etiology and/or progression of osteoarthritis (OA)Methods:We investigated the effects of mCRP in cultured human primary chondrocytes and in the chondrogenic ATDC5 mouse cell line. We determined mRNA and protein levels of relevant factors involved in inflammatory responses and the modulation of nitric oxide synthase type II (NOS2), an early inflammatory molecular target.Results:We demonstrate, for the first time, that monomeric C reactive protein increases NOS2, COX2, MMP13, VCAM1, IL-6, IL-8, and LCN2 expression in human and murine chondrocytes. We also demonstrated that NF-kB is a key factor in the intracellular signaling of mCRP-driven induction of pro-inflammatory and catabolic mediators in chondrocytes.Conclusion:mCRP exerts a sustained catabolic effect on human and murine chondrocytes, increasing the expression of inflammatory mediators and proteolytic enzymes, which can promote extracellular matrix (ECM) breakdown in healthy and OA cartilage. In addition, our results implicate the NF-kB signaling pathway in catabolic effects mediated by mCRP.

Nanoghosts: Mesenchymal Stem cells derived nanoparticles as a unique approach for cartilage regeneration

Osteoarthritis (OA) is a chronic degenerative disease, which affects the joints and is characterized by inflammation, cartilage loss and bone changes. Nowadays, there are no treatments for OA, and current therapies are focused on relieving the symptoms. As a new therapy approach, micro and nanoparticles have been extensively explored and among all the studied particles, the use of cell-membrane-based particles is expanding. Another promising approach studied to treat OA, is the use of mesenchymal stem cells (MSCs) which play an important role modulating inflammation. We developed a novel kind of MSCs' cytoplasmic-membrane-based nanoparticles, termed nano-ghosts (NGs).Retaining MSCs' surface properties and lacking cells' internal machinery allow the NGs to have immunomodulatory capacity and to be immune-evasive while not susceptible to host-induced changes.In this study, we demonstrate NGs' ability to target cartilage tissues, in vitro and in vivo, while modulating the inflammatory process. In vivo studies demonstrated NGs ability to act as an immunomodulatory drug slowing down cartilage degeneration process.Our proof-of-concept experiments show that NGs system is a versatile nano-carrier system, capable of therapeutics loading, with targeting capabilities towards healthy and inflamed cartilage cells. Our results, along with previously published data, clearly reveal the NGs system as a promising nano-carrier platform and as a potential immunomodulatory drug for several inflammation-related diseases.

Investigation of the Effects of Intra-articular Tranexamic Acid on Intact Cartilage Tissue and Cartilage Formation in Osteochondral Defects of the Rabbit Knee: An Experimental Study.

BackgroundIn this study, the effects of tranexamic acid (TXA) and saline on intact cartilage and the recovery of experimental osteochondral lesions following microfracture in a rabbit model were compared.MethodsTwenty adult rabbits were divided into four groups (1A, 1B, 2A, and 2B) based on with or without TXA use and microfracture. In addition, these groups were categorized into two different subgroups based on the use of TXA in Groups 1 and 2 (Groups A and B). Full-thickness cartilage defects were created on the weight-bearing surface of the medial femoral condyles unilaterally in Group 2 for the effect of TXA or saline on healthy cartilage tissue while a repetitive injection was applied in Group 1 for the effect of TXA or saline on intact cartilage. A single dose of 10 mg/kg TXA was injected into the knee joints of Group A and 10 mg/kg 0.9% saline solution injected in Group B for three consecutive days. All animals were sacrificed for the extraction of the medial condyles for histologic evaluation eight weeks after surgery. The International Cartilage Repair Society (ICRS) II scoring system was used for histologic evaluation.ResultsNo complications or adverse effects related to surgery were observed in all rabbits. All ICRS II parameters were similar in the TXA and saline solution groups in the intact cartilage group except for chondrocyte clustering, formation of a tidemark, subchondral bone abnormalities, and mid/deep zone assessment. Moreover, these parameters were higher in the saline solution group in the cartilage group, but no significant difference was observed in the TXA group in the intact cartilage group. All ICRS II parameters were higher in the saline solution group than in the TXA group in the microfracture group, but no significant difference was observed in the TXA group in the microfracture group except for inflammation, which was similar in the TXA and saline solution groups in the microfracture group.ConclusionWe found that intra-articular TXA administration did not have a negative impact on healthy cartilage tissue and cartilage transformation and proliferation as compared to the saline infusion.

