Lesions of bone and bone marrow in myeloid leukosis occurring naturally in adult broiler breeders

Lesions of bone and bone marrow in myeloid leukosis (ML) occurring naturally in adult broiler breeders were investigated pathologically. During gross examination, nodules and protrusions were commonly observed on the surface of the sternum, ribs, vertebrae, and synsacrum. The bone marrow of all the bones of the body was pale in color. Histologically, granulated myelocytes proliferated in the bone marrow of various bones and in the periosteum of the sternum, ribs, vertebrae, and synsacrum. The first proliferation of tumor cells occurred in the bone marrow of epiphysis. The myelocytes invaded through haversian and Volkmann's canals from the bone marrow to periosteal areas. Hematopoiesis was suppressed by marked proliferation of tumor cells in the bone marrow of the whole bone. Atrophy was also seen in the bones, including medullary bones of the chickens suffering from ML. Proliferation of myelocytes was seen in the bone marrow and periosteum of ossified cartilaginous rings of the trachea and larynx. Marked proliferation of myelocytes was seen in the dura mater of spinal cords, and it subsequently depressed the spinal cords. Bone formation with cartilage was seen in the periosteum of the sternum having marked proliferation of myelocytes in the bone marrow and periosteum. Ultrastructurally, tumor cells showed large nuclei and cytoplasm with large round electron-dense lysosomes. The virus particles were rarely detected in the cytoplasm of tumor cells. The polymerase chain reaction test of tumor samples showed positive for subgroup J avian leukosis virus. This study indicates that the myelocytes can invade through the compact bones to the periosteum in the sternum, ribs, vertebrae, synsarcum, and ossified cartilage of trachea and larynx having thinner compact bones. In addition, the periosteal osteogenesis with cartilage in the sternum may be reactive change against the bone atrophy because of the marked proliferation of myelocytes.     

Chondrodystrophy in calves associated with manganese deficiency

A severe congenital chondrodystrophy in Charolais calves is described. Thirty-two calves were born on the same farm with shortened limbs and enlarged joints. Histologically, there was poor cartilage maturation with excessive amounts of rarefied cartilage matrix. There were degenerative changes in the chondrocytes and a severe reduction in the mucopolysaccharide content of all body hyaline cartilage. The cartilage matrix, when examined ultrastructurally, showed loose, disorganised fibres in a rarefied matrix, and extensive vacuolation of the chondrocytes. A low level of manganese was found in the liver of an affected calf. The pregnant cows were fed on apple pulp and corn silage, both of which are low in manganese.     

Biological properties of allogenic articular chondrocytes on the surface of bovine cartilage explants in vitro

Bovine cartilage explants were co-cultured with or without allogenic chondrocytes for 4 weeks. The attachment of the applied chondrocytes to cartilage after labelling with fluorescence was assessed using a confocal laser microscope. Morphological changes and the production of extracellular matrix (ECM) of co-cultured chondrocytes on intact and damaged surfaces of cartilage were evaluated by histological and immunohistochemical methods. Co-cultured chondrocytes attached to and proliferated on the intact and damaged areas of cartilage, and a new layer was created there. The defects were also filled with ECM produced by the co-cultured chondrocytes. Glycosaminoglycans and collagen type II were detected in the newly formed ECM, and large numbers of rounded chondrocytes were observed at primitive lacunae in this matrix at 4 weeks of culture. The results suggest that chondrocytes have the ability to attach to, to proliferate on and to establish a new matrix on the intact and damaged surfaces of cartilage explants.     

Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.     

Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells

OBJECTIVE: To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. SAMPLE POPULATION: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. PROCEDURE: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen. RESULTS: MSC cultures exposed to TGF-beta1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-beta1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF-beta1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-beta1 led to dose-related increases in area and intensity of type-II collagen immunoreaction. CONCLUSION: Results suggest that TGF-beta1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner.     

