Synovial membrane asks for independence.

Synovial membrane is traditionally considered as a part of the joint capsule. It, however, differs from fibrous part of the capsule in development, structure, function, vascularisation, innervation and involvement in pathological processes. Moreover, in some areas, it even does not contact with the fibrous capsule. Thus, it appears that the synovial membrane should be considered as an independent organ and not as the lining of the joint capsule.

Formation of synovial joints and articular cartilage.

Chondrocytes differentiate from mesenchymal progenitors and produce templates(anlagen) for the developing bones. Chondrocyte differentiation is controlled by Sox transcription factors. Templates for the neighbour bones are subsequently separated by conversion of differentiated chondrocytes into non-chondrogenic cells and emergence of interzone in which joints cavitation occurs. A central role in initiating synovial joint formation plays Wnt-14/beta-catenin signalling pathway.Moreover, bone morphogenetic proteins and growth and differentiation factors are expressed at the site of joint formation. Joint cavitation is associated with increased hyaluronic acid synthesis. Hyaluronic acid facilitates tissue separation and creation of a functional joint cavity. According to the traditional view articular cartilage represents part of cartilage anlage that is not replaced by bone through endochondral ossification. Recent studies indicate, however, that peri-joint mesenchymal cells take part in interzone formation and that these interzone cells subsequently differentiate into articular chondrocytes and synovial cells. Thus,anlage chondrocytes have a transient character and disappear after cessation of growth plate function while articular chondrocytes have stable and permanent phenotype and function throughout life.

Comparison of bone formed intramuscularly after transplantation of scapular and calvarial osteoblasts.

Previous work suggested that osteoblasts determine the size of the bone marrow area within the bone and that calvarial osteoblasts differ from those induced intramuscularly by cartilage formed by transplanted epiphyseal chondrocytes. This study reports morphological observations of bone formed by transplanted scapular and calvarial osteoblasts isolated from bones of young rats. In intact scapulas of 28-day-old rats the percentage area occupied by bone tissue in relation to bone marrow was 6 times larger than in parietal bones of comparable age. Isolated syngeneic scapular osteoblasts usually produced an ossicle with similar general structure and ratio bone tissue/bone marrow area as in intact scapulas. In transplants of calvarial osteoblasts numerous islands of bone tissue with a small amount of bone marrow appeared. Bone formed in allogenic transplants was rejected. These results suggest that osteoblasts from endochondral scapular bone may have different properties than those from intramembranous calvarial bones. Alternatively, the large amount of medullary space in bone produced by transplanted scapular osteoblasts could result from their contamination with bone marrow stromal cells.

Partitioning of cytoplasmic organelles during mitosis with special reference to the Golgi complex.

During mitosis, not only the genetic material stored in the nucleus but also the constituents of the cytoplasm should be equally partitioned between the daughter cells. For this sake, the dividing cell goes through an extensive structural reorganization and transport along the endocytic and exocytic pathways is temporarily arrested. Early in prophase, the radiating array of cytoplasmic microtubules disassembles and the membrane systems of the secretory apparatus start to split up. In metaphase, the nuclear envelope fragments and the condensing chromosomes associate with the forming mitotic spindle. The cisternal and tubular elements of the endoplasmic reticulum and the Golgi complex break down into small vesicles, presumably as the result of an imbalance between vesicle budding and fusion. In anaphase, the two sets of chromosomes are pulled apart and a cleavage furrow forms halfway between the spindle poles. Since most organelles occur in multiple and widely dispersed copies at this stage, they will be evenly distributed between the daughter cells. During telophase and cytokinesis, the preceding fragmentation process is reversed. A nuclear envelope reappears around the chromosomes and cytoplasmic microtubules reassemble. The endoplasmic reticulum is rebuilt as a continuous system of flattened cisternae and tubules. Stacks of Golgi cisternae arise from small vesicles and are rearranged in an interconnected network. In parallel, the biosynthetic functions of the cell are normalized and intracellular membrane traffic is resumed.

Rejection of cartilage formed by transplanted allogeneic chondrocytes: evaluation with monoclonal antibodies.

