Differential regulation of immature articular cartilage compressive moduli and Poisson’s ratios by in vitro stimulation with IGF-1 and TGF-β1

Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-β1). This study tested the hypothesis that IGF-1 and TGF-β1 regulate immature cartilage compressive moduli and Poisson’s ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-β1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson’s ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-β1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-β1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson’s ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-β1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering.     

A cartilage growth mixture model for infinitesimal strains: solutions of boundary-value problems related to in vitro growth experiments

A cartilage growth mixture (CGM) model is linearized for infinitesimal elastic and growth strains. Parametric studies for equilibrium and nonequilibrium boundary-value problems representing the in vitro growth of cylindrical cartilage constructs are solved. The results show that the CGM model is capable of describing the main biomechanical features of cartilage growth. The solutions to the equilibrium problems reveal that tissue composition, constituent pre-stresses, and geometry depend on collagen remodeling activity, growth symmetry, and differential growth. Also, nonhomogeneous growth leads to nonhomogeneous tissue composition and constituent pre-stresses. The solution to the nonequilibrium problem reveals that the tissue is nearly in equilibrium at all time points. The results suggest that the CGM model may be used in the design of tissue engineered cartilage constructs for the repair of cartilage defects; for example, to predict how dynamic mechanical loading affects the development of nonuniform properties during in vitro growth. Furthermore, the results lay the foundation for future analyses with nonlinear models that are needed to develop realistic models of cartilage growth.     

Articular cartilage tensile integrity: modulation by matrix depletion is maturation-dependent

Articular cartilage function depends on the molecular composition and structure of its extracellular matrix (ECM). The collagen network (CN) provides cartilage with tensile integrity, but must also remodel during growth. Such remodeling may depend on matrix molecules interacting with the CN to modulate the tensile behavior of cartilage. The objective of this study was to determine the effects of increasingly selective matrix depletion on tensile properties of immature and mature articular cartilage, and thereby establish a framework for identifying molecules involved in CN remodeling. Depletion of immature cartilage with guanidine, chondroitinase ABC, chondroitinase AC, and Streptomyces hyaluronidase markedly increased tensile integrity, while the integrity of mature cartilage remained unaltered after depletion with guanidine. The enhanced tensile integrity after matrix depletion suggests that certain ECM components of immature matrix serve to inhibit CN interactions and may act as modulators of physiological alterations of cartilage geometry and tensile properties during growth/maturation.     

Articular cartilage mechanical and biochemical property relations before and after in vitro growth

The aim of this study was to design in vitro growth protocols that can comprehensively quantify articular cartilage structure–function relations via measurement of mechanical and biochemical properties. Newborn bovine patellofemoral groove articular cartilage explants were tested sequentially in confined compression (CC), unconfined compression (UCC), and torsional shear before (D0, i.e. day zero) and after (D14, i.e. day 14) unstimulated in vitro growth. The contents of collagen (COL), collagen-specific pyridinoline (PYR) crosslinks, glycosaminoglycan, and DNA significantly decreased during in vitro growth; consequently, a wide range of biochemical properties existed for investigating structure–function relations when pooling the D0 and D14 groups. All D0 mechanical properties were independent of compression strain while only Poisson's ratios were dependent on direction (i.e. anisotropic). Select D0 and D14 group mechanical properties were correlated with biochemical measures; including (but not limited to) results that CC/UCC moduli and UCC Poisson's ratios were correlated with COL and PYR. COL network weakening during in vitro growth due to reduced COL and PYR was accompanied by reduced CC/UCC moduli and increased UCC Poisson's ratios.     

