Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering

Articular cartilage injuries and degeneration affect a large proportion of the population in developed countries world wide. Stem cells can be differentiated into chondrocytes by adding transforming growth factor-beta1 and dexamethasone to a pellet culture, which are unfeasible for tissue engineering purposes. We attempted to achieve stable chondrogenesis without any requirement for exogenous growth factors. Human mesenchymal stem cells were transduced with an adenoviral vector containing the SRY-related HMG-box gene 9 (SOX9), and were cultured in a three-dimensional (3D) hydrogel scaffold composite. As an additional treatment, mechanical stimulation was applied in a custom-made bioreactor. SOX9 increased the expression level of its known target genes, as well as its cofactors: the long form of SOX5 and SOX6. However, it was unable to increase the synthesis of sulfated glycosaminoglycans (GAGs). Mechanical stimulation slightly enhanced collagen type X and increased lubricin expression. The combination of SOX9 and mechanical load boosted GAG synthesis as shown by (35)S incorporation. GAG production rate corresponded well with the amount of (endogenous) transforming growth factor-beta1. Finally, cartilage oligomeric matrix protein expression was increased by both treatments. These findings provide insight into the mechanotransduction of mesenchymal stem cells and demonstrate the potential of a transcription factor in stem cell therapy.

Cartilage tissue engineering for degenerative joint disease

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

Cartilage tissue engineering by expanded goat articular chondrocytes

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.

The structural architecture of adult mammalian articular cartilage evolves by a synchronized process of tissue resorption and neoformation during postnatal development

OBJECTIVE: During postnatal development, mammalian articular cartilage acts as a surface growth plate for the underlying epiphyseal bone. Concomitantly, it undergoes a fundamental process of structural reorganization from an immature isotropic to a mature (adult) anisotropic architecture. However, the mechanism underlying this structural transformation is unknown. It could involve either an internal remodelling process, or complete resorption followed by tissue neoformation. The aim of this study was to establish which of these two alternative tissue reorganization mechanisms is physiologically operative. We also wished to pinpoint the articular cartilage source of the stem cells for clonal expansion and the zonal location of the chondrocyte pool with high proliferative activity. METHODS: The New Zealand white rabbit served as our animal model. The analysis was confined to the high-weight-bearing (central) areas of the medial and lateral femoral condyles. After birth, the articular cartilage layer was evaluated morphologically at monthly intervals from the first to the eighth postnatal month, when this species attains skeletal maturity. The overall height of the articular cartilage layer at each juncture was measured. The growth performance of the articular cartilage layer was assessed by calcein labelling, which permitted an estimation of the daily growth rate of the epiphyseal bone and its monthly length-gain. The slowly proliferating stem-cell pool was identified immunohistochemically (after labelling with bromodeoxyuridine), and the rapidly proliferating chondrocyte population by autoradiography (after labelling with (3)H-thymidine). RESULTS: The growth activity of the articular cartilage layer was highest 1 month after birth. It declined precipitously between the first and third months, and ceased between the third and fourth months, when the animal enters puberty. The structural maturation of the articular cartilage layer followed a corresponding temporal trend. During the first 3 months, when the articular cartilage layer is undergoing structural reorganization, the net length-gain in the epiphyseal bone exceeded the height of the articular cartilage layer. This finding indicates that the postnatal reorganization of articular cartilage from an immature isotropic to a mature anisotropic structure is not achieved by a process of internal remodelling, but by the resorption and neoformation of all zones except the most superficial (stem-cell) one. The superficial zone was found to consist of slowly dividing stem cells with bidirectional mitotic activity. In the horizontal direction, this zone furnishes new stem cells that replenish the pool and effect a lateral expansion of the articular cartilage layer. In the vertical direction, the superficial zone supplies the rapidly dividing, transit-amplifying daughter-cell pool that feeds the transitional and upper radial zones during the postnatal growth phase of the articular cartilage layer. CONCLUSIONS: During postnatal development, mammalian articular cartilage fulfils a dual function, viz., it acts not only as an articulating layer but also as a surface growth plate. In the lapine model, this growth activity ceases at puberty (3-4 months of age), whereas that of the true (metaphyseal) growth plate continues until the time of skeletal maturity (8 months). Hence, the two structures are regulated independently. The structural maturation of the articular cartilage layer coincides temporally with the cessation of its growth activity - for the radial expansion and remodelling of the epiphyseal bone - and with sexual maturation. That articular cartilage is physiologically reorganized by a process of tissue resorption and neoformation, rather than by one of internal remodelling, has important implications for the functional engineering and repair of articular cartilage tissue.