Cellular infiltrates participating in rejection of cartilage formed by transplanted allogeneic rat epiphyseal chondrocytes were evaluated immunohistochemically using a panel of different monoclonal antibodies. One week after transplantation, the grafts were surrounded by numerous class II MHC+ (OX6+, OX17+), CD4+ (W3/25+), and W3/13+ cells as well as some ED1+ monocytes/macrophages. Only a few T (OX19+) and B (HIS14+) cells were present. The number of class II MHC+ cells and ED1+ monocytes/macrophages did not change significantly in the course of rejection whereas the number of CD4+ and W3/13+ cells gradually decreased. On the other hand, there was a significant increase in the number of CD8+ (OX8+) cells. CD8+ cells accumulated close to the transplants and some of them penetrated cartilage matrix suggesting that they might be involved in chondrocyte killing. After 3 months, cartilage was almost completely destroyed and the intensity of infiltrations was markedly decreased. Fibrous connective tissue predominated, however, some class II+ as well as few ED1+, CD4+ and CD8+ cells were still present adjacent to the cartilage remnants. At the time of transplantation, chondrocytes were endowed with RT1.D class II antigen (OX17+), but they did not react with OX6 mAb (monoclonal antibody) recognizing the RT1.B class II molecule. However, after 1 week, some chondrocytes reacted with OX6 mAb and the number of RT1.B positive chondrocytes increased in the course of cartilage rejection.

Differences in cartilage formed intramuscularly or in joint surface defects by syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex.

Syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex were suspended in hyaluronic acid and transplanted intramuscularly or into joint surface defects. Transplants were fixed in ruthenium hexammonium trichloride and embedded in glycol methacrylate. In cartilage nodules produced intramuscularly, chondrocyte hypertrophy and matrix calcification were observed after 2 wk. Partial ossification occurred after 4 wk and the cartilage was almost completely replaced by an ossicle after 8 wk. Only small, dispersed groups of chondrocytes remained within the ossicle. In cartilage formed in joint surface defects a superficial and a deep zone were distinguished. Chondrocytes in the superficial zone did not hypertrophy and cartilage remained unossified. In the deep zone matrix calcification and bone formation occurred. These processes were, however, retarded in comparison with intramuscular transplants. Thus, either intraarticular environment exerted an inhibitory effect on chondrocyte hypertrophy and matrix calcification or articular chondrocytes present among transplanted cells accumulated close to the joint lumen and reconstructed normal articular cartilage.

Immunosuppression and rejection of cartilage formed by allogeneic chondrocytes in rats.

Rat syngeneic and allogeneic chondrocytes were transplanted intramuscularly or into defects prepared in articular cartilage (intracartilaginous transplants). Recipients of allogeneic transplants received cyclosporin A (CsA), cladribine (2-chlorodeoxyadenosine, 2-CdA), or both drugs in combination. Transplants were taken for examination after 5 weeks. Cartilage formed intramuscularly by syngeneic chondrocytes was ossified. Allogeneic cartilage was resorbed by infiltrating cells. CsA or 2-CdA partially suppressed, and both these agents in combination strongly suppressed, formation of infiltrations. Both syngeneic and allogeneic chondrocytes formed cartilage in joint surface defects but only allogeneic cartilage was attacked by infiltrating cells. CsA + 2-CdA treatment slightly decreased intensity of infiltrations but did not prevent cartilage resorption. Antichondrocyte response was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte-chondrocyte cultures and by detection of antichondrocyte cytotoxic antibodies. SMC stimulation index (SI) was calculated separately for syngeneic and allogeneic chondrocytes. Comparison of SMC SI for syngeneic and allogeneic chondrocytes indicated lack of stimulation of SMC from control or syngeneic transplant recipients and significant stimulation of SMC from recipients of allogeneic transplants. SMC from animals treated with CsA + 2-CdA were not stimulated. Additional experiments aiming at an explanation of the lack of stimulation of SMC from intact animals by syngeneic chondrocytes reported in this work and contrary to other findings disclosed that it was caused by the use of collagenase solution containing N alpha-p-tosyl-l-lysine chloromethyl ketone for chondrocyte isolation. Spontaneous antichondrocyte cytotoxic antibody activity was found in intact rats raised only in sera from recipients of allogeneic intramuscular transplants without immunosuppression. Thus, strong immunosuppressive treatment of rats with allogeneic chondrocyte transplants was more effective in relation to the general immunological response than to the local reaction.

Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats.

Rat chondrocytes isolated from the articular-epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte-chondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.

Cartilage produced after transplantation of syngeneic chondrocytes is rejected in rats presensitized with allogeneic chondrocytes.

