Improved Understanding of the Inflammatory Response in Synovial Fluid and Serum after Traumatic Knee Injury, Excluding Fractures of the Knee: A Systematic Review.

BACKGROUND: Traumatic knee injury results in a 4- to 10-fold increased risk of post-traumatic osteoarthritis (PTOA). Currently, there are no successful interventions for preventing PTOA after knee injury. The aim of this study is to identify inflammatory proteins that are increased in serum and synovial fluid after acute knee injury, excluding intra-articular fractures. METHODS: A literature search was done according to the PRISMA guidelines. Articles reporting about inflammatory proteins after knee injury, except fractures, up to December 8, 2021 were collected. Inclusion criteria were as follows: patients younger than 45 years, no radiographic signs of knee osteoarthritis at baseline, and inflammatory protein measurement within 1 year after trauma. Risk of bias was assessed of the included studies. The level of evidence was determined by the Strength of Recommendation Taxonomy. RESULTS: Ten studies were included. All included studies used a healthy control group or the contralateral knee as healthy control. Strong evidence for interleukin 6 (IL-6) and limited evidence for CCL4 show elevated concentrations of these proteins in synovial fluid (SF) after acute knee injury; no upregulation in SF for IL-2, IL-10, CCL3, CCL5, CCL11, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) was found. Limited evidence was found for no difference in serum concentration of IL-1ß, IL-6, IL-10, CCL2, and tumor necrosis factor alpha (TNF-α) after knee injury. CONCLUSION: Interleukin 6 and CCL4 are elevated in SF after acute knee injury. Included studies failed to demonstrate increased concentration of inflammatory proteins in SF samples taken 6 weeks after trauma. Future research should focus on SF inflammatory protein measurements taken less than 6 weeks after injury.

Proteomic Profile Analysis of Synovial Fluid in Patients With Anterior Cruciate Ligament Tears.

BACKGROUND: Anterior cruciate ligament (ACL) tears are associated with posttraumatic osteoarthritis, but the early biological changes that initiate joint degeneration after this injury are not well characterized. ACL tears typically result in effusion in the knee, which may provide insight into the initial response of the joint to injuries. HYPOTHESIS: Patient- and injury-specific factors are associated with the proteomics of synovial fluid in knees with ACL tears. STUDY DESIGN: Descriptive laboratory study. METHODS: Synovial fluid was collected from 105 patients (38 male, 67 female) with an acute traumatic ACL tear. Patient- and injury-specific factors such as age, sex, body mass index, time from injury, presence/absence of concomitant meniscal tears, and location of concomitant bone bruises (if present) were recorded. The protein concentration of synovial fluid was measured, followed by benchmarking of samples for multi-affinity high-abundance protein depletion. An isotropically labeled high-resolution nano-liquid chromatography with tandem mass spectrometry-based proteomic approach was used to determine the synovial fluid protein profile. Data were processed, quality controlled, and analyzed computationally for each patient and injury factor. RESULTS: The proteomics of synovial fluid from ACL tears was associated with patient sex, injury pattern, and location of bone bruises but not with patient age, body mass index, or time from injury. Knees with an isolated ACL tear had higher glutathione peroxidase 1 (GPX1) and plastin 3 levels than knees with an ACL tear and meniscal tear. A bone bruise on the lateral femoral condyle was associated with elevated leptin and glucose-6-phosphate dehydrogenase (G6PD) levels. A bone bruise on the lateral tibial plateau was associated with decreased GPX1 levels. Male patients had higher matrix metalloproteinase 9 and lower G6PD levels than female patients. CONCLUSION: Patient sex, injury pattern, and bone bruise location were important determinants of the proteomic profile of effusion resulting from ACL tears. CLINICAL RELEVANCE: Longitudinal follow-ups to see if and how proteomic differences relate to clinical outcomes and mechanistic studies to assess the role that specific proteins play in the joint are warranted. Ultimately, these investigations could lead to better approaches to predict clinical outcomes and identify possible interventions to optimize outcomes in these patients.

