Glycosaminoglycan turn-over in articular cartilage.

Glycosaminoglycan turn-over has been studied both in vivo and in vitro, by using sodium [35S]sulphate as a precursor. The in vivo experiments were performed on rabbits and dogs, taking special care to monitor the 35S radioactivity in the serum throughout the experiment and to measure the radioactivity due to unincorporated inorganic [35S]sulphate in cartilage at the end of each experiment, in addition to that due to incorporated sulphate. The inorganic sulphate content of the serum was also determined as well as the distribution coefficient for the inorganic sulphate ion between cartilage and serum. From this information it was possible to calculate accurately the rate of sulphate uptake by cartilage in vivo and hence the turn-over rate. Experiments were then performed in vitro on cartilage from rabbits and dogs and the in vivo and in vitro results were compared. A very good agreement was obtained between the two sets of results. Studies were then carried out under exactly the same in vitro conditions on human articular cartilage and it was thus possible to obtain a turn-over rate for the latter which one could trust was close to the actual in vivo value. The mean half-lives thus obtained varied from 45 days for the young rabbit to 150 days for the adult dog and 800 days for the human femoral head. In human cartilage there were considerable variations in turn-over rate within a single joint as a function of depth below the surface, and between different joints. Thus, while the mean half-life for the human femoral head is 800 days, that for the femoral condyle is 300 days. Cartilage from osteoarthrosic femoral heads did not appear to differ much with respect to sulphate uptake from the normal specimens although the turn-over rates were somewhat higher.     

Effect of insulin on the altered production of proteoglycans in rib cartilage of experimentally diabetic rats

Costal cartilage from experimentally diabetic rats, labeled in vivo or in vitro with [35S]sulfate, was shown to incorporate less label into proteoglycans than cartilage from nondiabetic rats. Analyses of guanidine HCl cartilage extracts by gel chromatography on Sepharose CL-2B showed two major peaks at Kav approximately 0.4 and 0.8 (peaks I and II, respectively). Cartilage extracts from the diabetic rats contained predominantly peak II proteoglycans, while 60 and 55%, respectively, of the total 35S-labeled proteoglycans extracted from control cartilage labeled in vivo and in vitro with [35S]sulfate were present in peak I. After insulin treatment of the diabetic rats, the relative amount of peak I 35S-labeled proteoglycans synthesized in vivo was increased to 70%. The overall in vivo incorporation of [35S]sulfate into proteoglycans was also stimulated in diabetic rats treated with insulin to levels above those found for control rats. Thus, diabetes-induced changes in the biosynthesis of rat costal cartilage proteoglycans may be alleviated by normalization of the diabetic state by insulin treatment. However, addition of insulin (10(-5)-10(-9) M) to the culture medium did not affect the amount of 35S-labeled proteoglycans synthesized in vitro or the relative amounts of peak I proteoglycans produced by control or diabetic cartilage, suggesting that insulin does not have a direct effect on proteoglycan production. Moreover, no decrease in the amount of 35S-labeled proteoglycans produced was found when glucose at high concentrations was present in the culture medium. However, the presence of rat serum resulted in an increase in the amount of 35S-labeled proteoglycans produced by both control and diabetic cartilage, demonstrating that the cartilage explants were metabolically responsive to stimulatory factors.     

Immunohistochemical, autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein.

Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using 35S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for 35S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for 35S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.     

Immunohistochemical,autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein

Summary Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using ³⁵S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for ³⁵S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for ³⁵S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

Mode of degradation of the chondroitin sulphate proteoglycan in rat costal cartilage

1. Chondroitin sulphate was isolated from different regions of rat costal cartilage after extensive proteolysis of the tissues. The molecular weight, determined by gel chromatography, of the polysaccharide obtained from an actively growing region (lateral zone) near the osteochondral junction was higher than that of the polysaccharide isolated from the remaining portion of the costal cartilage (medial zone). 2. In both types of cartilage the molecular weight of chondroitin sulphate, labelled with [(35)S]sulphate, remained unchanged in vivo over a period of 10 days, approximately corresponding to the half-life of the chondroitin sulphate proteoglycan. The molecular-weight distribution of chondroitin [(35)S]sulphate, labelled in vivo or in vitro, was invariably identical with that of the bulk polysaccharide from the same tissue. It is concluded that the observed regional variations in molecular-weight distribution were established at the time of polysaccharide biosynthesis. 3. In tissue culture more than half of the (35)S-labelled polysaccharide-proteins of the two tissues was released into the medium within 10 days of incubation. The released materials were of smaller molecular size than were the corresponding native proteoglycans. In contrast, the molecular-weight distribution of the chondroitin [(35)S]sulphate (single polysaccharide chains) remained constant throughout the incubation period. 4. A portion (about 20%) of the total radioactive material released from (35)S-labelled cartilage in tissue culture was identified as inorganic [(35)S]sulphate. No corresponding decrease in the degree of sulphation of the labelled polysaccharide could be detected. These findings suggest that a limited fraction of the proteoglycan molecules had been extensively desulphated. 5. It is suggested that the initial phase of degradation involves proteolytic cleavage of the proteoglycan, but the constituent polysaccharide chains remain intact. The partially degraded proteoglycan may be eliminated from the cartilage by diffusion into the circulatory system. An additional degradative process, which may occur intracellularly, includes desulphation of the polysaccharide, probably in conjunction with a more extensive breakdown of the polymer.     

Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo.

Articular cartilage exhibits little intrinsic repair capacity, and new tissue engineering approaches are being developed to promote cartilage regeneration using cellular therapies. The goal of this study was to examine the chondrogenic potential of adipose tissue-derived stromal cells. Stromal cells were isolated from human subcutaneous adipose tissue obtained by liposuction and were expanded and grown in vitro with or without chondrogenic media in alginate culture. Adipose-derived stromal cells abundantly synthesized cartilage matrix molecules including collagen type II, VI, and chondroitin 4-sulfate. Alginate cell constructs grown in chondrogenic media for 2 weeks in vitro were then implanted subcutaneously in nude mice for 4 and 12 weeks. Immunohistochemical analysis of these samples showed significant production of cartilage matrix molecules. These findings document the ability of adipose tissue-derived stromal cells to produce characteristic cartilage matrix molecules in both in vitro and in vivo models, and suggest the potential of these cells in cartilage tissue engineering.     

Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage

The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0-50% static compression, and the transient and steady-state incorporation of [(3)H]proline and [(35)S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0-300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [(3)H]proline and [(35)S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of (125)I-labeled IGF-I into cartilage explants. Static compression decreased both [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (tau) for IGF-I stimulation of cartilage explants was approximately 24 h, while tau for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter tau for (125)I-labeled IGF-I transport into cartilage but decreased the concentration of (125)I-labeled IGF-I in the tissue at equilibrium.     

Cyclic nucleotides during chondrogenesis: concentration and distribution in vivo and in vitro

This study correlates endogenous levels of cAMP and cGMP with their immunohistochemical localization during chondrogenic differentiation of C57B1/6J mouse limb mesenchyme in vivo and in vitro. A transient decrease in cGMP but not cAMP was found from days 12 to 13 in vivo correlating with early stages of chondrogenesis in the developing limb. Intracellular levels of both cAMP and cGMP in high density limb mesenchyme cultures increased 25% after 24 hr in culture when aggregate and nodule formation was detectable. When cells were seeded at different initial plating densities to delay the onset of aggregate and nodule formation, increased levels of intracellular cAMP correlated temporally with the appearance of nodules. Both cyclic AMP and cGMP were immunohistochemically localized in perichondrial cells and chondrocytes in vivo and in vitro. Therefore, (1) cAMP levels correlated temporally with the appearance of chondrogenic cells and (2) cAMP and cGMP were immunohistochemically localized to chondrogenic cells. These data indicate that fluctuations of both cAMP and cGMP levels may be involved in limb cartilage differentiation. Although increases in both nucleotides were found to correlate with the onset of chondrogenesis in vitro, in vivo data suggest that the amount of cAMP relative to cGMP rather than the absolute amount of an individual cyclic nucleotide may be more significant in modulating differentiation.     

Hormonal control of sulfate uptake by branchial cartilage of coho salmon: role of IGF-I.

The direct hormonal control of sulfate uptake by cartilage matrix of coho salmon was examined by exposing branchial cartilage to 1 microCi.ml-1 35SO4 for 48 hours at 15 degrees C in a defined medium. Sulfate uptake occurred primarily in cartilage (rather than bone) and the amount of specific uptake was similar in epibranchial and ceratobranchial cartilages. Intact and hypophysectomized coho salmon starved for 22 days had equivalent in vitro sulfate uptake, which in both cases were 30% of the uptake seen in branchial cartilage of fed, intact controls. In branchial cartilage from starved coho salmon, in vitro exposure to recombinant bovine insulin-like growth factor I (rbIGF-I) at 1, 10, 100, and 1,000 ng.ml-1 caused a dose-dependent increase in sulfate uptake, with a maximum 3-fold increase over control at 1,000 ng.ml-1 rbIGF-I. Coho salmon insulin (1, 10, 100, and 1,000 ng.ml-1) resulted in a maximum 30% increase in sulfate uptake at the highest dose. Growth hormone and triiodo-L-thyronine had no direct effect on in vitro sulfate uptake. The results indicate that IGF-I has direct effects on coho salmon cartilage and may be an important regulator of growth in salmon and other teleosts.     

