Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.     

The rationale for using microscopic units of a donor matrix in cartilage defect repair

The efficacy of existing articular cartilage defect repair strategies are limited. Native cartilage tissue forms via a series of exquisitely orchestrated morphogenic events spanning through gestation into early childhood. However, defect repair must be achieved in a non-ideal microenvironment over an accelerated time-frame compatible with the normal life of an adult patient. Scaffolds formed from decellularized tissues are commonly utilized to enable the rapid and accurate repair of tissues such as skin, bladder and heart valves. The intact extracellular matrix remaining following the decellularization of these relatively low-matrix-density tissues is able to rapidly and accurately guide host cell repopulation. By contrast, the extraordinary density of cartilage matrix limits both the initial decellularization of donor material as well as its subsequent repopulation. Repopulation of donor cartilage matrix is generally limited to the periphery, with repopulation of lacunae deeper within the matrix mass being highly inefficient. Herein, we review the relevant literature and discuss the trend toward the use of decellularized donor cartilage matrix of microscopic dimensions. We show that 2-μm microparticles of donor matrix are rapidly integrate with articular chondrocytes, forming a robust cartilage-like composites with enhanced chondrogenic gene expression. Strategies for the clinical application of donor matrix microparticles in cartilage defect repair are discussed.     

The asymmetry of transient response in compression versus release for cartilage in unconfined compression

Observations in compression tests of articular cartilage have revealed unequal load increments for compression and release of the same amplitude applied to a disk with an identical previously imposed compression (in equilibrium). The mechanism of this asymmetric transient response is investigated here using a nonlinear fibril-reinforced model. It is found that the asymmetry is predominantly produced by the fibril stiffening with its tensile strain. In addition, allowing the hydraulic permeability to decrease significantly with compressive dilatation of cartilage increases the transient fibril strain, resulting in a stronger asymmetry. Large deformation also enhances the asymmetry as a consequence of stronger fibril stiffening.     

A fibril reinforced nonhomogeneous poroelastic model for articular cartilage: inhomogeneous response in unconfined compression

The depth dependence of material properties of articular cartilage, known as the zonal differences, is incorporated into a nonlinear fibril-reinforced poroelastic model developed previously in order to explore the significance of material heterogeneity in the mechanical behavior of cartilage. The material variations proposed are based on extensive observations. The collagen fibrils are modeled as a distinct constituent which reinforces the other two constituents representing proteoglycans and water. The Young's modulus and Poisson's ratio of the drained nonfibrillar matrix are so determined that the aggregate compressive modulus for confined geometry fits the experimental data. Three nonlinear factors are considered, i.e. the effect of finite deformation, the dependence of permeability on dilatation and the fibril stiffening with its tensile strain. Solutions are extracted using a finite element procedure to simulate unconfined compression tests. The features of the model are then demonstrated with an emphasis on the results obtainable only with a nonhomogeneous model, showing reasonable agreement with experiments. The model suggests mechanical behaviors significantly different from those revealed by homogeneous models: not only the depth variations of the strains which are expected by qualitative analyses, but also, for instance, the relaxation-time dependence of the axial strain which is normally not expected in a relaxation test. Therefore, such a nonhomogeneous model is necessary for better understanding of the mechanical behavior of cartilage.     

Borna disease: association with a maturation defect in the cellular immune response.

Borna disease virus (BDV) is a negative-strand RNA virus which produces persistent infection in a variety of experimental animals. In the rat, the presence or absence of clinical signs of Borna disease, a characteristic, biphasic neurobehavioral illness, depends on host-related factors. A window of opportunity exists after birth wherein inoculation with BDV produces a persistently infected rat without signs of Borna disease or encephalitis (persistent, tolerant infection-newborn [PTI-NB] rat). Although immunopathological destruction of the nervous system does not occur in the PTI-NB rat, significant alterations in the development of the nervous system were noted, including site-specific lysis of neurons. Unlike the case with other pharmacologically produced, persistent, tolerant BDV infections, adoptive transfer of spleen cells from BDV-infected rats did not produce disease in the PTI-NB rats. PTI-NB rats developed Borna disease after being connected by parabiosis to rats with Borna disease. Bone marrow transplantation experiments revealed that bone marrow cells from PTI-NB rats produced Borna disease in lethally irradiated, BDV-infected recipient rats. Bone marrow from PTI-NB rats contained a complement of inflammatory cells capable of inducing Borna disease. Thus, the loss of BDV-specific cellular immunity appeared to occur after the release of cells from the bone marrow.     