The alarmins high mobility group box protein 1 and S100A8/A9 display different inflammatory profiles after acute knee injury.

OBJECTIVE: To compare the concentrations of high mobility group box 1 protein (HMGB1) and S100A8/A9 in synovial fluid between patients with knee injuries and osteoarthritis (OA), and knee healthy subjects. To investigate associations of alarmin levels with different joint injuries and with biomarkers of inflammation, Wnt signaling, complement system, bone and cartilage degradation. METHODS: HMGB1 and S100A8/A9 were measured in synovial fluid by immunoassays in patients with knee injuries, with OA and from knee healthy subjects, and were related to time from injury and with biomarkers obtained from previous studies. Hierarchical cluster and enrichment analyses of biomarkers associated to HMGB1 and S100A8/A9 were performed. RESULTS: The synovial fluid HMGB1 and S100A8/A9 concentrations were increased early after knee injury; S100A8/A9 levels were negatively associated to time after injury and was lower in the old compared to recent injury group, while HMGB1 was not associated to time after injury. The S100A8/A9 levels were also increased in OA. The initial inflammatory response was similar between the alarmins, and HMGB1 and S100A8/A9 shared 9 out of 20 enriched pathways. The alarmins displayed distinct response profiles, HMGB1 being associated to cartilage biomarkers while S100A8/A9 was associated to proinflammatory cytokines. CONCLUSIONS: HMGB1 and S100A8/A9 are increased as an immediate response to knee trauma. While they share many features in inflammatory and immunoregulatory mechanisms, S100A8/A9 and HMGB1 are associated to different downstream responses, which may have impact on the OA progression after acute knee injuries.

E8002 Reduces Adhesion Formation and Improves Joint Mobility in a Rat Model of Knee Arthrofibrosis.

Knee arthrofibrosis is a common complication of knee surgery, caused by excessive scar tissue, which results in functional disability. However, no curative treatment has been established. E8002 is an anti-adhesion material that contains L-ascorbic acid, an antioxidant. We aimed to evaluate the efficacy of E8002 for the prevention of knee arthrofibrosis in a rat model, comprising injury to the surface of the femur and quadriceps muscle 1 cm proximal to the patella. Sixteen male, 8-week-old Sprague Dawley rats were studied: in the Adhesion group, haemorrhagic injury was induced to the quadriceps and bone, and in the E8002 group, an adhesion-preventing film was implanted between the quadriceps and femur after injury. Six weeks following injury, the restriction of knee flexion owing to fibrotic scarring had not worsened in the E8002 group but had worsened in the Adhesion group. The area of fibrotic scarring was smaller in the E8002 group than in the Adhesion group (p < 0.05). In addition, the numbers of fibroblasts (p < 0.05) and myofibroblasts (p < 0.01) in the fibrotic scar were lower in the E8002 group. Thus, E8002 reduces myofibroblast proliferation and fibrotic scar formation and improves the range of motion of the joint in a model of knee injury.

Metabolism of Subchondral Bone Tissue in Posttraumatic Osteoarthrosis in Rats.

In rats with modeled posttraumatic knee osteoarthrosis, negative changes in subchondral bone metabolism were revealed: a tendency to an increase in osteocalcin concentration, a decrease in sclerostin and osteoprotegerin levels, and a significant increase in FGF-23 concentration accompanied by a slight elevation of inorganic phosphorous and significant increase in total calcium levels in comparison with the corresponding parameters in intact controls. These findings demonstrate crucial importance of structural integrity of the subchondral bone, because its protection improves the results of reconstructive therapy for local cartilage defects.

Morphological Changes in Articular Cartilage and Free-Radical Lipid Peroxidation in Rats with Posttraumatic Osteoarthrosis.

