Roxadustat Attenuates Adverse Remodeling Following Myocardial Infarction in Mice.

Activation of the CXCL12/CXCR4/ACKR3 axis is known to aid myocardial repair through ischemia-triggered hypoxia-inducible factor-1α (HIF-1α). To enhance the upregulation of HIF-1α, we administered roxadustat, a novel prolyl hydroxylase inhibitor (PHI) clinically approved by the European Medicines Agency 2021 for the treatment of renal anemia, with the purpose of improving LV function and attenuating ischemic cardiomyopathy. METHODS: We evaluated roxadustat's impact on HIF-1 stimulation, cardiac remodeling, and function after MI. Therefore, we analyzed nuclear HIF-1 expression, the mRNA and protein expression of key HIF-1 target genes (RT-PCR, Western blot), inflammatory cell infiltration (immunohistochemistry), and apoptosis (TUNEL staining) 7 days after MI. Additionally, we performed echocardiography in male and female C57BL/6 mice 28 days post-MI. RESULTS: We found a substantial increase in nuclear HIF-1, associated with an upregulation of HIF-1α target genes like CXCL12/CXCR4/ACKR3 at the mRNA and protein levels. Roxadustat increased the proportion of myocardial reparative M2 CD206+ cells, suggesting beneficial alterations in immune cell migration and a trend towards reduced apoptosis. Echocardiography showed that roxadustat treatment significantly preserved ejection fraction and attenuated subsequent ventricular dilatation, thereby reducing adverse remodeling. CONCLUSIONS: Our findings suggest that roxadustat is a promising clinically approved treatment option to preserve myocardial function by attenuating adverse remodeling.

The Combination of Glucocorticoids and Hyaluronic Acid Enhances Efficacy in IL-1ß/IL-17-Treated Bovine Osteochondral Grafts Compared with Individual Application.

Patients with knee osteoarthritis often receive glucocorticoid (GC) or hyaluronic acid (HA) injections to alleviate symptoms. This study evaluated the impact of Triamcinolone Hexacetonide (a GC), HA, and a combination of both on bovine osteochondral grafts exposed to IL-1ß and IL-17 in an ex vivo culture. Metabolic activity increased with GC treatment. GCs and GCs/HA counteracted cytokine effects, with gene expressions similar to untreated controls, while HA alone did not. However, HA improved the coefficient of friction after two weeks. The highest friction values were observed in GC-containing and cytokine-treated groups. Cytokine treatment reduced tissue proteoglycan content, which HA could mitigate, especially in the GC/HA combination. This combo also effectively controlled proteoglycan release, supported by reduced sGAG release. Cytokine treatment led to surface cell death, while GCs, HA, or their combination showed protective effects against inflammation. The GC/HA combination had the best overall results, suggesting its potential as a superior treatment option for osteoarthritis.

Increased Chondroprotective Effect of Combining Hyaluronic Acid with a Glucocorticoid Compared to Separate Administration on Cytokine-Treated Osteoarthritic Chondrocytes in a 2D Culture.

Intra-articular injections of glucocorticoids (GC) or hyaluronic acid (HA) are commonly used interventions for patients suffering from knee osteoarthritis (OA). Both substances are combined to achieve a chondroprotective and anti-inflammatory effect. Clinical studies have shown benefits, but data on the cellular level are still lacking. This study aimed to investigate the effect of the GC triamcinolone hexacetonide, HA, and a mix of both substances on cytokine-treated chondrocytes in vitro. Chondrocytes isolated from human articular cartilage were seeded on 6- and 24-well plates. Mimicking OA's inflammatory state, cells were treated with IL-1ß and IL-17 for six days, whereby, after three days, test substances (10%) were added to the culture medium. Chondrocytes were analyzed on days three and six concerning their actin polymerization, expression of anabolic and catabolic genes, metabolic activity, cytokine release, and reactive oxygen species (ROS). Adding HA or GC/HA to the inflammatory culture medium increased the metabolic activity of chondrocytes, while groups containing GC reduced catabolic gene expression and the release of TNF-α. In addition, enhanced F-actin content was shown supplementing HA or GC/HA to the culture medium. Supplementing GC with HA leads to an anti-inflammatory and chondroprotective effect by diminishing the side effects of GC supplementation alone.

