Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of approximately 6.25 microm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm(-1)/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.     

Pathology of degenerative osteoarthrosis of the thoracic vertebrae in rats

Degenerative osteoarthrosis was observed in the thoracic vertebrae in specific pathogen-free Sprague-Dawley rats at the ages of eight and 19 weeks. Histological changes seen in the lesions were degenerated matrix intermixed with collagen fibers, erosion or ulceration, and formation of cysts in the articular cartilage, and degeneration and necrosis in the subchondral bone.     

Effect of interleukin-1 on the size distribution of cartilage proteoglycans as determined by sedimentation field flow fractionation

The effect of interleukin-1 (IL-1) on the size distribution of cartilage proteoglycans was studied using sedimentation field flow fractionation (SdFFF), a rapid, high-resolution technique for the separation of proteoglycan monomers and aggregates. During incubation of cartilage in control media, 35S-prelabeled proteoglycan was lost primarily from proteoglycan present in the monomer form; aggregates were conserved. In the presence of IL-1, both 35S-proteoglycan monomers and aggregates were lost, suggesting that IL-1 increases the susceptibility of aggregates to loss from the cartilage matrix. Evaluation of uronic acid as a measure of net change in proteoglycan content indicated that IL-1 causes a net decrease in both monomers and aggregates. Kinetic studies suggested that aggregates are degraded to monomers which then diffuse out of the matrix. Incorporation of [35S]sulfate into cartilage proteoglycans following exposure to IL-1 showed that synthesis of monomers and aggregates is inhibited similarly. SdFFF is a valuable technique for studying proteoglycan metabolism. With its use, changes in proteoglycan monomer and aggregate populations can be detected in response to cytokines such as IL-1.     

Matrix-associated heparan sulfate proteoglycan: core protein-specific monoclonal antibodies decorate the pericellular matrix of connective tissue cells and the stromal side of basement membranes

Cultured human lung fibroblasts produce a large, nonhydrophobic heparan sulfate proteoglycan that accumulates in the extracellular matrix of the monolayer (Heremans, A., J. J. Cassiman, H. Van den Berghe, and G. David. 1988. J. Biol. Chem. 263: 4731-4739). A panel of four monoclonal antibodies, specific for four distinct epitopes on the 400-kD core protein of this extracellular matrix heparan sulfate proteoglycan, detects similar proteoglycans in human epithelial cell cultures. Immunohistochemistry of human tissues with the monoclonal antibodies reveals that these proteoglycans are concentrated at cell-matrix interfaces. Immunogold labeling of ultracryosections of human skin indicates that the proteoglycan epitopes are nonhomogeneously distributed over the width of the basement membrane. Immunochemical investigations and amino acid sequence analysis indicate that the proteoglycan from the fibroblast matrix shares several structural features with the large, low density heparan sulfate proteoglycan isolated from the Engelbreth-Holm-Swarm sarcoma. Thus, both epithelial cell sheets and individual mesenchymal cells accumulate a large heparan sulfate proteoglycan(s) at the interface with the interstitial matrix, where the proteoglycan may adopt a specific topological orientation with respect to this matrix.     

Fourier transform infrared imaging spectroscopy investigations in the pathogenesis and repair of cartilage

