Collagen IX-deficiency seriously compromises growth cartilage development in mice.

For a large part, skeletal development, growth, and repair occur by endochondral ossification which comprises an orderly sequence of consecutive steps of proliferation and late differentiation of chondrocytes. After vascular invasion into hypertrophic cartilage, the tissue is remodelled into bone. At all stages, the process is under tight environmental control exerted by a combination of regulators, including nutritional supply and signalling through growth factors, hormones, and cell-matrix-interactions. Therefore, genetic elimination of collagen IX, a stabilizing component of the periphery of thin cartilage fibrils, is expected to compromise extracellular matrix properties and, hence, the chondrocyte environment required for normal cartilage development and homeostasis. Here, we have shown that growth plate cartilage morphology is markedly disturbed in mice lacking collagen IX. Abnormalities were most prominent in late proliferative, pre-hypertrophic, and hypertrophic zones whereas resting and early proliferative zones were less affected. In central epiphyseal regions of long bones, newborn animals show grossly abnormal areas with strongly reduced cell numbers, irregular distribution of glycosaminoglycans in the extracellular matrix, and a profoundly disturbed columnar arrangement of chondrocytes with an irregular beta1 integrin immunostaining. As a result, all long bones are shorter and broader in newborn Col9a1-/- mice. Remarkably, these abnormalities are attenuated in adult mice, but the number of cells per area still is too low due to reduced cell proliferation.     

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.     

Formation of cartilage by non-chondrogenic cell types

Freshly excised embryonic rat skeletal muscle has been shown to form hyaline cartilage when organ cultured upon demineralized rat bone (bone matrix). Since skeletal muscle is composed of fibrous connective tissue (C.T.) as well as muscle cells, the cartilage could arise from either of these sources. The object of this study was to determine whether cartilage arose from fibrous connective tissue or muscle cells, or both, and whether the ability to form cartilage is limited to tissues derived from somatic mesoderm. Control experiments demonstrated that 19-day embryonic rat skeletal muscle formed cartilage when organ cultured on bone matrix after dissociation and cultivation in vitro, and that 11-day embryonic chick muscle also formed cartilage, although less reproducibly (3 out of 10 cases). Fibroblasts and skeletal muscle were cloned from similar suspensions of dissociated muscle in order to test these purified cell types. Dermis, vascular tissue, and tendons were mechanically removed prior to dissociation in order to eliminate fibroblasts from contaminant sources. Cloned fibroblasts, derived from rat skeletal muscle, formed cartilage in three out of three cases. It was not possible to clone sufficient rat skeletal muscle to place an aggregate onto bone matrix. An aggregate of several hundred chick skeletal muscle clones formed cartilage on bone matrix. The freshly excised C.T. capsules of embryonic chick thyroid and lung were tested for the ability to form cartilage as nonskeletal C.T. derivatives. The epithelial rudiments of thyroid and lung were also tested as endodermal derivatives. Chick cornea was similarly tested as an ectodermal derivative. Of these tissues, only the C.T. capsules formed cartilage. The results demonstrate that various C.T. cell types may alter their phenotype well after that stage at which their differentiation is thought to be stabilized, and that the ability to differentiate as cartilage may be common to all C.T. cells. The option of differentiating along a certain variety of pathways may depend more upon local conditions than on a predetermined pattern.     

Perlecan displays variable spatial and temporal immunolocalisation patterns in the articular and growth plate cartilages of the ovine stifle joint

Perlecan is a modular heparan sulphate and/or chondroitin sulphate substituted proteoglycan of basement membrane, vascular tissues and cartilage. Perlecan acts as a low affinity co-receptor for fibroblast growth factors 1, 2, 7, 9, binds connective tissue growth factor and co-ordinates chondrogenesis, endochondral ossification and vascular remodelling during skeletal development; however, relatively little is known of its distribution in these tissues during ageing and development. The aim of the present study was to immunolocalise perlecan in the articular and epiphyseal growth plate cartilages of stifle joints in 2-day to 8-year-old pedigree merino sheep. Perlecan was prominent pericellularly in the stifle joint cartilages at all age points and also present in the inter-territorial matrix of the newborn to 19-month-old cartilage specimens. Aggrecan was part pericellular, but predominantly an extracellular proteoglycan. Perlecan was a prominent component of the long bone growth plates and displayed a pericellular as well as a strong ECM distribution pattern; this may indicate a so far unrecognised role for perlecan in the mineralisation of hypertrophic cartilage. A significant age dependant decline in cell number and perlecan levels was evident in the hyaline and growth plate cartilages. The prominent pericellular distribution of perlecan observed indicates potential roles in cell-matrix communication in cartilage, consistent with growth factor signalling, cellular proliferation and tissue development.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Expression of a Truncated, Kinase-Defective TGF-β Type II Receptor in Mouse Skeletal Tissue Promotes Terminal Chondrocyte Differentiation and Osteoarthritis

