Primary Bovine Chondrocytes Research Articles

Coaxial Bioprinting of Enzymatically Crosslinkable Hyaluronic Acid-Tyramine Bioinks for Tissue Regeneration.

Three-dimensional (3D) bioprinting has emerged as an important technique for fabricating tissue constructs with precise structural and compositional control. However, developing suitable bioinks with biocompatible crosslinking mechanisms remains a significant challenge. This study investigates extrusion-based bioprinting (EBB) using uniaxial or coaxial nozzles with enzymatic crosslinking (EC) to produce 3D tissue constructs in vitro. Initially, low-molecular-weight dextran-tyramine and hyaluronic acid-tyramine (LMW Dex-TA/HA-TA) bioink prepolymers were evaluated. Enzymatically pre-crosslinking these prepolymers, achieved by the addition of horseradish peroxidase and hydrogen peroxide, produced viscous polymer solutions. However, this approach resulted in inconsistent bioprinting outcomes (uniaxial) due to inhomogeneous crosslinking, leading to irreproducible properties and suboptimal shear recovery behavior of the hydrogel inks. To address these challenges, we explored a one-step coaxial bioprinting system consisting of enzymatically crosslinkable high-molecular-weight hyaluronic acid-tyramine conjugates (HMW HA-TA) mixed with horseradish peroxidase (HRP) in the inner core and a mixture of Pluronic F127 and hydrogen peroxide in the outer shell. This configuration resulted in nearly instantaneous gelation by diffusion of the hydrogen peroxide into the core. Stable hydrogel fibers with desirable properties, including appropriate swelling ratios and controlled degradation rates, were obtained. The optimized bioink and printing parameters included 1.3% w/v HMW HA-TA and 5.5 U/mL HRP (bioink, inner core), and 27.5% w/v Pluronic F127 and 0.1% H2O2 (sacrificial ink, outer shell). Additionally, optimal pressures for the inner core and outer shell were 45 and 80 kPa, combined with a printing speed of 300 mm/min and a bed temperature of 30 °C. The extruded HMW HA-TA core filaments, containing bovine primary chondrocytes (BPCs) or 3T3 fibroblasts (3T3 Fs), exhibited good cell viabilities and were successfully cultured for up to seven days. This study serves as a proof-of-concept for the one-step generation of core filaments using a rapidly gelling bioink with an enzymatic crosslinking mechanism, and a coaxial bioprinter nozzle system. The results demonstrate significant potential for developing designed, printed, and organized 3D tissue fiber constructs.

Metabolomic Profiling and Characterization of a Novel 3D Culture System for Studying Chondrocyte Mechanotransduction.

Articular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction, and PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels have been used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. Here, we demonstrate that primary chondrocytes cultured in alginate will form a pericellular matrix and display a phenotype similar to in vivo conditions. We found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism. Our data suggest that 3D preculture of chondrocytes in alginate before encapsulation in physiologically-stiff agarose leads to a pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This novel model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.

13C Metabolic Flux Analysis in Chondrocytes Reveals a Novel Switch in Metabolic Phenotype.

Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through 13C-metabolic flux analysis (13C-MFA), as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-13C] glucose labeling experiments were combined with a tissue-specific metabolic network model to carry out 13C-MFA under varying levels of nutrient availability. 13C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic-anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow toward the tricarboxylic acid cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect ("inverse Crabtree effect"). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes-a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.

Co-Electrospun Poly(ε-Caprolactone)/Zein Articular Cartilage Scaffolds.

Osteoarthritis scaffold-based grafts fail because of poor integration with the surrounding soft tissue and inadequate tribological properties. To circumvent this, we propose electrospun poly(ε-caprolactone)/zein-based scaffolds owing to their biomimetic capabilities. The scaffold surfaces were characterized using Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, static water contact angles, and profilometry. Scaffold biocompatibility properties were assessed by measuring protein adsorption (Bicinchoninic Acid Assay), cell spreading (stained F-actin), and metabolic activity (PrestoBlue™ Cell Viability Reagent) of primary bovine chondrocytes. The data show that zein surface segregation in the membranes not only completely changed the hydrophobic behavior of the materials, but also increased the cell yield and metabolic activity on the scaffolds. The surface segregation is verified by the infrared peak at 1658 cm-1, along with the presence and increase in N1 content in the survey XPS. This observation could explain the decrease in the water contact angles from 125° to approximately 60° in zein-comprised materials and the decrease in the protein adsorption of both bovine serum albumin and synovial fluid by half. Surface nano roughness in the PCL/zein samples additionally benefited the radial spreading of bovine chondrocytes. This study showed that co-electrospun PCL/zein scaffolds have promising surface and biocompatibility properties for use in articular-tissue-engineering applications.