Comparison of sulfate metabolism in costal cartilage of normal and 'little' mice

C57BL/6J and mutant 'little' (lit/lit) mice c. 50 days of age were injected with doses of [35S]sulfate proportional to their body weight. Despite the diminished growth rate of lit/lit mice compared with normal mice at this age, uptake of radioactivity per unit mass of cartilage was similar for both mouse types, confirming previous data. Additional experiments with these mice established that the similarity of sulfate uptake could not be accounted for by differences in the location of bound sulfate or (for females) by differences in cartilage cellularity. Investigation of sulfate loss by costal cartilage in vivo indicated that cartilage degradation occurred at a greater rate in lit/lit mice than in normally growing mice. These latter data suggest that growth hormone, which is lacking in lit/lit mice, may in part regulate skeletal growth (at least for female mice) by inhibiting degradation of cartilage.     

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo . When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo , the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo . These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo , however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.     

Regulatory mechanisms in cell-mediated immune responses. II. Comparison of culture-induced and alloantigen-induced suppressor cells in MLR and CML.

Two antigen-nonspecific T cell-dependent suppressor systems were compared for their effects upon CML and MLR. Suppressor cells generated by an in vitro culture of spleen cells were compared with suppressor cells generated by in vivo priming with alloantigen. Culture-induced suppressor cells were themselves unable to respond in CML or MLR; were able to suppress actively the CML and MLR responses of untreated responding cells; were mitomycin-sensitive; and, produced no easily demonstrable suppressive supernatant. Alloantigen-primed cells were able to respond in CML and LR; could suppress proliferation in MLR, but were able to suppress CML only after mitomycin treatment; and, produced suppressive supernatants active in suppressing both CML and MLR. In addition to cataloging the differences and similarities between these suppressor populations, the data have been employed to analyze the mechanisms by which suppression occurs in CML and MLR.     

A tissue culture analysis of the steps in limb chondrogenesis

Summary Tissue-culture methods can be used to test the developmental capacity of embryonic cells. In micro-mass cultures, derived from wing cells of stages 21 through 24 chick embryos, aggregates of cells form and then differentiate into cartilage nodules, as judged by the presence of an Alcian blue staining extracellular matrix. Wing cells derived from embryos as young as stage 17 can form aggregates. However, unless they are treated with db cyclic AMP and theophylline, it is not until stage 20 that these aggregates can produce cartilage in culture. In clonal cell culture, cartilage colonies are not produced by primary cell suspensions of limb cells until stage 25 when overt cartilage differentiation is occurring in vivo. It is possible to obtain clonable cartilage cells from limb cells from embryos between stages 20 and 24 if the cells are either treated with db cyclic AMP and theophylline or maintained in suspension culture for 12 to 48 hr. On the basis of these in vitro results a multiple step model for the conversion of limb mesenchyme into cartilage cells is proposed. The model involves the appearance of cells with a predisposition to form aggregates, development of the capacity to form cartilage in response to elevated levels of cyclic AMP, the appearance of receptors that translate changes in either cell shape or cell cycle parameters into elevated levels of cyclic AMP, aggregation, elevated levels of cyclic AMP, cartilage cell determination, and differentiation. This model can serve as the basis for further tests. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by USPHS Training Grant HD00152 from the National Institute of Child Health and Human Development, while P.B.A. was a postdoctoral trainee, and by NIH Grant HD05505 to M.S.     

Ontogenetic development of an exceptionally preserved Devonian cartilaginous skeleton

Cartilaginous vertebrate skeletons leave few records as fossils, unless mineralized. Here, we report outstanding preservation of early stages of cartilage differentiation, present in the Devonian vertebrate Palaeospondylus gunni. In large specimens of Palaeospondylus, enlarged, hypertrophic cell spaces (lacunae) are dominant in the cartilage matrix, each defined by thin mineralized matrix, where phosphorus and calcium co-occur. This is comparable to living endochondral cartilage, where cell hypertrophy and matrix mineralization mark the end of an ontogenetic process of cell growth and division before bone formation. New information from small individuals of Palaeospondylus demonstrates that the skeleton comprises mostly unmineralized organic matrix with fewer hypertrophic cell spaces, these occurring only in the central regions of each element. Only here has the surrounding matrix begun to mineralize, differing from the larger specimens in that phosphorus is dominant with little associated calcium at these earlier stages. This reflects cellular control of mineralization in living tissues through phosphate accumulation around hypertrophic cells, with later increase in calcium in the cartilaginous matrix. These features are always associated with endochondral bone development, but in the Palaeospondylus skeleton, this bone never develops. This skeletal state is thus far unique among vertebrates, with two alternative explanations: either later stages of endochondral bone development have been lost in Palaeospondylus, or, in a stepwise acquisition of the mineralized skeleton, these late stages have not yet evolved.     