Members of the TGF-β superfamily are important regulators of skeletal development. TGF-βs signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-βs in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space.

We then tested the hypothesis that TGF-β is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-β may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-β promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

     

Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development

Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf(-/-) mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.     

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.     

Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytes

Calcifying cartilages show a restricted expression of tissue transglutaminase. Immunostaining of newborn rat paw bones reveals expression only in the epiphyseal growth plate. Tissue transglutaminase appears first intracellularly in the proliferation/maturation zone and remains until calcification of the tissue in the lower hypertrophic zone. Externalization occurs before mineralization. Subsequently, the enzyme is present in the interterritorial matrix during provisional calcification and in the calcified cartilage cores of bone trabeculae. In trachea, mineralization occurring with maturation in the center of the cartilage is accompanied by expression of tissue transglutaminase at the border of the hydroxyapatite deposits. Transglutaminase activity also shows a restricted distribution in cartilage, similar to the one observed for tissue transglutaminase protein. Analysis of tissue homogenates showed that the enzyme is present in growth plate cartilage, but not in articular cartilage, and recognizes a limited set of substrate proteins. Osteonectin is coexpressed with tissue transglutaminase both in the growth plate and in calcifying tracheal cartilage and is a specific substrate for tissue transglutaminase in vitro. Tissue transglutaminase expression in skeletal tissues is strictly regulated, correlates with chondrocyte differentiation, precedes cartilage calcification, and could lead to cross-linking of the mineralizing matrix.     

Growth factors in cartilage tissue engineering

Tissue engineering of cartilage consists of two steps. Firstly, the cells from a small biopsy of patient's own tissue have to be multiplied. During this multiplication process they lose their cartilage phenotype. In the second step, these cells have to be stimulated to re-express their cartilage phenotype and produce cartilage matrix. Growth factors can be used to improve cell multiplication, redifferentiation and production of matrix. The choice of growth factors should be made for each phase of the tissue engineering process separately, taking into account cell phenotype and the presence of extracellular matrix. This paper demonstrates some examples of the use of growth factors to increase the amount, the quality and the assembly of the matrix components produced for cartilage tissue engineering. In addition it shows that the "culture history" (e.g., addition of growth factors during cell multiplication or preculture period in a 3-dimensional environment) of the cells influences the effect of growth factor addition. The data demonstrate the potency as well as the limitations of the use of growth factors in cartilage tissue engineering.     

Bone growth retardation in mouse embryos expressing human collagenase 1

Cellular growth and differentiation are readouts of multiple signaling pathways from the intercellular and/or extracellular milieu. The extracellular matrix through the activation of cellular receptors transmits these signals. Therefore, extracellular matrix proteolysis could affect cell fate in a variety of biological events. However, the biological consequence of inadequate extracellular matrix degradation in vivo is not clear. We developed a mouse model expressing human collagenase (matrix metalloproteinase-1, MMP-1) under the control of Col2a1 promoter. The mice showed significant growth retardation during embryogenesis and a loss of the demarcation of zonal structure and columnar array of the cartilage. Immunological examination revealed increased degradation of type II collagen and upregulation of fibronectin and alpha(5)-integrin subunit in the transgenic cartilage. The resting zone and proliferating zone of the growth plate cartilage exhibited a simultaneous increase in bromodeoxyuridine (BrdU)-incorporated proliferating cells and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling-positive apoptotic cells, respectively. Chondrocyte differentiation was not disturbed in the transgenic mice as evidenced by normal expression of the Ihh and type X collagen expression. These data demonstrate that type II collagen proteolysis is an important determinant for the skeletal outgrowth through modulation of chondrocyte survival and cartilagenous growth.     

