Safe and anti-inflammatory plant-based natural products present an increasing focus in the treatment of chronic inflammatory diseases such as osteoarthritis or inflammatory bowel diseases. Among them, saffron, a spice derived from the stigma of Crocus sativus, could have anti-inflammatory properties and would be therefore a promising therapeutic agent for the treatment of such conditions. However, the anti-inflammatory molecular mechanisms of saffron in humans are still understudied and unclear. In this study, combining human serum metabolites and cell cultures, we evaluated the effect of circulating metabolites from the consumption of a patented saffron extract (Safr\'InsideTM) on the chondrocytes and colon epithelial cell responses to inflammatory stress. Parametric or non-parametric Analysis of Variance with post hoc tests was performed. We demonstrated that human serum containing metabolites from saffron intake attenuated IL-1β-stimulated production of PGE2 and MMP-13 in chondrocyte cells and limited the increase in ICAM-1, MCP-1, iNOS, and MMP-3 in human epithelial cells following combined IL-1β and TNF-α inflammatory stimulation. Altogether, these data provide new findings into the mechanisms underlying the beneficial effects of saffron on chondrocytes and enterocyte cells at the cellular level and in the context of chronic inflammatory disorders.

BACKGROUND: CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources.
METHODS: We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β).
RESULTS: We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions.
CONCLUSIONS: We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.

The molecular niche of an osteoarthritic microenvironment comprises the native chondrocytes, the circulatory immune cells, and their respective inflammatory mediators. Although M2 macrophages infiltrate the joint tissue during osteoarthritis (OA) to initiate cartilage repair, the mechanistic crosstalk that dwells underneath is still unknown. Our study established a co-culture system of human OA chondrocytes and M2 macrophages in 3D spheroids and 3D bioprinted silk-gelatin constructs. It is already well established that Silk fibroin-gelatin bioink supports chondrogenic differentiation due to upregulation of the Wnt/β-catenin pathway. Additionally, the presence of anti-inflammatory M2 macrophages significantly upregulated the expression of chondrogenic biomarkers (COL-II, ACAN) with an attenuated expression of the chondrocyte hypertrophy (COL-X), chondrocyte dedifferentiation (COL-I) and matrix catabolism (MMP-1 and MMP-13) genes even in the absence of the interleukins. Furthermore, the 3D bioprinted co-culture model displayed an upper hand in stimulating cartilage regeneration and OA inhibition than the spheroid model, underlining the role of silk fibroin-gelatin in encouraging chondrogenesis. Additionally, the 3D bioprinted silk-gelatin constructs further supported the maintenance of stable anti-inflammatory phenotype of M2 macrophage. Thus, the direct interaction between the primary OAC and M2 macrophages in the 3D context, along with the release of the soluble anti-inflammatory factors by the M2 cells, significantly contributed to a better understanding of the molecular mechanisms responsible for immune cell-mediated OA healing.

BACKGROUND: Osteoarthritis (OA) is an inflammatory response in chondrocytes, causing extracellular matrix (ECM) degradation and cartilage destruction, affecting millions of people worldwide. Chinese herbal formulae BuShen JianGu Fang (BSJGF) has been clinically applied for treating OA-related syndromes, but the underlying mechanism still unclear.
METHODS: The components of BSJGF were analyzed by liquid chromatography-mass spectrometry (LC-MS). To make a traumatic OA model, the anterior cruciate ligament of 6-8-week-old male SD rats were cut and then the 0.4 mm metal was used to destroy the knee joint cartilage. OA severity was assessed by histological and Micro-CT. Mouse primary chondrocytes were utilized to investigate the mechanism of BSJGF alleviate osteoarthritis, which was examined by RNA-seq technology combined with a series of functional experiments.
RESULTS: A total 619 components were identified by LC-MS. In vivo, BSJGF treatment result in a higher articular cartilage tissue area compared to IL-1β group. Treatment also significantly increased Tb.Th, BV/TV and BMD of subchondral bone (SCB), which implied a protective effect on maintaining the stabilization of SCB microstructure. In vitro results indicated BSJGF promoted chondrocyte proliferation, increased the expression level of cartilage-specific genes (Sox9, Col2a1, Acan) and synthesized acidic polysaccharide, while inhibiting the release of catabolic enzymes and production of reactive oxygen species (ROS) induced by IL-1β. Transcriptome analysis showed that there were 1471 and 4904 differential genes between IL-1β group and blank group, BSJGF group and IL-1β group, respectively, including matrix synthesis related genes (Col2a1, H19, Acan etc.), inflammation related genes (Comp, Pcsk6, Fgfr3 etc.) and oxidative stress related genes (Gm26917, Bcat1, Sod1 etc.). Furthermore, KEGG analysis and validation results showed that BSJGF reduces OA-mediated inflammation and cartilage damaged due to modulation of NF-κB/Sox9 signaling axis.
CONCLUSIONS: The innovation of the present study was the elucidation of the alleviating cartilage degradation effect of BSJGF in vivo and in vitro and discovery of its mechanism through RNA-seq combined with function experiments, which provides a biological rationale for the clinical application of BSJGF for OA treatment.

