Aims. Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant (gynura bicolor), has demonstrated anti-inflammatory properties in various diseases. We aimed to explore the chondroprotective effect of DHCA on OA and its potential mechanism. Methods. In vitro, interleukin-1 beta (IL-1β) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of
Aims. To evaluate the effect of ultrasound-targeted simvastatin-loaded microbubble destruction (UTMDSV) for alleviation of the progression of osteoarthritis (OA) in rabbits through modulation of the peroxisome proliferator-activated receptor (PPARγ). Methods. In vitro, OA chondrocytes were treated with ultrasound (US), US-targeted microbubble destruction (UTMD), simvastatin (SV), and UTMDSV on alternate days for four weeks. Chondrocytes were also treated with PPARγ inhibitor, PPARγ inhibitor+ UTMDSV, and UTMDSV. The cholesterol efflux rate and triglyceride levels were measured using an assay kit and oil red O staining, respectively. In vivo, the OA rabbits were treated with a single intra-articular injection of UTMD, SV, and UTMDSV every seven days for four weeks. Cartilage histopathology was assessed by safranin-O staining and the Mankin score. Total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) in rabbit knee synovial fluid were detected by enzyme-marker assay. Aggrecan,
Aims. Osteoarthritis (OA) is the most common chronic pathema of human joints. The pathogenesis is complex, involving physiological and mechanical factors. In previous studies, we found that ferroptosis is intimately related to OA, while the role of Sat1 in chondrocyte ferroptosis and OA, as well as the underlying mechanism, remains unclear. Methods. In this study, interleukin-1β (IL-1β) was used to simulate inflammation and Erastin was used to simulate ferroptosis in vitro. We used small interfering RNA (siRNA) to knock down the spermidine/spermine N1-acetyltransferase 1 (Sat1) and arachidonate 15-lipoxygenase (Alox15), and examined damage-associated events including inflammation, ferroptosis, and oxidative stress of chondrocytes. In addition, a destabilization of the medial meniscus (DMM) mouse model of OA induced by surgery was established to investigate the role of Sat1 inhibition in OA progression. Results. The results showed that inhibition of Sat1 expression can reduce inflammation, ferroptosis changes, reactive oxygen species (ROS) level, and lipid-ROS accumulation induced by IL-1β and Erastin. Knockdown of Sat1 promotes nuclear factor-E2-related factor 2 (Nrf2) signalling. Additionally, knockdown Alox15 can alleviate the inflammation-related protein expression induced by IL-1β and ferroptosis-related protein expression induced by Erastin. Furthermore, knockdown Nrf2 can reverse these protein expression alterations. Finally, intra-articular injection of diminazene aceturate (DA), an inhibitor of Sat1, enhanced type
Porcine and fish by-products in particular are rich sources for collagen, which is the main component of the extracellular matrix (ECM). Although there are studies investigating different collagen derived from various tissue sources for the purpose of creating biomaterials, the comparison of biophysical, biochemical and biological properties of type
The anterior cruciate ligament (ACL) is the connective tissue located at the end of long bones providing stability to the knee joint. After tear or rupture clinical reconstruction of the tissue remains a challenge due to the particular mechanical properties required for proper functioning of the tissue. The outstanding mechanical properties of the ACL are characterized by a viscoelastic behavior responsible of the dissipation of the loads that are transmitted to the bone. These mechanical properties are the result of a very specialized graded extracellular matrix that transitions smoothly between the heterotypic cells, stiffness and composition of the ACL and the adjacent bone. Thus, mimicking the zonal biochemical composition, cellular phenotype and organization are key to reset the proper functioning of the ACL. We have previously shown how the biochemical composition presented to cells in electrospun scaffolds results in haptokinesis, reverting contact-guidance effects. [1]. Here, we demonstrate that contact guidance can also be disrupted by structural parameters in aligned wavy scaffolds. The presentation of a wavy fiber arrangement affected the cell organization and the deposition of a specific ECM characteristic of fibrocartilage. Cells cultured in wavy scaffolds grew in aggregates, deposited an abundant ECM rich in fibronectin and
In the native articular cartilage microenvironment, chondrocytes are constantly subjected to dynamic physical stimuli that maintains tissue homeostasis. They produce extra cellular matrix (ECM) components such as
Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. Quality of cartilaginous repair tissue following BMSC transplantation has been shown to correlate with functional outcome. Therefore, tissue-engineering variables, such as cell expansion environment and seeding density of scaffolds, are currently under investigation. The objectives of this study were to demonstrate chondrogenic differentiation of BMSCs seeded within a collagen I scaffold following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments, and assess the impact of seeding density on in vitro chondrogenesis. It was hypothesised that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 million cells/cm3. Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing expansion medium, and seeded within collagen I scaffolds (6 mm diameter, 3.5 mm thickness and 0.115 ± 0.020 mm pore size; Integra LifeSciences Corp.) at densities of 50, 10, 5, 1, and 0.5 million BMSCs/cm3. For 3D isolation and expansion, bone marrow aspirates containing known quantities of mononucleated cells (BMNCs) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 million BMNCs/cm3 and cultured in expansion medium for an equivalent duration to 2D expansion. All cell-scaffold constructs were differentiated in vitro in chondrogenic medium containing transforming growth factor-beta three for 21 days and assessed with RT-qPCR, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and
The biological understanding for the disease progression osteoarthritis (OA) has uncovered specific biomarkers from either synovial fluid, articular chondrocytes or synoviocytes that can be used to diagnose the disease. Examples of these biomarkers include interleukin-1β (IL-1β) or
In a healthy joint, mechanical loading increases matrix synthesis and maintains cell phenotype, while reducing catabolic activities. It activates several pathways, most of them yet largely unknown, with integrins, TGF-β, canonical (Erk 1/2) and stress-activated (JNK) MAPK playing a key role. Degenerative joint diseases are characterized by Wnt upregulation and by the presence of proteolytic fibronectin fragments (FB-fs). Despite they are known to impair some of the aforementioned pathways, little is known on their modulatory effect on cartilage mechanoresponsiveness. This study aims at investigating the effect of mechanical loading in healthy and in vitro diseased cartilage models using pro-hypertrophic Wnt agonist CHIR99021 and the pro-catabolic FB-fs 30 kDa. Human primary chondrocytes from OA patients have been grown in alginate hydrogels for one week, prior to be incubated for 4 days with 3μM CHIR99021 or 1 μM FB-fs. Human cartilage explants isolated from OA patients have incubated 4 days with 3 μM CHIR99021 or 1 μM FB-fs. Both groups have then been mechanically stimulated (unconfined compression, 10% displacement, 1.5 hours, 1 Hz), using a BioDynamic bioreactor 5270 from TA Instruments. Expression of collagen type I, II and X, aggrecan, ALK-1, ALK-5, αV, α5 and β1 integrins, TGF-β1 have been assessed by Real Time-PCR and normalized with the expression of S29. Percentage of phosphorylated Smad2, Smad1 and JNK were determined through western blot. TGF-β1 content was quantified by sandwich ELISA; MMP-13 and GAG by western blot and DMMB assay, respectively. At least three biological replicates were used. ANOVA test was used for parametric analysis; Kruskal-Wallis and Mann-Whitney post hoc test for non-parametric. Preliminary data show that compression increased
There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides osteoinduction, there is growing evidence of also anti-inflammatory, angiogenetic and neuroprotective effects of biodegradable magnesium-based biomaterials. Their use for the treatment of cartilage lesions in contrast is not well-evaluated yet. Mg-cylinders were analysed in an in vitro and in vivo OA model. In vitro, SCP-1 stem cell line was analysed under inflammatory conditions and Mg-impact. In vivo, small Mg- and WE43 alloy-cylinders (1mm × 0,5mm) were implanted into the subchondral bone of the knee joint of 24 NZW rabbits after establishment of OA. As control, another 12 rabbits received only drill-holes. µCT-scan were performed and assessed for changes in bone volume and density. After euthanasia, cartilage was evaluated macroscopically and histologically after Safranin-O-staining. Furthermore, staining with CD271 directed antibody was performed to assess neuro-reactivity. In vitro, an increased gene expression of extracellular matrix proteins as