Hydroquinone, an Environmental Pollutant, Affects Cartilage Homeostasis through the Activation of the Aryl Hydrocarbon Receptor Pathway.

Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

Characterization of Migratory Cells From Bioengineered Bovine Cartilage in a 3D Co-culture Model

Background: Chondrocyte migration in native cartilage is limited and has been implicated as one of the reasons for the poor integration of native implants. Through use of an in vitro integration model, it has previously been shown that cells from bioengineered cartilage can migrate into the native host cartilage during integration. Platelet-rich plasma (PRP) treatment further enhanced integration of bioengineered cartilage to native cartilage in vitro. However, it is not known how PRP treatment of the bioengineered construct promotes this. Hypothesis: PRP supports cell migration from bioengineered cartilage and these migratory cells have the ability to accumulate cartilage-like matrix. Study Design: Controlled laboratory study. Methods: Osteochondral-like constructs were generated by culturing primary bovine chondrocytes on the top surface of a porous bone substitute biomaterial composed of calcium polyphosphate. After 1 week in culture, the constructs were submerged in PRP and placed adjacent, but 2 mm distant, to a native bovine osteochondral plug in a co-culture model for 2 weeks. Cell migration was monitored using phase-contrast imaging. Cell phenotype was determined by evaluating the gene expression of matrix metalloprotease 13 (MMP-13), Ki67, and cartilage matrix molecules using quantitative polymerase chain reaction. When tissue formed, it was assessed by histology, immunohistochemistry, and quantification of matrix content. Results: PRP treatment resulted in the formation of a fiber network connecting the bioengineered cartilage and native osteochondral plug. Cells from both the bioengineered cartilage and the native osteochondral tissue migrated onto the PRP fibers and formed a tissue bridge after 2 weeks of culture. Migratory cells on the tissue bridge expressed higher levels of collagen types II and I (COL2, COL1), Ki67 and MMP-13 mRNA compared with nonmigratory cells in the bioengineered cartilage. Ki67 and MMP-13–positive cells were found on the edges of the tissue bridge. The tissue bridge accumulated COL1 and COL2 and aggrecan and contained comparable collagen and glycosaminoglycan content to the bioengineered cartilage matrix. The tissue bridge did not reliably develop in the absence of cells from the native osteochondral plug. Conclusion: Bioengineered cartilage formed by bovine chondrocytes contains cells that can migrate on PRP fibers and form cartilaginous tissue. Clinical Relevance: Migratory cells from bioengineered cartilage may promote cartilage integration. Further studies are required to determine the role of migratory cells in integration in vivo.

Strontium Regulates the Proliferation and Differentiation of Isolated Primary Bovine Chondrocytes via the TGFβ/SMAD Pathway.

The present study evaluated the effects of strontium (Sr) on proliferation and differentiation of chondrocytes isolated from dairy cows, and whether Sr exerts its effects via transforming growth factor β (TGFβ) signaling. The chondrocytes were isolated from patellar cartilage from newborn Holstein bull calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting) within 15 min after euthanasia, and treated with different concentrations of Sr (0, 0.1, 1, and 10 μg/ml, as SrCl2·6H2O). After pretreatment with or without activin receptor-like kinase 5 (ALK5) inhibitor (10 μM SB-505124) for 4 h, chondrocytes were incubated with Sr for another 4 h. Overall effects of Sr were evaluated relative to NaCl as the control. In contrast, the 1 μg/ml Sr-treated group served as the control to determine effects of preincubating with SB-505124. Western blot and qRT-PCR were used for measuring expression of proliferation-, differentiation-, and TGFβ1-responsive factors. Data were analyzed using one-way ANOVA in GraphPad Prism 7.0. Incubation with all doses of Sr increased TGFβ1/ALK5-induced SMAD3 phosphorylation, and at 10 μg/ml it inhibited ALK1-induced SMAD1/5/9 phosphorylation. Expression of mRNA and protein of the proliferation-responsive factors type Ⅱ Collagen α1 (COL2A1) and aggrecan (ACAN) was induced by Sr at 1 μg/ml. In contrast, Sr at 10 μg/ml inhibited the expression of differentiation-responsive factors type Ⅹ Collagen α1 (COL10A1) and secreted phosphoprotein 1 (SPP1), and at 1 μg/ml it had the same effect on alkaline phosphatase (ALPL) mRNA and protein levels. Cells were stained with PI/RNase Staining buffer to assess cell cycle activity using flow-cytometry. Incubation with Sr at 1 and 10 μg/ml induced an increase in the number of cells in the S-phase, leading to an increase in the proliferation index. Incubation with SB-505124 inhibited phosphorylation of SMAD3. Abundance of ACAN and COL2A1 mRNA and protein was lower when cells were pre-incubated with SB-505124. Overall, data indicated that Sr promotes proliferation and inhibits differentiation of primary chondrocytes by directing TGFβ1 signaling towards SMAD3 phosphorylation rather than SMAD1/5/9 phosphorylation. Whether these effects occur in vivo remains to be determined and could impact future application of Sr as an experimental tool in livestock.