Using the rat model of posttraumatic osteoarthrosis of the knee joint induced by surgical transection of their anterior cruciate ligaments, we showed that irreversible loss of hyaluronan by the extracellular matrix of the joint cartilage tissue against the background of oxidative stress accompanied by accumulation of intermediate LPO products in blood serum and formation of thiol system incompetence was one of the key patterns of dystrophic degeneration of the cartilage tissue. Considerable metabolic shifts were associated with structural modification of the articular hyaline cartilage: its thinning and a decrease of chondrocyte density and their abnormal spatial distribution in the matrix with predominance of solitary isolated cells with signs of karyopyknosis and karyolysis.

Anti-Inflammatory Therapeutic Approaches to Prevent or Delay Post-Traumatic Osteoarthritis (PTOA) of the Knee Joint with a Focus on Sustained Delivery Approaches.

Inflammation plays a central role in the pathogenesis of knee PTOA after knee trauma. While a comprehensive therapy capable of preventing or delaying post-traumatic osteoarthritis (PTOA) progression after knee joint injury does not yet clinically exist, current literature suggests that certain aspects of early post-traumatic pathology of the knee joint may be prevented or delayed by anti-inflammatory therapeutic interventions. We discuss multifaceted therapeutic approaches that may be capable of effectively reducing the continuous cycle of inflammation and concomitant processes that lead to cartilage degradation as well as those that can simultaneously promote intrinsic repair processes. Within this context, we focus on early disease prevention, the optimal timeframe of treatment and possible long-lasting sustained delivery local modes of treatments that could prevent knee joint-associated PTOA symptoms. Specifically, we identify anti-inflammatory candidates that are not only anti-inflammatory but also anti-degenerative, anti-apoptotic and pro-regenerative.

Evaluation of CCL21 role in post-knee injury inflammation and early cartilage degeneration.

The expression of some chemokines and chemokine receptors is induced during the development of post-traumatic osteoarthritis (PTOA), but their involvement in the pathogenesis of the disease is unclear. The goal of this study was to test whether CCL21 and CXCL13 play a role in PTOA development. For this purpose, we evaluated the expression profiles of the chemokines Ccl21 and Cxcl13, matrix metalloproteinase enzymes Mmp3 and Mmp13, and inflammatory cell markers in response to partial medial meniscectomy and destabilization (MMD). We then assessed the effect of local administration of CCL21 neutralizing antibody on PTOA development and post-knee injury inflammation. The mRNA expression of both Ccl21 and Cxcl13 was induced early post-surgery, but only Ccl21 mRNA levels remained elevated 4 weeks post-surgery in rat MMD-operated knees compared to controls. This suggests that while both CXCL13 and CCL21 are involved in post-surgery inflammation, CCL21 is necessary for development of PTOA. A significant increase in the mRNA levels of Cd4, Cd8 and Cd20 was observed during the first 3 days post-surgery. Significantly, treatment with CCL21 antibody reduced post-surgical inflammation that was accompanied by a reduction in the expression of Mmp3 and Mmp13 and post-MMD cartilage degradation. Our findings are consistent with a role for CCL21 in mediating changes in early inflammation and subsequent cartilage degeneration in response to knee injury. Our results suggest that targeting CCL21 signaling pathways may yield new therapeutic approaches effective in delaying or preventing PTOA development following injury.

Synovial Fluid Biomarkers in Knee Osteoarthritis: A Systematic Review and Quantitative Evaluation Using BIPEDs Criteria.

