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Pulsed Electromagnetic Field (PEMF) has shown efficacy in bone repair and yet the optimum characteristics of this modality and its molecular mechanism remain unclear. To determine the effects of timing of PEMF treatment, we present a novel three-dimensional culture model of osteogenesis that demonstrates strong de novo generation of collagen and mineral matrix and exhibits stimulation by PEMF in multiple stages over 62 days of culture. Mouse postnatal day 2 calvarial pre-osteoblasts were cast within and around Teflon rings by polymerization of fibrinogen and cultured suspended without contact with tissue culture plastic. Ring constructs were exposed to PEMF for 4h/day for the entire culture (Daily), or just during Day1-Day10, Day11-Day 27, or Day28-Day63 and cultured without PEMF for the preceding or remaining days, and compared to no-PEMF controls. PEMF was conducted as HF Physio, 40.85 kHz frequency with a 67 ms burst period and an amplitude of 1.19 mT. Osteogenesis was kinetically monitored by repeated fluorescence measurements of continuously present Alizarin Red S (ARS) and periodically confirmed by micro-CT. PEMF treatment induced early-onset and statistically significant transient stimulation (~4-fold) of the mineralization rate when PEMF was applied Daily, or during D1-D10 and D11-D27. Stimulation was apparent but not significant between D28-D63 by ARS but was significant at D63 by micro-CT. PEMF also shifted the micro-CT density profiles to higher densities in each PEMF treatment group. Ring culture generated tissue with a mineral:matrix ratio of 2.0 by thermogravimetric analysis (80% of the calvaria control), and the deposited crystal structure was 50% hydroxyapatite by X-ray diffraction (63% of the calvaria and femur controls), independent of PEMF. These results were consistent with backscatter, secondary electron, and elemental analysis by scanning electron microscopy. Thus, in a defined, strong osteogenic environment, PEMF applied at different times was capable of further stimulation of osteogenesis with the potential to enhance bone repair.

Keloids are wounding-induced tumor-like human scars. Unclear etiology and lack of animal models to reveal disease mechanisms and invent therapies deepen the grievous health and psychosocial state of vulnerable individuals. Epitomizing the injury-repair environment which triggers and fosters keloid formation and essential dermal/epidermal interactions in disease development, the novel animal model was established by implanting porous polyethylene ring-supported plasma/fibrin-based epidermal-dermal skin constructs on the dorsum of athymic NU/J mice. The implants were stable to 18 weeks, contained abundant human cells, and remodeled to yield scar architecture characteristic of keloid fibrosis compared with normal implants and clinical specimens: (1) macroscopic convex or nodular scar morphology; (2) morphogenesis and accumulation of large collagen bundles from collagen-null initial constructs; (3) epidermal hyperplasia, aberrant epidermal-dermal patency, and features of EMT; (4) increased vasculature, macrophage influx, and aggregation; and (5) temporal-spatial increased collagen-inducing PAI-1 and its interactive partner uPAR expression. Development of such pathology in the NU/J host suggests that T-cell participation is less important at this stage than at keloid initiation. These accessible implants also healed secondary excisional wounds, enabling clinically relevant contemporaneous wounding and treatment strategies, and evaluation. The model provides a robust platform for studying keloid formation and testing knowledge-based therapies.

Cartilage injury represents one of the most significant clinical conditions. Implantation of expanded autologous chondrocytes from noninjured compartments of the joint is a typical strategy for repairing cartilage. However, two-dimensional culture causes dedifferentiation of chondrocytes, making them functionally inferior for cartilage repair. We hypothesized that functional exclusion of dedifferentiated chondrocytes can be achieved by the selective mapping of collagen molecules deposited by chondrogenic cells in a three-dimensional environment. Freshly isolated and in vitro expanded human fetal or adult articular chondrocytes were cultured in a thermoreversible hydrogel at density of 1 × 10(7) cells/mL for 24 h. Chondrocytes were released from the gel, stained with antibodies against collagen type 2 (COL II) or COL I or COL X and sorted by fluorescence activated cell sorting. Imaging flow cytometry, immunohistochemistry, quantitative polymerase chain reaction, and glycosaminoglycan (GAG) assays were performed to evaluate the differences between COL II domain forming and COL II domain-negative cells. Freshly dissected periarticular chondrocytes robustly formed domains that consisted of the extracellular matrix surrounding cells in the hydrogel as a capsule clearly detectable by imaging flow cytometry (ImageStream) and confocal microscopy. These domains were almost exclusively formed by COL II. In contrast to that, a significant percentage of freshly isolated growth plate pre-hypertrophic and hyperdrophic chondrocytes deposited matrix domains positive for COL II, COL I, and COL X. The proportion of the cells producing COL II domains decreased with the increased passage of in vitro expanded periarticular fetal or adult articular chondrocytes. Sorted COL II domain forming cells deposited much higher levels of COL II and GAGs in pellet assays than COL II domain-negative cells. COL II domain forming cells expressed chondrogenic genes at higher levels than negative cells. We report a novel method that allows separation of functionally active chondrogenic cells, which deposit high levels of COL II from functionally inferior dedifferentiated cells or hypertrophic chondrocytes producing COL X. This approach may significantly improve current strategies used for cartilage repair.

Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with > 80% type I collagen and < 5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%-0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.

