Immature bovine cartilage wear is due to fatigue failure from repetitive compressive forces and not reciprocating frictional forces.

OBJECTIVE: Wear of articular cartilage is not well understood. We hypothesize that cartilage wears due to fatigue failure in repetitive compression instead of reciprocating friction. DESIGN: This study compares reciprocating sliding of immature bovine articular cartilage against glass in two testing configurations: (1) a stationary contact area configuration (SCA), which results in static compression, interstitial fluid depressurization, and increasing friction coefficient during reciprocating sliding, and (2) a migrating contact area configuration (MCA), which maintains pressurization and low friction while producing repetitive compressive loading in addition to reciprocating sliding. Contact pressure, sliding duration, and sliding distance were controlled to be similar between test groups. RESULTS: SCA tests exhibited an average friction coefficient of µ=0.084±0.032, while MCA tests exhibited a lower average friction coefficient of µ=0.020±0.008 (p<10-4). Despite the lower friction, MCA cartilage samples exhibited clear surface damage with a significantly greater average surface deviation from a fitted plane after wear testing (Rq=0.125±0.095 mm) than cartilage samples slid in a SCA configuration (Rq=0.044±0.017 mm, p=0.002), which showed minimal signs of wear. Polarized light microscopy confirmed that delamination damage occurred between the superficial and middle zones of the articular cartilage in MCA samples. CONCLUSIONS: The greatest wear was observed in the group with lowest friction coefficient, subjected to cyclical instead of static compression, implying that friction is not the primary driver of cartilage wear. Delamination between superficial and middle zones implies the main mode of wear is fatigue failure under cyclical compression, not fatigue or abrasion due to reciprocating frictional sliding.

Peri-discharge complex interventions for reducing 30-day hospital readmissions among heart failure patients: overview of systematic reviews and network meta-analysis.

AIMS: An overview of systematic reviews (SRs) and network meta-analysis (NMA) was conducted to synthesize evidence of comparative effectiveness of different peri-discharge complex interventions for reducing 30-day hospital readmissions among heart failure (HF) patients. METHODS: We searched five databases for SRs from their inception to August 2019 and conducted additional search for randomized controlled trials (RCTs) published between 2003 and 2020. We used random-effect pairwise meta-analysis with pooled risk ratios (RRs) and 95% confidence intervals (CIs) to quantify the effect of complex interventions, and NMA to evaluate comparative effectiveness among complex interventions. Primary outcome was 30-day all-cause hospital readmissions, while secondary outcomes were 30-day HF-related hospital readmissions, 30-day mortality, and 30-day emergency department visits. RESULTS: From 20 SRs and additional RCT search, 21 eligible RCTs (n = 5362) assessing eight different peri-discharge complex interventions were included. Pairwise meta-analysis showed no significant difference between peri-discharge complex interventions and controls on all outcomes, except that peri-discharge complex interventions were significantly more effective than controls in reducing 30-day mortality (pooled RR = 0.68, 95% CI: 0.49-0.95, 5 RCTs). NMA indicated that for reducing 30-day all-cause hospital readmissions, supportive-educative intervention had the highest probability to be the best intervention, followed by disease management; while for reducing 30-day HF-related hospital readmissions, disease management is likely to be the best intervention. CONCLUSIONS: Our results suggest that disease management has the best potential to reduce 30-day all-cause and HF-related hospital readmissions. Benefits of the interventions may vary across health system contexts. Evidence-based complex interventions require local adaptation prior to implementation.

Toward understanding the role of cartilage particulates in synovial inflammation.

OBJECTIVE: Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. METHOD: In this study sub-10 µm cartilage particles or 1 µm latex particles were co-cultured with FLS ±10 ng/mL interleukin-1α (IL-1α) or tumor necrosis factor-α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen, and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. RESULTS: Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. CONCLUSION: The proliferative response of FLS to cartilage wear particles resulted in an overall increase in extracellular matrix (ECM) content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

A hospital-wide screening programme to control an outbreak of vancomycin-resistant enterococci in a large tertiary hospital in Hong Kong.