Cartilage matrix changes in the developing epiphysis: early events on the pathway to equine osteochondrosis?

REASONS FOR PERFORMING STUDY: The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth. OBJECTIVE: To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions. METHODS: Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix. RESULTS: Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur. CONCLUSION: Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.     

Occurrence and Distribution of Lactate Dehydrogenase in Knee Joint Carilage in Healthy and Osteoarthrotic Dogs

According to clinical studies, degenerative diseases of canine joints lead to higher lactate dehydrogenase (LDH) levels in synovial fluid. The goal of the present study was to examine the intraarticular distribution of LDH in healthy and osteoarthrotic knee joints in order to identify possible sources of LDH in synovial fluid. As synovial LDH concentrations neither correlate with the number of leukocytes nor with synovitis, our investigation focused on the articular cartilage. Samples from healthy and osteoarthrotic knee joints were fixed and processed for transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunocytochemistry (ICC). In addition, fresh cartilage samples were investigated cytochemically by the tetrazolium‐formazan reaction. Analyses of blood and synovial fluid samples were used to confirm the absence of inflammatory disease. Morphology of articular cartilage was assessed macroscopically and by means of TEM. IHC revealed highest levels of LDH in chondrones and a diffuse labelling of the matrix with a distinctive decrease in signal from superficial to deeper cartilage layers. Ultrastructural localization by ICC showed LDH to be present in the cytoplasm of all chondrocytes and confirmed the density gradient in the matrix. Labelling was absent from nuclei and from pericellular rims. Cytochemistry confirmed the distribution pattern and, thus, expanded our findings beyond immunological evidence by providing proof of enzymatic activity of LDH in articular cartilage. The present results indicate that LDH is transferred from chondrocytes to the cartilaginous matrix. We suggest, therefore, that LDH found in synovial fluid originates from the articular cartilage and that osteoarthrotic processes promote LDH release from the cartilaginous matrix.     

Structure of the Seminal Pathway in the European Chub (Leuciscus cephalus)

According to clinical studies, degenerative diseases of canine joints lead to higher lactate dehydrogenase (LDH) levels in synovial fluid. The goal of the present study was to examine the intraarticular distribution of LDH in healthy and osteoarthrotic knee joints in order to identify possible sources of LDH in synovial fluid. As synovial LDH concentrations neither correlate with the number of leukocytes nor with synovitis, our investigation focused on the articular cartilage. Samples from healthy and osteoarthrotic knee joints were fixed and processed for transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunocytochemistry (ICC). In addition, fresh cartilage samples were investigated cytochemically by the tetrazolium-formazan reaction. Analyses of blood and synovial fluid samples were used to confirm the absence of inflammatory disease. Morphology of articular cartilage was assessed macroscopically and by means of TEM. IHC revealed highest levels of LDH in chondrones and a diffuse labelling of the matrix with a distinctive decrease in signal from superficial to deeper cartilage layers. Ultrastructural localization by ICC showed LDH to be present in the cytoplasm of all chondrocytes and confirmed the density gradient in the matrix. Labelling was absent from nuclei and from pericellular rims. Cytochemistry confirmed the distribution pattern and, thus, expanded our findings beyond immunological evidence by providing proof of enzymatic activity of LDH in articular cartilage. The present results indicate that LDH is transferred from chondrocytes to the cartilaginous matrix. We suggest, therefore, that LDH found in synovial fluid originates from the articular cartilage and that osteoarthrotic processes promote LDH release from the cartilaginous matrix.     

Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

Passive role of articular chondrocytes in the pathogenesis of acute meniscectomy-induced cartilage degeneration

The importance of viable articular chondrocytes and enzymes or factors from surgically traumatized synovium in the pathogenesis of acute meniscectomy-induced cartilage degeneration was examined in guinea pigs (nine groups of five animals each). Iodoacetate injected intra-articularly was used to kill articular chondrocytes of guinea pigs prior to meniscectomy to determine if they were active participants in the acute lesion induction. Lesions of similar severity to those occurring in animals with viable chondrocytes at the time of surgery were observed, suggesting that the chondrocytes were not actively involved in the pathogenesis in this group. In an additional group of guinea pigs in which chondrocytes were killed by iodoacetate, the medial collateral ligament was transected to determine if acute degenerative changes could be induced in acellular cartilage exposed to a surgical manipulation that does not by itself induce lesions but does expose cartilage to enzymes/factors from traumatized synovium. Transecting the medial collateral ligament and entering the joint space without induction of instability via meniscectomy did not result in histologic evidence of cartilage damage. This suggests that synovial trauma and mild inflammation were insufficient to induce matrix degeneration in the absence of abnormal load bearing. In further support of this, guinea pigs subjected to unilateral sciatic neurectomy at the time of meniscectomy were protected against development of acute cartilage degeneration. Results of this study suggest that articular cartilage devoid of viable chondrocytes at the time of meniscectomy responds acutely in much the same way as intact cartilage subjected to this procedure.     

肉鸡骨骼发育的调控机制

肉鸡骨骼发育主要是通过软骨内骨化完成的。在软骨内骨化的进程中,生长板软骨细胞经历增殖、肥大、转分化和软骨基质矿化等,最终成骨逐渐取代软骨原基,实现骨骼的线性延长。软骨内成骨是一个复杂精密的过程,由SOX9、RUNX2、MEF2C、OSX、TGF-β、BMP2、FGFs、IHH和PTHrP等多种信号因子和转录因子协调调控,这些调控因子由生长板不同区的软骨细胞表达或特异性的调控软骨细胞的增殖、分化及血管侵入等过程。在家禽养殖中,肉鸡常发腿病且治疗难度大,而有关肉鸡腿病发病机制的研究报道相对较少。本文综述了骨形成过程及具体的分子调控机制,为了解肉鸡腿病的发生以及提供有效治疗方案提供参考。     

Influence of serotype, cell type, tissue composition, and time after inoculation on gene expression in recombinant adeno-associated viral vector-transduced equine joint tissues

Objective-To evaluate transduction efficiency of gene therapy for treatment of osteoarthritis in horses. Sample-Cartilage and synovial tissues were aseptically collected from the stifle joints of 3 Thoroughbreds; horses were 3, 7, and 12 years old and free from sepsis and long-term drug treatment and were euthanized for reasons unrelated to joint disease. Procedures-Gene transfer experiments were performed with 8 recombinant adeno-associated viral vector (rAAV) serotypes in monolayer-cultured equine chondrocytes, synovial cells, and mesenchymal stromal cells and in cartilage and synovial tissues. Results-Serotypes rAAV2/5 and rAAV2/2 yielded the highest transduction efficiency in cultured cells 6 days after transduction. Synovial cells and mesenchymal stromal cells were more readily transduced than were chondrocytes. Serotype rAAV2/6.2 yielded the highest rate of gene expression in both cartilage and synovial tissues at 6 days after inoculation. However, at 30 and 60 days after inoculation, gene expression of serotypes rAAV2/2 and rAAV2/5 surpassed that of rAAV2/6.2 and all other serotypes. Conclusions and Clinical Relevance-Maximally expressing serotypes changed between 6 and 30 days in tissues; however, the most efficient serotypes for transduction of joint cells over time were also the most efficient serotypes for transduction of joint tissues. In addition, the low transduction efficiency of articular cartilage tissue was paralleled by a low transduction efficiency of isolated chondrocytes. This suggested that the typically low transduction efficiency of articular cartilage may be attributable in part to the low transduction efficiency of the chondrocytes and not solely a result of the dense cartilage matrix.     