The importance of wear particle characterization for orthopaedic implants has long been established in the hip and knee arthroplasty literature. With the increasing use of motion preservation implants in the spine, the characterization of wear debris, particularly metallic nature, is gaining importance. An accurate morphological analysis of wear particles provides for both a complete characterization of the biocompatibility of the implant material and its wear products, and an in-depth understanding of the wear mechanisms, ion release, and associated corrosive activity related to the wear particles. In this paper, we present an overview of the most commonly-used published protocols for the isolation and characterization of metal wear particles, and highlight the limitations and uncertainties inherent to metal particle analysis.

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.

Keloids are wounding-induced tumor-like human scars. Unclear etiology and lack of animal models to reveal disease mechanisms and invent therapies deepen the grievous health and psychosocial state of vulnerable individuals. Epitomizing the injury-repair environment which triggers and fosters keloid formation and essential dermal/epidermal interactions in disease development, the novel animal model was established by implanting porous polyethylene ring-supported plasma/fibrin-based epidermal-dermal skin constructs on the dorsum of athymic NU/J mice. The implants were stable to 18 weeks, contained abundant human cells, and remodeled to yield scar architecture characteristic of keloid fibrosis compared with normal implants and clinical specimens: (1) macroscopic convex or nodular scar morphology; (2) morphogenesis and accumulation of large collagen bundles from collagen-null initial constructs; (3) epidermal hyperplasia, aberrant epidermal-dermal patency, and features of EMT; (4) increased vasculature, macrophage influx, and aggregation; and (5) temporal-spatial increased collagen-inducing PAI-1 and its interactive partner uPAR expression. Development of such pathology in the NU/J host suggests that T-cell participation is less important at this stage than at keloid initiation. These accessible implants also healed secondary excisional wounds, enabling clinically relevant contemporaneous wounding and treatment strategies, and evaluation. The model provides a robust platform for studying keloid formation and testing knowledge-based therapies.

Transforming growth factor (TGF)-beta, bone morphogenetic protein (BMP), and interleukin-1beta activate TGF-beta-activated kinase 1 (TAK1), which lies upstream of the p38 MAPK, JNK, and NF-kappaB pathways. Our knowledge remains incomplete of TAK1 target genes, requirement for cooperative signaling, and capacity for shared or segregated ligand-dependent responses. We show that adenoviral overexpression of TAK1a in articular chondrocytes stimulated type II collagen protein synthesis 3-6-fold and mimicked the response to TGF-beta1 and BMP2. Both factors activated endogenous TAK1 and its activating protein, TAB1, and the collagen response was inhibited by dominant-negative TAK1a. Isoform-specific antibodies to TGF-beta blocked the response to endogenous and exogenous TGF-beta but not the response to TAK1a. Expression of Smad3 did not stimulate type II collagen synthesis or enhance that caused by TGF-beta1 or TAK1a, in contrast to its effects on its endogenous targets, CTGF and plasminogen-activated inhibitor-1. TAK1a, overexpressed alone and immunoprecipitated, phosphorylated MKK6 and stimulated the plasminogen-activated inhibitor-1 promoter following transient transfection; both effects were enhanced by TAB1 coexpression, but type II collagen synthesis was not. Stimulation by TAK1a or TGF-beta did not require increased Col2a1 mRNA, and TAK1 actually reduced Col2a1 mRNA in parallel with the cartilage markers, SRY-type HMG box 9 (Sox9) and aggrecan. Thus, TAK1 increased target gene expression (Col2a1) by translational or posttranslational mechanisms as a Smad3-independent response shared by TGF-beta1 and BMP2.

Hip simulator tests of femoral balls of cobalt-chromium alloy or zirconia against acetabular cups of UHMW polyethylene were run with and without a coolant circulated inside the femoral balls. Without cooling, the wear of polyethylene against zirconia was about 48% lower than with cobalt-chromium alloy, but the steady-state temperature of the zirconia ball was higher (55 degrees C vs. 41 degrees C), and there was more precipitation of protein from the serum, which sometimes formed an adherent layer on the surface of the zirconia. Circulating coolant at 1-20 degrees C markedly reduced the bearing temperatures and the protein precipitation. With coolant at 4 degrees C, wear of the polyethylene against cobalt-chromium alloy was about 26% lower than against zirconia, but the macroscopic and microscopic appearance of the worn polyethylene surfaces were unlike that typically generated in vivo. With or without coolant, the morphology of the polyethylene wear debris was comparable to that generated in vivo, but the ratio of fibrillar to granular debris was higher at the reduced temperature. These results suggested that circulating coolant at an appropriate temperature could avoid overheating (due to non-stop running of the simulator), preventing excessive protein precipitation while providing wear surfaces and wear debris with morphologies closely comparable to those generated in vivo.

Frictional heat was monitored during wear tests of ultrahigh molecular weight polyethylene acetabular cups bearing against femoral balls of metal or ceramic in a hip simulator, using bovine serum as a lubricant. About 1 to 2 h of continuous cycling were required for the temperature in the zone of contact between the cup and ball to rise to its maximum steady value, and this equilibrium temperature was markedly higher with increased load and/or cycling rate. Frictional heating caused substantial precipitation of the proteins from the serum and, in some of the tests running at 1.5 or 2 Hz, an adherent proteinaceous layer was observed attached to the surface of the balls. The maximum temperature was also substantially higher in tests run with the cup mounted above the ball rather than below. Surprisingly, the tests running at higher frictional torque and temperature (i.e., those with the most protein precipitation and/or adherent layers) produced the least wear of the polyethylene. This might have been due to the solid proteins that formed a protective layer between the ball and cup. Because patients with hip prostheses typically do not walk for hours without rest, the maximum temperatures in vivo are likely to be much lower than those reached in the hip simulator. Therefore, the affects of protein precipitation on the resultant wear properties of the materials should be considered potential artifacts of the hip simulator tests. Increasing the volume of the lubricant bath reduced the maximum temperatures for tests running at 1.5 Hz but had little affect at 2 Hz. Reducing the cycling rate is an effective way to avoid overheating of the specimens, but this necessarily extends the time required to complete a test.