The use of antifreeze proteins to modify pore structure in directionally frozen alginate sponges for cartilage tissue engineering

It is thought that osteoarthritis is one of the world’s leading causes of disability, with over 8.75 million people in the UK alone seeking medical treatment in 2013. Although a number of treatments are currently in use, a new wave of tissue engineered structures are being investigated as potential solutions for early intervention. One of the key challenges seen in cartilage tissue engineering is producing constructs that can support the formation of articular cartilage, rather than mechanically inferior fibrocartilage. Some research has suggested that mimicking structural properties of the natural cartilage can be used to enhance this response. Herein directional freezing was used to fabricate scaffolds with directionally aligned pores mimicking the mid-region of cartilage, anti-freeze proteins were used to modify the porous structure, which in turn effected the mechanical properties. Pore areas at the tops of the scaffolds were 180.46 ± 44.17 μm2 and 65.66 ± 36.20 μm2 for the AFP free and the AFP scaffolds respectively, and for the bases of the scaffolds were 91.22 ± 19.05 μm2 and 69.41 ± 21.94 μm2 respectively. Scaffolds were seeded with primary bovine chondrocytes, with viability maintained over the course of the study, and regulation of key genes was observed.

Microtubules and Vimentin Fiber Stability during Parabolic Flights

Adequate mechanical stimulation is essential for cellular health and tissue maintenance, including articular cartilage, which lines the articulating bones in joints. Chondrocytes, which are the sole cells found in articular cartilage, are responsible for matrix synthesis, maintenance and degradation. It is generally believed that chondrocytes require mechanical stimuli through daily physical activity for adequate cartilage homeostasis. However, to date, the molecular mechanisms of cellular force sensing (mechanotransduction) are not fully understood. Among other mechanisms, the cytoskeleton is thought to play a key role. Despite that gravity is a very small force at the cellular level, cytoskeletal adaptations have been observed under altered gravity conditions of a parabolic flight in multiple cell types. In this study, we developed a novel hardware which allowed to chemically fix primary bovine chondrocytes at 7 time points over the course of a 31-parabola flight. The samples were subsequently stained for the microtubules and vimentin network and microscopic images were acquired. The images showed a large heterogeneity among the cells in morphology as well as in the structure of both networks. In all, no changes or adaptions in cytoskeleton structure could be detected over the course of the parabolic flight.

Enhanced chondrogenic phenotype of primary bovine articular chondrocytes in Fibrin-Hyaluronan hydrogel by multi-axial mechanical loading and FGF18