Chondrocytes From Osteoarthritic and Chondrocalcinosis Cartilage Represent Different Phenotypes.

Basic calcium phosphate (BCP)-based calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease and hypertrophic differentiation of chondrocytes. Chondrocalcinosis (CC) is associated with calcium pyrophosphate dihydrate (CPPD) deposition disease in the joint inducing OA-like symptoms. There is only little knowledge about the effect of CPPD crystals on chondrocytes and the signaling pathways involved in their generation. The aim of this study was to investigate the chondrocyte phenotype in CC cartilage and the effect of CPPD crystals on chondrocytes. Cartilage samples of patients with CC, patients with severe OA, and healthy donors were included in this study. The presence of CC was evaluated using standard X-ray pictures, as well as von Kossa staining of cartilage sections. OA severity was evaluated using the Chambers Score on cartilage sections, as well as the radiological Kellgren–Lawrence Score. Patients with radiologically detectable CC presented calcification mainly on the cartilage surface, whereas OA patients showed calcification mainly in the pericellular matrix of hypertrophic chondrocytes. OA cartilage exhibited increased levels of collagen X and matrix metalloproteinase 13 (MMP13) compared with CC and healthy cartilage. This observation was confirmed by qRT-PCR using cartilage samples. No relevant influence of CPPD crystals on hypertrophic marker genes was observed in vitro, whereas BCP crystals significantly induced hypertrophic differentiation of chondrocytes. Interestingly, we observed an increased expression of p16 and p21 in cartilage samples of CC patients compared with OA patients and healthy controls, indicating cellular senescence. To investigate whether CPPD crystals were sufficient to induce senescence, we incubated chondrocytes with BCP and CPPD crystals and quantified senescence using β-gal staining. No significant difference was observed for the staining, but an increase of p16 expression was observed after 10 days of culture. Primary chondrocytes from CC patients produced CPPD crystals in culture. This phenotype was stabilized by mitomycin C-induced senescence. Healthy and OA chondrocytes did not exhibit this phenotype. BCP and CPPD crystals seem to be associated with two different chondrocyte phenotypes. Whereas BCP deposition is associated with chondrocyte hypertrophy, CPPD deposition is associated with cellular senescence.

Resilience to height loss of articular cartilage of osteoarthritic stifle joints of old pigs, compared with healthy cartilage from young pigs in a tribological pin-on-plate exposure, revealing similar friction forces.

IntroductionWe saw a lack of data on the biomechanical behavior of degenerated articular cartilage (OA) compared with that of healthy cartilage, even though the susceptibility to wear and tear of articular cartilage plays a key role in the progression of osteoarthritis (OA). Therefore, we performed a comparison between naturally occurring OA and healthy cartilage from pigs, before and after tribological stress.AimThe aim of the study was to compare OA-cartilage with healthy cartilage and to analyze the resilience to tribological shear stress, which will be measured as height loss (HL), and to friction forces of the cartilage layers. The findings will be substantiated in macro- and microscopical evaluations before and after tribological exposure.MethodsWe assessed stifle joints of fifteen old and sixteen young pigs from the local abattoir radiologically, macroscopically and histologically to determine possible OA alterations. We put pins from the femoral part of the joints and plates from the corresponding tibial plateaus in a pin-on-plate tribometer under stress for about two hours with about 1108 reciprocating cycles under a pressure of approximately 1 MPa. As a surrogate criterion of wear and tear, the HL was recorded in the tribometer. The heights of the cartilage layers measured before and after the tribological exposure were compared histologically. The condition of the cartilage before and after the tribological exposure was analyzed both macroscopically with an adapted ICRS score and microscopically according to Little et al. (2010). We assessed the friction forces acting between the surfaces of the cartilage pair–specimens.ResultsArticular cartilage taken from old pigs showed significant degenerative changes compared to that taken from the young animals. The macroscopic and microscopic scores showed strong alterations of the cartilage after the tribological exposure. There was a noticeable HL of the cartilage specimens after the first 100 to 300 cycles. The HL after tribological exposure was lower in the group of the old animals with 0.52 mm ± 0.23 mm than in the group of the young animals with 0.86 mm ± 0.26 mm (p < 0.0001). The data for the HL was validated by the histological height measurements with 0.50 mm ± 0.82 mm for the old and 0.79 mm ±0.53 mm for the young animals (p = 0.133). The friction forces measured at the cartilage of the old animals were 2.25 N ± 1.15 N and 1.89 N ± 1.45 N of the young animals (p = 0.3225).ConclusionUnlike articular cartilage from young pigs, articular cartilage from old pigs showed OA alterations. Tribological shear stress exposure revealed that OA cartilage showed less HL than healthy articular cartilage. Tribological stress exposure in a pin–on–plate tribometer seemed to be an appropriate way to analyze the mechanical stability of articular cartilage, and the applied protocol could reveal weaknesses of the assessed cartilage tissue. Friction and HL seemed to be independent parameters when degenerated and healthy articular cartilage were assessed under tribological exposure in a pin–on- plate tribometer.