The effects of pre-dilution and other modifications of bovine serum lubricants on the wear properties of UHMW polyethylene acetabular cups were evaluated in a hip joint simulator. The wear rate increased, and a nonphysiological type of surface-pitting occurred, when the serum was pre-diluted to 40% or lower concentration. During the wear test, the equilibrium temperature and the precipitation of proteins were substantially greater with zirconia balls than with cobalt-chromium. Protein precipitation, a potential modulator of in vitro wear, was shown to be temperature, concentration, and time-dependent in water-bath tests, which indicated that ball-cup interface temperatures in the simulator must be above 60 degrees C, i.e., well above the bulk lubricant temperature, to account for wear test protein precipitation. Several modifications of serum that were, in part, intended to decrease the tendency for protein precipitation were found to markedly affect the wear properties of the two combinations of materials. In particular, modified serum, which lacked some of the higher molecular weight proteins, produced a much higher wear rate than a control serum with the same initial protein concentration. The results indicated directions for further research to clarify the lubricating properties of serum, and for developing a universal standard test lubricant.

The less intense tissue reaction around metal on metal total hip replacements (THRs) compared to metal on polyethylene (PE) THRs may be explained by the differences in the characteristics of metal wear particles. In this study, transmission electron microscopy was used to study metal wear particles that were either in situ in cells or had been extracted from the cells by a new technique based on enzymatic tissue digestion. The tissues were obtained from 13 patients undergoing revision of metal on metal THRs with cobalt-chromium-molybdenum (CoCrMo) bearing couples. Most of the CoCrMo wear particles were smaller than 50 nm (range 6-834 nm) and round to oval in shape with irregular boundaries. This size range is considerably smaller than that reported for PE particles. While even a small volume of metal wear will produce high numbers of particles, the apparently less severe local tissue reaction to metal particles may be due to the possibility that corrosion, dissolution, and dissemination of metal particles may result in fewer local biological effects than the long-term retention of PE particles in the periprosthetic tissues.

PGs play an important role in regulating articular chondrocyte function in both normal and pathological states. However, the mechanisms of the effects of PG on chondrocyte function remain undefined. We, therefore, examined the effects of PGE1, PGE2, and PGE2 alpha on second messenger generation in relation to DNA and aggrecan synthesis in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line. RCJ cells were grown under minimal attachment conditions on a composite collagen-agarose (0.15%/0.8%) gel to maintain a differentiated phenotype. PGE1 and PGE2 (0.001-100 microM) produced a similar dose-related increase in cAMP accumulation, with a maximal 8-fold increase over basal values, whereas PGF2 alpha produced a minimal 1.3-fold increase in cAMP levels only at 100 microM. On the other hand, both PGE2 and PGE2 alpha raised the intracellular free calcium ([Ca2+]i) concentration, derived primarily from extracellular sources, whereas PGE1 was without effect on [Ca2+]i. These three PGs also had divergent effects on DNA synthesis, as measured by [3H]thymidine ([3H]TdR) incorporation. PGF2 alpha (0.001-5 microM) produced a dose-related increase in [3H]TdR incorporation, with a maximal 1.6-fold increase over baseline values at 5 microM and a slight decline to below maximal levels at 10 microM. PGE2 exhibited a contrasting inverse biphasic response, with an initial small suppressive effect that was maximal at 0.1 microM and a secondary stimulatory phase producing a small increase over control values at 5 microM. PGE1 had a uniformly suppressive effect, producing a 30% decrease at 10 microM. Despite the divergent effects of PGE1, PGE2, and PGE2 alpha on second messenger generation and DNA synthesis, all three PGs produced a dose-related stimulation of aggrecan synthesis. PGF2 alpha was the most potent, producing significant stimulation at 0.001 microM and a maximal 104% increase at 5 microM. PGE1 and PGE2 were approximately equipotent and approximately 60% as effective as PGF2 alpha in stimulating aggrecan synthesis. Northern analysis demonstrated that the effects of PG on aggrecan synthesis were not accompanied by changes in aggrecan core protein steady state messenger RNA levels. Thus, the effects of PG on aggrecan production in RCJ cells appear to be regulated at the posttranscriptional level. Forskolin and (Bu)2cAMP mimicked the suppressive effects of PGE1 on [3H]TdR incorporation, as well as the stimulatory effect of PGE1 on aggrecan synthesis. In addition, the phorbol ester 12-O-tetradecanoyl phorbol acetate mimicked PGF2 alpha stimulation of [3H]TdR incorporation and aggrecan synthesis, and the effects of PGE2 alpha on these processes were blocked by protein kinase C inhibitors. Therefore, it appears that in mammalian chondrocytes, PGE1 primarily activates the cAMP-protein kinase A second messenger system, PGE2 alpha affects primarily the Ca2(+)-protein kinase C system, and PGE2 activates both pathways. Moreover, PG posttranscriptional regulation of aggrecan synthesis in chondrocytes involves both the cAMP-protein kinase A and Ca2(+)-protein kinase C second messenger systems.