Hyaluronic Acid as a Carrier Supports the Effects of Glucocorticoids and Diminishes the Cytotoxic Effects of Local Anesthetics in Human Articular Chondrocytes In Vitro.

The current study aimed to investigate the cytotoxicity of co-administrating local anesthetics (LA) with glucocorticoids (GC) and hyaluronic acid (HA) in vitro. Human articular cartilage was obtained from five patients undergoing total knee arthroplasty. Chondrocytes were isolated, expanded, and seeded in 24-well plates for experimental testing. LA (lidocaine, bupivacaine, ropivacaine) were administered separately and co-administered with the following substances: GC, HA, and GC/HA. Viability was confirmed by microscopic images, flow cytometry, metabolic activity, and live/dead assay. The addition of HA and GC/HA resulted in enhanced attachment and branched appearance of the chondrocytes compared to LA and LA/GC. Metabolic activity was better in all LA co-administered with HA and GC/HA than with GC and only LA. Flow cytometry revealed the lowest cell viability in lidocaine and the highest cell viability in ropivacaine. This finding was also confirmed by live/dead assay. In conclusion, HA supports the effect of GC and reduces chondrotoxic effects of LA in vitro. Thereby, the co-administration of HA to LA and GC offers an alternative less chondrotoxic approach for treating patients with symptomatic osteoarthritis of the knee.

Decellularized Cartilage Extracellular Matrix Incorporated Silk Fibroin Hybrid Scaffolds for Endochondral Ossification Mediated Bone Regeneration.

Tissue engineering strategies promote bone regeneration for large bone defects by stimulating the osteogenesis route via intramembranous ossification in engineered grafts, which upon implantation are frequently constrained by insufficient integration and functional anastomosis of vasculature from the host tissue. In this study, we developed a hybrid biomaterial incorporating decellularized cartilage extracellular matrix (CD-ECM) as a template and silk fibroin (SF) as a carrier to assess the bone regeneration capacity of bone marrow-derived mesenchymal stem cells (hBMSC's) via the endochondral ossification (ECO) route. hBMSC's were primed two weeks for chondrogenesis, followed by six weeks for hypertrophy onto hybrid CD-ECM/SF or SF alone scaffolds and evaluated for the mineralized matrix formation in vitro. Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs, and retention of sGAG's were observed only in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC's under all conditions. Compressive modulus reached a maximum of 40 KPa after eight weeks of hypertrophic induction. µCT scanning at eight weeks indicated a cloud of denser minerals in groups after hypertrophic induction in CD-ECM/SF constructs than SF constructs. Gene expression by RT-qPCR revealed that hBMSC's expressed hypertrophic markers VEGF, COL10, RUNX2, but the absence of early hypertrophic marker ChM1 and later hypertrophic marker TSBS1 and the presence of osteogenic markers ALPL, IBSP, OSX under all conditions. Our data indicate a new method to prime hBMSC'S into the late hypertrophic stage in vitro in mechanically stable constructs for ECO-mediated bone tissue regeneration.

Biotribological Tests of Osteochondral Grafts after Treatment with Pro-Inflammatory Cytokines.