Current treatments for cartilage lesions are often associated with fibrocartilage formation and donor site morbidity. Mechanical and biochemical stimuli play an important role in hyaline cartilage formation. Biocompatible scaffolds capable of transducing mechanical loads and delivering bioactive instructive factors may better support cartilage regeneration.In this study we aimed to test the interplay between mechanical and FGF-18 mediated biochemical signals on the proliferation and differentiation of primary bovine articular chondrocytes embedded in a chondro-conductive Fibrin-Hyaluronan (FB/HA) based hydrogel.Chondrocytes seeded in a Fibrin-HA hydrogel, with or without a chondro-inductive, FGFR3 selective FGF18 variant (FGF-18v) were loaded into a joint-mimicking bioreactor applying controlled, multi-axial movements, simulating the natural movements of articular joints. Samples were evaluated for DNA content, sulphated glycosaminoglycan (sGAG) accumulation, key chondrogenic gene expression markers and histology.Under moderate loading, samples produced particularly significant amounts of sGAG/DNA compared to unloaded controls. Interestingly there was no significant effect of FGF-18v on cartilage gene expression at rest. Following moderate multi-axial loading, FGF-18v upregulated the expression of Aggrecan (ACAN), Cartilage Oligomeric Matrix Protein (COMP), type II collagen (COL2) and Lubricin (PRG4). Moreover, the combination of load and FGF-18v, significantly downregulated Matrix Metalloproteinase-9 (MMP-9) and Matrix Metaloproteinase-13 (MMP-13), two of the most important factors contributing to joint destruction in OA. Biomimetic mechanical signals and FGF-18 may work in concert to support hyaline cartilage regeneration and repair. Statement of significanceArticular cartilage has very limited repair potential and focal cartilage lesions constitute a challenge for current standard clinical procedures. The aim of the present research was to explore novel procedures and constructs, based on biomaterials and biomechanical algorithms that can better mimic joints mechanical and biochemical stimulation to promote regeneration of damaged cartilage.Using a hydrogel-based platform for chondrocyte 3D culture revealed a synergy between mechanical forces and growth factors. Exploring the mechanisms underlying this mechano-biochemical interplay may enhance our understanding of cartilage remodeling and the development of new strategies for cartilage repair and regeneration.

Inhibition of CD44 intracellular domain production suppresses bovine articular chondrocyte de-differentiation induced by excessive mechanical stress loading

CD44 fragmentation is enhanced in chondrocytes of osteoarthritis (OA) patients. We hypothesized that mechanical stress-induced enhancement of CD44-intracellular domain (CD44-ICD) production plays an important role in the de-differentiation of chondrocytes and OA. This study aimed to assess the relationship between CD44-ICD and chondrocyte gene expression. Monolayer cultured primary bovine articular chondrocytes (BACs) were subjected to cyclic tensile strain (CTS) loading. ADAM10 inhibitor (GI254023X) and γ-secretase inhibitor (DAPT) were used to inhibit CD44 cleavage. In overexpression experiments, BACs were electroporated with a plasmid encoding CD44-ICD. CTS loading increased the expression of ADAM10 and subsequent CD44 cleavage, while decreasing the expression of SOX9, aggrecan, and type 2 collagen (COL2). Overexpression of CD44-ICD also resulted in decreased expression of these chondrocyte genes. Both GI254023X and DAPT reduced the production of CD44-ICD upon CTS loading, and significantly rescued the reduction of SOX9 expression by CTS loading. Chemical inhibition of CD44-ICD production also rescued aggrecan and COL2 expression following CTS loading. Our findings suggest that CD44-ICD is closely associated with the de-differentiation of chondrocytes. Excessive mechanical stress loading promoted the de-differentiation of BACs by enhancing CD44 cleavage and CD44-ICD production. Suppression of CD44 cleavage has potential as a novel treatment strategy for OA.

Manganese dioxide nanoparticles modulate oxidative stress and protect cartilage from interleukin-1β induced degradation