The transforming growth factor beta (TGF-beta) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-beta type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors. The type-II receptor immuno-precipitated from human osteosarcoma cells exhibited autophosphorylation and casein kinase activity that was markedly stimulated by polylysine yet was insensitive to heparin. Affinity cross-linking of 125I-TGF-beta 1 ligand to cellular receptors followed by specific immunoprecipitation demonstrated that type-II receptors form stable complexes with both type-I and type-III receptors expressed on the surfaces of both human osteosarcoma cells and rabbit chondrocytes. Pretreatment of the cultured cells with an antibody directed against a distinct extracellular segment of the type-II receptor (anti-TGF-beta-IIR-NT) effectively blocked the 125I-TGF-beta labelling of type-I receptors without preventing the affinity labelling of type-II or type-III receptors, indicating a selective disruption of the type-I/type-II hetero-oligomers. The anti-TGF-beta-IIR-NT antibodies also blocked the TGF-beta-dependent induction of the plasminogen activator inhibitor (PAI-1) promoter observed in mink lung epithelial cells. However, the same anti-TGF-beta-IIR-NT antibodies did not prevent the characteristic inhibition of cellular proliferation by TGF-beta 1, as determined by [3H]thymidine incorporation into DNA. The selective perturbation of PAI-1 promoter induction versus cell-cycle-negative regulation suggests that strategic disruption of TGF-beta type-I and -II receptor interactions can effectively alter specific cellular responses to TGF-beta signalling.

Thirteen adult female rabbits underwent unilateral osteotomy of the proximal tibia. In nine animals, 30 degrees of valgus angulation was created; in four animals, osteotomy without angulation was performed. After a 12-week survival period, the knee joints were processed for histology by staining with hematoxylin and eosin and safranin O. Additionally, the chondroitin sulphate epitopes 3-B-3(-) and 7-D-4 were evaluated immunohistochemically as markers of osteoarthritis. Changes of the articular surface of the tibia were visualized by scanning electron microscopy. Light microscopic evaluation by the Mankin et al. scoring system revealed mild or moderate damage of the cartilage in the lateral compartment of angulated extremities when compared with the control side. Immunohistology with the monoclonal 3-B-3 and 7-D-4 antibodies showed no increased expression of these epitopes in the lateral compartments of the knee. Scanning electron microscopic evaluation of the tibial surfaces revealed slight surface damage localized to the central, weight-bearing portion of the lateral tibial plateau of angulated extremities. Angulation of 30 degrees led to only mild degenerative changes of the cartilage. These results indicate that, in the short term, cartilage has considerable capacity to withstand the effects caused by severe angulation of the limb.

Numerous studies indicate highly crosslinked polyethylenes reduce the wear debris volume generated by hip arthroplasty acetabular liners. This, in turns, requires new methods to isolate and characterize them.We describe a method for extracting polyethylene wear particles from bovine serum typically used in wear tests and for characterizing their size, distribution, and morphology.Serum proteins were completely digested using an optimized enzymatic digestion method that prevented the loss of the smallest particles and minimized their clumping. Density-gradient ultracentrifugation was designed to remove contaminants and recover the particles without filtration, depositing them directly onto a silicon wafer. This provided uniform distribution of the particles and high contrast against the background, facilitating accurate, automated, morphometric image analysis. The accuracy and precision of the new protocol were assessed by recovering and characterizing particles from wear tests of three types of polyethylene acetabular cups (no crosslinking and 5 Mrads and 7.5 Mrads of gamma irradiation crosslinking).The new method demonstrated important differences in the particle size distributions and morphologic parameters among the three types of polyethylene that could not be detected using prior isolation methods.The new protocol overcomes a number of limitations, such as loss of nanometer-sized particles and artifactual clumping, among others.The analysis of polyethylene wear particles produced in joint simulator wear tests of prosthetic joints is a key tool to identify the wear mechanisms that produce the particles and predict and evaluate their effects on periprosthetic tissues.

Rabbit articular chondrocytes were treated with retinoic acid (RA) to eliminate the differentiated phenotype marked by the synthesis of type II collagen and high levels of proteoglycan. Exposure of such cells to transforming growth factor-beta 1 (TGF-beta 1) in secondary culture under serum-free and RA-free, defined conditions led to reexpression of the differentiated phenotype. The microfilament modifying drug, dihydrocytochalasin B (DHCB), enhanced the effectiveness of TGF-beta 1 and produced a threefold stimulation of type II collagen reexpression (measured by 2-D CNBr peptide mapping) at 0.3 ng/ml TGF-beta 1 without altering total collagen synthesis. Type II collagen reexpression was maximal from 1 to 5 ng/ml TGF-beta 1, with or without DHCB. The effect of DHCB on proteoglycan synthesis was maximal at 1 ng/ml TGF-beta 1. At this dose TGF-beta alone produced no increase in 35SO4 incorporation, while simultaneous treatment with DHCB caused a sevenfold stimulation of proteoglycan synthesis. DHCB-independent stimulation proteoglycan reexpression occurred between 5 and 15 ng/ml TGF-beta 1. In contrast, TGF-beta 1-dependent stimulation of proteoglycan synthesis in differentiated chondrocytes in primary monolayer culture was not substantially affected by DHCB. The collagen data suggest that TGF-beta 1 utilizes separate pathways to control phenotypic change and collagen (matrix) synthesis. Microfilament modification by DHCB selectively enhances the effectiveness of the TGF-beta 1-dependent signaling pathway that controls reexpression of the differentiated phenotype.