OBJECTIVE: During osteoarthritis progression, cartilage degrades in a manner that influences its biomechanical and biotribological properties, while chondrocytes reduce the synthesis of extracellular matrix components and become apoptotic. This study investigates the effects of inflammation on cartilage under biomechanical stress using biotribological tests. METHODS: Bovine osteochondral grafts from five animals were punched out from the medial condyle and treated with or without pro-inflammatory cytokines (interleukin-1ß [IL-1ß], tumor necrosis factor-α [TNF-α], IL-6) for 2 weeks. After incubation, biotribological tests were performed for 2 hours (alternating 10 minutes test and pause respectively at 39°C, 180 N, 1 Hz, and 2 mm stroke). Before and after testing, the cartilage surface was imaged with a 3-dimensional microscope. During testing, the coefficient of friction (COF) was measured, while gene expression analysis and investigation of metabolic activity of chondrocytes were carried out after testing. Histological sections of the tissue and wear debris from the test fluid were also analyzed. RESULTS: After biotribological tests, surface cracks were found in both treated and untreated osteochondral grafts. In treated grafts, the COF increased, and the proteoglycan content in the cartilage tissue decreased, leading to structural changes. Chondrocytes from treated grafts showed increased expression of genes encoding for degradative enzymes, while cartilage-specific gene expression and metabolic activity exhibited no significant differences between treated and untreated groups. No measurable difference in the wear debris in the test fluid was found. CONCLUSIONS: Treatment of osteochondral grafts with cytokines results in a significantly increased COF, while also leading to significant changes in cartilage proteoglycan content and cartilage matrix compression during biotribological tests.

Concentration-Dependent Effects of Cobalt and Chromium Ions on Osteoarthritic Chondrocytes.

OBJECTIVE: Cobalt and chromium (CoCr) ions from metal implants are released into the joint due to biotribocorrosion, inducing apoptosis and altering gene expression in various cell types. Here, we asked whether CoCr ions concentration-dependently changed viability, transcriptional activity, and inflammatory response in human articular chondrocytes. DESIGN: Human articular chondrocytes were exposed to Co (1.02-16.33 ppm) and Cr (0.42-6.66 ppm) ions and cell viability and early/late apoptosis (annexin V and 7-AAD) were assessed in 2-dimensional cell cultures using the XTT assay and flow cytometry, respectively. Changes in chondrocyte morphology were assessed using transmitted light microscopy. The effects of CoCr ions on transcriptional activity of chondrocytes were evaluated by quantitative polymerase chain reaction (qPCR). The inflammatory responses were determined by measuring the levels of released pro-inflammatory cytokines (interleukin-1ß [IL-1ß], IL-6, IL-8, and tumor necrosis factor-α [TNF-α]). RESULTS: CoCr ions concentration-dependently reduced metabolic activity and induced early and late apoptosis after 24 hours in culture. After 72 hours, the majority of chondrocytes (>90%) were apoptotic at the highest concentrations of CoCr ions (16.33/6/66 ppm). SOX9 expression was concentration-dependently enhanced, whereas expression of COL2A1 linearly decreased after 24 hours. IL-8 release was enhanced proportionally to CoCr ions levels, whereas IL-1ß, IL-6, and TNF-α levels were not affected by the treatments. CONCLUSIONS: CoCr ions showed concentration- and time-dependent effects on articular chondrocytes. Fractions of apoptotic articular chondrocytes were proportional to CoCr ion concentrations. In addition, metabolic activity and expression of chondrocyte-specific genes were decreased by CoCr ions. Furthermore, exposure to CoCr ions caused a release of pro-inflammatory cytokines.

Effect of osteochondral graft orientation in a biotribological test system.

Autologous osteochondral transplantation (AOT) utilizing autografts is a widely used technique for the treatment of small-to-medium cartilage defects occurring in knee and ankle joints. The application of viable cartilage and bone ensures proper integration, early weight bearing, as well as restoration of biomechanical and biotribological properties. However, alignment of the autografts onto the defect site remains a pivotal aspect of reinstating the properties of the joint toward successful autograft integration. This is the first study to perform tests with different orientations of osteochondral grafts in a cartilage-on-cartilage test system. The objective was to estimate if there are differences between aligned and 90°-rotated grafts concerning molecular biological and biomechanical parameters. Tissue viability, assessed by XTT assay indicated lower metabolic activity in tested osteochondral grafts (aligned, p = 0.0148 and 90°-rotated, p = 0.0760) in favor of a higher anabolic gene expression (aligned, p = 0.0030 and 90°-rotated, 0.0027). Tissue structure was evaluated by Safranin O histology and microscopic images of the surface. Aligned and 90°-rotated grafts revealed no apparent differences between proteoglycan content or cracks and fissures on the cartilage surface. Test medium analyzed after tribological tests for their sulfated glycosaminoglycan content revealed no differences (p = 0.3282). During the tests, both the friction coefficient and the relative displacement between the two cartilage surfaces were measured, with no significant difference in both parameters (COF, p = 0.2232 and relative displacement, p = 0.3185). From the methods we deployed, this study can infer that there are no differences between aligned and 90°-rotated osteochondral grafts after tribological tests in the used ex vivo tissue model. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res.

