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.

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.

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.

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.

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.

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.

CCL22 is a biomarker of cartilage injury and plays a functional role in chondrocyte apoptosis.

BACKGROUND: Osteoarthritis (OA) is one of the leading causes of disability worldwide. Previous history of knee injury is a significant risk factor for OA. It has been established that low-level chronic inflammation plays a pivotal role in the onset and pathogenesis of OA. The primary aim of this research was to determine if a history of knee joint injury is associated with systemic inflammation. A secondary aim was to determine if systemic inflammation is related to knee pain and joint structure. METHODS: Differences in serum cytokine association networks, knee joint structural changes (MRI), and self-reported pain (i.e., Knee Injury and Osteoarthritis Outcome Score Pain subscale, KOOSPAIN and Intermittent and Constant Osteoarthritis Pain score, ICOAP) between individuals who had sustained a youth (aged 15-26 years) sport-related knee injury 3-10 years previously and age- and sex-matched controls were examined. Proteins of interest were also examined in an OA rat model. RESULTS: Cytokine association networks were found to differ significantly between study groups, yet no significant associations were found between networks and KOOSPAIN or MRI-defined OA. A group of cytokines (MCP1/CCL2, CCL22 and TNFα) were differentially associated with other cytokines between study groups. In a pre-clinical rat OA model, serum CCL22 levels were associated with pain (r = 0.255, p = 0.045) and structural changes to the cartilage. CCL22 expression was also observed in human OA cartilage and furthermore, CCL22 induced apoptosis of isolated human chondrocytes. DISCUSSION: These results suggest that CCL22 may be an early factor in the onset/pathogenic process of cartilage degeneration and/or related to pain OA.

Distribution of bone tracer uptake in symptomatic knees after ACL reconstruction compared to asymptomatic non-operated knees: a method for better differentiating patient-specific from disease-specific bone tracer uptake in SPECT/CT.

OBJECTIVE: To evaluate the differences of bone tracer uptake (BTU) in symptomatic and asymptomatic contralateral knees in patients after reconstruction of the anterior cruciate ligament (ACL-R) and to identify typical BTU patterns and threshold values to differentiate pathological from physiological BTU. METHODS: 53 patients after unilateral ACL-R were retrospectively included in the study. The population was subdivided into a group of symptomatic operated knees and a group of contralateral asymptomatic non-operated knees. BTU was measured in SPECT/CT using a validated anatomical localization-scheme and normalized mean BTU values were calculated in both knees. Wilcoxon signed rank-test and Pearson's rank-correlation coefficient were used (p < 0.05). RESULTS: Symptomatic knees after ACL-R showed significantly more BTU than asymptomatic ones (p < 0.01).Based on the measured BTU activity in SPECT/CT in symptomatic operated and asymptomatic non-operated knees, intensity thresholds of pathological BTU were established. A BTU threshold of greater than the Median + 1 SD of the asymptomatic non-operated knee was defined as pathological. In both groups the highest mean BTU was found on the femoral, tibial and patellar articular surfaces, the lowest BTU in femoral and tibial regions far from the joint. CONCLUSIONS: The established BTU thresholds for SPECT/CT in knees after ACL-R help to differentiate disease-specific from patient-specific BTU. It could be speculated that BTU in asymptomatic knees equates to the preoperative condition of the knee joint before ACL-R. Therefore, the results of this study help to understand in-vivo loading of the knee and ultimately lead to prediction of development of osteoarthritis in an early stage.

IGF-1 and BMP-7 synergistically stimulate articular cartilage repairing in the rabbit knees by improving chondrogenic differentiation of bone-marrow mesenchymal stem cells.

Knee injury is known as a frequently occurred damage related to sports, which may affect the function of cartilage. This study aims to explore whether Insulin-like growth factor 1 (IGF-1) and bone morphogenetic protein-7 (BMP-7)-modified bone-marrow mesenchymal stem cells (BMSCs) affect the repair of cartilage damage found in the knee. Primarily, BMSCs were treated with a series of pEGFP-C1, IGF-1, and BMP-7, followed by determination of IGF-1 and BMP-7 expression. A rabbit cartilage defect model was also established. Afterfward, cell morphology, viability, cartilage damage repair effect, and expression of collagen I and collagen II at the 6th and the 12th week were measured. BMSCs treated with pEGFP-C1/IGF-1, pEGFP-C1/BMP-7, and pEGFP-C1/BMP-7-IGF-1 exhibited elevated expression of BMP-7 and IGF-1. Besides, BMSCs in the P10 generation displayed decreased cell proliferation. Moreover, BMSCs treated with IGF-1, BMP-7, and IGF-1-BMP-7 showed reduced histological score and collagen I expression while elevated collagen II expression, as well as better repair effect, especially in those treated with IGF-1-BMP-7. Collectively, these results demonstrated a synergistic effect of IGF-1 and BMP-7 on the BMSC chondrogenic differentiation on the articular cartilage damage repair in the rabbit knees, highlighting its therapeutic potential for the treatment of articular cartilage damage.