Significant complications in the management of osteoarthritis (OA) are the inability to identify early cartilage changes during the development of the disease, and the lack of techniques to evaluate the tissue response to therapeutic and tissue engineering interventions. In recent studies several spectroscopic parameters have been elucidated by Fourier transform infrared imaging spectroscopy (FT-IRIS) that enable evaluation of molecular and compositional changes in human cartilage with progressively severe OA, and in repair cartilage from animal models. FT-IRIS permits evaluation of early-stage matrix changes in the primary components of cartilage, collagen and proteoglycan on histological sections at a spatial resolution of ∼6.25 μm. In osteoarthritic cartilage, the collagen integrity, monitored by the ratio of peak areas at 1338 cm⁻¹/Amide II, was found to correspond to the histological Mankin grade, the gold standard scale utilized to evaluate cartilage degeneration. Apparent matrix degradation was observable in the deep zone of cartilage even in the early stages of OA. FT-IRIS studies also found that within the territorial matrix of the cartilage cells (chondrocytes), proteoglycan content increased with progression of cartilage degeneration while the collagen content remained the same, but the collagen integrity decreased. Regenerative (repair) tissue from microfracture treatment of an equine cartilage defect showed significant changes in collagen distribution and loss in proteoglycan content compared to the adjacent normal cartilage, with collagen fibrils demonstrating a random orientation in most of the repair tissue. These studies demonstrate that FT-IRIS is a powerful technique that can provide detailed ultrastructural information on heterogeneous tissues such as diseased cartilage and thus has great potential as a diagnostic modality for cartilage degradation and repair.     

Immunogenicity of xenogeneic cartilage matrix components in a rabbit model

Purified xenogeneic cartilage matrix components, including proteoglycan subunits, chondroitin 4 sulfate, and chondroitin 6 sulfate, were inoculated into the knee joint of rabbits, and local as well as systemic responses were evaluated. proteoglycan was associated with synovial hyperplasia and infiltrates of eosinophils and lymphocytes and with rising titers of antiproteoglycan antibodies in a tanned sheep rbc hemagglutination assay over a six-week period of weekly intra-articular injections and observations. Chondroitin sulfates failed to evoke detectable changes in the joint or serum. Immunogenicity of cartilage matrix components may play a role in allogeneic and xenogeneic osteochondral grafts, and it is also possible that autogenous matrix immunogens exist and contribute to progression of degenerative joint disease. The immunogenicity of allogeneic and autogenous cartilage matrix components remains undefined.     

Proteoglycan-collagen associations in the non-lactating human breast connective tissue during the menstrual cycle

The human mammary gland undergoes a sequence of histological changes in both epithelial and stromal compartments during the menstrual cycle. Swelling and unswelling of the breast stromal tissue is a characteristic feature of the two phases of the cycle and is mediated by changes in the water content of sulfated proteoglycans in the matrix between the fibrils. In an ultrastructural study we investigated the distribution of sulfated proteoglycans identified as cupromeronic blue-positive needle-like structures and measured the distance between the dermatan sulfate-proteoglycan attachment sites at the d-bands of the collagen fibrils in the loose intralobular connective tissue and in the dense interlobular connective tissue. We characterized the dermatan sulfate proteoglycan by enzyme digestion and by immunogold-labeled antibody. In the follicular phase a relatively constant distance of 46 nm between neighboring proteoglycan attachment sites was found, while in the luteal phase the measured distances are strikingly variable and exceed the follicular value by up to 9 nm. This difference of the two cycle phases is more evident in the loose than in the dense connective tissue. Possibly the changes of the fibril-attached proteoglycans in the luteal phase reflect an influence of the higher water content of the matrix leading to a probably torsional swelling of the collagen fibril.     

Differentiation of the secondary elastic cartilage in the external ear of the rat.

The cartilage in the external ear of the rat belongs to the group of secondary cartilages and it has a unique structural organization. The chondrocytes are transformed into typical adipose cells, the proteoglycan cartilage matrix is reduced to thin capsules around the cells and the rest of the extracellular matrix is occupied by a network of coarse elastic fibers. It appears late in development (16-day fetus) and needs more than one month for final development. The differentiation proceeds in several steps which partly overlap: the appearance of collagen fibrils, elastin fibers, the proteoglycan matrix, and the adipose transformation of chondrocytes. The phenotype of this cartilage and the course of its differentiation are very stable, even in very atypical experimental environmental conditions. The only exceptions are explants in organ culture in vitro and perichondrial regenerates. In these conditions the development of elastic fibers is slow and poor while the production of the proteoglycan matrix is abundant. The resulting cartilage then displays structural characteristics of hyaline cartilage rather than those of the initial elastic one.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin.

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [35S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin.