INTRODUCTION: Apart from individual small-scale outbreaks, infections with vancomycin-resistant enterococci are uncommon in Hong Kong. A major outbreak of vancomycin-resistant enterococci, however, occurred at a large tertiary hospital in 2013. We describe the successful control of this outbreak and share the lessons learned. METHODS: In 2013, there was an abnormal increase in the incidence of vancomycin-resistant enterococci carriage compared with baseline in multiple clinical departments at Queen Elizabeth Hospital. A multipronged approach was adopted that included a 10-week hospital-wide active screening programme, which aimed to identify and isolate hidden vancomycin-resistant enterococci carriers among all in-patients. The identified carriers were completely segregated in designated wards where applicable. Other critical infection control measures included directly observed hand hygiene and environmental hygiene. A transparent and open disclosure approach was adopted throughout the outbreak. RESULTS: The infection control measures were successfully implemented. The active screening of vancomycin-resistant enterococci was conducted between 30 September and 10 November 2013. A total of 7053 rectal swabs were collected from patients in 46 hospital wards from 11 departments. The overall carriage rate of vancomycin-resistant enterococci was 2.8% (201/7053). Pulsed-field gel electrophoresis showed a predominant outbreak clone. We curbed the outbreak and kept the colonisation of vancomycin-resistant enterococci among patients at a pre-upsurge low level. CONCLUSIONS: We report the largest cohesive effort to control spread of vancomycin-resistant enterococci in Hong Kong. Coupled with other infection control measures, we successfully controlled vancomycin-resistant enterococci to the pre-outbreak level. We have demonstrated that the monumental tasks can be achieved with meticulous planning, and thorough communication and understanding between all stakeholders.

Passage-dependent relationship between mesenchymal stem cell mobilization and chondrogenic potential.

OBJECTIVE: Galvanotaxis, the migratory response of cells in response to electrical stimulation, has been implicated in development and wound healing. The use of mesenchymal stem cells (MSCs) from the synovium (synovium-derived stem cells, SDSCs) has been investigated for repair strategies. Expansion of SDSCs is necessary to achieve clinically relevant cell numbers; however, the effects of culture passage on their subsequent cartilaginous extracellular matrix production are not well understood. METHODS: Over four passages of SDSCs, we measured the expression of cell surface markers (CD31, CD34, CD49c, CD73) and assessed their migratory potential in response to applied direct current (DC) electric field. Cells from each passage were also used to form micropellets to assess the degree of cartilage-like tissue formation. RESULTS: Expression of CD31, CD34, and CD49c remained constant throughout cell expansion; CD73 showed a transient increase through the first two passages. Correspondingly, we observed that early passage SDSCs exhibit anodal migration when subjected to applied DC electric field strength of 6 V/cm. By passage 3, CD73 expression significantly decreased; these cells exhibited cell migration toward the cathode, as previously observed for terminally differentiated chondrocytes. Only late passage cells (P4) were capable of developing cartilage-like tissue in micropellet culture. CONCLUSIONS: Our results show cell priming protocols carried out for four passages selectively differentiate stem cells to behave like chondrocytes, both in their motility response to applied electric field and their production of cartilaginous tissue.

Animal models of cartilage repair.

Cartilage repair in terms of replacement, or regeneration of damaged or diseased articular cartilage with functional tissue, is the 'holy grail' of joint surgery. A wide spectrum of strategies for cartilage repair currently exists and several of these techniques have been reported to be associated with successful clinical outcomes for appropriately selected indications. However, based on respective advantages, disadvantages, and limitations, no single strategy, or even combination of strategies, provides surgeons with viable options for attaining successful long-term outcomes in the majority of patients. As such, development of novel techniques and optimisation of current techniques need to be, and are, the focus of a great deal of research from the basic science level to clinical trials. Translational research that bridges scientific discoveries to clinical application involves the use of animal models in order to assess safety and efficacy for regulatory approval for human use. This review article provides an overview of animal models for cartilage repair. Cite this article: Bone Joint Res 2014;4:89-94.

Time and dose-dependent effects of chondroitinase ABC on growth of engineered cartilage.