Cartilage produced in 2-week-old intramuscular transplants of syngeneic chondrocytes in rats did not display any signs of rejection. Cartilage produced by similar transplants in animals presensitized with intramuscular transplants of allogeneic chondrocytes was surrounded by infiltrations composed mainly of lymphocytes and was partially resorbed. Spleen mononuclear cells (SMC) from recipients of syngeneic transplants alone were not stimulated in mixed splenocyte-chondrocyte cultures by syngeneic or allogeneic chondrocytes. SMC from recipients of allogeneic and subsequent syngeneic transplants were strongly stimulated by both syngeneic and allogeneic chondrocytes, although stimulation by the latter was significantly more pronounced. Sera from naive rats usually contained cytotoxic antichondrocyte antibodies but their level varied considerably in various individuals. In rats chosen as transplant recipients on the basis of low antichondrocyte cytotoxicity of their sera, this toxicity was markedly raised after sensitization with allo- and syngeneic chondrocytes. Absorption with thymocytes or fibroblasts decreased but did not abrogate cytotoxicity. These observations support previous reports suggesting expression of tissue-specific antigen(s) by chondrocytes.

Mechanical barrier as a protection against rejection of allogeneic cartilage formed in joint surface defects in rats.

Cartilage formed in transplants of allogeneic chondrocytes into joint cartilage defects in rats was infiltrated by immune cells migrating from the bone marrow while the surface on the side of the joint cavity remained free of infiltrations. This suggested that immunization occurred via bone marrow and not via joint cavity. Because articular cartilage is nourished exclusively by the synovial fluid, we have attempted to prevent cartilage rejection by protecting transplants from the contact with bone marrow. Defects in articular surface were filled with bone cement and chondrocytes were transplanted into a cavity prepared within the bone cement plug. Cartilage formed within the cement shell remained free of infiltrations and did not evoke systemic immunological response. However, distribution of glycosaminoglycans in the matrix of protected transplants was irregular. Cultures of chondrocytes growing in vitro on cement contained less glycosaminoglycans than the controls. This suggests that some factor(s) released from the cement unfavorably influenced chondrocytes and matrix production in protected transplants.

Regenerating rib cartilage tentatively used as a source of chondrocytes for transplantation.

In search for cartilage which could serve as a source of chondrocytes for autogeneic transplantation in mature individuals we studied the morphological appearance and digestibility of normal rib cartilage and regenerated cartilage formed after subperichondrial removal of the former in adult dogs. Cells liberated after collagenase digestion were transplanted intramuscularly to see whether they will reconstruct cartilage similarly as it happens after transplantation of fetal chondrocytes. Chondrocytes in normal rib cartilage were arranged in three zones and lay in large isogenous groups. Collagenase dissolved only the peripheral zone. Isolated cells did not reconstruct cartilage after transplantation. No distinct zones could be seen in regenerated cartilage. Chondrocytes lay singly or in pairs but were not numerous. Regenerated cartilage could be completely dissolved by collagenase, but the yield of cells was low owing to their low content in digested material. After transplantation chondrocytes from regenerated cartilage reconstructed cartilage in one out of ten transplants. The possibility of increasing the cellularity of regenerated cartilage by stimulation of the perichondrium with factors known to promote chondrocyte growth in vitro is discussed.

Occurrence of large groups of mast cells in subcutaneous connective tissue in the mouse.

Skin from the mouse trunk together with panniculus adiposus and panniculus carnosus and, separately, trunk muscles, were fixed, stained with Astra blue at pH 1.0, made translucent in methyl salicylate and whole-mounted. In the connective tissue on the surface of panniculus carnosus directed towards the trunk muscles or on the surface of trunk muscles rounded and oval mast cells occurred singly or in groups from two to several dozen cells. These groups had no association with blood vessels or hair follicles. Mast cell groups were scarse in 1-month-old, clearly recognizable in 2-months-old and conspicuous in 4-months-old mice of both sexes. The number of mast cells and their number per group was larger in CFW/Ll and C3H than in Balb/c mice. Accumulation of mast cells in subcutaneous connective tissue was noted in animals from two separate breeding centers. The animals were free of ectoparasites and dermatophytes but contained some pinworms and protozoa. Elimination of these parasites, change of diet and drinking water as well as cage lining did not prevent the appearance of mast cell accumulations. These accumulations occurred in all studied mice (over 100) at the age of 2 months or older, and were also found in 1 out of 6 four-month-old hamsters and in 2 out of 6 four-month-old rats. It is suggested that mast cells accumulate in subcutaneous connective tissue in response to some undefined noxious agent. Whatever the reason of their presence, large groups of mast cells could considerably influence the results of tests performed in the skin-hypodermis area.