Evaluation of Three Growth Factors with the Most Controversial Expression Pattern in Osteoarthritic and Non-osteoarthritic Cartilage

There is no clear consensus regarding the expression pattern of many growth factors in healthy and osteoarthritic cartilage. In this study, the expression pattern of three controversial cartilage growth factors was compared between osteoarthritic (n=27) ‎and non-osteoarthritic cartilage samples (n=14). The candidate growth factor included Insulin-like ‎growth factor 1 (IGF-1), Fibroblast Growth Factor 2 (FGF-2), and Bone Morphogenetic Protein 2 (BMP-2). The candidate markers' gene and protein expression were analyzed using relative quantification relative to real-time PCR and ELISA, respectively. The mean IGF-I ‎mRNA and protein expression were 11.1, and 2 folds greater in osteoarthritic cartilage, respectively (p&lt;0.001 for both). The mean FGF-2 ‎mRNA and protein expression were 6.7 and 1.3 folds greater in osteoarthritic cartilage, respectively (p&lt;0.001 and p=0.003). The mean BMP-2 ‎mRNA and protein expression were 3.48 and 1.51 folds greater in osteoarthritic cartilage, respectively (p=0.001 and p&lt;0.001). A significant positive correlation was found between the mRNA and protein expression of all candidate growth factors. In conclusion, the gene and protein expression of IGF-I, FGF-2, and BMP-2 were significantly higher in the ‎osteoarthritic cartilages when compared with non-osteoarthritic, suggesting them as‎‏ a therapeutic ‏target in OA treatment.