OBJECTIVE: The aim of this systematic review was to analyze the evidence about the efficacy of the several synovial fluid (SF) biomarkers proposed for knee osteoarthritis (OA), categorizing them by both molecular characteristics and clinical use according to the BIPEDs criteria, to provide a comprehensive and structured overview of the current literature. DESIGN: A systematic review was performed in May 2020 on PubMed, Cochrane Library, and Embase databases about SF biomarkers in patients with knee OA. The search was limited to articles in the last 20 years on human studies, involving patients with knee OA, reporting SF biomarkers. The evidence for each selected SF biomarker was quantified according to the 6 categories of BIPEDs classification. RESULTS: A total of 159 articles were included in the qualitative data synthesis and 201 different SF biomarkers were identified. Among these, several were investigated multiple times in different articles, for a total of 373 analyses. The studies included 13,557 patients with knee OA. The most promising SF biomarkers were C4S, IL-6, IL-8, Leptin, MMP-1/3, TIMP-1, TNF-α, and VEGF. The "burden of disease" and "diagnostic" categories were the most represented with 132 and 106 different biomarkers, respectively. CONCLUSIONS: The systematic review identified numerous SF biomarkers. However, despite the high number of studies on the plethora of identified molecules, the evidence about the efficacy of each biomarker is supported by limited and often conflicting findings. Further research efforts are needed to improve the understanding of SF biomarkers for a better management of patients with knee OA.

Characterization of Synovial Cytokine Patterns in Bucket-Handle and Posterior Horn Meniscal Tears.

The specific etiology of meniscal tears, including the mechanism of lesion, location, and orientation, is considered for its contribution to subsequent joint cytokine responsiveness, healing outcomes, and by extension, appropriate lesion-specific surgical remediation. Meniscal repair is desirable to reduce the probability of development of posttraumatic osteoarthritis (PTOA) which is strongly influenced by the coordinate generation of pro- and anti-inflammatory cytokines by the injured cartilage. We now present biochemical data on variation in cytokine levels arising from two particular meniscal tears: bucket-handle (BH) and posterior horn (PH) isolated meniscal tears. We selected these two groups due to the different clinical presentations. We measured the concentrations of TNF-α, IL-1ß, IL-6, IL-8, and IL-10 in knee synovial fluid of 45 patients with isolated meniscal lesions (BH tear, n = 12; PH tear, n = 33). TNF-α levels were significantly (p < 0.05) greater in the BH group compared with the PH group, whereas IL-1ß levels were significantly greater (p < 0.05) in the PH group compared with the BH group. Both BH and PH groups were consistent in presenting a positive correlation between concentrations of IL-6 and IL-1ß. A fundamental difference in IL-10 responsiveness between the two groups was noted; specifically, levels of IL-10 were positively correlated with IL-6 in the BH group, whereas in the PH group, levels of IL-10 were positively correlated with IL-1ß. Collectively, our data suggest a possible influence of the meniscal tear pattern to the articular cytokine responsiveness. This differential expression of inflammatory cytokines may influence the risk of developing PTOA in the long term.

Lubricin in experimental and naturally occurring osteoarthritis: a systematic review.

OBJECTIVE: Lubricin is increasingly being evaluated as an outcome measure in studies investigating post-traumatic and naturally occurring osteoarthritis. However, there are discrepancies in results, making it unclear as to whether lubricin is increased, decreased or unchanged in osteoarthritis. The purpose of this study was to review all papers that measured lubricin in joint injury or osteoarthritis in order to draw conclusions about lubricin regulation in joint disease. DESIGN: A systematic search of the Pubmed, Web of Knowledge, and EBSCOhost databases for papers was performed. Inclusion criteria were in vivo studies that measured lubricin in humans or animals with joint injury, that investigated lubricin supplementation in osteoarthritic joints, or that described the phenotype of a lubricin knock-out model. A methodological assessment was performed. RESULTS: Sixty-two studies were included, of which thirty-eight measured endogenous lubricin in joint injury or osteoarthritis. Nineteen papers found an increase or no change in lubricin and nineteen reported a decrease. Papers that reported a decrease in lubricin were cited four times more often than those that reported an increase. Fifteen papers described lubricin supplementation, and all reported a beneficial effect. Eleven papers described lubricin knock-out models. CONCLUSIONS: The human literature reveals similar distributions of papers reporting increased lubricin as compared to decreased lubricin in osteoarthritis. The animal literature is dominated by reports of decreased lubricin in the rat anterior cruciate ligament transection model, whereas studies in large animal models report increased lubricin. Intra-articular lubricin supplementation may be beneficial regardless of whether lubricin increases or decreases in OA.