Small leucine-rich proteoglycans (SLRPs), for example, decorin, biglycan, fibromodulin, and lumican, are extracellular matrix organizers and binding partners of TGF-b. Decorin is also involved in growth control and angiogenesis. Hence, these proteoglycans are likely of importance in the pathogenesis of diabetic glomerulosclerosis. In normal kidney, SLRPs were preferentially expressed in the tubulointerstitium. Weak expression occurred in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, by distal tubular cells and in collecting ducts. In all stages of diabetic nephropathy, there was a marked up-regulation of the proteoglycans in tubulointerstitium and glomeruli. Decorin and lumican became expressed in tubuli. However, in glomeruli, overexpression was not mirrored by local proteoglycan accumulation except in advanced nephropathy. In severe glomerulosclerosis, increased decorin concentrations were found in plasma and urine, and urinary TGF-b/decorin complexes could be demonstrated indirectly. The failure to detect an increased glomerular proteoglycan quantity during the development of nephropathy could be explained by assuming that they are secreted into the mesangial matrix, but cleared via the vasculature or the urinary tract, in part as complexes with TGF-b. They could thereby counteract the vicious circle being characterized by increased TGF-b production and increased matrix deposition in diabetic nephropathy.     

Changes in the extracellular matrix of the normal human breast during the menstrual cycle

Summary The normal human mammary gland undergoes a well defined sequence of histological changes in both epithelial and stromal compartments during the menstrual cycle. Studies in vitro have suggested that the extracellular matrix surrounding the individual cells plays a central role in modulating a wide variety of cellular events, including proliferation, differentiation and gene expression. We therefore investigated the distribution of a number of extracellular matrix molecules in the normal breast during the menstrual cycle. By use of indirect immunofluorescence, with specific antibodies, we demonstrated that laminin, heparan sulphate proteoglycan, type IV collagen, type V collagen, chondroitin sulphate and fibronectin undergo changes in distribution during the menstrual cycle, whereas collagen types I, III, VI and VII remain unchanged. These changes were most marked in the basement membrane, sub-basement membrane zone and delimiting layer of fibroblasts surrounding the ductules where basement membrane markers such as laminin, heparan sulphate proteoglycan, and type IV and V collagens appear greatly reduced during the mid-cycle period (days 8 to 22). These results suggest that some extracellular matrix molecules may act as medittors in the hormonal control of the mammary gland, whereas others may have a predominantly structural role.     

Extracellular Matrix Deposition in Engineered Micromass Cartilage Pellet Cultures: Measurements and Modelling

This article explores possible mechanisms governing extracellular matrix deposition in engineered cartilaginous cell pellets. A theoretical investigation is carried out alongside an experimental study measuring proteoglycan and collagen volume fractions within murine chondrogenic (ATDC-5) cell pellets. The simple mathematical model, which adopts a nutrient-dependent proteoglycan production rate, successfully reproduces the periphery-dominated proteoglycan deposition, characteristic of the growth pattern observed experimentally within pellets after 21 days of culture. The results suggest that this inhomogeneous proteoglycan production is due to nutrient deficiencies at the pellet centre. Our model analysis further indicates that a spatially uniform distribution of proteoglycan matrix could be maintained by initiating the culture process with a smaller-sized pellet. Finally, possible extensions are put forward with an aim to improve the model predictions for the early behaviour, where different mechanisms appear to dominate the matrix production within the pellets.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [³⁵S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells

The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross- linked by antibodies, they initially assimilate into detergent- resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets.     