Optimization of knee cartilage texture analysis of quantitative MRI T2 maps

Optimization of knee cartilage texture analysis of quantitative MRI T2 maps

Articular cartilage regeneration in rats using human bone marrow stromal cells

Purpose: Injury to articular cartilage found in knee joints is often disabling and affects both younger and older individuals. Injured cartilage rarely heals, leading to a progressive loss of normal function of the joint in many cases. Current treatments are far from satisfactory. One treatment is surgery (such as drilling through the cartilage to the bone, abrasion to remove diseased cartilage and microfracture). These procedures can result in the formation of a mixture of fibrous tissue and cartilage within the injury, which does not lead to a total recovery of joint function. Autologous human bone marrow stromal cell (hBMSC) transplantation maybe a potential avenue to regenerate articular cartilage. We previously determined that naive (untreated) hBMSC were able to form stable, non-hypertrophic cartilage when transplanted subcutaneously in conjunction with fibrin microbeads covalently coated with hyaluronic acid (FMBs). In our current study, we are investigating the ability of hBMSC/FMB constructs to generate native-like cartilage in an articular defect in immunocompromised rodents. Methods: hBMSCs were obtained from surgical bone waste and grown in culture for 2-3 passages. Approval for this study was given by the institutional Animal Care and Use Committee (ACUC). To create the articular cartilage defects in immunocompromised SRG rats (Hera/Charles River), the patella was laterally displaced and the distal femur was immobilized using a clamp. The defect was made in the trochlear groove of the distal femur using a 1.8mm diameter microdrill. The defect was either left unrepaired (sham), or subsequently filled with either FMBs alone (FMBs Alone), hBMSCs attached to FMBs (FMBs+cells), or with a pellet created by incubating BMSCs with FMBs for 10 days in chondrogenic medium. At the 4-week and 8-week timepoints following surgery, the rats were euthanized and femurs were harvested and fixed in 4% paraformaldehyde for 24 hours and then decalcified using a 10% EDTA solution for 30 days. After decalcification, the femurs were paraffin embedded and sectioned at a thickness of 6 μm. Histological staining was performed using hematoxylin and eosin, toluidine blue, and safranin O methods to assess cartilage formation. Immunohistochemistry for aggrecan within the cartilage was also performed to assess cartilage proteoglycan formation. Image analysis using Image J was performed on sections stained with safranin O. Cartilage thickness was approximated based on the thickness of the intact cartilage surrounding the defect, and a region of interest (ROI) was drawn surrounding the defect with the same depth as the intact cartilage. The image color channels were separated into red, green, and blue, and the blue channel was used to threshold for the stained proteoglycan. The “analyze particles” module of Image J was used to calculate the percentage of the ROI that stained positive for proteoglycan. Second-harmonic generation microscopy was also used to evaluate collagen fibril structure and organization. Results: Toluidine blue staining revealed cartilage formation in defects at the 4-week and 8-week timepoints in both the FMBs+cells and pellet groups. No cartilage was detected in the FMBs alone or sham groups (Figure 1). Percentages of defect area positive for cartilage proteoglycan in the FMBs+cells, pellet, FMBs alone, and sham group defect areas were 8.95%, 3.03%, 0.008%, and 0.094% respectively. A comparable ROI in healthy rat cartilage showed proteoglycan positivity of 58.43% of the area (Figure 2). Second harmonic generation imaging of the defect area showed that from sham to FMB alone to pellet to FMBs+cells groups, the organization of the collagen fibrils begin to more closely approximate healthy rat cartilage (Figure 3). Long-term time points are currently being analyzed. Conclusions: At the early timepoints presented in this abstract, there is evidence of cartilage formation in the drilled articular cartilage defects in both the FMBs+cells and pellet groups. This initial data shows the potential for our method to regenerate articular cartilage in vivo. Analysis of our long-term time points will reveal the extent and quality of healing induced by hBMSCs transplanted in conjunction with FMBs.

Spectral CT imaging of human osteoarthritic cartilage via quantitative assessment of glycosaminoglycan content using multiple contrast agents.

Detection of early osteoarthritis to stabilize or reverse the damage to articular cartilage would improve patient function, reduce disability, and limit the need for joint replacement. In this study, we investigated nondestructive photon-processing spectral computed tomography (CT) for the quantitative measurement of the glycosaminoglycan (GAG) content compared to destructive histological and biochemical assay techniques in normal and osteoarthritic tissues. Cartilage-bone cores from healthy bovine stifles were incubated in 50% ioxaglate (Hexabrix®) or 100% gadobenate dimeglumine (MultiHance®). A photon-processing spectral CT (MARS) scanner with a CdTe-Medipix3RX detector imaged samples. Calibration phantoms of ioxaglate and gadobenate dimeglumine were used to determine iodine and gadolinium concentrations from photon-processing spectral CT images to correlate with the GAG content measured using a dimethylmethylene blue assay. The zonal distribution of GAG was compared between photon-processing spectral CT images and histological sections. Furthermore, discrimination and quantification of GAG in osteoarthritic human tibial plateau tissue using the same contrast agents were demonstrated. Contrast agent concentrations were inversely related to the GAG content. The GAG concentration increased from 25 μg/ml (85 mg/ml iodine or 43 mg/ml gadolinium) in the superficial layer to 75 μg/ml (65 mg/ml iodine or 37 mg/ml gadolinium) in the deep layer of healthy bovine cartilage. Deep zone articular cartilage could be distinguished from subchondral bone by utilizing the material decomposition technique. Photon-processing spectral CT images correlated with histological sections in healthy and osteoarthritic tissues. Post-imaging material decomposition was able to quantify the GAG content and distribution throughout healthy and osteoarthritic cartilage using Hexabrix® and MultiHance® while differentiating the underlying subchondral bone.