Lysosomal autophagy promotes recovery in rats with acute knee injury through TFEB mediation.

BACKGROUND: To study the role of lysosomal decomposition and elimination of old bone matrix, as well as the mechanism of promoting chondrocyte growth and bone recovery through the perspective of TFEB-mediated lysosomal autophagy. METHODS: Rat models of acute knee injury were designed, and autophagy flow was detected by injection of autophagy inhibitors 3-methyladenine. Autophagy flow was detected by RFP-GFP-LC3 double fluorescence molecule. The expression of TFEB, DRAM, MAPLC3, and MITF were analyzed by Western blot, and the expression of genes NITF, Bcl2, and TYR in rat cartilage tissues were detected by RT-PCR. RESULTS: The number of autophagosomes was increasing in the auto group compared with the inhibitor-auto group and normal group. There was a significant difference of LC3 levels in the auto group and inhibitor-auto group compared with the normal control. The expression of TFEB, DRAM, MAPLC3, and MITF proteins by Western blot analysis were significantly increased in the auto group and decreased in the inhibitor-auto group. The expression of NITF, Bcl2, and TYR by RT-PCR determination were higher in the auto group and inhibitor-auto group than the normal group. CONCLUSIONS: Autophagy can inhibit apoptosis, promote chondrocyte growth and bone regeneration, and restore knee joint injury of rats. The main mechanism is to promote the effect of TFEB-mediated lysosomal autophagy.

Microarray analysis of cartilage: comparison between damaged and non-weight-bearing healthy cartilage.

AIM: A limited healing response to focal cartilage lesions is frequently encountered in the clinical cartilage pathology. This study compares the gene expression patterns of damaged and undamaged regions of cartilage obtained from the same patient with focal cartilage lesions. The aim of this study is to provide new genes and proteins, which may be a potential future target of research. METHODS: During the autologous chondrocyte implantation (MACI) surgery, cartilage tissues (healthy non-weight bearing and Damaged-lesion side) were obtained from 10 patients with knee focal cartilage lesions. The degeneration status of the cartilage was characterized according to ICRS criteria. Whole genome microarray gene expression profiling was performed and some of the differentially regulated genes were validated with RT-PCR. RESULTS: Damaged and undamaged non-weight bearing cartilage showed distinct gene expression profiles. Genes involved in cell signaling, matrix degradation, hypoxia, and the inflammatory response showed significant up- or down-regulation. In the focal lesions, expression of genes such as HIF1α, TIMP-2, EID1, EID2, NCOA3, NBR1, SP100, and HSP90AA1 was significantly higher compared to healthy non-weight bearing cartilage from the same joint, whereas TIMP-4 was lower. CONCLUSION: The genes examined in this study differ distinctly between focal cartilage (ICRS 3-4) lesions and undamaged sites of the same joint. We believe that the data set forth in this study may be used for clinical purposes and be a guide in the development of new biological approaches for therapy.

T1ρ, T2 mapping, and EPIC-µCT Imaging in a Canine Model of Knee Osteochondral Injury.