The distribution of type I and type III collagens in rat, bovine and human skin were examined by a quantitative 2-D CNBr peptide mapping method. The procedure involved the solubilization of tissues by digestion with CNBr, radioactive labeling in vitro by [3H]-NaBH4 in dimethylformamide, reduction by mercaptoethanol, a second CNBr digestion and 2-D (isoelectric focusing and NaDodSO4 electrophoresis) mapping. The amounts of type I and type III collagen peptide spots in the fluorographs of 2-D maps were analyzed by 2-D scanning densitometer/analyzer. Mixtures containing various ratios of purified type I and type III collagen were used to obtain a standard curve. Using this procedure we were able to determine that in adult human skin (age range 35-65 years) 22% (+1.3%) of the labelled collagen is type III. This value is significantly higher than that was previously estimated by less accurate methods.

Coronal stepoffs of 0.5 mm (equal to the cartilage height) were created on the medial femoral condyles of adult, skeletally mature rabbits as a model for articular surface incongruity. After 3, 6, 12, and 24 weeks, tissue was analyzed histologically using hematoxylin and eosin and Safranin O staining, autoradiographs were made of the femoral condyles, and immunohistologic analysis was done for 3-B-3(-) and 7-D-4 chondroitin sulfate epitopes. An overlapping flap from the high toward the low side and an increase of the cartilage height on the low side of the defect were observed as permanent features of adaptation throughout the entire followup. Significant degeneration was not seen around the lesion or in the tibial cartilage opposing a stepoff defect. Autoradiography showed a three-phase response to the lesion: an early increase in radiolabeled sulfate (35SO4) uptake, a sharp decline of 35SO4 uptake, and finally a late recovery of the autoradiographic signal indicating partial recovery of proteoglycan synthetic activity. After an early increase, immunohistologic analysis for 3-B-3(-) showed a subsiding tendency by 24 weeks, and the staining with 7-D-4 remained elevated uniformly in the vicinity of the lesion. A rabbit femoral stepoff defect with an offset of 0.5 mm may remodel and not lead to degeneration within the first 6 months after injury in a stable joint.

Cell lines were established from rabbit articular chondrocytes following transfection with a plasmid encoding SV40 early function genes. This resulted in cell immortalization (130 passages have been completed for the oldest cell line) with acquisition of characteristics of partial transformation such as reduced serum requirements for normal and clonal growth. The immortalized chondrocytes, called SVRAC, did not form multilayer foci when maintained in postconfluent culture. Their ability to form colonies in soft agar was not increased in comparison with normal chondrocytes, but they were weakly tumorigenic in nude mice. SVRAC lost the ability to synthesize type II collagen and Alcian blue-stainable matrix, which are markers of the differentiated chondrocyte phenotype, and synthesized predominantly type I collagen. Studies of collagen gene expression showed that pro alpha 1 (II) mRNA was undetectable, whereas pro alpha 1 (I) collagen mRNA was expressed even in late passage cultures. Unlike normal dedifferentiated chondrocytes, SVRAC were unable to re-express the differentiated phenotype in response to tridimensional culture or microfilament depolymerization. Cell lines obtained from chondrocytes transfected either in primary culture or just after release of cells from cartilage displayed the same behaviour. Thus SV40 early genes were able to immortalize rabbit articular chondrocytes, but the resulting cell lines displayed an apparently irreversibly dedifferentiated phenotype. These cell lines can be used as models to identify regulatory pathways that are required for the maintenance or reexpression of differentiated function in chondrocytes.

Radioactive proline-labeled type VIII collagen was biosynthesized in the presence of beta-aminoproprionitrile by rabbit corneal endothelial cells and isolated from the culture medium. Type VIII was purified in the presence of protease inhibitors and at neutral pH by ultrafiltration, precipitation with 3.9 M NaCl, sedimentation in sucrose gradients, and DEAE-Sephacel chromatography. The major components of this collagen, VIII-1, -2, and -3, exhibited apparent molecular weights of greater than 194,000, 124,000, and 61,000, respectively, and were shown to contain identical CNBr peptides. Following separation of VIII-1, -2, and -3 from each other and any residual proteases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exposure to acetic acid led to the conversion of VIII-1 to VIII-2 and VIII-3. Thus, VIII-1 is not a continuous single peptide chain, and the preliminary interrupted-helix model of the type VIII structure (Benya, P. D. (1980) Renal Physiol. 3, 30-35) was revised. VIII-3 appears to be the parent alpha 1 (VIII)-chain, with VIII-2 and VIII-1 representing beta- and gamma-chain configurations stabilized by strong noncovalent acid-labile interactions and beta-aminoproprionitrile-insensitive covalent cross-links. Based on two-dimensional CNBr peptide mapping, the alpha-chain is composed of six peptides. Mr 5,300-19,600. The terminal peptides are pepsin sensitive and correlate with two noncollagenous domains, NC1 (Mr 14,700) and NC2 (Mr 4-5,000). NC1 contains the site of acid-labile chain association.

The differentiated phenotype of rabbit articular chondrocytes was modulated in primary culture by treatment with 1 microgram/ml retinoic acid (RA) and reexpressed in secondary culture by treatment with the microfilament-disruptive drug dihydrocytochalasin B (DHCB) in the absence of RA. Because the effective dose of DHCB (3 microM) did not elicit detectable cell rounding or retraction, the nature and extent of microfilament modification responsible for induction of reexpression was evaluated. The network of microfilament stress fibers detected with rhodamine-labeled phalloidin in primary control chondrocytes was altered by RA to a "cobblestone" pattern of circularly oriented fibers at the cell periphery. Subsequent treatment with DHCB resulted in rapid changes in this pattern before overt reexpression. Stress fibers decreased in number and were reoriented. Parallel arrays of long fibers that traversed the cell were evident, in addition to fiber fragments and focal condensations of staining. Immunofluorescent staining of intermediate filaments revealed a marked decrease in complexity and intensity during RA treatment but no change during reexpression. An extended microtubular architecture was present throughout the study. These results clearly identify microfilaments as the principal affected cytoskeletal element and demonstrate that their modification, rather than complete disruption, is sufficient for reexpression. The specificity of DHCB and the reorientation of these filaments before reexpression of the differentiated phenotype suggests a causative role in the mechanism of reexpression.