Tissue engineering techniques have been effective in developing cartilage-like tissues in vitro. However, many scaffold-based approaches to cultivating engineered cartilage have been limited by low collagen production, an impediment for attaining native functional load-bearing tensile mechanical properties. Enzymatic digestion of glycosaminoglycans (GAG) with chondroitinase ABC (chABC) temporarily suppresses the construct's GAG content and compressive modulus and increases collagen content. Based on the promising results of these early studies, the aim of this study was to further promote collagen deposition through more frequent chABC treatments. Weekly dosing of chABC at a concentration of 0.15 U/mL resulted in a significant cell death, which impacted the ability of the engineered cartilage to fully recover GAG and compressive mechanical properties. In light of these findings, the influence of lower chABC dosage on engineered tissue (0.004 and 0.015 U/mL) over a longer duration (one week) was investigated. Treatment with 0.004 U/mL reduced cell death, decreased the recovery time needed to achieve native compressive mechanical properties and GAG content, and resulted in a collagen content that was 65 % greater than the control. In conclusion, the results of this study demonstrate that longer chABC treatment (one week) at low concentrations can be used to improve collagen content in developing engineered cartilage more expediently than standard chABC treatments of higher chABC doses administered over brief durations.

Tuberculosis increases the subsequent risk of acute coronary syndrome: a nationwide population-based cohort study.

OBJECTIVE: To evaluate the effects of pulmonary tuberculosis (PTB) on the risk of subsequent acute coronary syndrome (ACS) development. METHODS: The incidence and risk factors of ACS were investigated in 10 168 newly diagnosed tuberculosis (TB) patients from Taiwan's National Health Insurance Research Database between 1997 and 2010, and 40 672 controls without TB from the general population. The follow-up period ran from the diagnosis of new TB to the date of the ACS event, censoring or 31 December 2010. RESULTS: During the follow-up period, the overall incidence of ACS was higher in TB patients than in non-TB patients (2.10 vs. 1.51 per 1000 person-years). The incidence of ACS increased by 40% in TB patients after adjusting for age, sex and co-morbidities. Male sex, age, hypertension and diabetes were independent factors for the risk of ACS development. The probability of ACS increased in the years following the TB diagnosis. CONCLUSION: This nationwide population-based cohort study provides compelling evidence that TB patients are at higher risk of developing ACS, and that the risk increases with age. Clinicians should be aware of this and strive to reduce ACS risk factors in TB patients.

Shearing of synovial fluid activates latent TGF-ß.

OBJECTIVE: TGF-ß is synthesized in an inactive latent complex that is unable to bind to membrane receptors, thus unable to induce a cellular biological response until it has been activated. In addition to activation by chemical mediators, recent studies have demonstrated that mechanical forces may activate latent TGF-ßvia integrin-mediated cellular contractions, or mechanical shearing of blood serum. Since TGF-ß is present in synovial fluid in latent form, and since normal diarthrodial joint function produces fluid shear, this study tested the hypothesis that the native latent TGF-ß1 of synovial fluid can be activated by shearing. DESIGN: Synovial fluid from 26 bovine joints and three adult human joints was sheared at mean shear rates up to 4000 s(-1) for up to 15 h. RESULTS: Unsheared synovial fluid was found to contain high levels of latent TGF-ß1 (4.35 ± 2.02 ng/mL bovine, 1.84 ± 0.89 ng/mL human; mean ± radius of 95% confidence interval) and low amounts (<0.05 ng/mL) of the active peptide. Synovial fluid concentrations of active TGF-ß1 increased monotonically with shear rate and shearing duration, reaching levels of 2.64 ± 1.22 ng/mL for bovine and 0.60 ± 0.39 ng/mL for human synovial fluid. Following termination of shearing, there was no statistical change in these active levels over the next 8 h for either species, demonstrating long-term stability of the activated peptide. The unsheared control group continued to exhibit negligible levels of active TGF-ß1 at all times. CONCLUSIONS: Results confirmed the hypothesis of this study and suggest that shearing of synovial fluid might contribute an additional biosynthetic effect of mechanical loading of diarthrodial joints.

Response of engineered cartilage to mechanical insult depends on construct maturity.

