Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes

Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.     

Isolation of elastin from bovine auricular cartilage.

Clostridium histolyticum collagenase (clostridiopeptidase A, EC 3.4.24.3), purified by affinity chromatography, was applied to the isolation of insoluble elastin from bovine auricular cartilage. The low level of N-terminal residues (2.8 mol per 10⁶g of protein) present in this preparation indicated the almost complete lack of hydrolytic damage caused by the isolation procedure. The amino acid composition of the preparation showed an overall two-fold increase in polar residues, and a 20% reduction in valine, when compared to those of aortic and ligamentum nuchae elastin, while the concentration of cross-links was almost identical in the three preparations. Analysis of peptides, isolated by gel-exclusion chromatography after digestion of auricular elastin with elastase (pancreatopeptidase E, EC 3.4.21.11) in the presence of sodium dodecyl sulfate, revealed elevated levels of polar residues in all fractions examined, with no correlation between the concentration of these amino acids and that of the lysine-derived cross-links. Comparison of auricular and ligamentum nuchae elastin by fingerprint analysis of their elastase digests also suggested that the two proteins were compositionally distinct. Finally, treatment of auricular elastin with either trypsin or chymotrypsin produced no significant reduction in the level of polar residues. It is concluded that elastin exhibits tissue-related compositional variability.     

Tension lines of the auricular cartilage

Utilizing Langer's technique for skin tension lines, we punctured the auricular cartilage of 10 human cadavers and 2 mature rabbits and 24 immature rabbits with a conical awl to determine their tension lines. Deformation of the holes into straight lines indicated the direction of maximum tension. A regular pattern was observed in adult rabbits and humans: Tension was at right angles to the long axis at the auricular base and parallel at the apex. This pattern was not present at birth, but developed at 1 to 2 weeks of age in rabbits. It is felt that these tension lines are responsible for the clinical results of auricular reconstruction. In children less than 1 week of age, tension lines are disorganized so that nonsurgical correction will be successful as long as hypoplasia is not present. In older children, correction will be successful when the deformity is parallel to the tension lines.     

Engineered cartilage covered ear implants for auricular cartilage reconstruction

Cartilage tissues are often required for auricular tissue reconstruction. Currently, alloplastic ear-shaped medical implants composed of silicon and polyethylene are being used clinically. However, the use of these implants is often associated with complications, including inflammation, infection, erosion, and dislodgement. To overcome these limitations, we propose a system in which tissue-engineered cartilage serves as a shell that entirely covers the alloplastic implants. This study investigated whether cartilage tissue, engineered with chondrocytes and a fibrin hydrogel, would provide adequate coverage of a commercially used medical implant. To demonstrate the in vivo stability of cell-fibrin constructs, we tested variations of fibrinogen and thrombin concentration as well as cell density. After implantation, the retrieved engineered cartilage tissue was evaluated by histo- and immunohistochemical, biochemical, and mechanical analyses. Histomorphological evaluations consistently showed cartilage formation over the medical implants with the maintenance of dimensional stability. An initial cell density was determined that is critical for the production of matrix components such as glycosaminoglycans (GAG), elastin, type II collagen, and for mechanical strength. This study shows that engineered cartilage tissues are able to serve as a shell that entirely covers the medical implant, which may minimize the morbidity associated with implant dislodgement.     

In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction

To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.     

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.     

Modeling the dynamic composition of engineered cartilage

Mathematical models to describe extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs for the design of engineered cartilage were developed and validated. The ECM deposition model characterized a product-inhibition mechanism in the concentration of cartilage molecules, collagen and glycosaminoglycans (GAG). The scaffold degradation model used first-order kinetics to describe hydrolysis (not limited by diffusion) of biodegradable polyesters, polyglycolic acid and polylactic acid. Each model was fit to published accumulation and degradation data. As experimental validation, cell-polymer constructs (n=24) and unseeded scaffolds (n=24) were cultured in vitro. Biochemical assays for ECM content and measurements of scaffold mass were performed at 1, 2, 4, 6, 8, or 10 weeks (n=8 per time point). The models demonstrated a strong fit with published data and experimental results (R(2)=0.75 to 0.99) and predicted the temporal total construct mass with reasonable accuracy (30% RMS error). This approach can elucidate mechanisms governing accumulation/degradation and may be coupled with structure-function relationships to describe time-dependent changes in construct elastic properties.     

Development of large engineered cartilage constructs from a small population of cells

Confronted with articular cartilage's limited capacity for self‐repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large‐sized engineered constructs (≥3 cm²) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm² tissue construct). Using rabbit donor tissue, the bioreactor‐cultivated constructs were hyaline‐like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid‐prototyped defect tissue culture molds. Similar hyaline‐like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large‐sized osteochondral constructs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Characteristics of stem cells derived from the degenerated human intervertebral disc cartilage endplate

Mesenchymal stem cells (MSCs) derived from adult tissues are an important candidate for cell-based therapies and regenerative medicine due to their multipotential differentiation capability. MSCs have been identified in many adult tissues but have not reported in the human intervertebral disc cartilage endplate (CEP). The initial purpose of this study was to determine whether MSCs exist in the degenerated human CEP. Next, the morphology, proliferation capacity, cell cycle, cell surface epitope profile and differentiation capacity of these CEP-derived stem cells (CESCs) were compared with bone-marrow MSCs (BM-MSCs). Lastly, whether CESCs are a suitable candidate for BM-MSCs was evaluated. Isolated cells from degenerated human CEP were seeded in an agarose suspension culture system to screen the proliferative cell clusters. Cell clusters were chosen and expanded in vitro and were compared with BM-MSCs derived from the same patient. The morphology, proliferation rate, cell cycle, immunophenotype and stem cell gene expression of the CESCs were similar to BM-MSCs. In addition, the CESCs could be induced into osteoblasts, adipocytes, chondrocytes, and are superior to BM-MSCs in terms of osteogenesis and chondrogenesis. This study is first to demonstrate the presence of stem cells in the human degenerated CEP. These results may improve our understanding of intervertebral disc (IVD) pathophysiology and the degeneration process, and could provide cell candidates for cell-based regenerative medicine and tissue engineering.     