Cartilaginous extracellular matrix derived from decellularized chondrocyte sheets for the reconstruction of osteochondral defects in rabbits.

Cartilaginous extracellular matrix (ECM) materials derived from decellularized native articular cartilage are widely used in cartilage regeneration. However, it is difficult for endogenous cells to migrate into ECM derived from native cartilage owing to its nonporous structure and dense nature. Moreover, current decellularization approaches frequently lead to architectural breakdown and potential loss of surface composition of ECM. To solve this problem, we aimed to establish a novel biological ECM scaffold from chondrocyte sheets for cartilage regeneration. We cultured chondrocytes harvested from the auricular cartilage of 4-week-old New Zealand rabbits and enabled them to form cell sheets. These sheets were decellularized using sodium dodecyl sulfate (SDS) with three different concentrations, namely, 1%, 5%, and 10%, followed by 1% Triton X-100 and deoxyribonuclease enzyme solution. In vitro microstructural examination and mechanical tests demonstrated that 1% SDS not only removed chondrocytes completely but also maintained the native architecture and composition of ECM, thus avoiding the use of high-concentration SDS. Application of decellularized chondrocyte sheets for osteochondral defects in rabbits resulted in substantial host remodeling and variant regeneration of osteochondral tissues. One percent SDS-treated decellularized chondrocyte sheets contributed to the superior reconstruction of osteochondral defects as compared with 5% and 10% SDS groups, which includes vascularized subchondral bone, articular cartilage with adequate thickness, and integration with host tissues. Furthermore, ECM from 1% SDS significantly increased the migrating potential of bone marrow mesenchymal stem cells (BMSCs) in vitro. RT-PCR and western blot also revealed that ECM increased the expression of SOX-9 in BMSCs, whereas it decreased COL-X expression. In conclusion, our results suggested that the chondrocyte sheets decellularized with 1% SDS preserved the integrity and bioactivity, which favored cell recruitment and enabled osteochondral regeneration in the knee joints of rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. STATEMENT OF SIGNIFICANCE: Although biological extracellular matrix (ECM) derived from decellularized native cartilage has been widely used in cartilage regeneration, it is difficult for endogenous cells to migrate into ECM owing to its dense nature. Moreover, current decellularization approaches lead to architectural breakdown of ECM. This study established a novel biological ECM from decellularized chondrocyte sheets for cartilage regeneration. Our results suggested that cartilaginous ECM favored cell recruitment and enabled osteochondral regeneration in rabbits, thus offering a promising approach for articular cartilage reconstruction without cell transplantation. SDS 1% adequately decellularized the chondrocytes in cell sheets, whereas it maintained the native architecture and composition of ECM, thereby avoiding the use of high-concentration SDS and providing a new way to acquire cartilaginous ECM.

Intra-articular administration of EP2 enhances the articular cartilage repair in a rabbit model.

We have reported the usefulness of chondrocyte sheets on articular cartilage repair in animal experiments. Here, we investigated the regenerative effects of EP2 signalling with or without chondrocyte sheets. Forty-five rabbits were used, with six rabbits in each of the six groups and nine rabbits for chondrocytes and synovial cells harvesting to fabricate triple-layered chondrocyte sheets: osteochondral defect only (control, Group A), EP2 agonist (Group B), EP2 antagonist (Group C), chondrocyte sheets (Group D), EP2 agonist and chondrocyte sheets (Group E), and EP2 antagonist and chondrocyte sheets (Group F). After surgery, the weight distribution ratio was measured as an indicator of pain alleviation. Injections of the EP2 agonist or EP2 antagonist were given from 4 weeks after surgery. The rabbits were sacrificed at 12 weeks, and the repaired tissues were evaluated for histology. The weight distribution ratio and International Cartilage Repair Society grading were as follows: Group A: 40.5% ± 0.2%, 14.8 ± 0.5; Group B: 43.4% ± 0.7%, 25.4 ± 0.8; Group C: 38.7% ± 0.7%, 13.7 ± 0.3; Group D: 48.6% ± 0.6%, 40.2 ± 0.5; Group E: 49.1% ± 0.3%, 40.5 ± 0.4; and Group F; 46.8% ± 0.4%, 38.7 ± 0.5. Significant differences in histology and pain alleviation were observed between groups except between Groups A and C, between Groups D and E, and between Groups D and F. These findings show that the intra-articular administration of an EP2 agonist achieved pain alleviation and tissue repair. However, no synergistic effect with chondrocyte sheets was observed.

Effects of TGF-ß Overexpression via rAAV Gene Transfer on the Early Repair Processes in an Osteochondral Defect Model in Minipigs.