The dog is the most commonly used large animal model for the study of osteoarthritis. Optimizing methods for assessing cartilage health would prove useful in reducing the number of dogs needed for a valid study of osteoarthritis and cartilage repair. Twelve beagles had critical-sized osteochondral defects created in the medial femoral condyle of both knees. Eight dogs had T1ρ and T2 magnetic resonance imaging (MRI) performed approximately 6 months after defect creation. Following MRI evaluations, all 12 dogs were humanely euthanatized and cartilage samples were obtained from the medial and lateral femoral condyles, medial and lateral tibial plateaus, trochlear groove, and patella for proteoglycan and collagen quantification. Equilibrium partitioning of an ionic contrast (EPIC)-µCT was then performed followed by the histologic assessment of the knees. Correlations between T1ρ, T2, EPIC-µCT and proteoglycan, collagen, and histology scores were assessed using a multivariate analysis accounting for correlations from samples within the same knee and in the same dog. Pearson's correlation coefficients were calculated to assess the strength of significant relationships. Correlations between µCT values and biochemical or histologic assessment were weak to moderately strong (0.09-0.41; p < 0.0001-0.66). There was a weak correlation between the T2 values and cartilage proteoglycan (-0.32; p = 0.04). The correlation between T1ρ values and cartilage proteoglycan were moderately strong (-0.38; p < 0.05) while the strongest correlation was between the T1ρ values and histological assessment of cartilage with a correlation coefficient of 0.58 (p < 0.0001). These data suggest that T1ρ shows promise for possible utility in the translational study of cartilage health and warrants further development in this species. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:368-377, 2020.

The effects of age on the severity of joint damage and intra-articular inflammation following a simulated medial meniscus injury in 3, 6, and 9 month old male rats.

Purpose: Aging is a known risk factor for osteoarthritis (OA). Several transgenic rodent models have been used to investigate the effects of accelerated or delayed aging in articular joints. However, age-effects on the progression of post-traumatic OA are less frequently evaluated. The objective of this study is to evaluate how animal age affects the severity of intra-articular inflammation and joint damage in the rat medial collateral ligament plus medial meniscus transection (MCLT+MMT) model of knee OA.Methods: Forty-eight, male Lewis rats were aged to 3, 6, or 9 months old. At each age, eight rats received either an MCLT+MMT surgery or a skin-incision. At 2 months post-surgery, intra-articular evidence of CTXII, IL1ß, IL6, TNFα, and IFNγ was evaluated using a multiplex magnetic capture technique, and histological evidence of OA was assessed via a quantitative histological scoring technique.Results: Elevated levels of CTXII and IL6 were found in MCLT+MMT knees relative to skin-incision and contralateral controls; however, animal age did not affect the severity of joint inflammation. Conversely, histological investigation of cartilage damage showed larger cartilage lesion areas, greater width of affected cartilage, and more evidence of hypertrophic cartilage damage in MCLT+MMT knees with age.Conclusions: These data indicate the severity of cartilage damage subsequent to MCLT+MMT surgery is related to the rat's age at the time of injury. However, despite greater levels of cartilage damage, the level of intra-articular inflammation was not necessarily affected in 3, 6, and 9 month old male rats.

Explant models for meniscus metabolism, injury, repair, and healing.

Purpose/Aim: Knee meniscus is a wedge-shaped fibrocartilaginous tissue, playing important roles in maintaining joint stability and function. Injuries to the meniscus, particularly with the avascular inner third zone, hardly heal and frequently progress into structural breakdown, followed by the initiation of osteoarthritis. As the importance of meniscus in joint function and diseases is being recognized, the field of meniscus research is growing. Not only development, biology, and metabolism but also injury, repair, and healing of meniscus are being actively investigated. As meniscus functions as an integrated unit of a knee joint, in vivo models with various species have been the predominant method for studying meniscus pathophysiology and for testing healing/regeneration strategies. However, in vivo models for meniscus studies suffer from low reproducibility and high cost. To complement the limitations of in vivo animal models, several types of meniscus explants have been applied as highly controlled, standardized in vitro models to investigate meniscus metabolism, pathophysiology, and repair or regeneration process. This review summarizes and compares the existing meniscus explant models. We also discuss the advantages and disadvantages of each explant model.Conclusion: Despite few outstanding challenges, meniscus explant models have potential to serve as an effective tool for investigations of meniscus metabolism, injury, repair and healing.