Proteoglycan deposition around chondrocytes in agarose culture: construction of a physical and biological interface for mechanotransduction in cartilage

With a view towards the development of methods for cartilage tissue engineering, matrix deposition around individual chondrocytes was studied during de novo matrix synthesis in agarose suspension culture. At a range of times in culture from 2 days to 1 month (long enough for cartilage-like material properties to begin to emerge), pericellular distributions of proteoglycan and matrix protein deposition were measured by quantitative autoradiography, while matrix accumulation and cell volumes were estimated by stereological methods. Consistent with previous work, tissue-average rates of matrix synthesis generally decreased asymptotically with time in culture, as de novo matrix accumulated. Cell-scale analysis revealed that this evolution was accompanied by a transition from predominantly pericellular matrix (within a few microm from the cell membrane) deposition early in culture towards proteoglycan and protein deposition patterns more similar to those observed in cartilage explants at later times. This finding may suggest a differential recruitment of different proteoglycan metabolic pools as matrix assembly progresses. Cell volumes increased with time in culture, suggestive of alterations in volume regulatory processes associated with changes in the microphysical environment. Results emphasize a pattern of de novo matrix construction which proceeds outward from the pericellular matrix in a progressive fashion. These findings provide cell-scale insight into the mechanisms of assembly of matrix proteins and proteoglycans in de novo matrix, and may aid in the development of tissue engineering methods for cartilage repair.     

Submicroscopic response of articular chondrocytes to a cholesterol-containing diet.

10 days' to 3 months' consumption of a diet containing 4% cholesterol causes a minor and transitory stimulation of articular chondrocytes of mice. The transitory disturbance is accompanied by a more permanent increase in glycogen, by abnormal deposition of cytoplasmic lipid and by an increase in the fibrillarity of the pericellular matrix. The changes are consistent with the failure of the cholesterol diet to influence the course of osteoarthrosis if fed to mice from an early age through life.     

The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro.

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.     

Influence of the pericellular environment on the cells. The role of mucopolysaccharides in the protection of cartilage cells against immune reactions.

Problems related to rheumatoid arthritis have been investigated by a group at Cambridge using the organ culture technique. Since auto-allergic reactions may be concerned in the chronicity of the disease, the effects of reactive complement-sufficient antisera (AS+C') on embryonic and post-foetal cartilage were examined. The cartilaginous limb bone rudiments enlarged to several times their original volume in control medium, but in the presence of AS+C' they gradually disintegrated, owing to the breakdown of the cartilage matrix; only the superficial cells of the enveloping soft connective tissue were killed, however. Provided breakdown had not advanced too far, the effects of AS+C' were reversible. It was not clear how AS+C' produced these changes, since cartilage matrix is impermeable to molecules as large as the immunoglobulins. To find whether there was a difference in permeability between embryonic and post-foetal cartilage, similar experiments were made on the articular cartilage of young pigs. AS+C' had no effect on pure articular cartilage, and it was shown immunohistochemically that IgG did not penetrate beyond the most superficial layer of cartilage. When, however, the explant was associated with soft connective tissue either as invading marrow or as an adjacent explant of synovium, the cartilage matrix was depleted of proteoglycan; IgG antibodies then entered the cartilage and reacted with the chondrocytes. After a lapse of 8-10 days, collagen also began to break down. If the degradation of collagen was not too extensive, the changes were reversible. Pure cartilage was depleted of proteoglycan by trypsinization and then cultivated in AS+C'. All the chondrocytes reacted with the IgG antibodies. The peripheral cells were killed, but those in the interior survived and rapidly secreted pericellular capsules rich in proteoglycan, which shielded them from further contact with antibodies. In other experiments, pure cartilage was associated with a synovial explant and cultivated in AS+C' for 10 days; this caused severe depletion of the matrix. The synovial tissue was then removed and the isolated cartilage cultured for a further 10 days in either AS+C' or control medium. If mainly proteoglycan had been lost during the primary culture period, breakdown did not continue in AS+C', and sometimes a little new matrix was regenerated, though less than in control medium; if, however, the collagen had been extensively degraded, breakdown continued even in control medium. It is suggested that in both the embryonic and post-foetal cartilage, degradation of the cartilage matrix was due to the enzymatic activity of the associated soft connective tissue which caused a loss first of proteoglycan, which enabled antibodies to reach the chondrocytes, and then of collagen. The possible relevance of these results to the pathogenesis of rheumatoid arthritis is discussed.