UNLABELLED: Injury to articular cartilage leads to degenerative changes resulting in a loss of mechanical and biochemical properties. In engineered cartilage, the injury response of developing constructs is unclear. OBJECTIVE: To characterize the cellular response of tissue-engineered constructs cultured in chemically-defined medium after mechanical insult, either by compression-induced cracking, or by cutting, as a function of construct maturity. METHODS: Primary immature bovine articular chondrocytes (4-6 weeks) were encapsulated in agarose hydrogel (2%, 30 millioncells/mL) and cultured in chemically-defined medium supplemented with Transforming growth factor (TGF)-ß3 (10ng/mL, first 2 weeks). At early (5 days) and late (35 days) times in culture, subsets of constructs were exposed to mechanical overload to produce a crack in the tissue or were exposed to a sharp wound with a perpendicular cut. Constructs were returned to culture and allowed to recover in static conditions. Mechanical and biochemical properties were evaluated at 2-week intervals to day 70, and cellular viability was assessed at 2-week intervals to day 85. RESULTS: Constructs injured early in culture recovered their mechanical stiffness back to control values, regardless of the mode of injury. Later in culture, when constructs exhibited properties similar to those of native cartilage, compression-induced cracking catastrophically damaged the bulk matrix of the tissue and resulted in permanent mechanical failure with persistent cell death. No such detrimental outcomes were observed with cutting. Biochemical content was similar across all groups irrespective of mode or time of injury. CONCLUSIONS: Unlike native cartilage, engineered cartilage constructs exhibit a reparative capacity when the bulk integrity of the developing tissue is preserved after injury.

Spatial regulation of human mesenchymal stem cell differentiation in engineered osteochondral constructs: effects of pre-differentiation, soluble factors and medium perfusion.

OBJECTIVE: The objective of the study was to investigate the combined effects of three sets of regulatory factors: cell pre-differentiation, soluble factors and medium perfusion on spatial control of human mesenchymal stem cell (hMSC) differentiation into cells forming the cartilaginous and bone regions in engineered osteochondral constructs. DESIGN: Bone-marrow derived hMSCs were expanded in their undifferentiated state (UD) or pre-differentiated (PD) in monolayer culture, seeded into biphasic constructs by interfacing agarose gels and bone scaffolds and cultured for 5 weeks either statically (S) or in a bioreactor (BR) with perfusion of medium through the bone region. Each culture system was operated with medium containing either chondrogenic supplements (C) or a cocktail (Ck) of chondrogenic and osteogenic supplements. RESULTS: The formation of engineered cartilage in the gel region was most enhanced by using undifferentiated cells and chondrogenic medium, whereas the cartilaginous properties were negatively affected by using pre-differentiated cells or the combination of perfusion and cocktail medium. The formation of engineered bone in the porous scaffold region was most enhanced by using pre-differentiated cells, perfusion and cocktail medium. Perfusion also enhanced the integration of bone and cartilage regions. CONCLUSIONS: (1) Pre-differentiation of hMSCs before seeding on scaffold was beneficial for bone but not for cartilage formation. (2) The combination of medium perfusion and cocktail medium inhibited chondrogenesis of hMSCs. (3) Perfusion improved the cell and matrix distribution in the bone region and augmented the integration at the bone-cartilage interface. (4) Osteochondral grafts can be engineered by differentially regulating the culture conditions in the two regions of the scaffold seeded with hMSCs (hydrogel for cartilage, perfused porous scaffold for bone).

Involvement of cyclooxygenase 2 and prostagladin E(2) in the effects of insulin on gastric emptying in male rats.