Redifferentiation of Articular Chondrocytes by Hyperacute Serum and Platelet Rich Plasma in Collagen Type I Hydrogels.

Matrix-assisted autologous chondrocyte transplantation (MACT) for focal articular cartilage defects often fails to produce adequate cartilage-specific extracellular matrix in vitro and upon transplantation results in fibrocartilage due to dedifferentiation during cell expansion. This study aimed to redifferentiate the chondrocytes through supplementation of blood-products, such as hyperacute serum (HAS) and platelet-rich plasma (PRP) in vitro. Dedifferentiated monolayer chondrocytes embedded onto collagen type I hydrogels were redifferentiated through supplementation of 10% HAS or 10% PRP for 14 days in vitro under normoxia (20% O2) and hypoxia (4% O2). Cell proliferation was increased by supplementing HAS for 14 days (p < 0.05) or by interchanging from HAS to PRP during Days 7⁻14 (p < 0.05). Sulfated glycosaminoglycan (sGAG) content was deposited under both HAS, and PRP for 14 days and an interchange during Days 7⁻14 depleted the sGAG content to a certain extent. PRP enhanced the gene expression of anabolic markers COL2A1 and SOX9 (p < 0.05), whereas HAS enhanced COL1A1 production. An interchange led to reduction of COL1A1 and COL2A1 expression marked by increased MMP13 expression (p < 0.05). Chondrocytes secreted less IL-6 and more PDGF-BB under PRP for 14 days (p < 0.0.5). Hypoxia enhanced TGF-ß1 and BMP-2 release in both HAS and PRP. Our study demonstrates a new approach for chondrocyte redifferentiation.

Platelet-Rich Plasma Supports Proliferation and Redifferentiation of Chondrocytes during In Vitro Expansion.

Articular cartilage regeneration is insufficient to restore sports injuries or defects that can occur from trauma. Treatment options for cartilage repair include autologous chondrocyte implantation (ACI) by isolation, expansion, and reimplantation of healthy donor chondrocytes. Chondrocyte expansion onto 2D substrates leads to dedifferentiation and loss of the cellular phenotype. We aimed to overcome the state of dedifferentiation by biochemical stimuli with platelet derivatives such as platelet-rich plasma (PRP) and hyperacute serum (HAS) to achieve sufficient cell numbers in combination with variable oxygen tension. Human articular chondrocytes from osteoarthritic (OA) cartilage chondrocytes were switched from 10% FCS supplementation to either 10% PRP or 10% HAS after initial passaging for further experiments under normoxic (20% O2) or hypoxic (1% O2) conditions. An XTT assay measured the effect of PRP or HAS on the cell proliferation at 3, 6, and 9 days. The chondrogenic redifferentiation potential of dedifferentiated chondrocytes was determined with reverse transcriptase quantitative real-time PCR for markers of expression for type II collagen (COL2A1), type I collagen (COL1A1), and matrix metalloproteinases MMP3, matrix metalloproteinase 13 (MMP13) at 24 and 72 h. Measured protein levels of 100% PRP or HAS by multiplex quantification revealed basic fibroblast growth factor, G-CSF, and PDGF were significantly higher in PRP than in HAS (p < 0.05) but LEPTIN levels did not differ. The quantified protein levels did not differ when isolated from same donors at a different time. Chondrocyte proliferation indicated that supplementation of 10% HAS enhanced the proliferation rate compared to 10% PRP or 10% FCS at 6 and 9 days significantly (p < 0.05). mRNA levels for expression of COL1A1 were significantly downregulated (p < 0.05) when cultured with 10% PRP than 10% HAS or 10% FCS under normoxic/hypoxic conditions. COL2A1 was significantly upregulated (p < 0.05) in PRP than 10% HAS or 10% FCS. MMP3 expression was downregulated after 72 h under all conditions. MMP13 was upregulated with 10% PRP at both 24 and 72 h but significantly downregulated under hypoxia (1% O2) for all circumstances. While HAS has its effect on chondrocyte proliferation, PRP enhances both proliferation and redifferentiation of dedifferentiated chondrocytes. PRP can replace standard usage of FCS for chondrogenic priming and expansion as implications for clinical use such as ACI procedures.

