Leon Sokoloff

Chondroid Expression by Lapine Articular Chondrocytes in Spinner Culture Following Monolayer Growth

When articular chondrocytes from rabbits 2 to 3 months old were transferred from confluent monolayer to spinner culture, they ceased to proliferate but deposited large quantities of metachromatic extracellular material. Return of the suspended cells to monolayer conditions resulted in renewal of DNA and marked depression of sulfated glycosaminoglycan (GAG) synthesis. The GAG of rabbit articular cartilage were heterogeneous and included 4.9% hyaluronate, 11.6% unsulfated chondroitin, 18.7%, disulfated chondroitin and 2.4% dermatan sulfate in addition to chondroitin sulfates 4 and 6. Monolayer cultured chondrocytes produced higher levels of sulfated GAG but much smaller quantities of hyaluronate than did dermal fibrocytes. The GAG synthesized by chondrocytes in spinner culture closely resembled those of whole cartilage or synthesized by whole cartilage in organ culture.

The effect of prostaglandins on cultured lapine articular chondrocytes

The effect of B protaglandins (PG) on growth and sulfate incorportation by monolayer and spinner-cultured rabbit articular chondrocytes has been measured. PGA1, PGB1, PGE1 and PGE2 reduced synthesis of sulfated glycosaminoglycans (GAG) but the PGF series did not. PGA1 was the most potent, being effective at a concentration of 2.5 microng/ml [6.8 micronM] while the others required 25 microng/ml. These compounds had no effect on degradation of GAG. All 8 PGs augmented growth slightly but significantly at 2.5 microng/ml. At the higher concentration, PGA1 was highly cytotoxic, and PGB1 as well as PGE2 reduced cell growth. The cytotoxicity of PGA1 was also observed in two additional types of cultured connective tissue cells, but the inhibition of sulfated-GAG synthesis by PGA1 and PGB1 was confined to the chondrocytes. The response of cultured chondrocytes to exogenous PGs, albeit at apparently unphysiologically high concentrations, together with other evidence, suggests that these compounds may conceivably play a direct role in cartilage metabolism in vivo.

The effect of serum on monolayer cell culture of mammalian articular chondrocytes.

Large variations were found in the ability of six species of sera to support growth of rabbit, human and dog articular chondrocytes in monolayer culture. In most instances the DNA content of the cell pellets increased directly as the serum concentration rose from 10 to 30%. Indications of inhibitory as well as growth-promoting actions were found in some sera. Stimulation of rabbit chondrocyte proliferation by increasing concentrations of serum was accompanied by a reduction of radiosulfate incorporation and cell protein content. There was no consistent relation between the response of chondrocytes from a given species and its homologous serum. However, the growth of human chondrocytes was greatly potentiated by human serum provided that interference with initial attachment of the cells to the culture flask by homologous serum was overcome by priming with fetal calf serum.

Xenografts of articular chondrocytes in the nude mouse

SummarySubcutaneous transplantation of articular chondrocytes isolated enzymatically from immature rabbits and dogs into athymic (nu/nu) mice resulted in the formation of hyaline cartilaginous nodules. Graft rejection was seen when the cells were injected into heterozygous (nu/+) mice. Radiosulfate-labeled proteoglycan extracted from the xenografts had a high buoyant density and was digested by chondroitinase ABC. A monomeric preparation of proteoglycan (A1-D1) contained a small quantity of aggregate as assessed by gel chromatography and gel electrophoresis. Almost no incorporation of3H-thymidine was found by autoradiography. The matrix did not become calcified over the course of 42 days. The nude mouse system lends itself to testing a variety of problems in the biology of cartilage. These include the redifferentiation of chondrocytes following dedifferentiation in vitro. Species differences were found in this regard. The nodules formed by rabbit articular chondrocytes, grown in monolayer culture for up to 14 days, had a hyaline chondroid character. Dog chondrocytes that had “dedifferentiated,” during 14 days of culture prior to transplantation, formed a graft that had a sparse fibrous rather than hyaline matrix.

Secondary osteonecrosis in osteoarthritis of the femoral head

Secondary osteonecrosis was found by pathologic examination in nine (6 per cent) of 150 femoral heads removed surgically for osteoarthritis. The changes were morphologically distinct from the remodeling deformities that supervene late in steroid-induced or idiopathic osteonecrosis. They appeared as infarcts confined to previously eburnated tissue. There were no distinctive clinical or radiologic characteristics. The findings do not lend weight to the hypothesis that unrecognized osteonecrosis is a major etiologic factor in apparently primary osteoarthritis.