Delayed gastric emptying in patients with both type 1 and type 2 diabetes mellitus (DM) occurs in approximately 50% of these patients. However, the role and the action mechanism of insulin on gastrointestinal (GI) motility are still unclear. The purpose of the present study was to investigate the involvement of cyclooxygenase-2 (COX-2) and prostaglandin E(2) in the effects of insulin on gastric emptying in male rats. The normal and streptozotocin (STZ)-pretreated rats were injected intraperitoneally with or without insulin, atropine and specific muscarinic receptor antagonists before examination of measurement of gastric emptying, spontaneous contractile activity of smooth muscle strips, plasma cholecystokinin (CCK), and prostaglandin E(2) (PGE(2)) analysis. Protein expression of COX-2 and insulin receptors (IRs) were analyzed by the technique of western blot. Acute different doses of insulin accelerated gastric emptying. Atropine interrupted the insulin effect on gastric emptying, and muscarnic M1/M3 receptor antagonists interrupted the insulin-reversed gastric emptying in normal and DM rats. Besides, we observed the expression of (IRs) in GI and found that IR was changed under the insulin and DM treatment, and was also different between STZ-pretreated rats and hyperglycemic rats. Expression of COX-2 in stomach was decreased in DM rats but restored by insulin. The COX inhibitor, indomethacin, decreased the gastric emptying which was induced or reversed by insulin in normal and DM rats, respectively. PGE(2) production in stomach corresponded to the COX-2 expression. The contraction of GI smooth muscle stimulated by PGE(2) was increased in insulin-pretreated normal and DM rats. We conclude that insulin changed the expression of IRs in stomach in DM rats. The delayed GI motility in diabetes was at least in part due to the COX-2 and PGE(2) pathway which associated with decreasing COX-2 and diminishing PGE(2) production in stomach. The attenuation of PGE(2) production was employed for the index of the reduction of smooth muscle contraction in stomach in diabetes. Insulin stimulated the smooth muscle contraction through the IRs and COX-2 expression plus PGE(2) production in rat stomach as well as reversed the delayed gastric emptying via the nervous actions of muscarinic M1 and M3 receptors in DM rats.

Analysis of radial variations in material properties and matrix composition of chondrocyte-seeded agarose hydrogel constructs.

OBJECTIVE: To examine the radial variations in engineered cartilage that may result due to radial fluid flow during dynamic compressive loading. This was done by evaluating the annuli and the central cores of the constructs separately. METHOD: Chondrocyte-seeded agarose hydrogels were grown in free-swelling and dynamic, unconfined loading cultures for 42 days. After mechanical testing, constructs were allowed to recover for 1-2h, the central 3mm cores removed, and the cores and annuli were retested separately. Histological and/or biochemical analyses for DNA, glycosaminoglycan (GAG), collagen, type I collagen, type II collagen, and elastin were performed. Multiple regression analysis was used to determine the correlation between the biochemical and material properties of the constructs. RESULTS: The cores and annuli of chondrocyte-seeded constructs did not exhibit significant differences in material properties and GAG content. Annuli possessed greater DNA and collagen content over time in culture than cores. Dynamic loading enhanced the material properties and GAG content of cores, annuli, and whole constructs relative to free-swelling controls, but it did not alter the radial variations compared to free-swelling culture. CONCLUSION: Surprisingly, the benefits of dynamic loading on tissue properties extended through the entire construct and did not result in radial variations as measured via the coring technique in this study. Nutrient transport limitations and the formation of a fibrous capsule on the periphery may explain the differences in DNA and collagen between cores and annuli. No differences in GAG distribution may be due to sufficient chemical signals and building blocks for GAG synthesis throughout the constructs.

Influence of decreasing nutrient path length on the development of engineered cartilage.

OBJECTIVE: Chondrocyte-seeded agarose constructs of 4mm diameter (2.34 mm thickness) develop spatially inhomogeneous material properties with stiffer outer edges and a softer central core suggesting nutrient diffusion limitations to the central construct region [Guilak F, Sah RL, Setton LA. Physical regulation of cartilage metabolism. In: Mow VC, Hayes WC, Eds. Basic Orthopaedic Biomechanics, Philadelphia 1997;179-207.]. The effects of reducing construct thickness and creating channels running through the depth of the thick constructs were examined. METHODS: In Study 1, the properties of engineered cartilage of 0.78 mm (thin) or 2.34 mm (thick) thickness were compared. In Study 2, a single nutrient channel (1 mm diameter) was created in the middle of each thick construct. In Study 3, the effects of channels on larger 10 mm diameter, thick constructs were examined. RESULTS: Thin constructs developed superior mechanical and biochemical properties than thick constructs. The channeled constructs developed significantly higher mechanical properties vs control channel-free constructs while exhibiting similar glycosaminoglycan (GAG) and collagen content. Collagen staining suggested that channels resulted in a more uniform fibrillar network. Improvements in constructs of 10 mm diameter were similarly observed. CONCLUSIONS: This study demonstrated that more homogeneous tissue-engineered cartilage constructs with improved mechanical properties can be achieved by reducing their thickness or incorporating macroscopic nutrient channels. Our data further suggests that these macroscopic channels remain open long enough to promote this enhanced tissue development while exhibiting the potential to refill with cell elaborated matrix with additional culture time. Together with reports that <3 mm defects in cartilage heal in vivo and that irregular holes are associated with clinically used osteochondral graft procedures, we anticipate that a strategy of incorporating macroscopic channels may aid the development of clinically relevant engineered cartilage with functional properties.

