RESUMO
The efforts to grow mechanically functional cartilage from human mesenchymal stem cells have not been successful. We report that clinically sized pieces of human cartilage with physiologic stratification and biomechanics can be grown in vitro by recapitulating some aspects of the developmental process of mesenchymal condensation. By exposure to transforming growth factor-ß, mesenchymal stem cells were induced to condense into cellular bodies, undergo chondrogenic differentiation, and form cartilagenous tissue, in a process designed to mimic mesenchymal condensation leading into chondrogenesis. We discovered that the condensed mesenchymal cell bodies (CMBs) formed in vitro set an outer boundary after 5 d of culture, as indicated by the expression of mesenchymal condensation genes and deposition of tenascin. Before setting of boundaries, the CMBs could be fused into homogenous cellular aggregates giving rise to well-differentiated and mechanically functional cartilage. We used the mesenchymal condensation and fusion of CMBs to grow centimeter-sized, anatomically shaped pieces of human articular cartilage over 5 wk of culture. For the first time to our knowledge biomechanical properties of cartilage derived from human mesenchymal cells were comparable to native cartilage, with the Young's modulus of >800 kPa and equilibrium friction coeffcient of <0.3. We also demonstrate that CMBs have capability to form mechanically strong cartilage-cartilage interface in an in vitro cartilage defect model. The CMBs, which acted as "lego-like" blocks of neocartilage, were capable of assembling into human cartilage with physiologic-like structure and mechanical properties.
Assuntos
Cartilagem Articular/citologia , Cartilagem Articular/fisiologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Animais , Fenômenos Biomecânicos/fisiologia , Biomimética/métodos , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Doenças das Cartilagens/terapia , Cartilagem Articular/crescimento & desenvolvimento , Bovinos , Diferenciação Celular/fisiologia , Células Cultivadas , Meios de Cultura/farmacologia , Módulo de Elasticidade/fisiologia , Fricção/fisiologia , Humanos , Células-Tronco Mesenquimais/fisiologiaRESUMO
Quantitative measurements of cartilage wear have been challenging, with no method having yet emerged as a standard. This study tested the hypothesis that latest-generation particle analyzers are capable of detecting cartilage wear debris generated during in vitro loading experiments that last 24 h or less, by producing measurable content significantly above background noise levels otherwise undetectable through standard biochemical assays. Immature bovine cartilage disks (4 mm diameter, 1.3 mm thick) were tested against glass using reciprocal sliding under unconfined compression creep for 24 h. Control groups were used to assess various sources of contamination. Results demonstrated that cartilage samples subjected to frictional loading produced particulate volume significantly higher than background noise and contamination levels at all tested time points (1, 2, 6, and 24 h, p < 0.042). The particle counter was able to detect very small levels of wear (less than 0.02% of the tissue sample by volume), whereas no significant differences were observed in biochemical assays for collagen or glycosaminoglycans among any of the groups or time points. These findings confirm that latest-generation particle analyzers are capable of detecting very low wear levels in cartilage experiments conducted over a period no greater than 24 h.
Assuntos
Cartilagem Articular/citologia , Fenômenos Mecânicos , Tamanho da Partícula , Animais , Fenômenos Biomecânicos , Cartilagem Articular/metabolismo , Bovinos , Teste de Materiais , Pressão , Fatores de TempoRESUMO
In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.
Assuntos
Cartilagem Articular , Mecanotransdução Celular , Fenômenos Biomecânicos , Biofísica , Reatores Biológicos , Cartilagem Articular/fisiologia , Fricção , Lubrificação , Estresse Mecânico , Líquido SinovialRESUMO
This study investigated wear damage of immature bovine articular cartilage using reciprocal sliding of tibial cartilage strips against glass or cartilage. Experiments were conducted in physiological buffered saline (PBS) or mature bovine synovial fluid (SF). A total of 63 samples were tested, of which 47 exhibited wear damage due to delamination of the cartilage surface initiated in the middle zone, with no evidence of abrasive wear. There was no difference between the friction coefficient of damaged and undamaged samples, showing that delamination wear occurs even when friction remains low under a migrating contact area configuration. No difference was observed in the onset of damage or in the friction coefficient between samples tested in PBS or SF. The onset of damage occurred earlier when testing cartilage against glass versus cartilage against cartilage, supporting the hypothesis that delamination occurs due to fatigue failure of the collagen in the middle zone, since stiffer glass produces higher strains and tensile stresses under comparable loads. The findings of this study are novel because they establish that delamination of the articular surface, starting in the middle zone, may represent a primary mechanism of failure. Based on preliminary data, it is reasonable to hypothesize that delamination wear via subsurface fatigue failure is similarly the primary mechanism of human cartilage wear under normal loading conditions, albeit requiring far more cycles of loading than in immature bovine cartilage.