Alternations of Metabolic Profiles in Synovial Fluids and the Correlation with T2 Relaxation Times of Cartilage and Meniscus-A Study on Anterior Cruciate Ligament- (ACL-) Injured Rabbit Knees at Early Stage.

OBJECTIVES: To examine the metabolic profiles alterations of synovial fluids from anterior cruciate ligament- (ACL-) injured rabbit knees at early stage and analyze the correlation with T2 relaxation times of cartilage and meniscus. METHODS: The right knees of 15 rabbits were selected for the construction of ACL injury models, whereas the contralateral knees served as control group. After 4 weeks, both knees were examined by MRI with quantitative T2 mapping sequence, and the T2 relaxation times of cartilage and meniscus were measured. Then, the synovial fluids were obtained from both knee capsules and performed liquid chromatography-mass spectrometry analysis (LC-MS). RESULTS: The T2 relaxation times of cartilage and meniscus in ACL-injured knees were significantly higher than those in control knees (Cartilage: 41.52 ± 2.98 ms vs 36.02 ± 2.71 ms, P < 0.001; Meniscus: 33.35 ± 3.57 ms vs 27.27 ± 2.10 ms, P < 0.001). Twenty-eight differential metabolites were identified based on a total of 1569 detected signatures between ACL-injured knees and control knees. These differential metabolites primarily implied perturbations in the fluxes of lipids and steroid-based compounds. The Linear regression analysis demonstrated satisfactory correlations between glycerophospholipid metabolism and T2 relaxation times of both cartilage and meniscus in ACL-injured knees (R2 = 0.8204 and 0.8197, respectively). CONCLUSION: ACL injury of rabbit knees resulted in elevated T2 relaxation times of cartilage and meniscus and perturbed metabolism of various lipids and steroids in synovial fluids, particularly glycerophospholipids. Glycerophospholipid metabolism related compounds could serve as potential biomarkers for early degenerative changes of cartilage and meniscus after ACL injury.

Polyhydroxybutyrate/Chitosan 3D Scaffolds Promote In Vitro and In Vivo Chondrogenesis.

The articular cartilage is an avascular and aneural tissue and its injuries result mostly in osteoarthritic changes and formation of fibrous tissue. Efforts of scientists worldwide are focused on restoration of cartilage with increase in life quality of patients. Novel polymeric polyhydroxybutyrate/chitosan (PCH) porous 3D scaffolds were developed and characterized. The rat mesenchymal stem cells (MSCs) were seeded in vitro on PCH scaffolds by a simple filtration of MSCs suspension over scaffolds using syringe. The chondrogenesis of cell-scaffold constructs was carried out in supplemented chondrogenic cultivation medium. After 2 and 4 weeks of in vitro culturing cell-scaffold constructs in chondrogenic differentiation medium, the cartilage extracellular matrix components like glycosaminoglycans and collagens were identified in scaffolds by biochemical assays and histological and immunohistochemical staining. Preliminary in vivo experiments with acellular scaffolds, which filled the artificially created cartilage defect in sheep knee were done and evaluated. Cells released from the bone marrow cavity have penetrated into acellular PCH scaffold in cartilage defect and induced tissue formation similar to hyaline cartilage. The results demonstrated that PCH scaffolds supported chondrogenic differentiation of MSCs in vitro. Acellular PCH scaffolds were successfully utilized in vivo for reparation of artificially created knee cartilage defects in sheep and supported wound healing and formation of hyaline cartilage-like tissue.

Synovial fluid proteome changes in ACL injury-induced posttraumatic osteoarthritis: Proteomics analysis of porcine knee synovial fluid.