Comparison of the equilibrium response of articular cartilage in unconfined compression,confined compression and indentation

At mechanical equilibrium, articular cartilage is usually characterized as an isotropic elastic material with no interstitial fluid flow. In this study, the equilibrium properties (Young's modulus, aggregate modulus and Poisson's ratio) of bovine humeral, patellar and femoral cartilage specimens (n=26) were investigated using unconfined compression, confined compression, and indentation tests. Optical measurements of the Poisson's ratio of cartilage were also carried out. Mean values of the Young's modulus (assessed from the unconfined compression test) were 0.80+/-0.33, 0.57+/-0.17 and 0.31+/-0.18MPa and of the Poisson's ratio (assessed from the optical test) 0.15+/-0.06, 0.16+/-0.05 and 0.21+/-0.05 for humeral, patellar, and femoral cartilages, respectively. The indentation tests showed 30-79% (p<0.01) higher Young's modulus values than the unconfined compression tests. In indentation, values of the Young's modulus were independent of the indenter diameter only in the humeral cartilage. The mean values of the Poisson's ratio, obtained indirectly using the mathematical relation between the Young's modulus and the aggregate modulus in isotropic material, were 0.16+/-0.06, 0.21+/-0.05, and 0.26+/-0.08 for humeral, patellar, and femoral cartilages, respectively. We conclude that the values of the elastic parameters of the cartilage are dependent on the measurement technique in use. Based on the similar values of Poisson's ratios, as determined directly or indirectly, the equilibrium response of articular cartilage under unconfined and confined compression is satisfactorily described by the isotropic elastic model. However, values of the isotropic Young's modulus obtained from the in situ indentation tests are higher than those obtained from the in vitro unconfined or confined compression tests and may depend on the indenter size in use.     

Strong hyaluronan expression in the full-thickness rat articular cartilage repair tissue

Articular cartilage lesions have a poor capacity to regenerate. In full-depth articular cartilage defects, the repair process involves an ingrowth of mesenchymal cells from the bone marrow to the injured area, and these cells attempt to restore the lesion with cartilage-like repair tissue. In this study, we investigated histologically the distribution of hyaluronan in the rat repair tissue in relation to other glycosaminoglycans. Full-depth lesions were drilled to the weight-bearing region of rat medical femoral condyle. The rats were divided into two groups: intermittent active motion (IAM) and running training (RT) groups. In the RT group, programmed exercise was started 1 week after surgery, while the rats in the IAM group could move freely in their cages. The lesions were investigated 4 and 8 weeks after the surgery. Semiquantitative histological grading showed no significant differences in the repair between the groups. In normal articular cartilage, hyaluronan was stained mainly around chondrocytes. During repair, strong hyaluronan staining was observed in loose mesenchymal tissue, while in the repair area undergoing endochondral ossification, hyaluronan was intensively stained mainly around the hypertrophic chondrocytes. Remarkably strong staining for hyaluronan was noticed in areas of apparent mesenchymal progenitor cell invasion, the areas being simultaneously devoid of staining for keratan sulphate. In conclusion, hyaluronan is strongly expressed in the early cartilage repair tissue, and its staining intensity and distribution shows very sensitively abnormal articular cartilage structure.     

The influence of the acetabular labrum on hip joint cartilage consolidation: a poroelastic finite element model

The goal of this study was to investigate the influence of the acetabular labrum on the consolidation, and hence the solid matrix strains and stresses, of the cartilage layers of the hip joint. A plane-strain finite element model was developed, which represented a coronal slice through the acetabular and femoral cartilage layers and the acetabular labrum. Elements with poroelastic properties were used to account for the biphasic solid/fluid nature of the cartilage and labrum. The response of the joint over an extended period of loading (10,000s) was examined to simulate the nominal compressive load that the joint is subjected to throughout the day. The model demonstrated that the labrum adds an important resistance in the flow path of the fluid being expressed from the cartilage layers of the joint. Cartilage layer consolidation was up to 40% quicker in the absence of the labrum. Following removal of the labrum from the model, the solid-on-solid contact stresses between the femoral and acetabular cartilage layers were greatly increased (up to 92% higher), which would increase the friction between the joint surfaces. In the absence of the labrum, the centre of contact shifted towards the acetabular rim. Subsurface strains and stresses were much higher without the labrum, which could contribute to fatigue damage of the cartilage layers. Finally, the labrum provided some structural resistance to lateral motion of the femoral head within the acetabulum, enhancing joint stability and preserving joint congruity.     