Assuntos
Cartilagem Articular , Animais , Bovinos , Fricção , Humanos , Estresse Mecânico , Líquido Sinovial , TíbiaRESUMO
Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Técnicas de Cultura de Células/métodos , Humanos , Microscopia de Fluorescência , Estresse MecânicoRESUMO
Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format. Silicone membranes were attached to 96 well plate tops to create stretchable, culture substrates. A custom-built device was designed and validated to apply repeatable, biofidelic strains and strain rates to these plates. A high content approach was used to measure injury in a hypothesis-free manner. These measurements are shown to provide a sensitive, dose-dependent, multi-modal description of the response to mechanical insult. hiPSCNs transition from healthy to injured phenotype at approximately 35% Lagrangian strain. Continued development of this model may create novel opportunities for drug discovery and exploration of the role of human genotype in TAI pathology.
RESUMO
The objective of this study was to measure the wear response of immature bovine articular cartilage tested against glass or alloys used in hemiarthroplasties. Two cobalt chromium alloys and a stainless steel alloy were selected for these investigations. The surface roughness of one of the cobalt chromium alloys was also varied within the range considered acceptable by regulatory agencies. Cartilage disks were tested in a configuration that promoted loss of interstitial fluid pressurization to accelerate conditions believed to occur in hemiarthroplasties. Results showed that considerably more damage occurred in cartilage samples tested against stainless steel (10 nm roughness) and low carbon cobalt chromium alloy (27 nm roughness) compared to glass (10 nm) and smoother low or high carbon cobalt chromium (10 nm). The two materials producing the greatest damage also exhibited higher equilibrium friction coefficients. Cartilage damage occurred primarily in the form of delamination at the interface between the superficial tangential zone and the transitional middle zone, with much less evidence of abrasive wear at the articular surface. These results suggest that cartilage damage from frictional loading occurs as a result of subsurface fatigue failure leading to the delamination. Surface chemistry and surface roughness of implant materials can have a significant influence on tissue damage, even when using materials and roughness values that satisfy regulatory requirements.
Assuntos
Cartilagem Articular/fisiologia , Fricção , Hemiartroplastia/instrumentação , Ligas , Animais , Cartilagem , Bovinos , Ligas de Cromo/química , Fêmur/cirurgia , Hemiartroplastia/métodos , Teste de Materiais , Próteses e Implantes , Falha de Prótese , Aço Inoxidável/química , Estresse Mecânico , Propriedades de Superfície , Tíbia/cirurgiaRESUMO
This study examined functional properties and biocompatibility of glutaraldehyde-fixed bovine articular cartilage over several weeks of incubation at body temperature to investigate its potential use as a resurfacing material in joint arthroplasty. In the first experiment, treated cartilage disks were fixed using 0.02, 0.20 and 0.60% glutaraldehyde for 24h then incubated, along with an untreated control group, in saline for up to 28d at 37°C. Both the equilibrium compressive and tensile moduli increased nearly twofold in treated samples compared to day 0 control, and remained at that level from day 1 to 28; the equilibrium friction coefficient against glass rose nearly twofold immediately after fixation (day 1) but returned to control values after day 7. Live explants co-cultured with fixed explants showed no quantitative difference in cell viability over 28d. In general, no significant differences were observed between 0.20 and 0.60% groups, so 0.20% was deemed sufficient for complete fixation. In the second experiment, cartilage-on-cartilage frictional measurements were performed under a migrating contact configuration. In the treated group, one explant was fixed using 0.20% glutaraldehyde while the apposing explant was left untreated; in the control group both explants were left untreated. From day 1 to 28, the treated group exhibited either no significant difference or slightly lower friction coefficient than the untreated group. These results suggest that a properly titrated glutaraldehyde treatment can reproduce the desired functional properties of native articular cartilage and maintain these properties for at least 28d at body temperature.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/fisiologia , Fixadores/farmacologia , Fricção/efeitos dos fármacos , Glutaral/farmacologia , Fatores Etários , Animais , Bioprótese , Temperatura Corporal , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Força Compressiva/efeitos dos fármacos , Teste de Materiais , Técnicas de Cultura de Órgãos , Osteoartrite/fisiopatologia , Resistência à Tração/efeitos dos fármacos , Suporte de CargaRESUMO