Scaffold degradation elevates the collagen content and dynamic compressive modulus in engineered articular cartilage.

OBJECTIVE: It was hypothesized that controlled, scaffold removal in engineered cartilage constructs would improve their collagen content and mechanical properties over time in culture. DESIGN: Preliminary experiments characterized the effects of agarase on cell-free agarose disks and cartilage explants. Immature bovine chondrocytes were encapsulated in agarose, cultured to day 42, and incubated with 100 units/mL agarase for 48 h. After treatment, constructs were cultured to day 91. The compressive Young's modulus and dynamic modulus of the constructs were determined every 2 weeks and immediately after agarase treatment. Post-mechanical testing, constructs were processed for biochemistry and histology. RESULTS: Agarase treatment on explants had no detrimental effect on the cartilage matrix. Treatment applied to engineered constructs on day 42 did not affect DNA or collagen content. Agarase treatment decreased tissue GAG content (via GAG loss to the media) and Young's modulus, both of which recovered to control values over time in culture. By day 91 agarase-treated constructs possessed approximately 25% more DNA, approximately 60% more collagen, and approximately 40% higher dynamic modulus compared to untreated controls. CONCLUSIONS: Scaffold degradation increased construct collagen content and dynamic mechanical properties, affirming the experimental hypothesis. The mechanism may lie in increased nutrient transport, increased space for collagen fibril formation, and cellular response to the loss of GAG with agarase treatment. The results highlight the role of the scaffold in retaining synthesized matrix during early and late tissue formation. This work also shows promise in developing an engineered tissue that may be completely free of scaffold material for clinical implantation.

High-mobility group box 1 protein activates hepatic stellate cells in vitro.

OBJECTIVES: Our previous study noticed remarkably elevated titers of anti-high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis. METHODS: The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure alpha-smooth muscle actin (alpha-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs). RESULTS: HMGB1 at concentrations > 1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. alpha-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment. CONCLUSION: Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts.

Mechanical and biochemical characterization of cartilage explants in serum-free culture.

Allografts of articular cartilage are both used clinically for tissue-transplantation procedures and experimentally as model systems to study the physiological behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Chemical mediators in poorly defined culture media can stimulate cells to quickly degrade their surrounding extracellular matrix. This is particularly true of juvenile cartilage which is generally more responsive to chemical stimuli than mature tissue. By carefully modulating the culture media, however, it may be possible to preserve allograft tissue over the long-term while maintaining its original mechanical and biochemical properties. In this study juvenile bovine cartilage explants (both chondral and osteochondral) were cultured in both chemically defined medium and serum-supplemented medium for up to 6 weeks. The mechanical properties and biochemical content of explants cultured in chemically defined medium were enhanced after 2 weeks in culture and thereafter remained stable with no loss of cell viability. In contrast, the mechanical properties of explants in serum-supplemented medium were degraded by ( approximately 70%) along with a concurrent loss of biochemical content (30-40% GAG). These results suggest that long-term maintenance of allografts can be extended significantly by the use of a chemically defined medium.

Amino acids supply in culture media is not a limiting factor in the matrix synthesis of engineered cartilage tissue.

Increased amino acid supplementation (0.5 x, 1.0 x, and 5.0 x recommended concentrations or additional proline) was hypothesized to increase the collagen content in engineered cartilage. No significant differences were found between groups in matrix content or dynamic modulus. Control constructs possessed the highest compressive Young's modulus on day 42. On day 42, compared to controls, decreased type II collagen was found with 0.5 x, 1.0 x, and 5.0 x supplementation and significantly increased DNA content found in 1.0 x and 5.0 x. No effects were observed on these measures with added proline. These results lead us to reject our hypothesis and indicate that the low collagen synthesis in engineered cartilage is not due to a limited supply of amino acids in media but may require a further stimulatory signal. The results of this study also highlight the impact that culture environment can play on the development of engineered cartilage.