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.     

Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit

We previously compared mesenchymal stem cells (MSCs) from a variety of mesenchymal tissues and demonstrated that synovium-MSCs had the best expansion and chondrogenic ability in vitro in humans and rats. In this study, we compared the in vivo chondrogenic potential of rabbit MSCs. We also examined other parameters to clarify suitable conditions for in vitro and in vivo cartilage formation. MSCs were isolated from bone marrow, synovium, adipose tissue, and muscle of adult rabbits. Proliferation potential and in vitro chondrogenic potential were compared. Toxicity of the tracer DiI for in vitro chondrogenesis was also examined. MSCs from each tissue were embedded in collagen gel and transplanted into full thickness cartilage defects of rabbits. Cartilage matrix production was compared histologically. The effects of cell density and periosteal patch on the in vivo chondrogenic potential of synovium-MSCs were also examined. Synovium- and muscle-MSCs had a higher proliferation potential than other cells. Pellets from synovium- and bone-marrow-MSCs showed abundant cartilage matrix. DiI had no significant influence on in vitro cartilage formation. After transplantation into cartilage defects, synovium- and bone-marrow-MSCs produced much more cartilage matrix than other cells. When synovium-MSCs were transplanted at a higher cell density and with a periosteal patch, more abundant cartilage matrix was observed. Thus, synovium- and bone-marrow-MSCs had greater in vivo chondrogenic potential than adipose- and muscle-MSCs, but synovium-MSCs had the advantage of a greater proliferation potential. Higher cell density and a periosteum patch were needed to obtain a high production of cartilage matrix by synovium-MSCs.     

Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development

Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.     

Further characterization of 4-bromomisonidazole as a potential detector of hypoxic cells

[14C]Bromomisonidazole was prepared by direct bromination of [ring-2] [14C]misonidazole in dioxane. The uptake and binding of the two labeled sensitizers were compared in vitro in 1-mm EMT-6 spheroids which contain a necrotic core. Using liquid scintillation counting it was shown that spheroids incubated with 50 microM [14C]bromomisonidazole concentrated drug above levels in the medium by 1 1/2 hr and achieved maximum concentration by 10 hr with no further increase at 23 hr. Spheroids incubated with 50 microM [14C]misonidazole may concentrate the sensitizer more slowly but ultimately reached the same fivefold increase over levels in the medium by 23 hr as was observed for bromomisonidazole. Autoradiographs prepared from spheroids after incubation with [14C]misonidazole or [14C]bromomisonidazole showed silver grains preferentially located over viable hypoxic cells in the inner half of the spheroid rim adjacent to the necrotic center, with lower grain density over nonviable necrotic areas and many fewer grains over oxic cells at the periphery of the spheroid. The results indicate that both severely and moderately hypoxic cells may preferentially bind [14C]bromomisondiazole. The data support the potential of radiolabeled bromomisonidazole for in vivo imaging pending additional studies of the metabolism of this agent.     

Autoradiographic localization of transported neutral amino acids in epithelia of cat submandibular and sublingual salivary glands

Summary Light-microscopic autoradiography was used to localize the cellular sites for neutral amino acid uptake in submandibular and sublingual salivary gland epithelia. The vasculature of isolated glands was perfused for 3–5 min with either L-(3-³H)serine or L-(4-³H)phenylalanine and then fixed by perfusion with buffered glutaraldehyde. In the submandibular gland the small neutral amino acid L-serine and the aromatic amino acid L-phenylalanine were localized to central acinar cells, demilunar cells and ductal cells. In the sublingual gland silver grains associated with each of these tritiated amino acids were localized to central acinar and ductal cells. Perfusion of both submandibular and sublingual glands with unlabelled L-serine (25 mM) or L-phenylalanine (30 mM) resulted in a significant decrease in the silver grain density associated with each labelled amino acid. The absence of silver grains in the lumina of acinar and ductal cells and the presence of tight junctions near the apical surface of the epithelium strongly suggest that the initial uptake of these amino acids was mediated by basolateral plasma membrane carriers.