Cartilage transplants in normal and preimmunized mice.

Syngeneic, H-Y incompatible and allogeneic rib and nasal cartilages from 5-day- and 8-week-old mice were transplanted into adult recipients. Some recipients of allogeneic transplants were preimmunized with splenocytes or chondrocytes from animals of the same strain as cartilage donors, and some with sheep red blood cells (SRBC). Syngeneic or allogeneic transplants independently of the age of the cartilage, did not evoke any detectable humoral or cellular response as judged by evaluation of the specific cytotoxic antibodies and indirect migration inhibition test, respectively. Allogeneic transplants of nasal cartilage were free of infiltrating lymphoid cells, but in the vicinity of rib cartilage grafts some lymphocytes were present. Animals preimmunized with allogeneic splenocytes or chondrocytes displayed both humoral and cellular response to donor cells. Allogeneic cartilage transplants from these animals were surrounded by heavy infiltrations. Preimmunization with SRBC did not evoke infiltrations around allogeneic cartilage grafts.

Structural differences between bone formed intramuscularly following the transplantation of isolated calvarial bone cells or chondrocytes.

Bone formed in intramuscular transplants of isolated syngeneic calvarial bone cells in mice, was compared with endochondral bone induced by cartilage produced by analogous transplants of isolated epiphyseal chondrocytes, as well as with parietal bones forming the bulk of the calvaria. Transplanted calvarial cells produced islands of bone, some of which contained intraosseous cavities. Osteoclasts inside these cavities were observed only in 14-day-old transplants and bone marrow cells in 28-day and older transplants. On the contrary, bone marrow appeared soon after formation of bone trabeculae in endochondral bone. The percentage area occupied by bone marrow in these specimens was about twentyfold larger than in the bone formed by transplanted bone cells. On the other hand, the bone marrow area in the latter type of bone was somewhat smaller but of similar order as in parietal bones. Moreover, both in parietal bones and in bone formed by isolated bone cells, the bone marrow was devoid of fat cells which were numerous in bone arising by endochondral ossification. It appears, therefore, that the ratio of bone marrow to the bone tissue area in parietal bones depends more on the intrinsic properties of osteoblasts than on the local factors in the environment of the developing bone. In the case of bone induced by cartilage, the bone marrow/bone tissue area could be determined both by the extent of cartilage resorption by vascularized tissue and by the properties of osteoblasts.

In vitro elastic fiber formation by aggregated arotic cells of newborn rabbits.

Cells isolated enzymatically from the aortas of newborn rabbits were aggregated and grown in organ culture. Bundles of microfibrils, some of them with an amouphous core (elastin), were evident in 3-day-old aggregates. Furthermore, droplets of elastin surrounded by darker dots and short filaments, corresponding to the elastic units were observed. In 6-day-old aggregates the number of elastic fibers composed of bundles of microfibrils with deposited elastin increased. Elastic elements which probably resulted from a coalescence of elastic units were also present. These two ways of elastic fiber formation in aggregates are compared with those described in papers on aortic development.

Difference in size of bone islands formed by isolated bone cells transplanted intramuscularly under various conditions.

Bone cells isolated from the whole calvaria (2 x 10(6)) from either central or peripheral parts of parietal bones (1 x 10(6)) and from scapulas (2 x 10(6)) were allowed to adhere to devitalized calvarial bones in the number indicated in brackets and transplanted intramuscularly (supported transplants). Whole calvaria bone cells (2.4 or 8 x 10(6) cells per transplant) were also injected intramuscularly as free transplants. Calvarial cells produced solid bone islands with small intraosseous cavities, while bone formed by scapular cells contained large medullary spaces. The size of bone islands formed in transplants and the shortest distance between the neighbor islands were measured. The results of these measurements were similar in all groups of free transplants. The size of bone islands formed in supported transplants of cells from the whole calvaria or from central and peripheral parts of parietal bones was also roughly similar, but the shortest distance between islands was larger than in the free transplants. Furthermore, in these groups of transplants bone islands considerably larger than the largest islands in free transplants were present. Scapular bone cells formed islands much larger than those produced by calvarial cells. Bone islands formed by calvarial cells in free transplants were separated by bands of fibrous tissue which was absent in supported transplants. It appears that this tissue could limit growth and/or fusion of neighbor bone islands and in this manner influence their size. The population of transplanted scapular cells contained numerous stromal elements which could form an exclusion area inaccessible to local cells from the site of transplantation and thus favour formation of large bone islands within this area.