Hyaluronan thiomer gel/matrix mediated healing of articular cartilage defects in New Zealand White rabbits-a pilot study.

BACKGROUND: Articular cartilage defects are limited to their regenerative potential in human adults. Our current study evaluates tissue regeneration in a surgically induced empty defect site with hyaluronan thiomer as a provisional scaffold in a gel/matrix combination without cells on rabbit models to restore tissue formation. METHODS: An osteochondral defect of 4 mm in diameter and 5 mm in depth was induced by mechanical drilling in the femoral center of the trochlea in 18 New Zealand White rabbits. Previously evaluated from an in vitro study hyaluronan thiomer matrix, and a hyaluronan thiomer gel was used to treat the defect. As a control, the defect was left untreated. During the whole study, rabbits were clinically examined and after 4 (n = 3) or 12 (n = 3) weeks, the rabbits were sacrificed. Joints were evaluated macroscopically (Brittberg score) and by histology (O'Driscoll score). Synovial cells from the synovial fluid smear were histopathologically evaluated. RESULTS: The healing of the defects varied intra-group wise at the first observation period. After 12 weeks the results concerning the cartilage repair score were inhomogeneous within each group, while the macroscopic analysis was more homogenous. In the synovial fluid smear, the mean score of infiltrated synovial and non-synovial cells was slightly increased after 4 weeks and slightly decreased after 12 weeks in both the treatment groups in comparison to the untreated control. CONCLUSIONS: Taken together with results from the in vivo study indicated that implantation of hyaluronan thiomer as a combination of gel and matrix might enhance articular cartilage regeneration in an empty defect. Despite their benefits, the intrinsic healing capacity of New Zealand rabbits is a limitation for comparative test subject in pre-clinical models of cartilage defects.

Chondrogenic Gene Expression Differences between Chondrocytes from Osteoarthritic and Non-OA Trauma Joints in a 3D Collagen Type I Hydrogel.

Objective The purpose of the current study was to compare the donor age variation of chondrocytes from non-OA (osteoarthritic) trauma joints in patients of young to middle age (20.5 ± 3.7, 31.8 ± 1.9, 41.9 ± 4.1 years) embedded in matrix-associated autologous chondrocyte transplantation (MACT) grafts (CaReS). The chondrocyte-specific gene expression of CaReS grafts were then compared to chondrocytes from OA joints (in patients aged 63.8 ± 10 years) embedded in a collagen type I hydrogel. Design OA chondrocytes and articular chondrocyte-laden grafts were cultured over 14 days in chondrogenic growth medium. We performed reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to evaluate the mRNA expression levels of chondrocyte-specific and hypertrophic markers. Results Gene expression analysis with RT-qPCR revealed no significant difference in chondrocyte-specific genes ( COL2A1, ACAN, SOX9, SOX5, SOX6) among 3 different age group of patients with CaReS grafts. In a comparative analysis of OA chondrocytes to articular chondrocytes, chondrogenic markers ( COL2A1, SOX6) exhibited higher expression in OA chondrocytes ( P < 0.05). Hypertrophic or OA cartilage pathogenesis marker ( MMP3, MMP13) expression was higher and COL1A1 had significantly lower expression ( P < 0.05) in OA chondrocytes than articular chondrocytes when cultivated in collagen type I hydrogels. Conclusion In summary, we identify that donor age variation does not influence the chondrogenic gene expression of the CaReS system. We also identified that freshly isolated OA chondrocytes embedded in collagen type I hydrogels can exhibit chondrogenic gene expression as observed in articular chondrocytes on the CaReS grafts. Transforming OA chondrocytes to articular chondrocytes can be regarded as an alternative option in the MACT technique.