In the field of tissue engineering (TE), mainly two approaches have been widely studied and utilised throughout the last two decades. Ovsianikov et al. proposed a third strategy for tissue engineering to combine the advantages of the scaffold-based and scaffold-free approach [1]. We utilise the third strategy for TE by fabrication of cell spheroids that are reinforced by microscaffolds, called tissue units (TUs). Aim of the presented study is to differentiate TUs towards a chondrogenic phenotype to show the self-assembly of a millimetre sized cartilage-like tissue in a bottom-up TE approach in vitro. Two-Photon polymerization (2PP) was utilised to fabricate highly porous microscaffolds with a diameter of 300 µm. The biocompatible and biodegradable, resin Degrad INX (supplied from Xpect INX, Ghent, Belgium) was used for 3D-printing. Each microscaffold was seeded with 4000 human adipose derived stem cells (hASCs) in low-adhesive 96-well plates to allow spheroid formation. TUs were differentiated towards the chondrogenic lineage by application of chondrogenic media, subsequently merged in a cylindrical agarose mold, to fuse into a connected tissue with a diameter of ~1.8 mm and a height of 8 mm. The characterization of TUs differentiated towards the chondrogenic phenotype included gene expression and protein analysis. Furthermore, immunohistochemically staining for
Meniscus tears have been treated using partial meniscectomy to relieve pain in patients, although this leads to the onset of early osteoarthritis (OA). Cell-based therapies can help preserve the meniscus, although the presence of inflammatory cytokines compromises clinical outcomes. Anti-inflammatory drugs (e.g. celecoxib), can help to reduce pain in patients and in vitro studies suggest a beneficial effect on cytokine inhibited matrix content. Previously, we have demonstrated that the inhibitory effects of IL-1β can be countered by culture under low oxygen tension or physioxia. The present study sought to understand whether physioxia, celecoxib or combined application can counter the inhibitory effects IL-1β inhibited meniscus cells. Human avascular and vascular meniscus cells (n =3) were isolated and expanded under 20% (hyperoxia) or 2% (physioxia) oxygen. Cells were seeded into collagen scaffolds (Geistlich, Wolhusen) and cultured for 28 days either in the presence of 0.1ng/mL IL-1β, 5µg/mL celecoxib or both under their expansion oxygen conditions. Histological (DMMB, collagen I and
Cartilage lesions often undergo irreversible progression due to low self-repair capability of this tissue. Tissue engineered approaches based in extrusion bioprinting of constructs loaded with stem cell spheroids may offer valuable alternatives for the treatment of cartilage lesions. Human mesenchymal stromal cell (hMSC) spheroids can be chondrogenically differentiated faster and more efficiently than single cells. This approach allows obtaining larger tissues in a rapid, controlled and reproducible way. However, it is challenging to control tissue architecture, construct stability, and cell viability during maturation. In this study we aimed at the development of a reproducible bioprinting process followed by post-bioprinting chondrogenic differentiation procedure using large quantities of hMSC spheroids encapsulated in a xanthan gum-alginate hydrogel. Multi-layered constructs were bioprinted, ionically crosslinked, and chondrogenically differentiated for 28 days. The expression of glycosaminoglycan,
Silk fibroin (SF) has been used as a scaffold for cartilage tissue engineering. Different silkworms strain produced different protein. Also, molecular weight of SF depends on extraction method. We hypothesised that strain of silkworm and method of SF extraction would effect biological properties of SF scaffold. Therefore, cell viability and chondrogenic gene expression of human chondrogenic progenitor cells (HCPCs) treated with SF from 10 silkworm strains and two common SF extraction methods were investigate in this study. Twenty g of 10 strains silk cocoons were separately degummed in 0.02M Na2CO3 solution and dissolved in 100๐C for 30 minutes. Half of them were then dissolved in CaCl2/Ethanol/H2O [1:2:8 molar ratio] at 70±5๐C (method 1) and other half was dissolved in 46% w/v CaCl2 at 105±5๐C (method 2) for 4 hours. HCPCs were cultured in SF added cultured medial according to strain and extraction method. Cell viability at day 1, 3, and 7, were determined. Expression of collagen I,
Aims. This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. Methods. This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis. Results. Western blot analysis confirmed the expression of cleaved CASP-1 and melatonin receptor (MT-1A-R) in NPCs. The cultured NPCs were identified by detecting the expression of CD24,