Influence of liver environment on the maturation of isolated epiphyseal chondrocyte transplants.

To study the phenomenon of chondrocyte hypertrophy, rat or mouse isolated epiphyseal chondrocytes were transplanted into the kidney, spleen or liver for 7 days. Each transplant had its own control transplanted intramuscularly. Rat chondrocytes were also placed on a chorioallantoic membrane of chick embryos, incubated for 11 days and transferred for the next 11 days either onto another chorioallantoic membrane or into rat muscle. The surface area of largest lacunae cross-sections in cartilage produced by transplants was measured as an indicator of chondrocyte hypertrophy. In cartilage from the chorioallantoic membrane chondrocytes remained small but hypertrophied after transfer into a muscle. Lacunae in seven-day-old cartilage nodules in the liver were considerably larger than in muscle, kidney or spleen. After 7 days matrix calcification was observed only in liver transplants. Thus, liver environment, stimulated chondrocyte hypertrophy. Taken together these results suggest that chondrocytes are unable to hypertrophy spontaneously and that the rate of hypertrophy is subjected to regulation by extra-cartilaginous factor(s).

Formation of lamellar bone on the surface of woven bone is not prevented by BAPN.

To study the possible effect of collagen cross-link formation on the lamellar bone deposition, isolated mouse bone cells were transplanted in the syngeneic system and the formation of bone was followed in control and beta-aminopropionitrile (BAPN) treated animals. Woven and lamellar bone were distinguished by shape of bone lacunae, PAS reaction and Sirius red staining of collagen fibers. BAPN was administered in various doses either subcutaneously or in drinking water. Bone formed in BAPN treated animals contained both woven and lamellar bone in similar proportion as in controls. Thus, cross-linking of collagen seems to be unnecessary for lamellar bone deposition.

Comparison of bone formed in transplants of isolated scapular and vertebral osteoblasts.

To compare the properties of osteoblasts from various endochondrilia bones, scapular and calvarial osteoblasts were intramuscularly transplanted in "sandwiches" made of devitalized calvarial vaults. The structure of transplants produced by both types of bone cells appeared similar. In 4 week-old transplants woven bone with numerous osteoclasts predominated. The area occupied on the cross-sections of transplants by bone tissue was considerably larger than that of the bone marrow cavities. Transplants of 8-week-duration contained mainly cancellous bone, the number of osteoblasts was low and the area taken by medullary space was larger than that of bone tissue. This finding indicates that either osteoblasts from various endochondrlia bones have similar properties or that the possible differences in intrinsic features of these osteoblasts were masked by the conditions of transplantation.

Difference in shape of bone formed by isolated calvarial and scapular osteoblasts transplanted under various conditions.

To study the influence of transplantation conditions on early stages of osteogenesis, isolated calvarial or scapular osteoblasts were injected into the leg or dorsal muscles (free transplants) or implanted after seeding on fragments of devitalized parietal bones (supported transplants) into dorsal muscles. The cross-sections of bone islands formed by calvarial osteoblasts in the different types of transplants were then compared according to their maximal breadth and length. Moreover, the same dimensions of pieces of bone formed by scapular osteoblasts in supported transplants were compared with those of bones formed in free transplants into leg muscles. Finally, comparison of the dimensions of cross-sections of supported transplants of calvarial and scapular osteoblasts was done. Calvarial osteoblasts in dorsal muscles produced a slightly higher percentage of wider and longer islands than those in leg muscles. In supported transplants of calvarial osteoblasts the percentage of narrow bone islands (breadth less than 100 microns) was considerably higher than in free transplants. Similarly, the percentage of narrow cross-sections in bones formed by scapular osteoblasts was higher in supported than in free transplants. In supported transplants of calvarial osteoblasts the percentage of narrow islands was higher than in similar transplants of scapular bone cells. It is suggested that the differences in shape of pieces of bone formed in supported and free transplants reflect the difference in mechanical conditions to which the bone cells were subjected. Furthermore, in supported transplants devitalized parietal bones could form a barrier for diffusion of nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)