Effect of PRP and PPP on proliferation and migration of human chondrocytes and synoviocytes in vitro

Cartilage tissue engineering can provide substantial relief to people suffering from degenerative cartilage disease, such as osteoarthritis. The autologous platelet-rich plasma (PRP) application appears to improve cartilage healing due to its ability to positively influence cellular mechanisms, mainly in cells from synovium and cartilage. Primary cultures of human synovial fluid stem cells (synoviocytes, SCs) and chondrocytes (CCs) were exposed to various concentrations of non-activated PRP and plateletpoor plasma (PPP) prepared by apheresis. Cell proliferation and migration were evaluated in real-time with the non-invasive xCELLigence System. It was found that PRP had a similar effect on the growth of cells as fetal bovine serum (FBS). Surprisingly, our proliferation assay results indicated that 50% PPP had the largest effect on both cell types, with a statistically significant increase in cell number (P<0.001) compared to the (0% FBS) in vitro control. The migratory ability of SCs was significantly enhanced with 10% PRP and 0.8% hyaluronic acid (HA). HA also augmented migration of CCs. In summary, these results demonstrate that directed cell proliferation and migration are inducible in human articular CCs and SCs, and that both platelet-derived fractions may exert a positive effect and modulate several cell responses that are potentially involved in tissue integration during cartilage repair.     

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.     

Gene expression profile of rabbit cartilage by expressed sequence tag analysis

Although the rabbit is commonly used as an animal model for the in vivo study of cartilage formation or regeneration, genetic approaches to the rabbit cartilage are rare. We constructed an expressed sequence tag (EST) library from rabbit cartilage tissue for the first time to establish the foundations for genetic study on rabbit cartilage. From our results, we identified 2387 unique genes among 4885 clones, corresponding to 1839 matched to characterized genes including 1618 genes with known function and 548 uncharacterized and novel genes. Gene expression profiles based on EST frequency show that type II collagen (COL2A1) and type X collagen (COL10A1) among collagen clones, proteoglycan 4 (PRG4) and decorin (DCN) among proteoglycan clones, and cartilage oligomeric matrix protein (COMP) and matrix Gla protein (MGP) among other extracellular matrix clones, are highly expressed in rabbit cartilage. In addition, gene expression analysis based on real-time PCR of these major extracellular matrix constituents showed that expression of col2a1 and col10a1 remains constant whereas the expression of prg4, dcn, and comp reveals substantial change with rabbit age. This EST library will provide a valuable resource with which to identify genes involved in the biochemical and physiological functions of rabbit cartilage, and will contribute to establishing the rabbit as an animal model for cartilage research.     

A poroelastic finite element model of the bone–cartilage unit to determine the effects of changes in permeability with osteoarthritis

The changes experienced in synovial joints with osteoarthritis involve coupled chemical, biological, and mechanical processes. The aim of this study was to investigate the consequences of increasing permeability in articular cartilage (AC), calcified cartilage (CC), subchondral cortical bone (SCB), and subchondral trabecular bone (STB) as observed with osteoarthritis. Two poroelastic finite element models were developed using a depth-dependent anisotropic model of AC with strain-dependent permeability and poroelastic models of calcified tissues (CC, SCB, and STB). The first model simulated a bone-cartilage unit (BCU) in uniaxial unconfined compression, while the second model simulated spherical indentation of the AC surface. Results indicate that the permeability of AC is the primary determinant of the BCU’s poromechanical response while the permeability of calcified tissues exerts no appreciable effect on the force-indentation response of the BCU. In spherical indentation simulations with osteoarthritic permeability properties, fluid velocities were larger in magnitude and distributed over a smaller area compared to normal tissues. In vivo, this phenomenon would likely lead to chondrocyte death, tissue remodeling, alterations in joint lubrication, and the progression of osteoarthritis. For osteoarthritic and normal tissue permeability values, fluid flow was predicted to occur across the osteochondral interface. These results help elucidate the consequences of increases in the permeability of the BCU that occur with osteoarthritis. Furthermore, this study may guide future treatments to counteract osteoarthritis.     

Mechanical properties of human tracheal cartilage.