BACKGROUND: Application of the chondrogenic transforming growth factor beta (TGF-ß) is an attractive approach to enhance the intrinsic biological activities in damaged articular cartilage, especially when using direct gene transfer strategies based on the clinically relevant recombinant adeno-associated viral (rAAV) vectors. PURPOSE: To evaluate the ability of an rAAV-TGF-ß construct to modulate the early repair processes in sites of focal cartilage injury in minipigs in vivo relative to control (reporter lacZ gene) vector treatment. STUDY DESIGN: Controlled laboratory study. METHODS: Direct administration of the candidate rAAV-human TGF-ß (hTGF-ß) vector was performed in osteochondral defects created in the knee joint of adult minipigs for macroscopic, histological, immunohistochemical, histomorphometric, and micro-computed tomography analyses after 4 weeks relative to control (rAAV- lacZ) gene transfer. RESULTS: Successful overexpression of TGF-ß via rAAV at this time point and in the conditions applied here triggered the cellular and metabolic activities within the lesions relative to lacZ gene transfer but, at the same time, led to a noticeable production of type I and X collagen without further buildup on the subchondral bone. CONCLUSION: Gene therapy via direct, local rAAV-hTGF-ß injection stimulates the early reparative activities in focal cartilage lesions in vivo. CLINICAL RELEVANCE: Local delivery of therapeutic (TGF-ß) rAAV vectors in focal defects may provide new, off-the-shelf treatments for cartilage repair in patients in the near future.

Effects of fibroblast growth factors 2 and low intensity pulsed ultrasound on the repair of knee articular cartilage in rabbits.

OBJECTIVE: To investigate the effect of low intensity ultrasound irradiation combine with fibroblast growth factors (FGF2) on the repair of the knee articular cartilage and to explore its mechanism in rabbit. MATERIALS AND METHODS: The model of the rabbit knee joint injury was established. 40 rabbits were divided into four groups, including control group, model group, FGF2 group and FGF2 + low intensity pulsed ultrasound group (FGF2 + LIPU). The knee joints of rabbits were taken at 4 and 8 weeks, respectively. Histopathological changes were detected by Hematoxylin and Eosin stain (HE) and evaluated by Wakitani score. The expression of FGF2 mRNA was detected by Real-time polymerase chain reaction (RT-PCR) and the levels of Collagen I and Collagen II protein were analyzed by Western blotting. RESULTS: In FGF2 group and FGF2 + LIPU group, it was found that the tissues of knee joint were gradually repaired following the change of time. Further, the recovery was better in FGF2 + LIPU group. Cartilage defect areas were filled with cartilage-like cells and the repair surface was fused with surrounding cartilage in FGF2 and FGF2 + LIPU groups. Wakitani scores were consistent with HE results. The expressions of FGF2 mRNA were higher in FGF2 and FGF2 + LIPU group than the model group. Western blotting results showed that the levels of Collagen I and Collagen II protein in FGF2 and FGF2 + LIPU groups were significantly increased compared with that in model group. CONCLUSIONS: FGF2 and LIPU combined application on the rabbit knees joint repair is better than FGF2 alone. FGF2 and LIPU combination can promote the synthesis and secretion of collagen in chondrocytes, promote the differentiation and maturation of chondrocytes during the repair of cartilage defects.

Transcriptomic analysis of synovial extracellular RNA following knee trauma: A pilot study.

Traumatic knee injuries often result in damage to articular cartilage and other joint structures. Such trauma is a strong risk factor for the future development and progression of osteoarthritis (OA). The molecular mechanisms and signaling pathways modulating response to knee joint trauma remain unclear. Moreover, investigations of biomarkers influencing responses have been targeted rather than broad, unbiased discovery studies. Herein, we characterize the complete complement of extracellular RNA (exRNA) in the synovial fluid of 14 subjects following knee injury. Fluid was collected during surgery from the injured knees, and from the contralateral knee in a subset, undergoing surgical repair of the ACL and/or meniscal repair/debridement. Arthroscopic grading of chondral damage in four knee compartments was performed using the Outerbridge classification. exRNA was extracted and subjected to massively parallel total RNA sequencing. Differential abundance of RNA was calculated between the subject cohorts of injured and non-injured knee, average Outerbridge score ≥0.5 and less, and chronic and acute injury duration defined as ≤4 months till surgery or longer. Overall, expression of several thousand genes was identified in the synovial fluid. Furthermore, differential expression analysis suggests a role of exRNA fragments of matrix metalloproteinases and skeletal muscle fiber genes in the response to traumatic injury. Together, these data suggest that high-throughput approaches can indicate exRNA molecular signatures following knee trauma. Future studies are required to more fully characterize the biological roles of these exRNA and the cadence of their respective release that may lead to translational treatment options for post-traumatic OA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1659-1665, 2018.

Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.

Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5CreERT2 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.