The differentiated phenotype of rabbit articular chondrocytes consists primarily of type II collagen and cartilage-specific proteoglycan. During serial monolayer culture this phenotype is lost and replaced by a complex collagen phenotype consisting predominately of type I collagen and a low level of proteoglycan synthesis. Such dedifferentiated chondrocytes reexpress the differentiated phenotype during suspension culture in firm gels of 0.5% low Tm agarose. Approximately 80% of the cells survive this transition from the flattened morphology of anchorage-dependent culture to the spherical morphology of anchorage-independent culture and then deposit characteristic proteoglycan matrix domains. The rates of proteoglycan and collagen synthesis return to those of primary chondrocytes. Using SDS-polyacrylamide gel electrophoresis of intact collagen chains and two-dimensional cyanogen bromide peptide mapping, we demonstrated a complete return to the differentiated collagen phenotype. These results emphasize the primary role of cell shape in the modulation of the chondrocyte phenotype and demonstrate a reversible system for the study of gene expression.

Primary monolayers of rabbit articular chondrocytes synthesize high levels of type II collagen and proteoglycan. This capacity was used as a marker for the expression of the differentiated phenotype. Such cells were treated with 1 microgram/ml retinoic acid (RA) for 10 d to produce a modulated collagen phenotype devoid of type II and consisting of predominantly type I trimer and type III collagen. After transfer to secondary culture in the presence of RA, the stability of the RA-modulated phenotype was investigated by culture in the absence of RA. Little reexpression of type II collagen synthesis occurred in this period unless cultures were treated with 3 X 10(-6) M dihydrocytochalasin B to modify microfilament structures. Reexpression of the differentiated phenotype began between days 6-8 and was essentially complete by day 14. Substantial reexpression occurred by day 8 without a detectable increase in cell rounding. Colony formation, characteristic of primary chondrocytes, was infrequent even after reexpression was complete. These data suggest that the integrity of microfilament cytoskeletal structures can be a source of regulatory signals that mechanistically appear to be more proximal to phenotypic change than the overt changes in cell shape that accompany reexpression of subculture-modulated chondrocytes in agarose culture.

Chondrocytes isolated from 15-day-old embryonic chick sterna were cultured as monolayers for 7 days in control medium or in medium supplemented with retinoic acid or 5-bromo-2'-deoxyuridine. Control cells exhibited characteristic polygonal morphology and maintained the synthesis of cartilage-specific collagens, i.e. type II, type IX, 1 alpha, 2 alpha, and 3 alpha chains, and 45 K (presumptive type X). Type IX was the second most prevalent collagen and represented 12-15% of the phenotype. When exposed to retinoic acid, chrondrocytes displayed a fibroblast-like morphology and decreased collagen synthesis by day 2. The synthesis of collagen types II and IX declined in parallel along with that of the other cartilage collagens and ceased by day 7. During the same period, the synthesis of collagen types I, III, and V and two unidentified collagen chains was initiated and stimulated. Similar changes in collagen expression were caused by 5-bromo-2'-deoxyuridine but were delayed, beginning after day 4. Type III collagen, however, was never detected in 5-bromo-2'-deoxyuridine or control cultures. Because two different agents and two rates of modulation produced parallel changes in the synthesis of collagen types II and IX, these collagens appear to be coordinately regulated.

Chondrocytes isolated from rabbit articular cartilage undergo a progressive ‘derepression’ in culture and synthesize Type I, Type III and an unidentified collagen designated Peak ‘X’. In contrast, cartilage slices synthesize predominantly Type II collagen with increasing amounts of Peak ‘X’ during prolonged culture.

The pathogenesis of many corneal diseases involves alteration in endothelium and subjacent Descemet’s membrane. A common manifestation of these disorders is the appearance of a retrocorneal fibrous membrane (RCM) composed of dissociated endothelial cells interposed between layers of Descemet’s membrane-like materials. The stimulus for the endothelium to lose its epithelioid characteristics and assume this more fibroblastic behavior is unknown. In the present study we describe tissue culture conditions which stimulate isolated corneal endothelial cells to undergo a transition similar to that seen in RCM formation. Accordingly, a monolayer of hexagonal endothelial cells become multilayered metaplastic cells and produce abundant Descemet’s membrane-like material. Cells were isolated by enzyme digestion and modification of thiol groups to yield single or small 2- to 3-cell aggregates which could be evenly dispersed on plastic tissue culture dishes. Contaminating stromal fibroblasts were not present in these preparations as verified morphologically. Cultures were confluent within 5–7 days after plating and at 28 days of incubation, cultures displayed features similar to regenerating rabbit endothelium. These included loss of contact inhibition leading to multilayering of cells, intracytoplasmic densities and extracellular matrix (ECM) which accumulated not only between the basal layer of cells and plastic substratum but also in the cellular interstices. The fibrillar and amorphous components of this ECM accumulated in thin layers and were ultrastructurally similar to the anterior banded and posterior unbanded portions of Descemet’s membrane, respectively. This in vitro lamination of cells and their ECM is highly reminiscent of the RCM accompanying various corneal diseases. Therefore, our in vitro system, leading to the formation of multilaminar corneal endothelial cells, provides an advantageous experimental model for studies of cellular activity leading to RCM formation.