Surgical transection of the anterior cruciate ligament (ACL) in the porcine model leads to posttraumatic osteoarthritis if left untreated. However, a recently developed surgical treatment, bridge-enhanced ACL repair, prevents further cartilage damage. Since the synovial fluid bathes all the intrinsic structures of knee, we reasoned that a comparative analysis of synovial fluid protein contents could help to better understand the observed chondroprotective effects of the bridge-enhanced ACL repair. We hypothesized that post-surgical changes in the synovial fluid proteome would be different in the untreated and repaired knees, and those changes would correlate with the degree of cartilage damage. Thirty adolescent Yucatan mini-pigs underwent unilateral ACL transection and were randomly assigned to either no further treatment (ACLT, n = 14) or bridge-enhanced ACL repair (BEAR, n = 16). We used an isotopically labeled high resolution LC MS/MS-based proteomics approach to analyze the protein profile of synovial fluid at 6 and 12 months after ACL transection in untreated and repaired porcine knees. A linear mixed effect model was used to compare the normalized protein abundance levels between the groups at each time point. Bivariate linear regression analyses were used to assess the correlations between the macroscopic cartilage damage (total lesion area) and normalized abundance levels of each of the identified secreted proteins. There were no significant differences in cartilage lesion area or quantitative abundance levels of the secreted proteins between the ACLT and BEAR groups at 6 months. However, by 12 months, greater cartilage damage was seen in the ACLT group compared to the BEAR group (p = 0.005). This damage was accompanied by differences in the abundance levels of secreted proteins, with higher levels of Vitamin K-dependent protein C (p = 0.001), and lower levels of Apolipoprotein A4 (p = 0.021) and Cartilage intermediate layer protein 1 (p = 0.049) in the ACLT group compared to the BEAR group. There were also group differences in the secreted proteins that significantly changed in abundance between 6 and 12 months in ACLT and BEAR knees. Increased concentration of Ig lambda-1 chain C regions and decreased concentration of Hemopexin, Clusterin, Coagulation factor 12 and Cartilage intermediate layer protein 1 were associated with greater cartilage lesion area. In general, ACLT knees had higher concentrations of pro-inflammatory proteins and lower concentrations of anti-inflammatory proteins than BEAR group. In addition, the ACLT group had a lower and declining synovial concentrations of CILP, in contrast to a consistently high abundance of CILP in repaired knees. These differences suggest that the knees treated with bridge-enhanced ACL repair may be maintaining an environment that is more protective of the extracellular matrix, a function which is not seen in the ACLT knees.

Repair of cartilage defects with devitalized osteochondral tissue: A pilot animal study.

Devitalization using high hydrostatic pressure (HHP) treatment inactivates cells while matrix structure and biomechanical properties are maintained. Because of strong chondroinductive potential of HHP-devitalized cartilage matrix, it may be used as scaffold for reconstruction of (osteo-)chondral lesions. In this pilot study, we evaluated the feasibility of HHP-devitalized osteochondral tissue to repair osteochondral defects in a rabbit model. Removal and reimplantation of osteochondral plugs were performed in 12 female New Zealand White rabbits. From the knee joint of each animal, osteochondral plugs (diameter = 4 mm; depth = 2.5 mm) were harvested and devitalized by HHP (452 MPa for 10 min). Afterward, the plugs were reimplanted into the respective cavity, from where they were taken. Animals were sacrificed 12 weeks postoperatively and the integration of osteochondral plugs was examined using µ-CT, MRI, and histological staining. Furthermore, revitalization of HHP-treated osteochondral plugs was characterized by gene expression analyses. Macroscopic evaluation of tissue repair at implantation sites of HHP-treated osteochondral plugs showed an adequate defect filling 12 weeks after implantation. Plug margins were hardly detectable indicating successful tissue integration. Additionally, gene expression analyses demonstrated initial revitalization of the HHP-treated tissue 12 weeks postoperatively. Our preliminary data revealed that HHP-treated osteochondral plugs could be used to refill osteochondral defects in the knee joint and promote cell migration into defect site. Data indicated that HHP-treated tissue has the potential to act as functional scaffolds for reconstruction of cartilage defects. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2354-2364, 2019.