Biomechanical changes in airway cartilage could influence the mechanics of maximal expiratory flow and cough and the degree of shortening of activated airway smooth muscle. We examined the tensile stiffness of small samples of human tracheal cartilage rings in specimens obtained at autopsy from 10 individuals who ranged in age from 17 to 81 yr. The tensile properties of the cartilage were compared with its content of water (%water), glycosaminoglycans (chondroitin sulfate equivalents, mg/mg dry wt), and hydroxyproline content (mg hydroxyproline/mg dry weight). The average values for tensile stiffness ranged between 1 and 15 MPa and increased significantly with increasing age [tensile stiffness = 0.19 x (age in yr) + 2.02; r = 0.83, P less than 0.05]. The outermost layer of cartilage was the most stiff in all individuals, and the deeper layers were progressively less stiff. Water content and hydroxyproline content both decreased with increasing age. Thus tensile stiffness correlated inversely with water content and hydroxyproline content [tensile stiffness = -0.83 x (%water) + 16.4; r = 0.82, P less than .05 and tensile stiffness = -342 x (hydroxyproline content) + 25; r = 0.87, P less than 0.05]. Total tissue content of glycosaminoglycans did not change with age, although changes in glycosaminoglycan type and proteoglycan structure with increasing age have been described. We conclude that there are age-related changes in the biomechanical properties and biochemical composition of airway cartilage that could influence airway dynamics.     

Bioreactor studies of native and tissue engineered cartilage

Functional tissue engineering of cartilage involves the use of bioreactors designed to provide a controlled in vitro environment that embodies some of the biochemical and physical signals known to regulate chondrogenesis. Hydrodynamic conditions can affect in vitro tissue formation in at least two ways: by direct effects of hydrodynamic forces on cell morphology and function, and by indirect flow-induced changes in mass transfer of nutrients and metabolites. In the present work, we discuss the effects of three different in vitro environments: static flasks (tissues fixed in place, static medium), mixed flasks (tissues fixed in place, unidirectional turbulent flow) and rotating bioreactors (tissues dynamically suspended in laminar flow) on engineered cartilage constructs and native cartilage explants. As compared to static and mixed flasks, dynamic laminar flow in rotating bioreactors resulted in the most rapid tissue growth and the highest final fractions of glycosaminoglycans and total collagen in both tissues. Mechanical properties (equilibrium modulus, dynamic stiffness, hydraulic permeability) of engineered constructs and explanted cartilage correlated with the wet weight fractions of glycosaminoglycans and collagen. Current research needs in the area of cartilage tissue engineering include the utilization of additional physiologically relevant regulatory signals, and the development of predictive mathematical models that enable optimization of the conditions and duration of tissue culture.     

Modelling cartilage mechanobiology

The growth, maintenance and ossification of cartilage are fundamental to skeletal development and are regulated throughout life by the mechanical cues that are imposed by physical activities. Finite element computer analyses have been used to study the role of local tissue mechanics on endochondral ossification patterns, skeletal morphology and articular cartilage thickness distributions. Using single-phase continuum material representations of cartilage, the results have indicated that local intermittent hydrostatic pressure promotes cartilage maintenance. Cyclic tensile strains (or shear), however, promote cartilage growth and ossification. Because single-phase material models cannot capture fluid exudation in articular cartilage, poroelastic (or biphasic) solid/fluid models are often implemented to study joint mechanics. In the middle and deep layers of articular cartilage where poroelastic analyses predict little fluid exudation, the cartilage phenotype is maintained by cyclic fluid pressure (consistent with the single-phase theory). In superficial articular layers the chondrocytes are exposed to tangential tensile strain in addition to the high fluid pressure. Furthermore, there is fluid exudation and matrix consolidation, leading to cell 'flattening'. As a result, the superficial layer assumes an altered, more fibrous phenotype. These computer model predictions of cartilage mechanobiology are consistent with results of in vitro cell and tissue and molecular biology experiments.     

Characterization of articular cartilage by combining microscopic analysis with a fibril-reinforced finite-element model

Load-bearing characteristics of articular cartilage are impaired during tissue degeneration. Quantitative microscopy enables in vitro investigation of cartilage structure but determination of tissue functional properties necessitates experimental mechanical testing. The fibril-reinforced poroviscoelastic (FRPVE) model has been used successfully for estimation of cartilage mechanical properties. The model includes realistic collagen network architecture, as shown by microscopic imaging techniques. The aim of the present study was to investigate the relationships between the cartilage proteoglycan (PG) and collagen content as assessed by quantitative microscopic findings, and model-based mechanical parameters of the tissue. Site-specific variation of the collagen network moduli, PG matrix modulus and permeability was analyzed. Cylindrical cartilage samples (n=22) were harvested from various sites of the bovine knee and shoulder joints. Collagen orientation, as quantitated by polarized light microscopy, was incorporated into the finite-element model. Stepwise stress-relaxation experiments in unconfined compression were conducted for the samples, and sample-specific models were fitted to the experimental data in order to determine values of the model parameters. For comparison, Fourier transform infrared imaging and digital densitometry were used for the determination of collagen and PG content in the same samples, respectively. The initial and strain-dependent fibril network moduli as well as the initial permeability correlated significantly with the tissue collagen content. The equilibrium Young's modulus of the nonfibrillar matrix and the strain dependency of permeability were significantly associated with the tissue PG content. The present study demonstrates that modern quantitative microscopic methods in combination with the FRPVE model are feasible methods to characterize the structure-function relationships of articular cartilage.     

In vitro articular cartilage growth with sequential application of IGF-1 and TGF-β1 enhances volumetric growth and maintains compressive properties

In vitro cultures with insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1) have previously been shown to differentially modulate the growth of immature bovine articular cartilage. IGF-1 stimulates expansive growth yet decreases compressive moduli and increases compressive Poisson's ratios, whereas TGF-β1 maintains tissue size, increases compressive moduli, and decreases compressive Poisson's ratios. The current study's hypothesis was that sequential application of IGF-1 and TGF-β1 during in vitro culture produces geometric and compressive mechanical properties that lie between extreme values produced when using either growth factor alone. Immature bovine articular cartilage specimens were harvested and either untreated (D0, i.e., day zero) or cultured in vitro for either 6 days with IGF-1 (D6 IGF), 12 days with IGF-1 (D12 IGF), or 6 days with IGF-1 followed by 6 days with TGF-β1 (D12 SEQ, i.e., sequential). Following treatment, all specimens were tested for geometric, biochemical, and compressive mechanical properties. Relative to D0, D12 SEQ treatment enhanced volumetric growth, but to a lower value than that for D12 IGF. Furthermore, D12 SEQ treatment maintained compressive moduli and Poisson's ratios at values higher and lower, respectively, than those for D12 IGF. Considering the previously described effects of 12 days of treatment with TGF-β1 alone, D12 SEQ induced both growth and mechanical property changes between those produced with either IGF-1 or TGF-β1 alone. The results suggest that it may be possible to vary the durations of select growth factors, including IGF-1 and TGF-β1, to more precisely modulate the geometric, biochemical, and mechanical properties of immature cartilage graft tissue in clinical repair strategies.     

Extracellular matrix integrity affects the mechanical behaviour of in-situ chondrocytes under compression

Cartilage lesions change the microenvironment of cells and may accelerate cartilage degradation through catabolic responses from chondrocytes. In this study, we investigated the effects of structural integrity of the extracellular matrix (ECM) on chondrocytes by comparing the mechanics of cells surrounded by an intact ECM with cells close to a cartilage lesion using experimental and numerical methods. Experimentally, 15% nominal compression was applied to bovine cartilage tissues using a light-transmissible compression system. Target cells in the intact ECM and near lesions were imaged by dual-photon microscopy. Changes in cell morphology (N_cell=32 for both ECM conditions) were quantified. A two-scale (tissue level and cell level) Finite Element (FE) model was also developed. A 15% nominal compression was applied to a non-linear, biphasic tissue model with the corresponding cell level models studied at different radial locations from the centre of the sample in the transient phase and at steady state. We studied the Green-Lagrange strains in the tissue and cells. Experimental and theoretical results indicated that cells near lesions deform less axially than chondrocytes in the intact ECM at steady state. However, cells near lesions experienced large tensile strains in the principal height direction, which are likely associated with non-uniform tissue radial bulging. Previous experiments showed that tensile strains of high magnitude cause an up-regulation of digestive enzyme gene expressions. Therefore, we propose that cartilage degradation near tissue lesions may be due to the large tensile strains in the principal height direction applied to cells, thus leading to an up-regulation of catabolic factors.     