Cartilage slices maintained in organ culture have been shown to develop an enhanced capacity to respond to serum. The response was measured at the initiation of culture and after 3 and 7 days of culture in medium containing an inhibitor of DNA synthesis and 0, 1, or 16% serum. At these times, cartilage slices were washed to remove serum and inhibitor, and then exposed to various concentrations of serum for evaluation of DNA and proteoglycan synthesis. The range of the derived dose-response curves and the indicated sensitivity to low serum concentrations were the parameters used to evaluate the response capacity. Response capacity increased gradually, reaching a maximum after 8 days of culture. Considerable enhancement was obtained after maintenance in the absence of serum using both DNA and proteoglycan synthesis as markers. Additional, graded enhancement of response capacity was obtained when the cartilage slices were maintained in 1 or 16% serum. The effects of maintenance in serum were much greater when DNA synthesis rather than proteoglycan synthesis was used to measure the response. However, this serum-dependent enhancement was only prominent when ascorbate was present during the dose-response assay. Ascorbate caused a similar but less-marked increase in sensitivity to serum when proteoglycan synthesis was measured. The possibility that ascorbate may function as a cofactor during the progression phase of cell proliferation is discussed.

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [ 3 H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X 2 Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.

In order to characterize a larger form of disulfide-bonded cartilage collagen, explants of 17-day embryonic chick sterna were cultured in the presence of [3H] proline. Radioactive collagen chains and fragments that were synthesized and secreted into the culture medium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. After limited pepsin digestion of the medium, two discrete disulfide-bonded collagen fragments were detected with Mr = 210,000 and 153,000. These fragments contained 28 and 17.5%, respectively, of the radioactivity in the alpha 1(II)-chains. The smaller fragment (called M) produced three components upon reduction (Mr = 104,000, 51,000, and 31,000) and seemed to represent the previously reported collagens, HMW and M1. The larger fragment (called N) has not been previously described and gave rise to three components upon reduction (Mr = 140,000, 69,000, and 49,000). Prolonged pepsin treatment resulted in the gradual decrease of N with a corresponding increase of M, suggesting the conversion of N to M. CNBr peptide mapping demonstrated that all M-derived peptides were present in N and that N contained extra peptides that account for its larger size. These observations suggest that N represents a larger more intact form of cartilage-derived disulfide-bonded collagen.

We have isolated two overlapping cDNA clones covering 2425 base pairs encoding a short type VIII collagen chain synthesized by rabbit corneal endothelial cells. The cDNAs encode an open reading frame of 744 amino acid residues containing a triple-helical domain of 454 residues flanked by 117- and 173-residue amino and carboxyl non-triple-helical domains (called NC2 and NC1, respectively). Based on the identity between the DNA-derived amino acid sequence and the amino acid sequence of a type VIII collagen CNBr peptide obtained from rabbit corneal Descemet's membrane, we conclude that the cDNAs code for a type VIII collagen chain. We give this chain the designation alpha 1(VIII). The alpha 1(VIII) triple-helical domain contains eight imperfections in the Gly-X-Y repeated structure with Gly-X instead of a full triplet. The length of the triple-helical domain and number and relative locations of these imperfections are remarkably similar to those of chicken alpha 1(X) collagen. The amino acid sequence of the carboxyl three-quarters of the NC1 domain has high sequence similarity to that of alpha 1(X) collagen. These data suggest that the triple-helix coding portions and carboxyl three-quarters of the NC1 domains of the alpha 1(VIII) and alpha 1(X) genes have a common evolutionary origin.

The radioactive collagens synthesized by the fourth subculture progeny of rabbit articular chondrocytes were extracted and purified after limited pepsin digestion by neutral and acid salt precipitation. In order to identify the different types of collagen present, denatured collagen chains were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5% gels, electrophoretically eluted, and cleaved with cyanogen bromide, and the resultant peptides were fractionated by a new sodium dodecyl sulfate electrophoresis system (tris(hydroxymethyl)aminomethane-borate buffer, 15% gels). Comparison of these separate peptide profiles with those from alpha1(I) and alpha1(III) collagen chains permitted the unambiguous identification of these chains in the radioactive collagen synthesized by chondrocytes. Although cartilage slices predominantly synthesized alpha1(II) chains, only alpha1(I) chains were made by cells in fourth subculture. A large fraction of these alpha1(I) chains could not be accounted for by the presence of type I collagen. While in a native, triple-helical conformation, some of these extra alpha1(I) chains were completely separated from type I collagen by their solubility at pH 8.0 in 2.6 M NaCl and therefore identified as [alpha1(I)]3, type I trimer. In addition to type I collagen and type I trimer, these chondrocyte progeny also synthesized type III collagen and two new collagen chains, X and Y. Each collagen type was further characterized by carboxymethylcellulose chromatography and its distribution between the medium and the cell layer. These findings support the idea that cultured chondrocytes assume a collagen phenotype similar to that of their undifferentiated mesenchymal cell precursors.