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.     

Computational modeling of nutrient utilization in engineered cartilage

This study presents a mathematical model for simulating cartilaginous culture of chondrocytes seeded in scaffolds and for investigating the effects of glucose and oxygen concentration and pH value on cell metabolic rates. The model can clearly interpret the unexplained experimental observation (Sengers BG, Heywood HK, Lee DA, Oomens CWJ, Bader DL. Nutrient utilization by bovine articular chondrocytes: A combined experimental and theoretical approach. J Biomech Eng. 2005;127:758–766.), which showed that the oxygen concentration within the scaffold may increase instead of continuously decreasing in static cartilaginous culture of chondrocytes. Results from simulation demonstrate that when cells metabolize glucose and form lactate under high glucose concentration conditions, the acidity in the culture environment increases, inhibiting cell metabolic rates in the process. Consequently, the rate of oxygen consumption decreases in later stages of cell culture. As oxygen can be replenished through the free surface of the culture medium, oxygen concentration within the scaffold increases rather than decreases over time in the acidic environment. Different initial glucose concentration yields different results. In low glucose concentration conditions, oxygen concentration basically keeps decreasing with culture time. This is because the pH in the environment does not significantly change because of slower glycolysis rate in low glucose concentration cases, forming less lactic acid. From the simulation results, additional information regarding in vitro culture of chondrocytes is obtained. The correlations between nutrient consumption, lactate secretion, and pH changes during cell culture are also understood and may serve as a reference for in vitro cell culture research of tissue engineering. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 452–462, 2013     

Elastic fiber formation in monolayer and organ cultures of chondrocytes isolated from auricular cartilage.

Chondrocytes were isolated from auricular cartilage of immature rabbits and maintained in monolayer or organ culture for 14 days. In both types of culture the chondrocytes formed conspicuous elastic fibers. In monolayer culture the fibers could be identified by orcein staining in the culture dish. Electron microscopy of organ cultures revealed the presence of two basic components of elastic fibers, i.e. microfibrils and elastin.     

Lumican inhibits collagen deposition in tissue engineered cartilage

Lumican is a glycoprotein that is found in the extracellular matrix of many connective tissues, including cartilage. It is a member of the small leucine-rich repeat proteoglycans family and along with two others, decorin and fibromodulin, has the capacity to bind to fibrillar collagens and limit their growth. Cartilage tissue engineering provides a potential method for the production of three-dimensional tissue for implantation into eroded joints. Many studies have demonstrated the growth of cartilage in vitro. However in all cases, biochemical analysis of the tissue revealed a significant deficit in the collagen content. We have now tested the hypothesis that the reduced collagen accumulation in engineered cartilage is a result of over-expression of decorin, fibromodulin or lumican. We have found that the lumican gene and protein are both over-expressed in engineered compared to natural cartilage whereas this is not the case for decorin or fibromodulin. Using a small hairpin lumican antisense sequence we were able to knockdown the lumican gene and protein expression in chondrocytes being used for tissue engineering. This resulted in increased accumulation of type II collagen (the major collagen of cartilage) whilst there was no significant alteration in the proteoglycan content. Furthermore, the antisense knockdown of lumican resulted in an increase in the average collagen fibril diameter measured by transmission electron microscopy. These results suggest that lumican plays a pivotal role in the development of tissue engineered cartilage and that regulation of this protein may be important for the production of high-quality implants.     

Engineering superficial zone features in tissue engineered cartilage

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion‐induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features. Biotechnol. Bioeng. 2013; 110: 1476–1486. © 2012 Wiley Periodicals, Inc.     

Modulation of the mechanical properties of tissue engineered cartilage

Cartilaginous constructs have been grown in vitro using chondrocytes, biodegradable polymer scaffolds, and tissue culture bioreactors. In the present work, we studied how the composition and mechanical properties of engineered cartilage can be modulated by the conditions and duration of in vitro cultivation, using three different environments: static flasks, mixed flasks, and rotating vessels. After 4-6 weeks, static culture yielded small and fragile constructs, while turbulent flow in mixed flasks induced the formation of an outer fibrous capsule; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels had the highest fractions of glycosaminoglycans and collagen (respectively 75% and 39% of levels measured in native cartilage), and the best mechanical properties (equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential were all about 20% of values measured in native cartilage). Chondrocytes in cartilaginous constructs remained metabolically active and phenotypically stable over prolonged cultivation in rotating bioreactors. The wet weight fraction of glycosaminoglycans and equilibrium modulus of 7 month constructs reached or exceeded the corresponding values measured from freshly explanted native cartilage. Taken together, these findings suggest that functional equivalents of native cartilage can be engineered by optimizing the hydrodynamic conditions in tissue culture bioreactors and the duration of tissue cultivation.