Purpose: Osteoarthritis (OA) is associated with chronic joint inflammation, whereby pro-inflammatory cytokines such as IL-1β upregulate reactive oxygen species (ROS) production while downregulating anti-oxidants in cells. The resulting oxidative stress leads to extracellular matrix degradation, joint inflammation, and chondrocyte death and senescence. The overall goal of this research is to develop a bioactive nanoparticle (NP) system that scavenges ROS in cartilage to modulate the impact of joint inflammation. Manganese dioxide (MnO2) catalyzes the breakdown of hydrogen peroxide (H2O2) and is currently being evaluated for scavenging ROS in inflammatory diseases such as atherosclerosis. We previously reported engineered MnO2 NPs with properties that enable them to penetrate cartilage and become endocytosed by chondrocytes. The objective of this study was to evaluate the chondroprotective mechanisms of MnO2 NPs and their in vivo retention and biocompatibility. Methods: MnO2 NPs were synthesized and characterized as previously described. Uptake of Alexa Fluor 488 conjugated NPs by bovine chondrocytes in 2D culture and cartilage explants were confirmed by fluorescent imaging after 24 hours of incubation. Gene expression analysis of both catabolic and ROS mediator genes was conducted on a monolayer of primary bovine chondrocytes challenged by human recombinant IL-1β in the presence of MnO2 NPs after 24-hour culture. Fresh bovine cartilage biopsies were evaluated for loss of glycosaminoglycans (GAG), the production of nitric oxide (NO) and cell viability over a 14-day period when challenged by IL-1β in the presence of MnO2NPs. Articular joint retention of Alexa Fluor 750 and 594 conjugated NPs in Lewis rats was monitored using an in vivo IVIS Imaging System. Long-term cytocompatibility of the particles 6 weeks after intraarticular injection in healthy rat joints was assessed histologically using GEKO©, a tool for quantitative grading of toluidine stained knees. Results: The PEG stabilized MnO2 NPs had a hydrodynamic diameter of 15 nm, TEM size of 8 nm and had a cationic surface charge. The NPs effectively scavenged H2O2, with 5μg/mL MnO2 NPs neutralizing 55% of 100uM H2O2 in PBS. Chondrocytes in monolayer showed uptake of MnO2 NPs without cytotoxicity. Furthermore, the NPs reduced the gene expression of catabolic mediator, heme oxygenase-1, catabolic factor matrix metalloproteinase 3, antioxidant mediator nuclear factor erythroid 2-related factor 2, antioxidants catalase, glutathione peroxidase and manganese superoxide dismutase by chondrocytes (Fig. 1A) indicating protection of the particles against oxidative stress. When incubated with cartilage explants, the MnO2 NPs penetrated through the cartilage depth and were found in the extracellular matrix as well as intracellularly in chondrocytes. In cytokine challenged cartilage explants, supplementation with MnO2 NPs decreased GAG loss by 50% and NO production by 40% compared to controls (Fig. 1B and C). Importantly, treatment with MnO2 NPs improved viability of chondrocytes in cartilage explants over the 2-week study. Upon in vivo injection, the MnO2 NPs persisted in the joint space over one week, with 20% of the initial fluorescent signal still observed by day 13 (Fig. 2A and B). Biodistribution and histological analysis (Fig. 2C) revealed accumulation of particles at the chondral surfaces, and colocalization of the NPs with the lacunae of chondrocytes, indicating particle penetration into the cartilage and uptake by chondrocytes following intraarticular injection. A gradient of NPs existed in cartilage with the highest levels of NPs on the articular surface which decreased with tissue depth. Histological analysis also revealed no adverse effects of intra-articular MnO2 NP injection into healthy animals. Histological parameters such as synovium thickness, total cartilage degeneration width and cartilage matrix loss width were comparable in knees injected with the NPs and those injected with saline after 6 weeks, indicating biocompatibility of the particles in vivo (Fig. 2D). Conclusions: MnO2 NPs have the potential to reduce oxidative stress in osteoarthritic cartilage. In vitro, the NPs protected chondrocytes in cartilage explants from IL-1β mediated oxidative stress and apoptosis. Further analysis of the joint confirmed penetration of MnO2 NPs into cartilage in vivo. Given their joint retention time and ROS scavenging capacity, these NPs could target oxidative stress to treat or prevent OA. As the NPs show intracellular localization in chondrocytes, they could also deliver other chondroprotective agents including nucleic acids to target multiple pathways in the OA pathology. Further studies will focus on the therapeutic impact of MnO2 NPs in OA disease models.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Self-assembled monolayers of phosphonates promote primary chondrocyte adhesion to silicon dioxide and polyvinyl alcohol materials

The optimal solution for articular cartilage repair has not yet been identified, in part because of the challenges in achieving integration with the host. Coatings have the potential to transform the adhesive features of surfaces, but their application to cartilage repair has been limited. Self-assembled monolayer of phosphonates (SAMPs) have been demonstrated to increase the adhesion of various immortalized cell types to metal and polymer surfaces, but their effect on primary chondrocyte adhesion has not been studied. The objective of this study was to investigate the response of primary chondrocytes to SAMP coatings. We hypothesized a SAMP terminated with an α,ω-bisphosphonic acid, in particular butane-1,4-diphosphonic acid, would increase the number of adherent primary chondrocytes to polyvinyl alcohol (PVA). To test our hypothesis, we first established our ability to successfully modify silicon dioxide (SiO2) surfaces to enable chondrocytes to attach to the surface, without substantial changes in gene expression. Secondly, we applied identical chemistry to PVA, and quantified chondrocyte adhesion. SAMP modification to SiO2 increased chondrocyte adhesion by ×3 after 4 hr and ×4.5 after 24 hr. PVA modification with SAMPs increased chondrocyte adhesion by at least ×31 after 4 and 24 hours. Changes in cell morphology indicated that SAMP modification led to improved chondrocyte adhesion and spreading, without changes in gene expression. In summary, we modified SiO2 and PVA with SAMPs and observed an increase in the number of adherent primary bovine chondrocytes at 4 and 24 hr post-seeding. Mechanisms of chondrocyte interaction with SAMP-modified surfaces require further investigation.

Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes

For both the incorporation of cells and future therapeutic applications the sterility of a biomaterial must be ensured. However, common sterilisation techniques are intense and often negatively impact on material physicochemical attributes, which can affect its suitability for tissue engineering and 3D printing. In the present study four sterilisation methods, autoclave, supercritical CO2 (scCO2) treatment, UV- and gamma (γ) irradiation were evaluated regarding their impact on material properties and cellular responses. The investigations were performed on methyl cellulose (MC) as a component of an alginate/methyl cellulose (alg/MC) bioink, used for bioprinting embedded bovine primary chondrocytes (BPCs). In contrast to the autoclave, scCO2 and UV-treatments, the γ-irradiated MC resulted in a strong reduction in alg/MC viscosity and stability after extrusion which made this method unsuitable for precise bioprinting. Gel permeation chromatography analysis revealed a significant reduction in MC molecular mass only after γ-irradiation, which influenced MC chain mobility in the Ca2+-crosslinked alginate network as well as gel composition and microstructure. With regard to cell survival and proteoglycan matrix production, the results determined UV-irradiation and autoclaving as the best candidates for sterilisation. The scCO2-treatment of MC resulted in an unfavourable cell response indicating that this method needs careful optimisation prior to application for cell encapsulation. As proven by consistent FT-IR spectra, chemical alterations could be excluded as a cause for the differences seen between MC treatments on alg/MC behaviour. This investigation provides knowledge for the development of a clinically appropriate 3D-printing-based fabrication process to produce bioengineered tissue for cartilage regeneration.

Effect of centrifugal force on the development of articular neocartilage with bovine primary chondrocytes

A lot has been invested into understanding how to assemble cartilage tissue in vitro and various designs have been developed to manufacture cartilage tissue with native-like biological properties. So far, no satisfactory design has been presented. Bovine primary chondrocytes are used to self-assemble scaffold-free constructs to investigate whether mechanical loading by centrifugal force would be useful in manufacturing cartilage tissue in vitro. Six million chondrocytes were laid on top of defatted bone disks placed inside an agarose well in 50-ml culture tubes. The constructs were centrifuged once or three times per day for 15 min at a centrifugal force of 771×g for up to 4 weeks. Control samples were cultured under the same conditions without exposure to centrifugation. The samples were analysed by (immuno)histochemistry, Fourier transform infrared imaging, micro-computed tomography, biochemical and gene expression analyses. Biomechanical testing was also performed. The centrifuged tissues had a more even surface covering a larger area of the bone disk. Fourier transform infrared imaging analysis indicated a higher concentration of collagen in the top and bottom edges in some of the centrifuged samples. Glycosaminoglycan contents increased along the culture, while collagen content remained at a rather constant level. Aggrecan and procollagen α1(II) gene expression levels had no significant differences, while procollagen α2(I) levels were increased significantly. Biomechanical analyses did not reveal remarkable changes. The centrifugation regimes lead to more uniform tissue constructs, whereas improved biological properties of the native tissue could not be obtained by centrifugation.

Induction of zonal-specific cellular morphology and matrix synthesis for biomimetic cartilage regeneration using hybrid scaffolds.

Cartilage is anisotropic in nature and organized into distinct zones. Our goal was to develop zonal-specific three-dimensional hybrid scaffolds which could induce the generation of zonal-specific cellular morphology and extracellular matrix (ECM) composition. The superficial and middle zones comprised two layers of hyaluronic acid (HA) hydrogel which enveloped specifically orientated or randomly arranged polylactic acid nanofibre meshes. The deep zone comprised a HA hydrogel with multiple vertical channels. Primary bovine chondrocytes were seeded into the individual zonal scaffolds, cultured for 14 days and then the ECM was analysed. The aligned nanofibre mesh used in the superficial zone induced an elongated cell morphology, lower glycosaminoglycan (GAG) and collagen II production, and higher cell proliferation and collagen I production than the cells in the middle zone scaffold. Within the middle zone scaffold, which comprised a randomly orientated nanofibre mesh, the cells were clustered and expressed more collagen II. The deep zone scaffold induced the highest GAG production, the lowest cell proliferation and the lowest collagen I expression of the three zones. Assembling the three zones and stabilizing the arrangement with a HA hydrogel generated aligned, randomly aggregated and columnar cells in the superficial, middle and deep zones. This study presents a method to induce zonal-specific chondrocyte morphology and ECM production.

Metabolomics and metabolic function analysis of the secretome of articular cartilage and isolated chondrocytes in response to pro-inflammatory cytokines

Purpose: Chondrocytes rely primarily on glycolysis to meet their energy requirements, but possess the metabolic flexibility to support cell survival and matrix synthesis during periods of nutrient stress, by enhancing glycolysis with mitochondrial respiration. Accessing this ‘spare respiratory capacity’ requires optimal mitochondrial function, but since impaired mitochondrial function is implicated in osteoarthritis (OA), this mechanism may be deficient or attenuated in joint disease. Metabolic adaptation is evident in early-stage OA, however cartilage from late-stage disease does not seem to have this flexibility. A deeper understanding of these complex metabolic pathways may identify new metabolic markers of disease stage, and support therapeutic strategies for treating OA. Metabolomics has the potential to identify underlying metabolic changes, reveal pathological pathways, novel biomarkers and therapeutic targets. The aim of this study was to identify metabolic processes involved in early stage disease by analysis of metabolites and metabolic function in two inflammatory models of cartilage degradation. Methods: Macroscopically normal articular cartilage was obtained from equine and bovine metacarpophalangeal joints. Six-millimetre diameter equine cartilage explants (n=6) and isolated primary equine chondrocytes (n=4), seeded at high density (105,000/cm2), were cultured for 7 days in serum-free low or high glucose Glutamax DMEM (Gibco), respectively, with or without 10ng/ml equine interleukin-1β (IL-1β) and 10ng/ml equine tumour necrosis factor-α (TNF-α). Spent media (secretome) was collected and subjected to metabolomic analysis. Secretome metabolite levels were measured using the AbsoluteIDQ® p180 targeted metabolomics kit (Biocrates), and a Waters Xevo TQ-S mass spectrometer coupled to an Acquity UPLC system. Multivariate analysis was performed by principal component analysis and orthogonalized partial least squares discriminant analysis using SIMCA-P v12.0 software (Umetrics). Metabolic function of primary equine (n=9) and bovine chondrocytes (n=3) was determined using Seahorse XFp and XFe24 analyzers (Agilent). Cells were seeded at high density, treated with species-specific 10ng/ml IL-1β and/or 10ng/ml TNF-α for 18 h, and metabolically challenged with the Mito Stress Test during analysis. Metabolite levels, and oxygen consumption rates, were normalised to total cell protein, and differences between means determined by one-way analysis of variance with Tukey’s multiple comparison post-tests. Results: Secretome metabolites which decreased with pro-inflammatory cytokine treatment were proline, ornithine and alpha-aminoadipic acid (p<0.0001). Citrulline increased with cytokine treatment (p<0.0001) and glutamate, present in DMEM, was elevated with cytokine treatment (p<0.0001). Metabolomic analysis of the chondrocyte secretome showed that glutamine decreased (p<0.02) with cytokine treatment whereas citrulline was elevated (p<0.003). Metabolic analysis showed that combined cytokine treatment reduced basal respiration, ATP production and negated spare respiratory capacity in chondrocytes (p<0.01), and the effect was due to IL-1β alone. Conclusions: Cytokines decreased proline, ornithine, alpha-aminoadipic acid and glutamine, which are all downstream metabolites of glutamate. Along with elevated glutamate, this suggests that cytokine treatment inhibits glutamate uptake and metabolism. A marked increase in citrulline observed in both cell and explant models may be attributed to cytokine induction of nitric oxide synthase, resulting in elevated citrulline production via the urea cycle. IL-1β alone prevented chondrocytes from accessing their spare respiratory capacity, and remained glycolytic. In conclusion, glutamate and citrulline metabolism, which are both tightly regulated by mitochondrial metabolism, are altered by cytokines, and IL-1β alone is responsible for the metabolic switch observed in this model. These metabolic pathways could provide markers of early-stage disease, and targets for therapeutic intervention.

Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review.