The differentiated phenotype of rabbit articular chondrocytes can be characterized by the synthesis of high levels of cartilage specific proteoglycan and collagen (type II). Treatment of these cells in primary monolayer culture for periods of up to 18 days with 0.03 to 3.0 micrograms/ml retinoic acid (RA) resulted in suppression of colony formation, altered morphology, and decreased (eightfold) proteoglycan and collagen synthesis. With the exception of collagen synthesis, these changes were complete with all doses after 4 days of treatment. Collagen synthesis declined more slowly; it was dose dependent after 4 days and maximally inhibited by all doses by 9 days. Detailed analysis of the collagen phenotype was performed using SDS-PAGE of intact chains and 2-D CNBr peptide analysis. RA caused cessation of type II synthesis, and transient stimulation of type III and type I trimer collagen synthesis, without induction of type I collagen. Essentially identical results were obtained with retinol. The resultant collagen phenotype differed significantly from the type I-containing phenotype induced by subculture. Thus, suppression of this differentiated program did not elicit a common modulated phenotype. The results are discussed in the context of direct and indirect mechanisms of RA-dependent modulation of chondrocyte gene expression.

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2−/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade. Electronic supplementary material The online version of this article (doi:10.1007/s12079-013-0201-y) contains supplementary material, which is available to authorized users.

A method for labeling proteins or insoluble protein matrices to high specific radioactivity using 3 H-NaBH 4 in anhydrous dimethylformamide without the presence of formaldehyde or other aldehydes and ketones is presented. Chemical analysis of the labeled proteins suggests that the labeling mechanism involves methylation of lysyl residues. The procedure is simple and the labeled protein products retain their size and charge characteristics.

CNBr peptides from the α1-chains of collagen types I, II and III have been fractionated by a high resolution two-dimensional mapping procedure. Radioactive collagen chains were synthesized without deamination of lysyl residues by rabbit cells in culture and were treated with pepsin and purified prior to cleavage by CNBr. The resultant peptides from each collagen type were separately fractionated by a two-dimensional mapping procedure consisting of non-equilibrium isoelectric focusing in the first dimension and sodium dodecyl sulfate electrophoresis in the second dimension. Unique peptide maps were obtained for each type of α-chain and only small amounts of sample were required. This method will be useful for unambiquous identification of isolated collagen chains, identification of collagens in complex mixtures, and the rapid identification of crosslinked peptides.

CNBr peptides from insoluble bovine cortical bone collagen were analyzed using a 2-D mapping technique. The major type 1 collagen CNBr peptides were detected by fluorography after general-labelling with 3H-NaBH4 in dimethyl-formamide. These maps were similar to those visualized by coomassie blue staining and demonstrated a proportional decrease of alpha 1CB6. New groups of peptides, different from those normally present in soluble type I collagen were detected. Some of these peptides were slightly larger and more acidic than alpha 1CB6 and were highly labelled when the demineralized bone was specifically labelled for the presence of aldehydes and crosslinks with 3H-NaBH4 in a phosphate buffer, pH 7.4. Based on the size and charge characteristics of these specifically labelled peptides, they were tentatively identified as crosslinking peptides containing different combinations of alpha 1CB6, alpha 1CB0,1 and alpha 1CB5. The specificity of the labelling method using 3H-NaBH4 in phosphate buffer was demonstrated by the detection of other known crosslinked peptides and by the virtual absence of label in alpha 1CB7, CB8, and CB3. We feel that this simple methodological approach developed in these experiments will prove to be very useful in the analysis of collagen crosslinks present in insoluble collagens derived from normal tissues of various ages as well as from pathological states.

Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X 2 Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X 2 Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X 2 Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X 2 Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final "de-differentiated" phenotype was 41% type I, 25% X 2 Y, 20% type I trimer, 13% type III and 1% type II.

Primary cultures of rabbit corneal endothelial cells were labeled with proline and the medium proteins were fractionated by sedimentation through sucrose density gradients. A new collagen, endothelial cell (EC) collagen, was completely separated from type IV collagen without denaturation by this technique. Sodium dodecyl sulfate electrophoresis demonstrated EC collagen fragments of 180K, 120K, and 60K daltons. Prior pepsin treatment produced only the 60K fragment after electrophoresis. The cyanogen bromide peptide pattern of the 180K EC peptide demonstrated that EC was different from type IV. A preliminary structural model for the largest helical form (∼ 540K daltons) of EC collagen is presented.

The effect of serial subculture upon collagen biosynthesis by rabbit corneal endothelial cells has been studied to determine if the collagen phenotype is stable. Nearly confluent first through fourth endothelial cell cultures were labeled for 48 hr with [3H]proline and newly synthesized radioactive collagens were treated with pepsin and purified by neutral and acid salt precipitation. Cellular production of collagen (c/min/μg DNA) was greatest in primary cultures and decreased with subsequent passage. No differences were observed in the distribution of radioactive peptides separated by sodium dodecyl sulfate gel electrophoresis. This suggests that the collagen phenotype is qualitatively stable during the first four serial cultures. The [3H]proline content and the electrophoretic patterns of these collagens clearly distinguish them from those produced by fetal rabbit fibroblasts (i.e. type I and III). In summary, the progeny of rabbit corneal endothelial cells produced decreased amounts of qualitatively similar type(s) of collagen synthesized by their parent primary cultures.

A method is presented for determining the amounts of malonaldehyde and malonaldehyde yielding compounds in the presence of a 20-fold excess of sialic acid. The assay is based on the malonaldehyde-2-thiobarbituric acid chromophore, which shifts its absorption maximum from 532 to 557 mμ upon the addition of base. The absorbance of 557 mμ was linear from 0.002 to 0.02 μmole of malonaldehyde per 0.2 ml of sample. The assay was sensitive (molar extinction coefficient 130,000) and extremely specific for malonaldehyde while unreactive for other dialdehydes or aldehydes. A wide variety of compounds were tested and were found to be poor sources of chromogen. By eliminating interfering chromophores this assay will facilitate the identification of the unknown malonaldehyde yielding compound(s) in insoluble collagen and connective tissue extracts both of which contain sialic acid.