Expression of cartilage-related genes in bovine synovial tissue

The synovium contains mesenchymal stem cells with chondrogenic potential. Although synovial and articular cartilage tissue develop from a common pool of mesenchymal cells, little is known about their genetic commonalities. In the present study, the mRNA levels for several cartilage-related proteins, namely, cartilage oligomeric matrix protein (COMP), Sox9, aggrecan, and collagen types I, II, IX, X, and XI, were measured using the real-time polymerase chain reaction. Our data reveal the synovium of calf metacarpal joints to physiologically express not only type I collagen but also COMP, Sox9, aggrecan, and collagen types X and XI. The mRNA levels for the latter five proteins lie between 2% and 15% of those in articular cartilage. We speculate that these genes are being expressed by chondroprogenitor cells, whose presence in the synovium reflects a common ontogenetic phase in the fetal development of this tissue and of articular cartilage.

Cell and matrix morphology in articular cartilage from adult human knee and ankle joints suggests depth-associated adaptations to biomechanical and anatomical roles

OBJECTIVE Marked differences exist between human knee and ankle joints regarding risks and progression of osteoarthritis (OA). Pathomechanisms of degenerative joint disease may therefore differ in these joints, due to differences in tissue structure and function. Focussing on structural issues which are design goals for tissue engineering, we compared cell and matrix morphologies in different anatomical sites of adult human knee and ankle joints. METHODS Osteochondral explants were acquired from knee and ankle joints of deceased persons aged 20 to 40 years and analyzed for cell, matrix and tissue morphology using confocal and electron microscopy and unbiased stereological methods. Variations associated with joint (knee versus ankle) and biomechanical role (convex versus concave articular surfaces) were identified by 2-way analysis of variance and post-hoc analysis. RESULTS Knee cartilage exhibited higher cell densities in the superficial zone than ankle cartilage. In the transitional zone, higher cell densities were observed in association with convex versus concave articular surfaces, without significant differences between knee and ankle cartilage. Highly uniform cell and matrix morphologies were evident throughout the radial zone in the knee and ankle, regardless of tissue biomechanical role. Throughout the knee and ankle cartilage sampled, chondron density was remarkably constant at approximately 4.2×10(6) chondrons/cm(3). CONCLUSION Variation of cartilage cell and matrix morphologies with changing joint and biomechanical environments suggests that tissue structural adaptations are performed primarily by the superficial and transitional zones. Data may aid the development of site-specific cartilage tissue engineering, and help identify conditions where OA is likely to occur.

Transforming growth factor beta signaling is essential for the autonomous formation of cartilage-like tissue by expanded chondrocytes.

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.

Variation of cell and matrix morphologies in articular cartilage among locations in the adult human knee

OBJECTIVE: Understanding of articular cartilage physiology, remodelling mechanisms, and evaluation of tissue engineering repair methods requires reference information regarding normal structural organization. Our goals were to examine the variation of cartilage cell and matrix morphology in different topographical areas of the adult human knee joint. METHODS: Osteochondral explants were acquired from seven distinct anatomical locations of the knee joints of deceased persons aged 20-40 years and prepared for analysis of cell, matrix and tissue morphology using confocal microscopy and unbiased stereological methods. Differences between locations were identified by statistical analysis. RESULTS: Medial femoral condyle cartilage had relatively high cell surface area per unit tissue volume in the superficial zone. In the transitional zone, meniscus-covered lateral tibia cartilage showed elevated chondrocyte densities compared to the rest of the knee while lateral femoral condyle cartilage exhibited particularly large chondrocytes. Statistical analyses indicated highly uniform morphology throughout the radial zone (lower 80% of cartilage thickness) in the knee, and strong similarities in cell and matrix morphologies among cartilage from the femoral condyles and also in the mediocentral tibial plateau. Throughout the adult human knee, the mean matrix volume per chondron was remarkably constant at approximately 224,000 microm(3), corresponding to approximately 4.6 x 10(6) chondrons per cm(3). CONCLUSIONS: The uniformity of matrix volume per chondron throughout the adult human knee suggests that cell-scale biophysical and metabolic constraints may place limitations on cartilage thickness, mechanical properties, and remodelling mechanisms. Data may also aid the evaluation of cartilage tissue engineering treatments in a site-specific manner. Results indicate that joint locations which perform similar biomechanical functions have similar cell and matrix morphologies; findings may therefore also provide clues to understanding conditions under which focal lesions leading to osteoarthritis may occur.

An educational review of cartilage repair: precepts & practice - myths & misconceptions - progress & prospects.

OBJECTIVE The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty clinical problem. Although research activity in this area has been indefatigably sustained, no significant progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair redivivus! DESIGN Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. RESULTS AND CONCLUSIONS Tissue engineering aims to reconstitute a tissue both structurally and functionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be surmounted for ultimate success are discussed.