Distribution of type VI collagen in the cartilaginous tissue of the proximal tibia in the domestic cat

To investigate the distribution of the early stage chondrocytes during the formation and closure of epiphyseal growth plate (EGP) of the domestic cat, we examined the EGP of proximal tibiae by immunohistochemistry for type VI collagen. In the epiphyseal cartilage without the secondary ossification center (SOC) and EGP in newborn cats aged 1 and 10 days, type VI collagen-positive chondrocytes were located around the cartilage canals and articular surface. In the epiphyseal cartilage with the SOC and EGP in young cats aged 1 to 3 months, type VI collagen-positive chondrocytes were located in the upper resting zone of the EGP, and then increased throughout the resting zone along with maturation. In the adult cats with the partially closed EGP, type VI collagen-positive chondrocytes were distributed throughout the remaining EGP. These findings indicate that the early stage chondrocytes characterized with type VI collagen are continuously located in the EGP during maturation. In addition, the increase of the early stage chondrocytes and the decrease of the reserve chondrocytes in the EGP along with maturation may cause the cessation of the longitudinal growth of the EGP, and finally bring about the EGP closure.     

Immunohistochemical study of matrix metalloproteinase‐3 (MMP‐3) at the articular cartilage in osteochondrotic pigs

In order to understand the mechanism of osteochondrosis in the pig, articular cartilage was taken from the distal femoral condyles of Duroc pigs exhibiting leg weakness and then examined immunohistochemically for the localization of matrix metalloproteinases‐3 (MMP‐3), one of the enzymes involved in the resolution of cartilage matrix. The articular cartilage had the typical characteristics of osteochondrotic lesions, such as abnormal calcification, clefts of cartilage, disappearance of proteoglycan, and necrotic chondrocytes. The immunoreaction of MMP‐3 was observed in chondrocytes at the boundary between normal cartilage and proteoglycan‐deficient area. Moreover, chondrocytes expressing MMP‐3 showed normal morphology, but the surrounding cartilage matrix did not stain with toluidine blue, which indicated a lack of glycosaminoglycans. These results suggest that MMP‐3 is highly involved in the appearance and expansion of osteochondrotic lesions.     

Characterisation of reconstituted equine cartilage formed in vitro

Lesions in cartilage of equine weightbearing joints commonly result in lameness. Cell-based resurfacing techniques are currently being developed for human and veterinary applications. Biopsies of stifle joint cartilage (1 g) were harvested aseptically and chondrocytes were isolated by sequential enzyme digestion. The cells were grown in vitro on filter inserts. Analysis of cultures 8 weeks later showed that the cells had accumulated extracellular matrix and formed a continuous layer of cartilagenous tissue as determined histologically. The cells maintained their phenotype as they synthesised type II collagen and proteoglycans similar in size to those synthesised by chondrocytes in native cartilage, but this reconstituted tissue had more sulphated glycosaminoglycan and lower collagen content than native cartilage. This experiment tests the feasibility of growing equine cartilagenous tissue in vitro. This tissue may be useful in the management of chondral injuries in the horse in a scenario where the patient donates cells, the cells are propagated under laboratory conditions and the resulting tissue becomes the therapeutic agent.     

Immunolocalization of cathepsin B in equinedyschondroplastic articular cartilage

A polyclonal antiserum raised in sheep against human cathepsin B was tested for specificity and cross-reactivity with the horse homologue by SDS-PAGE and Western blotting, prior to being used for immunolocalization of the enzyme in equine articular cartilage. In Western blots, the antiserum recognized the 30 kDa single chain and 25 kDa heavy chain of the mature enzyme in purified bovine cathepsin B, and corresponding bands at 32 and 27 kDa in equine chondrocyte and fibroblast lysates. This antiserum was then used to compare the expression and distribution of cathepsin B in normal and dyschondroplastic cartilage of young horses.In normal articular cartilage (n=6 animals), significant amounts of enzyme were detected only in hypertrophicchondrocytes in the deep zone. The enzyme was intracellular, located in the lysosomal granules. No extracellular matrix staining was observed. Levels of cathepsin B were increased slightly above normal in the deep zone in age-matched dyschondroplastic cartilage (n=5 animals). The most striking finding, however, was the abundance of the enzyme in chondrocyte clonal clusters associated with the lesions. Cathepsin B levels were low in chondrocytes isolated from normal cartilage (n=6), but increased progressively during serial subculture, reaching a maximum at passage 5–6. In contrast, primary cultures of dyschondroplastic chondrocytes (n=3) expressed abundant cathepsin B.     

Lipids of normal and osteochondritic cartilage of the immature canine humeral head

Specimens of cartilage removed from the shoulder joint of dogs with osteochondritis dissecans were compared with the articular-epiphyseal cartilage from the same location of healthy dogs. The pathologic cartilage lost the lamination pattern of articular-epiphyseal cartilage from healthy dogs. Chondrocytes of healthy and pathologic cartilage contained lipid inclusions. However, in pathologic samples, the lipid inclusions were more prominent, particularly in the superficial region of the presumptive articular cartilage. Lipids in the interterritorial matrix of the pathologic cartilage were observed. The pathologic cartilage contained large groups of chondrocytes and areas of mineralization of variable magnitude. Although lipid inclusions were found in the chondrocytes of cartilage of healthy dogs, the increase in magnitude of lipids in the cartilage from dogs with osteochondritis dissecans was considered to result from a metabolic response of the chondrocyte to an altered microenvironment. The loss of sudanophilic lipids in areas of chondrolysis may be used to explain the retardation of osteogenesis of chondrolytic cartilage.     

Systemic cartilage defect in a calf

A 6-month-old Ayrshire calf had a systemic growth defect of cartilage. The most severe changes occurred in articular cartilage and epiphyseal plates of long bones where disturbance of ossification, necrosis, erosion, vascular invasion, proliferation of cartilaginous and fibrous tissue were seen. The lesions probably were the result of a failure in the initial phases of ossification and mechanical pressure was responsible for their severity in the cartilage of the limbs.     

Assessment of cellular, biochemical, and histologic effects of bipolar radiofrequency treatment of canine articular cartilage

OBJECTIVE: To assess the cellular, biochemical, and histologic effects of bipolar radiofrequency-generated heat on canine articular cartilage. SAMPLE POPULATION: Articular cartilage explants (n = 72) from 6 canine cadavers and cultured articular chondrocytes from 5 canine cadavers. PROCEDURE: Cartilage explants were randomly assigned to receive no treatment or treatment with focal (3 seconds) or diffuse bipolar radiofrequency. Following treatment, methylene blue permeability assay was performed (n = 12) and remaining samples (60) were cultured. Immediately and 5, 10, and 20 days after treatment, cultured explants were assessed for glycosaminoglycan (GAG) and collagen contents, type II collagen and matrix metalloproteinase (MMP)-13 immunoreactivity, and modified Mankin histologic scores. Liquid culture media were collected every 4 days and GAG content measured. Additionally, cultured chondrocytes were exposed for 3 seconds to media preheated to 37 degrees, 45 degrees, or 55 degrees C. Cell viability was determined via 2 different assays immediately and 24 hours after treatment. RESULTS: Radiofrequency-treated cartilage had reduced permeability and considerable histologic damage, compared with control samples; most treated samples had reduced collagen II staining and increased MMP-13 immunostaining. Compared with other treatments, less GAGs were released from cartilage after diffuse radiofrequency treatment throughout the study period. Cell viability was significantly different between controls and cells treated at 55 degrees C immediately and 24 hours after heat treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, bipolar radiofrequency treatment had detrimental effects on normal articular cartilage cells and extracellular matrix with probable long-term clinical consequences. The usefulness of radiofrequency for treatment of osteoarthritic articular cartilage requires further investigation.     

Isolation, propagation, and cryopreservation of equine articular chondrocytes.

Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffered with HEPES, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at -196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.