The influence of the fixed negative charges on mechanical and electrical behaviors of articular cartilage under unconfined compression

Unconfined compression test has been frequently used to study the mechanical behaviors of articular cartilage, both theoretically and experimentally. It has also been used in explant and gel-cell-complex studies in tissue engineering. In biphasic and poroelastic theories, the effect of charges fixed on the proteoglycan macromolecules in articular cartilage is embodied in the apparent compressive Young's modulus and the apparent Poisson's ratio of the tissue, and the fluid pressure is considered to be the portion above the osmotic pressure. In order to understand how proteoglycan fixed charges might affect the mechanical behaviors of articular cartilage, and in order to predict the osmotic pressure and electric fields inside the tissue in this experimental configuration, it is necessary to use a model that explicitly takes into account the charged nature of the tissue and the flow of ions within its porous interstices. In this paper, we used a finite element model based on the triphasic theory to study how fixed charges in the porous-permeable soft tissue can modulate its mechanical and electrochemical responses under a step displacement in unconfined compression. The results from finite element calculations showed that: 1) A charged tissue always supports a larger load than an uncharged tissue of the same intrinsic elastic moduli. 2) The apparent Young's modulus (the ratio of the equilibrium axial stress to the axial strain) is always greater than the intrinsic Young's modulus of an uncharged tissue. 3) The apparent Poisson's ratio (the negative ratio of the lateral strain to the axial strain) is always larger than the intrinsic Poisson's ratio of an uncharged tissue. 4) Load support derives from three sources: intrinsic matrix stiffness, hydraulic pressure and osmotic pressure. Under the unconfined compression, the Donnan osmotic pressure can constitute between 13%-22% of the total load support at equilibrium. 5) During the stress-relaxation process following the initial instant of loading, the diffusion potential (due to the gradient of the fixed charge density and the associated gradient of ion concentrations) and the streaming potential (due to fluid convection) compete against each other. Within the physiological range of material parameters, the polarity of the electric potential depends on both the mechanical properties and the fixed charge density (FCD) of the tissue. For softer tissues, the diffusion effects dominate the electromechanical response, while for stiffer tissues, the streaming potential dominates this response. 6) Fixed charges do not affect the instantaneous strain field relative to the initial equilibrium state. However, there is a sudden increase in the fluid pressure above the initial equilibrium osmotic pressure. These new findings are relevant and necessary for the understanding of cartilage mechanics, cartilage biosynthesis, electromechanical signal transduction by chondrocytes, and tissue engineering.     

Breakdown of cartilage proteoglycan in a tissue culture model of rheumatoid arthritis

Proteoglycan breakdown was studied in a coculture model which mimics the confrontation between synovium and cartilage that occurs in rheumatoid arthritis. Bovine nasal-septum cartilage discs radioactively labeled (³⁵SO₄^2? with or without [³H]glucosamine) and ‘chased’ in non-radioactive medium were cultured in contact with minced rheumatoid synovial membranes for intervals up to 8 days. Synovium-stimulated (2–3 fold) cartilage breakdown was unaffected by ascorbate supplementation. Labeled products (small molecules plus proteoglycan complexes) in culture media were characterized by chromatographic, sedimentation and enzymic digestion methods. Breakdown was dominated by the release of a range of proteoglycan products, fully disaggregated and incapable of reaggregation with added hyaluronate. Because constituent glycosaminoglycans were of uniform size, proteoglycan polydispersity was attributed to differences in core protein length. Hydrocortisone inhibited degradation and partially prevented the shift of proteoglycans to lower average molecular weight. An additional breakdown pattern occasionally noted during the initial 48 h of coculture was characterized by release of a subpopulation of low charge-density proteoglycan bearing shortened glycosaminoglycan chains, consistent with glycosidase action. We conclude that rheumatoid synovia exhibit two distinct cartilage degradative potencies in vitro that may be important in vivo: (a) A variable hyaluronidase-like activity at early culture times, and (b) a dominant proteolytic activity generating an array of disaggregated proteoglycann products that differ largely on the basis of their core lengths. The response to hydrocortisone is consistent with inhibition of proteolysis through the stabilization of cellular membranes.     

A reaction-diffusion model to predict the influence of neo-matrix on the subsequent development of tissue-engineered cartilage

Extracellular matrix (ECM) in chondrocytes-seeded agarose aggregates to form islands of matrix. These islands need to coalesce to develop functional cartilage. Hence, macroscopic properties are determined by transport and aggregation of macromolecules at the microscale, which varies temporally and spatially. This study evaluates the importance of the mutual interaction between matrix components and matrix development. Fluorescence recovery after photobleaching measurements demonstrates that diffusivity depends on the presence and density of ECM. A reaction-diffusion model describing synthesis, transport and immobilisation of ECM predicts steep gradients in ECM around chondrocytes, resembling histology. Steric hindrance of diffusion by ECM is essential for the formation of these gradients. Finally, microscopic ECM concentration is linked with macroscopic mechanical properties. Construct softening is predicted when temporal and spatial variations in diffusivity are considered. In conclusion, non-constant diffusion renders significant effects on both the microscopic ECM development and the macroscopic mechanical properties of developing tissue-engineered cartilage.     