Chondroprotective effect of high-molecular-weight hyaluronic acid on osteoarthritic chondrocytes in a co-cultivation inflammation model with M1 macrophages.

BACKGROUND: Osteoarthritis (OA) is described by an imbalance between anabolic and catabolic processes in the affected joint. This dysregulation of metabolism affects not only chondrocytes within cartilage tissue but also the cells of the synovial membrane across the border of the joint. An important factor in OA is the low viscosity of the synovial fluid. High-molecular-weight hyaluronic acid (HA) can be used to increase the viscosity and also reduce inflammatory processes. The purpose was to establish an in vitro inflammation model and to evaluate the effects of high-molecular-weight HA in a co-cultivation inflammation model of osteoarthritic chondrocytes and M1 macrophages. METHODS: For the establishment of the inflammation model THP-1 cells were, at first, differentiated to M0 macrophages and then activated to the M1 subtype after 5 days of resting period. Surface markers, cytokine release, and gene expression, were analyzed to examine the successful differentiation. In the inflammation model, the defined M1 macrophages were co-cultivated with osteoarthritic chondrocytes for 2 days, with and without the addition of 10 % HA and further analyzed for chondrogenic gene expression markers and the release of cytokines in the supernatant. RESULTS: The differentiation and activation process was successful as M1 macrophages expressed higher levels of pro-inflammatory cytokines and specific genes. Similarly, the surface marker CD14 was significantly decreased compared to M0 macrophages. For the co-culture system, the analysis of gene expression showed that HA increased the expression of cartilage-specific genes while catabolic-encoding genes exhibited lower expression levels than the control group. This positive effect of HA was also demonstrated by the measurement of pro-inflammatory cytokines, as their level decreased. CONCLUSION: Our study implies that high-molecular-weight HA has a chondroprotective effect in the present co-cultivation inflammation model, as it decreases pro-inflammatory cytokines and increases anabolic factors.

Correlation Analysis of SOX9, -5, and -6 as well as COL2A1 and Aggrecan Gene Expression of Collagen I Implant-Derived and Osteoarthritic Chondrocytes.

OBJECTIVE: Matrix-assisted autologous chondrocyte implantation is frequently applied to replace damaged cartilage in order to support tissue regeneration or repair and to prevent progressive cartilage degradation and osteoarthritis. Its application, however, is limited to primary defects and contraindicated in the case of osteoarthritis that is partially ascribed to dedifferentiation and phenotype alterations of chondrocytes obtainable from patients' biopsies. The differentiation state of chondrocytes is reflected at the level of structural gene (COL2A1, ACAN, COL1A1) and transcription factor (SOX9, 5, 6) expression. METHODS/DESIGN: We determined the mRNA abundances of COL2A1, ACAN, and COL1A1as well as SOX9, -5, and -6 of freshly isolated and passaged collagen I implant-derived and osteoarthritic chondrocytes via reverse transcription-polymerase chain reaction. Moreover, we analyzed the correlation of structural and transcription factor gene expression. Thus, we were able to evaluate the impact of the mRNA levels of transcription factors on the expression of cartilage-specific structural genes. RESULTS: Significant differences were obtained (1) for freshly isolated osteoarthritic versus collagen I implant-derived chondrocytes, (2) due to passaging of the respective cell sources, (3) for osteoarthritic versus nonosteoarthritic chondrocytes, and (4) for COL2A1 versus ACAN expression with respect to the coherence with SOX9, -5, and -6 transcript levels. CONCLUSION: Our results might contribute to a better understanding of the transcriptional regulation of structural gene expression of chondrocytes with implications for their use in matrix-assisted autologous chondrocyte implantation.