The detailed biomechanical mechanism of annulus fibrosus under abnormal loading is still ambiguous, especially at the micro and nano scales. This study aims to characterize the alterations of modulus at the nano scale of individual collagen fibrils in annulus fibrosus after in-situ immobilization, and the corresponding micro-biomechanics of annulus fibrosus. An immobilization model was used on the rat tail with an external fixation device. Twenty one fully grown 12-week-old male Sprague-Dawley rats were used in this study. The rats were assigned to one of three groups randomly. One group was selected to be the baseline control group with intact intervertebral discs (n=7). In the other two groups, the vertebrae were immobilized with an external fixation device that fixed four caudal vertebrae (C7-C10) for 4 and 8 weeks, respectively. Four K-wires were fixed in parallel using two aluminum alloy cuboids which do not compress or stretch the target discs. The immobilized discs were harvested and then stained with hematoxylin/eosin, scanned using atomic force microscopy to obtain the modulus at both nano and micro scales, and analyzed the gene expression with real-time quantitative polymerase chain reaction. Significance of differences between the study groups was obtained using a two-way analysis of variance (ANOVA) with Fisher's Partial Least-Squares Difference (PLSD) to analyze the combined influence of immobilization time and scanning region. Statistical significance was set at P≤0.05. Compared to the control group, the inner layer of annulus fibrosus presented significant disorder and hyperplasia after immobilization for 8 weeks, but not in the 4 week group. The fibrils in inner layer showed an alteration in elastic modulus from 91.38±20.19MPa in the intact annulus fibrosus to 110.64±15.58MPa (P<0.001) at the nano scale after immobilization for 8 weeks, while the corresponding modulus at the micro scale also underwent a change from 0.33±0.04MPa to 0.47±0.04MPa (P<0.001). The upregulation of
Cartilage injuries often represent irreversible tissue damage because cartilage has only a low ability to regenerate. Thus, cartilage loss results in permanent damage, which can become the starting point for osteoarthritis. In the past, bioactive glass scaffolds have been developed for bone replacement and some of these variants have also been colonized with chondrocytes. However, the hydroxylapaptite phase that is usually formed in bioglass scaffolds is not very suitable for cartilage formation (chondrogenesis). This interdisciplinary project was undertaken to develop a novel slowly degrading bioactive glass scaffold tailored for cartilage repair by resembling the native extracellular cartilage matrix (ECM) in structure and surface properties. When colonized with articular chondrocytes, the composition and topology of the scaffolds should support cell adherence, proliferation and ECM synthesis as a prerequisite for chondrogenesis in the scaffold. To study cell growth in the scaffold, the scaffolds were colonized with human mesenchymal stromal cells (hMSCs) and primary porcine articular chondrocytes (pACs) (27,777.8 cells per mm. 3. ) for 7 – 35 d in a rotatory device. Cell survival in the scaffold was determined by vitality assay. Scanning electron microscopy (SEM) visualized cell ultramorphology and direct interaction of hMSCs and pACs with the bioglass surface. Cell proliferation was detected by CyQuant assay. Subsequently, the production of sulphated glycosaminoglycans (sGAGs) typical for chondrogenic differentiation was depicted by Alcian blue staining and quantified by dimethylmethylene blue assay assay. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of cartilage-specific aggrecan, Sox9,
Mesenchymal Stem Cells (MSCs) are a candidate cell type for treating osteoarthritic focal defects. In vivo, cartilage and bone marrow reside under a low oxygen tension, between 2–7% oxygen or physioxia, that has been shown to enhance MSC chondrogenesis. However, chondrogenesis is inhibited in the presence of IL-1. Here, it was hypothesized that physioxia reduces IL-1 inhibited chondrogenesis. Human MSCs (Mean age, 32 years; n = 9) were split equally for expansion under either 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2 × 10. 5. MSCs/pellet) were formed and cultured in the presence of 10 ng/ml TGF-b. 1. and in combination with either 0.1 or 0.5 ng/ml IL-1 under their respective expansion conditions. Pellets were assessed for their wet weight, GAG and
Purpose: Current techniques for articular cartilage repair remain suboptimal. The best technique involves the introduction of cultured chondrocytes into the injury site. Experimental results of current chondrocyte culture and expansion techniques (passaging) have shown phenotypic alteration resulting in fibroblast-like cells. Therefore, treatment methods that propose the transplantation of cultured chondrocytes might be transplanting fibroblast-like cells instead of chondrocytes. This experiment explored the difference in genetic expression of chondrocytes left at confluence compared to chondrocytes that were passaged as performed in current culture techniques. It was hypothesized that chondrocytes left at confluence would maintain their collagen I and
Aims. Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Methods. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of