The NK model of rugged fitness landscapes and its application to maturation of the immune response

Adaptive evolution is, to a large extent, a complex combinatorial optimization process. Such processes can be characterized as "uphill walks on rugged fitness landscapes". Concrete examples of fitness landscapes include the distribution of any specific functional property such as the capacity to catalyze a specific reaction, or bind a specific ligand, in "protein space". In particular, the property might be the affinity of all possible antibody molecules for a specific antigenic determinant. That affinity landscape presumably plays a critical role in maturation of the immune response. In this process, hypermutation and clonal selection act to select antibody V region mutant variants with successively higher affinity for the immunizing antigen. The actual statistical structure of affinity landscapes, although knowable, is currently unknown. Here, we analyze a class of mathematical models we call NK models. We show that these models capture significant features of the maturation of the immune response, which is currently thought to share features with general protein evolution. The NK models have the important property that, as the parameter K increases, the "ruggedness" of the NK landscape varies from a single peaked "Fujiyama" landscape to a multi-peaked "badlands" landscape. Walks to local optima on such landscapes become shorter as K increases. This fact allows us to choose a value of K that corresponds to the experimentally observed number of mutational "steps", 6-8, taken as an antibody sequence matures. If the mature antibody is taken to correspond to a local optimum in the model, tuning the model requires that K be about 40, implying that the functional contribution of each amino acid in the V region is affected by about 40 others. Given this value of K, the model then predicts several features of "antibody space" that are in qualitative agreement with experiment: (1) The fraction of fitter variants of an initial "roughed in" germ line antibody amplified by clonal selection is about 1-2%. (2) Mutations at some sites of the mature antibody hardly affect antibody function at all, but mutations at other sites dramatically decrease function. (3) The same "roughed in" antibody sequence can "walk" to many mature antibody sequences. (4) Many adaptive walks can end on the same local optimum. (5) Comparison of different mature sequences derived from the same initial V region shows evolutionary hot spots and parallel mutations. All these predictions are open to detailed testing by obtaining monoclonal antibodies early in the immune response and carrying out in vitro mutagenesis and adaptive hill climbing with respect to affinity for the immunizing antigen.     

A microstructural model of elastostatic properties of articular cartilage in confined compression

A microstructural model of cartilage was developed to investigate the relative contribution of tissue matrix components to its elastostatic properties. Cartilage was depicted as a tensed collagen lattice pressurized by the Donnan osmotic swelling pressure of proteoglycans. As a first step in modeling the collagen lattice, two-dimensional networks of tensed, elastic, interconnected cables were studied as conceptual models. The models were subjected to the boundary conditions of confined compression and stress-strain curves and elastic moduli were obtained as a function of a two-dimensional equivalent of swelling pressure. Model predictions were compared to equilibrium confined compression moduli of calf cartilage obtained at different bath concentrations ranging from 0.01 to 0.50 M NaCl. It was found that a triangular cable network provided the most consistent correspondence to the experimental data. The model showed that the cartilage collagen network remained tensed under large confined compression strains and could therefore support shear stress. The model also predicted that the elastic moduli increased with increasing swelling pressure in a manner qualitatively similar to experimental observations. Although the model did not preclude potential contributions of other tissue components and mechanisms, the consistency of model predictions with experimental observations suggests that the cartilage collagen network, prestressed by proteoglycan swelling pressure, plays an important role in supporting compression.     

Collagen IX is indispensable for timely maturation of cartilage during fracture repair in mice.

Fracture repair recapitulates in adult organisms the sequence of cell biological events of endochondral ossification during skeletal development and growth. After initial inflammation and deposition of granulation tissue, a cartilaginous callus is formed which, subsequently, is remodeled into bone. In part, bone formation is influenced also by the properties of the extracellular matrix of the cartilaginous callus. Deletion of individual macromolecular components can alter extracellular matrix suprastructures, and hence stability and organization of mesenchymal tissues. Here, we took advantage of the collagen IX knockout mouse model to better understand the role of this collagen for organization, differentiation and maturation of a cartilaginous template during formation of new bone. Although a seemingly crucial component of cartilage fibrils is missing, collagen IX-deficient mice develop normally, but are predisposed to premature joint cartilage degeneration. However, we show here that lack of collagen IX alters the time course of callus differentiation during bone fracture healing. The maturation of cartilage matrix was delayed in collagen IX-deficient mice calli as judged by collagen X expression during the repair phase and the total amount of cartilage matrix was reduced. Entering the remodeling phase of fracture healing, Col9a1(-/-) calli retained a larger percentage of cartilage matrix than in wild type indicating also a delayed formation of new bone. We concluded that endochondral bone formation can occur in collagen IX knockout mice but is impaired under conditions of stress, such as the repair of an unfixed fractured long bone.     

Informing future cartilage repair strategies: a comparative study of three different human cell types for cartilage tissue engineering

A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering.