Metabolic Activity Of Chondrocytes Research Articles

ARTICULAR CHONDROCYTES FROM DIABETIC AND NON-DIABETIC RATS EXPOSED TO NORMO- OR HYPERGLYCAEMIA RESPOND DIFFERENTIALLY TO THE COMPLEMENT SPLIT FRAGMENT ANAPHYLATOXIN C5A AND TNFA

Osteoarthritis (OA) and diabetis mellitus type 2 (DMT2) are pathogenetically linked. Complement dysregulation contributes to OA and could be involved in DMT2. The inflammatory anaphylatoxin C5a is released during complement activation. This study aims to understand the specific responses of chondrocytes isolated from diabetic and non-diabetic rats exposed to C5a and/or the proinflammatory cytokine TNFα in vitro dependent on the glucose supply. Articular chondrocytes of adult Zucker Diabetic Fatty (ZDF) rats (homozygous: fa/fa, diabetic, heterozygous: fa/+, lean controls) were exposed to 10 ng/mL TNFα and 25 ng/mL C5a alone or in combination, both, under normo- (NG, 1 g/L glucose) and hyperglycemic (HG, 4.5 g/L glucose) conditions (4 or 24 h). Chondrocyte survival, metabolic activity and gene expression of collagen type 2, suppressors of cytokine signaling (SOCS)1, −3 and anti-oxidative hemoxygenase-1 (HMOX1) were assessed. The complement regulatory protein CD46 and cell nuclei sizes were analyzed. Chondrocyte vitality remained unaffected by the treatment. Metabolic activity was impaired in chondrocytes of non-diabetic rats under HG conditions. Collagen type 2 transcription was suppressed by TNFα under HG condition in chondrocytes from nondiabetic donors and under both conditions in those of DMT2 rats (24 h)Except for DMT2 chondrocytes under HG (4 h), HMOX1 was generally induced by TNFα +/- C5a (NG, HG). C5a elevated HMOX1 only in chondrocytes of controls. The SOCS1/3 genes were increased by TNFα (NG, diabetic, non diabetic, 4 and 24 h). This could also be observed in chondrocytes of diabetic, but not of lean rats (24 h, HG). At 4 h, C5a induced SOCS1 only in non diabetic chondrocytes (NG, HG). Cytoprotective CD46 protein was suppressed by TNFα under NG condition. Nuclear volumes of chondrocyte were lower in chondrocytes from DMT2 rats compared to those from controls. The differential response suggests that chondrocytes are irreversibly compromised by DMT2.Achnowledgement: The authors are grateful for the support by the “Stiftung Edoprothetik (S 04/21)”

Disruption of Endochondral Ossification and Extracellular Matrix Maturation in an Ex Vivo Rat Femur Organotypic Slice Model Due to Growth Plate Injury.

Postnatal bone fractures of the growth plate (GP) are often associated with regenerative complications such as growth impairment. In order to understand the underlying processes of trauma-associated growth impairment within postnatal bone, an ex vivo rat femur slice model was developed. To achieve this, a 2 mm horizontal cut was made through the GP of rat femur prior to the organotypic culture being cultivated for 15 days in vitro. Histological analysis showed disrupted endochondral ossification, including disordered architecture, increased chondrocyte metabolic activity, and a loss of hypertrophic zone throughout the distal femur. Furthermore, altered expression patterns of Col2α1, Acan, and ColX, and increased chondrocyte metabolic activity in the TZ and MZ at day 7 and day 15 postinjury were observed. STEM revealed the presence of stem cells, fibroblasts, and chondrocytes within the injury site at day 7. In summary, the findings of this study suggest that the ex vivo organotypic GP injury model could be a valuable tool for investigating the underlying mechanisms of GP regeneration post-trauma, as well as other tissue engineering and disease studies.

Evaluation of articular cartilage wear against pyrolytic carbon in the context of spherical interposition shoulder arthroplasty