Articular cartilage functions to transmit and translate loads. In a classical structure–function relationship, the tissue resides in a dynamic mechanical environment that drives the formation of a highly organized tissue architecture suited to its biomechanical role. The dynamic mechanical environment includes multiaxial compressive and shear strains as well as hydrostatic and osmotic pressures. As the mechanical environment is known to modulate cell fate and influence tissue development toward a defined architecture in situ, dynamic mechanical loading has been hypothesized to induce the structure–function relationship during attempts at in vitro regeneration of articular cartilage. Researchers have designed increasingly sophisticated bioreactors with dynamic mechanical regimes, but the response of chondrocytes to dynamic compression and shear loading remains poorly characterized due to wide variation in study design, system variables, and outcome measurements. We assessed the literature pertaining to the use of dynamic compressive bioreactors for in vitro generation of cartilaginous tissue from primary and expanded chondrocytes. We used specific search terms to identify relevant publications from the PubMed database and manually sorted the data. It was very challenging to find consensus between studies because of species, age, cell source, and culture differences, coupled with the many loading regimes and the types of analyses used. Early studies that evaluated the response of primary bovine chondrocytes within hydrogels, and that employed dynamic single-axis compression with physiologic loading parameters, reported consistently favorable responses at the tissue level, with upregulation of biochemical synthesis and biomechanical properties. However, they rarely assessed the cellular response with gene expression or mechanotransduction pathway analyses. Later studies that employed increasingly sophisticated biomaterial-based systems, cells derived from different species, and complex loading regimes, did not necessarily corroborate prior positive results. These studies report positive results with respect to very specific conditions for cellular responses to dynamic load but fail to consistently achieve significant positive changes in relevant tissue engineering parameters, particularly collagen content and stiffness. There is a need for standardized methods and analyses of dynamic mechanical loading systems to guide the field of tissue engineering toward building cartilaginous implants that meet the goal of regenerating articular cartilage.

Co-culture of chondrons and mesenchymal stromal cells reduces the loss of collagen VI and improves extracellular matrix production.

Adult articular chondrocytes are surrounded by a pericellular matrix (PCM) to form a chondron. The PCM is rich in hyaluronan, proteoglycans, and collagen II, and it is the exclusive location of collagen VI in articular cartilage. Collagen VI anchors the chondrocyte to the PCM. It has been suggested that co-culture of chondrons with mesenchymal stromal cells (MSCs) might enhance extracellular matrix (ECM) production. This co-culture study investigates whether MSCs help to preserve the PCM and increase ECM production. Primary bovine chondrons or chondrocytes or rat MSCs were cultured alone to establish a baseline level for ECM production. A xenogeneic co-culture monolayer model using rat MSCs (20, 50, and 80%) was established. PCM maintenance and ECM production were assessed by biochemical assays, immunofluorescence, and histological staining. Co-culture of MSCs with chondrons enhanced ECM matrix production, as compared to chondrocyte or chondron only cultures. The ratio 50:50 co-culture of MSCs and chondrons resulted in the highest increase in GAG production (18.5±0.54pg/cell at day 1 and 11±0.38pg/cell at day 7 in 50:50 co-culture versus 16.8±0.61pg/cell at day 1 and 10±0.45pg/cell at day 7 in chondron monoculture). The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of high-temperature requirement serine protease A1 (HtrA1) demonstrated an inverse relationship to that of the collagen VI. Together, this implies that MSCs directly or indirectly inhibited HtrA1 activity and the co-culture of MSCs with chondrons enhanced ECM synthesis and the preservation of the PCM.

Local Heterogeneities Improve Matrix Connectivity in Degradable and Photoclickable Poly(ethylene glycol) Hydrogels for Applications in Tissue Engineering.

Hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels are promising platforms for cell encapsulation and tissue engineering. However, hydrolysis leads to bulk degradation and a decrease in hydrogel mechanical integrity. Despite these challenges, hydrolytically degradable hydrogels have supported macroscopic neotissue growth. The goal of this study was to combine experimental methods with a multiscale mathematical model to analyze hydrogel degradation concomitant with neocartilage growth in PEG hydrogels. Primary bovine chondrocytes were encapsulated at increasing densities (50, 100, and 150 million cells/mL of precursor solution) in a radical-mediated photoclickable hydrogel formed from 8-arm PEG-co-caprolactone end-capped with norbornene and cross-linked with PEG dithiol. Two observations were made in the experimental system: (1) the cell distribution was not uniform and cell clustering was evident, which increased with increasing cell density and (2) a significant decrease in the initial hydrogel compressive modulus was observed with increasing cell concentration. By introducing heterogeneities in the form of cell clusters and spatial variations in the network structure around cells, the mathematical model explained the drop in initial modulus and captured the experimentally observed spatial evolution of ECM and the construct modulus as a function of cell density and culture time. Overall, increasing cell density led to improved ECM formation, ECM connectivity, and overall modulus. This study strongly points to the importance of heterogeneities within a cell-laden hydrogel in retaining mechanical integrity as the construct transitions from hydrogel to neotissue.