Translation from research to applications

The article summarizes the collective views expressed at the fourth session of the workshop Tissue Engineering-the Next Generation, which was devoted to the translation of results of tissue engineering research into applications. Ernst Hunziker described the paradigm of a dual translational approach, and argued that tissue engineering should be guided by the dimensions and physiological setting of the bodily compartment to be repaired. Myron Spector discussed collagen-glycosaminoglycan (GAG) scaffolds for musculoskeletal tissue engineering. Jeanette Libera focused on the biological and clinical aspects of cartilage tissue engineering, and described a completely autologous procedure for engineering cartilage using the patient's own chondrocytes and blood serum. Arthur Gertzman reviewed the applications of allograft tissues in orthopedic surgery, and outlined the potential of allograft tissues as models for biological and medical studies. Savio Woo discussed a list of functional tissue engineering approaches designed to restore the biochemical and biomechanical properties of injured ligaments and tendons to be closer to that of the normal tissues. Specific examples of using biological scaffolds that have chemoattractants as well as growth factors with unique contact guidance properties to improve their healing process were shown. Anthony Ratcliffe discussed the translation of the results of research into products that are profitable and meet regulatory requirements. Michael Lysaght challenged the proposition that commercial and clinical failures of early tissue engineering products demonstrate a need for more focus on basic research. Arthur Coury described the evolution of tissue engineering products based on the example of Genzyme, and how various definitions of success and failure can affect perceptions and policies relative to the status and advancement of the field of tissue engineering.

BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel

OBJECTIVE: According to recent reports, the synovial membrane may contain mesenchymal stem cells with the potential to differentiate into chondrocytes under appropriate conditions. In order to assess the usefulness of synovium-derived progenitor cells for the purposes of cartilage tissue engineering, we explored their requirements for the expression of chondrocyte-specific genes after expansion in vitro. DESIGN: Mesenchymal progenitor cells were isolated from the synovial membranes of bovine shoulder joints and expanded in two-dimensions on plastic surfaces. They were then seeded either as micromass cultures or as single cells within alginate gels, which were cultured in serum-free medium. Under these three-dimensional conditions, chondrogenesis is known to be supported and maintained. Cell cultures were exposed either to bone morphogenetic protein-2 (BMP-2) or to isoforms of transforming growth factor-beta (TGF-beta). The levels of mRNA for Sox9, collagen types I and II and aggrecan were determined by RT-PCR. RESULTS: When transferred to alginate gel cultures, the fibroblast-like synovial cells assumed a rounded form. BMP-2, but not isoforms of TGF-beta, stimulated, in a dose-dependent manner, the production of messenger RNAs (mRNAs) for Sox9, type II collagen and aggrecan. Under optimal conditions, the expression levels of cartilage-specific genes were comparable to those within cultured articular cartilage chondrocytes. However, in contrast to cultured articular cartilage chondrocytes, synovial cells exposed to BMP-2 continued to express the mRNA for alpha1(I) collagen. CONCLUSIONS: This study demonstrates that bovine synovium-derived mesenchymal progenitor cells can be induced to express chondrocyte-specific genes. However, the differentiation process is not complete under the chosen conditions. The stimulation conditions required for full transformation must now be delineated.

Chondrocyte mechanotransduction: effects of compression on deformation of intracellular organelles and relevance to cellular biosynthesis

OBJECTIVE: The effects of mechanical deformation of intact cartilage tissue on chondrocyte biosynthesis in situ have been well documented, but the mechanotransduction pathways that regulate such phenomena have not been elucidated completely. The goal of this study was to examine the effects of tissue deformation on the morphology of a range of intracellular organelles which play a major role in cell biosynthesis and metabolism. DESIGN: Using chemical fixation, high pressure freezing, and electron microscopy, we imaged chondrocytes within mechanically compressed cartilage explants at high magnification and quantitatively and qualitatively assessed changes in organelle volume and shape caused by graded levels of loading. RESULTS: Compression of the tissue caused a concomitant reduction in the volume of the extracellular matrix (ECM), chondrocyte, nucleus, rough endoplasmic reticulum, and mitochondria. Interestingly, however, the Golgi apparatus was able to resist loss of intraorganelle water and retain a portion of its volume relative to the remainder of the cell. These combined results suggest that a balance between intracellular mechanical and osmotic gradients govern the changes in shape and volume of the organelles as the tissue is compressed. CONCLUSIONS: Our results lead to the interpretive hypothesis that organelle volume changes appear to be driven mainly by osmotic interactions while shape changes are mediated by structural factors, such as cytoskeletal interactions that may be linked to extracellular matrix deformations. The observed volume and shape changes of the chondrocyte organelles and the differential behavior between organelles during tissue compression provide evidence for an important mechanotransduction pathway linking translational and post-translational events (e.g., elongation and sulfation of glycosaminoglycans (GAGs) in the Golgi) to cell deformation.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.