Metal shoulder hemiarthroplasty is known to cause failure due to glenoid cartilage erosion. Pyrolytic carbon (PyC), a non-metallic biomaterial, may be an attractive alternative due to its good general tribological characteristics and biocompatibility. We investigated the in vitro biotribological properties of PyC articulated against living cartilage for use as spherical shoulder interposition implant. The evaluation includes a comparison with the typically used cobalt‑chromium‑molybdenum alloy (CoCrMo) and zirconia toughened alumina (ZTA), a more recent biomaterial for hemiarthroplasty. The rate of cartilage degradation was assessed by means of cartilage wear biomarkers (glycosaminoglycans and hydroxyproline) in lubricant and histological changes of the cartilage superficial zone. In addition, the percentage of cell viability and metabolic activity of chondrocytes was assessed. The data revealed that PyC has similar tribological performance in terms of cell viability, cell metabolic activity, and tissue degradation compared to ZTA. Although not significant, CoCrMo affected chondrocyte metabolism and induced a thinner superficial layer with structural defects. There was a combined adsorption of phospholipids and biomarkers on the biomaterials; sGAGs adsorbed between 23 and 43% of the total concentration, and the adsorption rate of phospholipids increased significantly with friction. Biomaterial analysis is required to obtain the full picture of cartilage biomarker release and lubrication mechanisms. Our results suggest that PyC has good tribological performance and compares well with established biomaterials for hemiarthroplasty. It supports the use of PyC in spherical shoulder interposition implant as a viable alternative to conventional metal hemiarthroplasty. Further evaluation of the clinical performance of PyC is required.

Research Progress on the Application of Icariin in Osteoarthritis Treatment

Osteoarthritis (OA) is a multifaceted bone and joint disease with degenerative changes of articular cartilage. Therefore, the current research on the etiology of OA mainly focuses on the etiology of articular cartilage degeneration. Clarifying the pathogenesis of OA from the molecular level has become an important research topic in the field of orthopedics. Its main pathological features are the decrease of chondrocyte metabolic activity, cartilage matrix degradation, and osteophyte formation. Icariin (ICA) is the main active monomer of epimedium, a traditional Chinese medicine, and the pharmacological research has revealed favorable effects of ICA on the bone system. ICA can promote the proliferation of chondrocytes and inhibit the apoptosis of chondrocytes, alleviate the degradation rate of cartilage matrix, and effectively protect articular cartilage. This review also expounds the mechanism of promoting cartilage repair.

Bio-orthogonal Click Chemistry Methods to Evaluate the Metabolism of Inflammatory Challenged Cartilage after Traumatic Overloading.

During traumatic joint injuries, impact overloading can cause mechanical damage to the cartilage. In the following inflammation phase, excessive inflammatory cytokines (e.g., interleukin-1β (IL-1β)) can act on chondrocytes, causing over-proliferation, apoptosis, and extracellular matrix (ECM) degradation that can lead to osteoarthritis. This study investigated the combined effects of traumatic overloading and IL-1β challenge on the metabolic activities of chondrocytes. Bovine cartilage explants underwent impact overloading followed by IL-1β exposure at a physiologically relevant dosage (1 ng/mL). New click chemistry-based methods were developed to visualize and quantify the proliferation of in situ chondrocytes in a nondestructive manner without the involvement of histological sectioning or antibodies. Click chemistry-based methods were also employed to measure the ECM synthesis and degradation in cartilage explants. As the click reactions are copper-free and bio-orthogonal, i.e., with negligible cellular toxicity, cartilage ECM was cultured and studied for 6 weeks. Traumatic overloading induced significant cell death, mainly in the superficial zone. The high number of dead cells reduced the overall proliferation of chondrocytes as well as the synthesis of glycosaminoglycan (GAG) and collagen contents, but overloading alone had no effects on ECM degradation. IL-1β challenge had little effect on cell viability, proliferation, or protein synthesis but induced over 40% GAG loss in 10 days and 61% collagen loss in 6 weeks. For the overloaded samples, IL-1β induced greater degrees of degradation, with 68% GAG loss in 10 days and 80% collagen loss in 6 weeks. The results imply a necessary immediate ease of inflammation after joint injuries when trauma damage on cartilage is present. The new click chemistry methods could benefit many cellular and tissue engineering studies, providing convenient and sensitive assays of metabolic activities of cells in native three-dimensional (3D) environments.

Vitrification of particulated articular cartilage via calculated protocols

Preserving viable articular cartilage is a promising approach to address the shortage of graft tissue and enable the clinical repair of articular cartilage defects in articulating joints, such as the knee, ankle, and hip. In this study, we developed two 2-step, dual-temperature, multicryoprotectant loading protocols to cryopreserve particulated articular cartilage (cubes ~1 mm3 in size) using a mathematical approach, and we experimentally measured chondrocyte viability, metabolic activity, cell migration, and matrix productivity after implementing the designed loading protocols, vitrification, and warming. We demonstrated that porcine and human articular cartilage cubes can be successfully vitrified and rewarmed, maintaining high cell viability and excellent cellular function. The vitrified particulated articular cartilage was stored for a period of 6 months with no significant deterioration in chondrocyte viability and functionality. Our approach enables high-quality long-term storage of viable articular cartilage that can alleviate the shortage of grafts for use in clinically repairing articular cartilage defects.

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

ObjectiveDuring 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.MethodsBovine osteochondral grafts from five animals were punched out from the medial condyle and treated with or without pro-inflammatory cytokines (interleukin-1β [IL-1β], 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.ResultsAfter 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.ConclusionsTreatment 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.

Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage

Vitrification is a cryopreservation technique for the long-term storage of viable tissue, but the success of this technique relies on multiple factors. In 2012, our group published a working vitrification protocol for intact human articular cartilage and reported promising chondrocyte recovery after using a four-step multi-cryoprotectant (CPA) loading method that required 570 min. However, this protocol requires further optimization for clinical practice. Herein, we compared three multi-step CPA loading protocols to investigate their impact on chondrocyte recovery after vitrification of porcine articular cartilage on a bone base, including our previous four-step protocol (original: 570 min), and two shorter three-step protocols (optimized: 420 min, and minimally vitrifiable: 310 min). Four different CPAs were used including glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. As vitrification containers, two conical tubes (50 ml and 15 ml) were evaluated for their heat transfer impact on chondrocyte recovery after vitrification. Osteochondral dowels were cored into two diameters of 10.0 mm and 6.9 mm with an approximately 10-mm thick bone base, and then allocated into the twelve experimental groups based on CPA loading protocol, osteochondral dowel size, and vitrification container size. After vitrification at −196 °C and tissue warming and CPA removal, samples in all groups were assessed for both chondrocyte viability and metabolic activity. The optimized protocol proposed based on mathematical modelling resulted in similar chondrocyte recovery to our original protocol and it was 150 min shorter. Furthermore, this study illustrated the role of CPA permeation (dowel size) and heat transfer (container size) on vitrification protocol outcome.

Exploring Penicillin G as an Intrawound Antibiotic Powder for Prevention of Postoperative Shoulder Infections: Does It Exhibit In Vitro Chondrotoxicity?

Cutibacterium acnes (C. acnes) is a significant insidious pathogen for postoperative infections in shoulder surgery. Studies have demonstrated that certain topical antibiotic powders used have the potential for chondrotoxicity. Benzylpenicillin, commonly referred to as Penicillin G (Pen G) has the lowest minimum inhibitory concentration (MIC) for C. acnes. There is no research regarding the topical application of Pen G during shoulder surgery, nor has its chondrocyte toxicity been previously investigated. This study sought to characterize the in vitro chondrocyte toxicity of Pen G. Culture-derived bovine chondrocytes were exposed to serial Pen G concentrations and compared with a positive and negative control. A negative control of growth medium and positive control of 1% Triton solution. The chondrocyte viability was assessed via spectrophotometer absorbance. The treatment groups were analyzed using one-way repeated measures analysis of variance and Pearson's correlation analysis. The chondrocyte viability was significantly higher for all Pen G concentrations as compared with the positive control (p < 0.001). All concentrations of Pen G exhibited continued chondrocyte metabolic activity over time. Analysis of variance, independent of time, demonstrated no significant decrease in chondrocyte viability for Pen G concentrations ≤6.25 mg/ml, as compared with the negative control (p > 0.05). Pen G demonstrated a significant negative correlation with its concentration and absorbance (r = 0.371, p < 0.001), however, concentrations ≤6.25 mg/ml did not demonstrate a significant decrease in chondrocyte viability (p = 0.063). Pen G in concentrations appropriate for C. acnes is not significantly chondrotoxic and may be safe for intrawound application. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:726-730, 2020.

Tribocorrosion of a CoCrMo alloy sliding against articular cartilage and the impact of metal ion release on chondrocytes

Partial knee replacement and hemiarthroplasty are some of the orthopedic procedures resulting in a metal on cartilage interface. As metal implant material, CoCrMo based alloys are commonly used. The aim of the present study is to assess the role of biotribocorrosion on the CoCrMo-cartilage interface with an emphasis on metal release during sliding contact. The biotribocorrosion experiments were performed under controlled electrochemical conditions using a floating cell with a three electrode set up coupled to a microtribometer. Throughout the experiment the coefficient of friction and the open circuit potential were monitored. Analyses of the electrolyte after the experiment show that metal release can occur during sliding contact of CoCrMo alloy against articular cartilage despite the extraordinary low coefficient of friction measured. Metal release is attributed to changes in passive layer caused at the onset of sliding. The released metal was found to be forming compounds with potential cytotoxicity. Since the presence of metal ions in the cartilage matrix can potentially lead to cell apoptosis, the metabolic activity of human osteoarthritic chondrocytes (2D-cultures) was investigated in the presence of phosphate buffered saline containing metal ions using XTT-assay. The experiments indicate that critical concentrations of Co ions lead to a significant decrease in chondrocyte metabolic activity. Therefore, biotribocorrosion is a mechanism that can occur in partial replacements and lead to chondrocyte apoptosis thus playing a role in the observed accelerated degradation of the remaining cartilage tissue after the mentioned orthopedic procedures. Statement of SignificancePartial replacements provide an alternative to total joint replacements. This procedure is less invasive, allows a faster rehabilitation and provides a better function of the joint. However, the remaining native cartilage experiences accelerated degradation when in contact with metallic implant components. This work investigates the role of tribocorrosion at the metal-cartilage interface during sliding. Tribocorrosion is a degradation process that can alter significantly the wear rates experienced by metallic implants and lead to the release of metal ions and particles. The released metal can form compounds with potential cytotoxicity on cartilage tissue. The knowledge gained in this work will serve to understand the mechanisms behind the failure of partial replacements and develop future biomaterials with an enhanced lifetime.

Human Engineered Cartilage and Decellularized Matrix as an Alternative to Animal Osteoarthritis Model.

(1) Objective: to obtain a reproducible, robust, well-defined, and cost-affordable in vitro model of human cartilage degeneration, suitable for drug screening; (2) Methods: we proposed 3D models of engineered cartilage, considering two human chondrocyte sources (articular/nasal) and five culture methods (pellet, alginate beads, silk/alginate microcarriers, and decellularized cartilage). Engineered cartilages were treated with pro-inflammatory cytokine IL-1β to promote cartilage degradation; (3) Results: articular chondrocytes have been rejected since they exhibit low cellular doubling with respect to nasal cells, with longer culture time for cell expansion; furthermore, pellet and alginate bead cultures lead to insufficient cartilage matrix production. Decellularized cartilage resulted as good support for degeneration model, but long culture time and high cell amount are required to obtain the adequate scaffold colonization. Here, we proposed, for the first time, the combined use of decellularized cartilage, as aggrecanase substrate, with pellet, alginate beads, or silk/alginate microcarriers, as polymeric scaffolds for chondrocyte cultures. This approach enables the development of suitable models of cartilaginous pathology. The results obtained after cryopreservation also demonstrated that beads and microcarriers are able to preserve chondrocyte functionality and metabolic activity; (4) Conclusions: alginate and silk/alginate-based scaffolds can be easily produced and cryopreserved to obtain a cost-affordable and ready-to-use polymer-based product for the subsequent screening of anti-inflammatory drugs for cartilage diseases.

NONSTEROID ANTI-INFLAMMATORY DRUGS FOR THE TREATEMENT OF OSTEOARTHROSIS: COMPARATIVE EFFICACY AND TOLERABILITY OF NIMESULIDE

Nonsteroid anti-inflammatory drugs are the fast-acting medications used widely to treat pain and inflammation for osteoarthritis. The pharmacological activity of most of them is characterized by the chondronegative action on osteoarthrosis cartilage, which manifests itself as inhibition of the chondrocyte metabolic activity and intensification of its apoptosis, which reduces the synthesis of proteoglycans, the main intercellular substance of the hyaline cartilage and thus its degeneration rates. In contrast, nimesulides show a chondronegative action. The article presents the results of a comparative randomized study evaluating the efficacy and tolerability of two Nimesulide generics, Nimesan and Nise. Nimesan was not only inferior to Nise by its characteristics, but also proved to be more efficient by the overall WOMAC index, positive dynamics of the functional capacity of the affected joints and the ESR index. Both drugs showed comparable tolerability and safety results.