BACKGROUND: Chondrocytes are the only cell components in the cartilage, which has the poor regeneration ability. Thus, repairing damaged cartilage remains a huge challenge. Sika deer antlers are mainly composed of cartilaginous tissues that have an astonishing capacity for repair and renewal. Our previous study has demonstrated the transforming growth factor β (TGF-β1) is considered to be a key molecule involved in rapid growth, with the strongest expression in the cartilage layer. However, it remains to be clarified whether deer TGF-β1 has significantly different function from other species such as mouse, and what is the molecular mechanism of regulating cartilage growth.
METHODS: Primary chondrocytes was collected from new born mouse rib cartilage. The effect of TGF-β1 on primary chondrocytes viability was elucidated by RNA sequencing (RNA-seq) technology combined with validation methods such as quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence assay (IFA). Differential expression genes were identified using the DEGseq package.
RESULTS: Our results demonstrated that the overexpression of deer TGF-β1 possibly promoted chondrocyte proliferation and extracellular matrix (ECM) synthesis, while simultaneously suppressing chondrocyte differentiation through regulating transcription factors, growth factors, ECM related genes, proliferation and differentiation marker genes, such as Comp, Fgfr3, Atf4, Stat1 etc., and signaling pathways such as the MAPK signaling pathway, inflammatory mediator regulation of TRP channels etc. In addition, by comparing the amino acid sequence and structures between the deer TGF-β1 and mouse TGF-β1, we found that deer TGF-β1 and mouse TGF-β1 proteins are mainly structurally different in arm domains, which is the main functional domain. Phenotypic identification results showed that deer TGF-β1 may has stronger function than mouse TGF-β1.
CONCLUSIONS: ​These results suggested that deer TGF-β1 has the ability to promote chondrogenesis by regulating chondrocyte proliferation, differentiation and ECM synthesis. This study provides insights into the molecular mechanisms underlying the effects of deer TGF-β1 on chondrocyte viability.

Osteoarthritis (OA) is associated with oxidative stress injury (OSI) and inflammatory responses in chondrocytes. This study sought to explore the mechanism of high mobility group A1 (HMGA1) in interleukin-1beta (IL-1β)-induced OSI and inflammatory responses in primary chondrocytes.
Primary chondrocytes were cultured and treated with IL-1β to establish an OA-cell model. Levels of HMGA1, Jumonji domain-containing 3 (JMJD3), and ZEB1 in cells were determined by real-time quantitative polymerase chain reaction and Western blot analysis. Cell viability, contents of tumor necrosis factor-α, IL-6, and IL-10, reactive oxygen species level, and glutathione peroxidase activity were assessed by the cell counting kit-8 assay, enzyme-linked immunosorbent assay, and assay kits. Enrichment levels of HMGA1 on the JMJD3 promoter and enrichment levels of JMJD3 and trimethylated histone H3 at lysine 27 (H3K27me3) on the ZEB1 promoter region were determined by chromatin immunoprecipitation. Functional rescue experiments were performed to analyze the impact of ZEB1 and JMJD3 on IL-1β-induced chondrocytes.
IL-1β treatment induced HMGA1 upregulation, OSI, and inflammatory responses in chondrocytes. HMGA1 downregulation reduced IL-1β-induced OSI and inflammatory responses in chondrocytes. Mechanically, HMGA1 was bound to the JMJD3 promoter to promote JMJD3 transcription, and JMJD3 induced demethylation of H3K27me3 on the ZEB1 promoter to promote ZEB1 transcription. Overexpression of JMJD3 or ZEB1 neutralized the protective role of silencing HMGA1 in IL-1β-induced chondrocytes.
HMGA1 aggravated IL-1β-induced OSI and inflammatory responses in chondrocytes through the promotion of JMJD3 and ZEB1.

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

OBJECTIVE: Obesity is a known risk factor for knee osteoarthritis (OA). Diabetes has been associated with progression of OA and metformin is the first-line treatment in type 2 diabetes. The effect of the body mass index (BMI) and metformin on the expression of certain matrix genes in human chondrocytes is unclear. The purpose of this study was to investigate the effect of BMI and metformin on the expression of matrix genes in primary human chondrocytes.
METHODS: Adult female patients undergoing knee arthroplasty for end-stage OA were enrolled. Primary chondrocytes were cultivated and stimulated with metformin. Matrix gene expression was analyzed using polymerase chain reaction. Clinical data were used in multivariable regression models to assess the influence of BMI and metformin stimulation on gene expression.
RESULTS: A total of 14 patients were analyzed. BMI was a predictor of increased expression in ADAMTS5 (β = -0.11, P = 0.03). Metformin slightly reduced expression in ADAMTS5 (β = 0.34, P = 0.04), HIF-1a (β = 0.39, P = 0.04), IL4 (β = 0.30, P = 0.02), MMP1 (β = 0.47, P < 0.01), and SOX9 (β = 0.37, P = 0.03). The hip-knee-ankle angle and proton pump inhibitors (PPIs) intake were associated with reduced SOX9 expression (β = 0.23, P < 0.01; β = 2.39, P < 0.01). Higher C-reactive protein (CRP) levels were associated with increased MMP1 expression (β = -0.16, P = 0.02).
CONCLUSIONS: We found that BMI exerts a destructive effect via induction of ADAMTS5. Metformin reduced the expression of catabolic genes ADAMTS5 and MMP1 and might play a role in disease prevention. Limb malalignment and PPI intake was associated with a reduced expression of SOX9, and higher CRP levels correlated with increased MMP1 expression, indicating a destructive process.

There is an unmet need for a single-stage cartilage repair treatment that is cost-effective and chondrocyte-based.
To evaluate the safety and preliminary efficacy of autologous freshly isolated primary chondrocytes and bone marrow mononucleated cells (MNCs) seeded into a PolyActive scaffold in patients with symptomatic cartilage lesions of the knee.
Case series; Level of evidence, 4.
A total of 40 patients with symptomatic knee cartilage lesions were treated with freshly isolated autologous chondrocytes combined with bone marrow MNCs delivered in a biodegradable load-bearing scaffold. The treatment requires only 1 surgical intervention and is potentially a cost-effective alternative to autologous chondrocyte implantation. The primary chondrocytes and bone marrow MNCs were isolated, washed, counted, mixed, and seeded into a load-bearing scaffold in the operating room. Patients were followed up at 3, 6, 12, 18, and 24 months. Primary endpoints were treatment-related adverse events up to 3 months, adverse implant effects between 3 and 24 months, and the implant success rate at 3 months as measured by lesion filling.
Successful lesion filling (≥67% on magnetic resonance imaging) was found in 40 patients at 3 months and in 32 of the 32 patients analyzed at 24 months. Significant improvement over baseline was found for visual analog scale for pain from 3 months onward; Knee injury and Osteoarthritis Outcome Score (KOOS)-Pain and KOOS-Activities of Daily Living from 6 months onward; for KOOS-Symptoms and Stiffness, KOOS-Quality of Life and International Knee Documentation Committee from 12 months onward; and for KOOS-Sport and Recreation from 18 months onward. Hyaline-like repair tissue was found in 22 of 31 patients available for biopsy. Arthralgia and joint effusion were the most common adverse events. Scaffold delamination and adhesions led to removal of the implant in 2 patients.
The treatment of knee cartilage lesions with autologous primary chondrocytes and bone marrow MNCs, both isolated and seeded into a load-bearing PolyActive scaffold within a single surgical intervention, is safe and clinically effective. Good lesion fill and sustained clinically important and statistically significant improvement in all patient-reported outcome scores were found throughout the 24-month study. Hyaline-like cartilage was observed on biopsy specimen in at least 22 of the 40 patients.
NCT01041885 (ClinicalTrials.gov identifier).

OBJECTIVE: Homeobox genes of the Hox class are required for proper patterning of skeletal elements and play a role in cartilage differentiation. In transgenic mice with overexpression of Hoxc8 and Hoxd4 during cartilage development, the authors observed severe defects, namely, physical instability of cartilage, accumulation of immature chondrocytes, and decreased maturation to hypertrophy. To define the molecular basis underlying these defects, the authors performed gene expression profiling using the Affymetrix microarray platform.
RESULTS: Primary chondrocytes were isolated from Hoxc8- and Hoxd4-transgenic mouse embryo rib cartilage at 18.5 days of gestation. In both cases, differentially expressed genes were identified that have a role in cell proliferation and cell cycle regulation. A comparison between the controls for both experimental groups did not reveal significant differences, as expected. However, the repertoires of differentially expressed genes were found not to overlap between Hoxc8- and Hoxd4-transgenic cartilage. This included different Wnt genes, cell cycle, and apoptosis regulators.
CONCLUSIONS: Overexpression of Hoxc8 and Hoxd4 transcription factors alters transcriptional profiles in chondrocytes at E18.5. The differences in repertoires of altered gene expression between the 2 transgenic conditions suggest that the molecular mechanisms underlying the cartilage defects may be different in both transgenic paradigms, despite apparently similar phenotypes.