Explant Culture of Human and Rabbit Articular Chondrocytes

Copious outgrowth of chondrocytes was obtained by explantation from each of three rabbit and one surgically-resected human articular cartilages pretreated briefly with trypsin. In lapine explants, ascorbate (40 micrograms/ml) increased DNA three-fold over control values and resulted in deposition of a chondroid matrix. It doubled radiosulfate incorporation by the outgrowths. Up to 56% of the sulfated glycosaminoglycan synthesized was located in the trypsin-digestible pericellular coat compared with about 15% in previous monolayer cultures. The collagens synthesized were characterized partially. In rabbit cell cultures, the alpha 1:alpha 2 ratio varied from 2.9 to 3.8. In human cultures, an unusual post-alpha 2 peak was observed. The findings suggest an uncoupling of the phenotypic expression of the major cartilaginous macromolecules in the cultures. There were no distinctive differences between chondrocytes derived from normal and fibrillated human cartilage of the same individual.

The Neuromuscular Pathology of the Eosinophilia-Myalgia Syndrome

The Eosinophilia-Myalgia Syndrome (EMS) is a recently recognized disorder in patients ingesting pharmacologic doses of L-tryptophan. We studied the lesions of skeletal muscle, peripheral nerve and skin in 12 cases of EMS. Perimyositis was severe in four, moderate in two, mild in three and absent in three cases. The lesions contained many eosinophils, T-helper cells, mast cells and activated macrophages. Type 2 myofiber atrophy was present in five cases and in one, this was the only pathologic finding. Severe epineurial inflammation was seen in the three sural nerve biopsies. Indirect evidence for peripheral neurologic involvement in three other cases consisted of inflammation surrounding intramuscular nerve twigs (two cases) and neurogenic atrophy (one case). Phlebitis accompanied the connective tissue inflammation in five cases and endarteritis in one. Fasciitis was present in three of four skin biopsies and dermal fibrosis in one.

Species differences in cell culture of mammalian articular chondrocytes

SummaryArticular chondrocytes from eight mammalian species (rabbit, opossum, woodchuck, cat, dog, sheep, rhesus and cebus monkeys) were grown in monolayer culture using a single regimen. The animals were immature or young adult. hams F12 medium supplemented with 10% fetal bovine serum was employed for the primary cultures and Dulbecco-Vogt medium, for the secondary. Marked species differences were found with respect to cell morphology, growth in primary and secondary cultures, incorporation of radiosulfate into macromolecules, adhesion to the flask surface, response to vitamin C, and chondroid expression in spinner bottles. Under these particular conditions, rabbit chondrocytes grew most rapidly and incorporated several times more sulfate than did the others. Additional experiments carried out with other media on four of the species indicate that optimal conditions for culturing mammalian chondrocytes must be determined for each species individually.

Cartilage Content of an Immature Dog

The near total mineral-free and elastin-free cartilage content of a three-month-old mongrel dog was 0.73% of the dry body weight. Extraarticular (costal, tracheobronchial, thyroid, cricoid, nasal septum) cartilage constituted 0.44%; articular, 0.06; intervertebral disc, 0.10; elastic, 0.12 and meniscal fibrocartilage, 0.01%. Articular tissue accounted for only 7.25% of the total cartilage uronic acid. This is the first estimate of the amount and distribution of cartilage in a vertebrate species.

Induction of bone by xenografts of rabbit growth plate chondrocytes in the nude mouse

SummarySubcutaneous transplantation of growth plate chondrocytes isolated enzymatically from the proximal tibia of 6-week-old rabbits into athymic (nu/nu) mice resulted in the formation of cartilaginous nodules. Calcification of the matrix was first seen after 48 hrs, and endochrondral ossification at 12 days. The mineral first occurred about hypertrophic cells. Histochemical alkaline phosphatase activity was concentrated in pericellular collars at the same location. Immunofluorescence examination with rabbit anti-mouse lymphocyte serum disclosed that the bulk of the osteoblasts was derived from the mouse. A small quantity of mouse antigen was present in the cartilage matrix at its junction with bone. It presumably diffused into the cartilaginous interface from the host, but the possibility that some chondrocytes were of murine origin has not been excluded. Five of six grafts of cells grown to confluence in monolayer culture for 10 to 14 days became ossified. The ability to induce mineralization declined in subculture. Chondrocytes killed by heating to 56° did not induce calcified cartilage or bone.