Characterization of layered chondrocyte sheets created in a co-culture system with synoviocytes in a hypoxic environment

Endeavouring to repair and regenerate articular cartilage using cell sheets, we have previously established a co-culture system of chondrocytes and synoviocytes, and have reported the successful and rapid production of chondrocyte sheets. In the present study, to examine the effects of oxygen concentration on the chondrocyte sheets, we co-cultured human articular chondrocytes and human synoviocytes in 2%, 5% and 21% oxygen, and measured chondrocyte metabolic activity and proliferation activities under each condition for 14days in culture. Layered chondrocyte sheets were also created under each condition and the proteoglycan (PG) level was compared with the gene expression of type I collagen (COL1), COL2, COL27, tissue metallopeptidase inhibitor 1 (TIMP1), fibronectin-1 (FN1), SRY-related HMG Box 9 (SOX9), aggrecan-1 (ACAN), integrin-α10 (ITGα10), matrix metalloproteinase 3 (MMP3), MMP13 and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5). Compared with 5% and 21% oxygen, the 2% condition caused significantly greater cell metabolic activity and proliferation (p<0.05). The 2% condition produced a 10% greater PG level compared with 21% oxygen (p<0.05). All conditions increased the expression of chondrocyte-specific genes, such as COL2, and were associated with low expression levels of catabolic factors, such as MMP3 and MMP13. These observations indicated that the specificity of the chondrocyte sheets was maintained under all conditions. The culture times did not differ between the 5% and 21% conditions. Compared with 21% oxygen, layered chondrocyte sheets rich in extracellular matrix were created 2.85days earlier in 2% oxygen, which is similar to the level found in deep cartilage. Copyright © 2016 John Wiley & Sons, Ltd.

36. An engineering perspective on toxicity

Toxicity plays a central role in cryopreservation, whether it be toxicity of added cryoprotectants or of naturally occurring salts and other solutes concentrated in the unfrozen solution by ice formation. Toxicity may be studied from the viewpoint of biochemical mechanisms—understanding what molecule, pathway, or structure is “poisoned”. However, an engineering perspective on toxicity is extremely relevant to cryopreservation. The toxicity of compounds to cells is concentration, temperature, and exposure-time dependent. Understanding the cryoprotectant exposure that a cell deep within a tissue experiences in a given loading/removal protocol requires an understanding of permeation/efflux kinetics with time. The dependence of toxicity on temperature can be used to advantage in designing cryopreservation strategies. Finally, even without knowledge of toxicity mechanisms, cryoprotectant toxicity and toxicity interactions can be modelled with empirical equations for use in protocol design. Our research group has explored these issues. The developed engineering approaches to toxicity were critical in design of a protocol to cryopreserve intact full-thickness human articular cartilage on a bone base (as required for transplantation) with high chondrocyte viability, metabolic activity and function.

Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation

Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1(-/-) chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.

Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis.

Nanocomposite Scaffold for Chondrocyte Growth and Cartilage Tissue Engineering: Effects of Carbon Nanotube Surface Functionalization

The goal of this study was to assess the long-term biocompatibility of single-wall carbon nanotubes (SWNTs) for tissue engineering of articular cartilage. We hypothesized that SWNT nanocomposite scaffolds in cartilage tissue engineering can provide an improved molecular-sized substrate for stimulation of chondrocyte growth, as well as structural reinforcement of the scaffold's mechanical properties. The effect of SWNT surface functionalization (-COOH or -PEG) on chondrocyte viability and biochemical matrix deposition was examined in two-dimensional cultures, in three-dimensional (3D) pellet cultures, and in a 3D nanocomposite scaffold consisting of hydrogels+SWNTs. Outcome measures included cell viability, histological and SEM evaluation, GAG biochemical content, compressive and tensile biomechanical properties, and gene expression quantification, including extracellular matrix (ECM) markers aggrecan (Agc), collagen-1 (Col1a1), collagen-2 (Col2a1), collagen-10 (Col10a1), surface adhesion proteins fibronectin (Fn), CD44 antigen (CD44), and tumor marker (Tp53). Our findings indicate that chondrocytes tolerate functionalized SWNTs well, with minimal toxicity of cells in 3D culture systems (pellet and nanocomposite constructs). Both SWNT-PEG and SWNT-COOH groups increased the GAG content in nanocomposites relative to control. The compressive biomechanical properties of cell-laden SWNT-COOH nanocomposites were significantly elevated relative to control. Increases in the tensile modulus and ultimate stress were observed, indicative of a tensile reinforcement of the nanocomposite scaffolds. Surface coating of SWNTs with -COOH also resulted in increased Col2a1 and Fn gene expression throughout the culture in nanocomposite constructs, indicative of increased chondrocyte metabolic activity. In contrast, surface coating of SWNTs with a neutral -PEG moiety had no significant effect on Col2a1 or Fn gene expression, suggesting that the charged nature of the -COOH surface functionalization may promote ECM expression in this culture system. The results of this study indicate that SWNTs exhibit a unique potential for cartilage tissue engineering, where functionalization with bioactive molecules may provide an improved substrate for stimulation of cellular growth and repair.