Assessment of hyaline cartilage matrix composition using near infrared spectroscopy

Changes in the composition of the extracellular matrix (ECM) are characteristic of injury or disease in cartilage tissue. Various imaging modalities and biochemical techniques have been used to assess the changes in cartilage tissue but lack adequate sensitivity, or in the case of biochemical techniques, result in destruction of the sample. Fourier transform near infrared (FT-NIR) spectroscopy has shown promise for the study of cartilage composition. In the current study NIR spectroscopy was used to identify the contributions of individual components of cartilage in the NIR spectra by assessment of the major cartilage components, collagen and chondroitin sulfate, in pure component mixtures. The NIR spectra were obtained using homogenous pellets made by dilution with potassium bromide. A partial least squares (PLS) model was calculated to predict composition in bovine cartilage samples. Characteristic absorbance peaks between 4000 and 5000 cm⁻¹ could be attributed to components of cartilage, i.e. collagen and chondroitin sulfate. Prediction of the amount of collagen and chondroitin sulfate in tissues was possible within 8% (w/dw) of values obtained by gold standard biochemical assessment. These results support the use of NIR spectroscopy for in vitro and in vivo applications to assess matrix composition of cartilage tissues, especially when tissue destruction should be avoided.     

Feasibility of noninvasive evaluation of biophysical properties of tissue-engineered cartilage by using quantitative MRI

The application of tissue-engineered cartilage in a clinical setting requires a noninvasive method to assess the biophysical and biochemical properties of the engineered cartilage. Since articular cartilage is composed of 70-80% water and has dense extracellular matrixes (ECM), it is considered that the condition of the water molecules in the tissue is correlated with its biomechanical property. Therefore, magnetic resonance imaging (MRI) represents a potential approach to assess the biophysical property of the engineered cartilage. In this study, we test the hypothesis that quantitative MRI can be used as a noninvasive assessment method to assess the biophysical property of the engineered cartilage. To reconstruct a model of cartilaginous tissue, chondrocytes harvested from the humeral head of calves were embedded in an agarose gel and cultured in vitro up to 4 weeks. Equilibrium Young's moduli were determined from the stress relaxation tests. After mechanical testing, MRI-derived parameters (longitudinal relaxation time T1, transverse relaxation time T2, and water self-diffusion coefficient D) were measured. The equilibrium Young's modulus of the engineered cartilage showed a tendency to increase with an increase in the culture time, whereas T1 and D decreased. Based on a regression analysis, T1 and D showed a strong correlation with the equilibrium Young's modulus. The results showed that T1 and D values derived from the MRI measurements could be used to noninvasively monitor the biophysical properties of the engineered cartilage.     

Characterization of pediatric microtia cartilage: a reservoir of chondrocytes for auricular reconstruction using tissue engineering strategies

The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.     

Cartilage biomechanics: A key factor for osteoarthritis regenerative medicine

Osteoarthritis (OA) is a joint disorder that is highly extended in the global population. Several researches and therapeutic strategies have been probed on OA but without satisfactory long-term results in joint replacement. Recent evidences show how the cartilage biomechanics plays a crucial role in tissue development. This review describes how physics alters cartilage and its extracellular matrix (ECM); and its role in OA development. The ECM of the articular cartilage (AC) is widely involved in cartilage turnover processes being crucial in regeneration and joint diseases. We also review the importance of physicochemical pathways following the external forces in AC. Moreover, new techniques probed in cartilage tissue engineering for biomechanical stimulation are reviewed. The final objective of these novel approaches is to create a cellular implant that maintains all the biochemical and biomechanical properties of the original tissue for long-term replacements in patients with OA.