This study investigated the potential use of static osmotic loading as a cartilage tissue engineering strategy for growing clinically relevant grafts from either synovium-derived stem cells (SDSCs) or chondrocytes. Bovine SDSCs and chondrocytes were individually encapsulated in 2% w/v agarose and divided into chondrogenic media of osmolarities 300 (hypotonic), 330 (isotonic), and 400 (hypertonic, physiologic) mOsM for up to 7 weeks. The application of hypertonic media to constructs comprised of SDSCs or chondrocytes led to increased mechanical properties as compared to hypotonic (300mOsM) or isotonic (330mOsM) media (p<0.05). Constant exposure of SDSC-seeded constructs to 400mOsM media from day 0 to day 49 yielded a Young's modulus of 513±89kPa and GAG content of 7.39±0.52%ww on day 49, well within the range of values of native, immature bovine cartilage. Primary chondrocyte-seeded constructs achieved almost as high a Young's modulus, reaching 487±187kPa and 6.77±0.54%ww (GAG) for the 400mOsM condition (day 42). These findings suggest hypertonic loading as a straightforward strategy for 3D cultivation with significant benefits for cartilage tissue engineering strategies. In an effort to understand potential mechanisms responsible for the observed response, cell volume measurements in response to varying osmotic conditions were evaluated in relation to the Boyle-van't Hoff (BVH) law. Results confirmed that chondrocytes behave as perfect osmometers; however SDSCs deviated from the BVH relation.
Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Módulo de Elasticidade , Pressão Osmótica/fisiologia , Engenharia Tecidual , Animais , Cartilagem/citologia , Bovinos , Células Cultivadas , Condrócitos/citologia , Fatores de TempoRESUMO
The potential influence of mechanical loading on transvascular transport in vascularized soft tissues has not been explored extensively. This experimental investigation introduced and explored the hypothesis that dynamic mechanical loading can pump solutes out of blood vessels and into the surrounding tissue, leading to faster uptake and higher solute concentrations than could otherwise be achieved under unloaded conditions. Immature epiphyseal cartilage was used as a model tissue system, with fluorescein (332 Da), dextran (3, 10, and 70 kDa) and transferrin (80 kDa) as model solutes. Cartilage disks were either dynamically loaded (± 10% compression over a 10% static offset strain, at 0.2 Hz) or maintained unloaded in solution for up to 20 h. Results demonstrated statistically significant solute uptake in dynamically loaded (DL) explants relative to passive diffusion (PD) controls for all solutes except unbound fluorescein, as evidenced by the DL:PD concentration ratios after 20 h (1.0 ± 0.2, 2.4 ± 1.1, 6.1 ± 3.3, 9.0 ± 4.0, and 5.5 ± 1.6 for fluorescein, 3, 10, and 70 kDa dextran, and transferrin). Significant uptake enhancements were also observed within the first 30s of loading. Termination of dynamic loading produced dissipation of enhanced solute uptake back to PD control values. Confocal images confirmed that solute uptake occurred from cartilage canals into their surrounding extracellular matrix. The incidence of this loading-induced transvascular solute pumping mechanism may significantly alter our understanding of the interaction of mechanical loading and tissue metabolism.
Assuntos
Vasos Sanguíneos/fisiologia , Lâmina de Crescimento/fisiologia , Animais , Fenômenos Biomecânicos , Biópsia , Cartilagem/química , Bovinos , Dextranos/farmacologia , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Fluoresceína/farmacologia , Microscopia Confocal/métodos , Soluções/metabolismo , Estresse Mecânico , Fatores de Tempo , Transferrina/químicaRESUMO
A cartilage growth mixture (CGM) model is proposed to address limitations of a model used in a previous study. New stress constitutive equations for the solid matrix are derived and collagen (COL) remodeling is incorporated into the CGM model by allowing the intrinsic COL material constants to evolve during growth. An analytical validation protocol based on experimental data from a recent in vitro growth study is developed. Available data included measurements of tissue volume, biochemical composition, and tensile modulus for bovine calf articular cartilage (AC) explants harvested at three depths and incubated for 13 days in 20% fetal borine serum (FBS) and 20% FBS+beta-aminopropionitrile. The proposed CGM model can match tissue biochemical content and volume exactly while predicting theoretical values of tensile moduli that do not significantly differ from experimental values. Also, theoretical values of a scalar COL remodeling factor are positively correlated with COL cross-link content, and mass growth functions are positively correlated with cell density. The results suggest that the CGM model may help us to guide in vitro growth protocols for AC tissue via the a priori prediction of geometric and biomechanical properties.