The study of the frequency effect of dynamic compressive loading on primary articular chondrocyte functions using a microcell culture system.

Compressive stimulation can modulate articular chondrocyte functions. Nevertheless, the relevant studies are not comprehensive. This is primarily due to the lack of cell culture apparatuses capable of conducting the experiments in a high throughput, precise, and cost-effective manner. To address the issue, we demonstrated the use of a perfusion microcell culture system to investigate the stimulating frequency (0.5, 1.0, and 2.0 Hz) effect of compressive loading (20% and 40% strain) on the functions of articular chondrocytes. The system mainly integrates the functions of continuous culture medium perfusion and the generation of pneumatically-driven compressive stimulation in a high-throughput micro cell culture system. Results showed that the compressive stimulations explored did not have a significant impact on chondrocyte viability and proliferation. However, the metabolic activity of chondrocytes was significantly affected by the stimulating frequency at the higher compressive strain of 40% (2 Hz, 40% strain). Under the two compressive strains studied, the glycosaminoglycans (GAGs) synthesis was upregulated when the stimulating frequency was set at 1 Hz and 2 Hz. However, the stimulating frequencies explored had no influence on the collagen production. The results of this study provide useful fundamental insights that will be helpful for cartilage tissue engineering and cartilage rehabilitation.

Influence of cryopreservation, cultivation time and patient’s age on gene expression in Hyalograft® C cartilage transplants

Our aim was to evaluate the impact of cryopreservation, cultivation time and patient's age on the expression of specific chondrogenic markers in Hyalograft® C transplants. Gene expression of chondrocyte markers [collagen type I (COL1A1), COL2A1, aggrecan, versican, melanoma inhibitory activity (MIA) and interleukin (IL)-1β] was analysed in cartilage biopsies (n = 17) and Hyalograft® C transplant samples (non-cryopreserved = 78, cryopreserved = 13) by quantitative real-time polymerase chain reaction (PCR). Correlation analyses were performed to evaluate the influence of the above-named parameters on the level of gene expression. Cryopreservation of cells was found to decrease COL2A1 and MIA significantly (4.6-fold, p < 0.01 and 2-fold, p < 0.045, respectively). The duration of cryopreservation had no further influence on the expression of these factors. No correlation was detected between cultivation time (75 ± 31 days) and the expression level of any gene. Cartilage transplants from older patients (>35 years) exhibited a significantly higher IL-1β expression (3.7-fold, p < 0.039) than transplants from younger patients (≤ 35 years). Our data demonstrate that cryopreservation has a profound impact on chondrocyte metabolic activity by decreasing the expression of COL2A1 and MIA in Hyalograft® C transplants, independent of the duration of cryopreservation.

Mesenchymal stromal cell-derived extracellular matrix influences gene expression of chondrocytes

Decellularized extracellular matrix (ECM) has recently gained a lot of interest as an instructive biomaterial for regenerative medicine applications. In this study, the ability of adult human mesenchymal stem cell (hMSC)-derived ECM to rescue the phenotype of osteoarthritic (OA) chondrocytes and to further stimulate the differentiation of healthy (HL) chondrocytes was evaluated. ECMs were prepared by decellularizing hMSCs cultured in basic medium (BM) and chondrogenic medium (CM). The obtained ECM was then combined with a polymeric solution of Poly (ε-caprolactone) (PCL) dissolved in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) and electrospun meshes were fabricated. Electrospun ECM scaffolds were characterized using scanning electron microscopy (SEM) and picrosirius red staining was used to confirm the presence of collagen. OA and HL chondrocytes were cultured on scaffolds containing hMSC ECM in BM or CM and compared to PCL electrospun scaffolds without ECM. Metabolic activity and chondrogenic gene expression were assessed by Alamar blue assay and quantitative PCR (qPCR) analysis, respectively. The ECM presence resulted in a significant difference in chondrocyte metabolic activity compared to PCL scaffolds alone. HL chondrocytes cultured for 21 days in chondrogenic medium on electrospun scaffolds containing hMSC ECM from BM showed a significant increase in collagen II and aggrecan expression compared to hMSC ECM from CM and PCL scaffolds without ECM incorporation. No significant influence of hMSC ECM presence on the chondrogenic signature of OA chondrocytes was found. The influence of decellularized hMSC ECM on HL chondrocytes suggests that hMSC-derived ECM scaffolds are promising candidates for cartilage tissue engineering applications.