Bone and cartilage formation by skeletal muscle derived cells

In adult individuals when most tissues have progressively lost the ability to regenerate, bone maintains the potential for a continuous self remodeling. The bone marrow has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The possibility though exists that a pool of osteoprogenitor cells resides within other adult tissues and in particular, as reported previously, in other connective tissues such as fat and skeletal muscle. In an attempt to identify an alternative source of osteoprogenitor cells other than bone marrow we looked into the skeletal muscle. A plastic adhering cell population, from now on referred to as skeletal muscle derived cells (SMDCs), was obtained from biopsies of human skeletal muscle. SMDCs were clonogenic and displayed a fibroblast-like morphology. The isolated cell population had a mesenchymal origin as indicated by abundant expression of type I collagen, fibronectin, and vimentin and appeared heterogeneous. SMDCs were positive for alpha smooth actin, and to a lesser extent for desmin and alpha sarcomeric myosin, two specific markers of the myogenic phenotype. Surprisingly though SMDCs expressed early markers of an osteogenic commitment as indicated by positive staining for alkaline phosphatase, osteopontin, and osteonectin. Under the appropriate stimuli, these cells deposited in vitro a mineralized bone matrix and a proteoglycan rich matrix. In addition, SMDCs cultured in the presence of low serum and insulin differentiated towards adipocytes developing abundant lipid droplets in the cytoplasm. Furthermore SMDCs formed three-dimensional bone tissue in vivo when implanted in an immunodeficient mouse, and a mature cartilage rudiment when maintained as a pellet culture. In summary, we report the isolation and characterization of a cell population from the human skeletal muscle not only able to express in vitro specific markers of distinct mesenchymal lineages (adipogenic, chondrogenic, and osteogenic), but most importantly, able to complete the differentiation pathway leading to the formation of bone and cartilage. In this respect SMDCs resemble bone marrow stromal cells (BMSCs).     

RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.

An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.     

Analysis of matrix vesicles and their role in the calcification of epiphyseal cartilage.

Extracellular matrix vesicles, which have been shown to be associated with initial calcification of cartilage, were isolated, characterized, and studied with 45calcium isotope to determine whether they could form mineral in vitro. It was found that the isolated matrix vesicles contain a phosphatase, active at neutral pH, which has a very wide specificity and will hydrolyze a variety of nucleotide triphosphates, diphosphates, monophosphates, and other phosphate-containing substrate and metabolites. Acid phosphatase, beta-glucuronidase, and cathepsin D were found to be in the cell fractions, in lysosomes; these enzymes are not present in matrix vesicles and this is additional evidence for the difference between matrix vesicles and lysosomes. Matrix vesicles were found to take up 45Ca even in the presence of low levels of Ca and P1 and also to facilitate precipitation of hydroxylapatite when incubated under physiological conditions in the presence of ATP and other phosphate-containing substrates. Systematic electron probe analysis of a septum of epiphyseal cartilage indicates that matrix vesicles gradually accumulate calcium and then phosphorus and thus facilitate the advance of the calcification front. Adjoinging nonvesicular matrix in the hypertrophic zone, cell cytoplasm, and cell processes had very low levels of calcium and phosphorus in a region where matrix vesicles showed high levels of these elements. New concepts are put forward that take accounts of these findings which provide a better understanding of the sequence of mineralization in growth cartilage.     

Effect of extracellular ph on matrix synthesis by chondrocytes in 3D agarose gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.     

Chondrodysplasias due to proteoglycan defects

The proteoglycans, especially the large chondroitin sulfate proteoglycan aggrecan, have long been viewed as important components of the extracellular matrix of cartilage. The drastic change in expression during differentiation from mesenchyme to cartilage, the loss of tissue integrity associated with proteoglycan degradation in several disease processes and, most important, the demonstration of abnormalities in proteoglycan production concomitant with the aberrant growth patterns exhibited by the brachymorphic mouse, the cartilage matrix deficient mouse, and the nanomelic chick provide the strongest evidence that the proteoglycan aggrecan is essential during differentiation and for maintenance of the skeletal elements. More recently, mutations associated with proteoglycans other than aggrecan, especially the heparan sulfate proteoglycans, glypican and perlecan, suggest an important role for these molecules in skeletal development as well. This review focuses on the molecular bases of the hereditary proteoglycan defects in animal models, as well as of some human chondrodysplasias, that collectively are providing a better understanding of the role of proteoglycans in the development and maintenance of the skeletal elements.