Requirements for maintaining the embryonic state of avian tendon cells in culture

Summary Primary avian tendon cells (PAT) maintain their embryonic state when cultured in medium F-12 with very low serum (0.2%) and ascorbate (50 μg per ml); that is, they retain the potential for devoting 20–30% of their total protein synthesis to collagen. However, if the cells are left at a confluent cell density or are derived from confluent cultures, this potential is irreversibly decreased. This effect, along with poor medium formulations, probably accounts for the “dedifferentiation” process that occurs when fibroblasts are cultured. In contrast, PAT cells kept at subconfluent cell densities retain the ability to synthesize high levels of collagen. The one limitation in obtaining long-term cultures of high collagen-producing tendon cells in the inability of serum at low concentrations to remain a potent mitogen after a few subcultures. The quantitative loss of function has long been considered to be a cell culture artifact; however, we propose that this drop in collagen synthesis is a reflection of the developmental programing of these cells. In separate series of experiments using organ cultures, we show that tendon tissue from the embryo makes over 30% collagen, whereas, “young” tendons make 18% and “older” tendons from the adult make less than 1%. Therefore, a quantitative drop in collagen synthesis would be expected if normal development were to occur in culture. Our data are consistent with the idea that cultures of embryonic tendon cells are triggered to mature by a mechanism that correlates with high cell density. This investigation was supported in part by National Science Foundation Grant PCM 77-14982; in part by the Division of Biomedical and Environmental Research of the Department of Energy under contract W-7405-ENG-48; and by a National Institutes of Health Fellowship IF32 CA 05807-01, from the National Cancer Institute to R. I. S.     

Relative content of isoaccepting tRNAs for glycine and proline in avian tendon cells with different rates of procollagen synthesis

The relative amounts of iso-tRNAsGly and iso-tRNAsPro existing in chick embryo tendon are indicative of a specialization of the tRNA population for collagen synthesis. These amounts are not modified (i) in primary avian tendon (PAT) cells in culture for which the procollagen production varies from about 10% of total protein synthesis to 60% and (ii) in tendons from immature chicks, which show a 3-fold decrease of procollagen production with increasing age. The characteristic tRNA pattern was not maintained in cells which had lost the ability to make high levels of collagen as observed in the cases of: (i) PAT cells reaching confluency; (ii) virus-transformed PAT cells and (iii) tendon from adult chick. Our data are consistent with the idea that tendon tRNA specialization for collagen synthesis is a differentiation feature independent of the expression level of the collagenic function but related to its maintenance.     

Ascorbate induction of collagen synthesis as a means for elucidating a mechanism of quantitative control of tissue-specific function.

Ascorbic acid displays the characteristics of an ideal inducer of tissue-specific function in primary avian tendon cells in culture. It is a highly specific, potent stimulator of collagen synthesis, it demonstrates slow reversible kinetics, and it has no effect on growth rate of the cultured cells. Kinetic analysis of ascorbate induction of collagen synthesis was used to determine the critical steps in this complex biosynthetic pathway. Full hydroxylation of the proline residues in collagen, although probably a necessary step for collagen induction, was in itself not sufficient for achieving either increased secretion or increased synthesis. On the other hand, an increase in secretion rate, which required both the presence of ascorbate and a high cell density, did correlate with the later stimulation in procollagen production. The process of procollagen secretion, therefore, meets the minimal requirements for the rate-limiting step. The fact that the cells maintained a large pool of intracellular procollagen despite changes in the rates of translation or secretion led us to postulate a possible feedback between the level of the internal procollagen pool and the rate of procollagen synthesis.     

Primary avian tendon cells in culture. An improved system for understanding malignant transformation

Primary avian tendon (PAT) cells which maintain their differentiated state in culture are rapidly transformed by Rous sarcoma virus. By criteria of morphology, increased rate of 2-deoxyglucose uptake, and loss of density dependent growth control, PAT cells transform as well as their less differentiated counterpart, chick embryo fibroblasts. In addition, the percentage of collagen produced by PAT cells drops on transformation by an order of magnitude, from 23 to 2.5%, but is unaffected by viral replication of a transformation-defective mutant. The responsiveness of normal and transformed PAT cells to various environmental factors changes dramatically upon transformation. Normal PAT cells respond to the presence of ascorbate and high cell density by raising the level of collagen synthesis from 5 to 23%. Transformed PAT cells are totally unresponsive. These and previously reported results lead us to postulate that the break-down in the normal regulatory mechanisms used by the cell to maintain the differentiated state is related to or is responsible for the onset of malignant transformation.     

Relative content of isoaccepting tRNAs for glycine and proline in avian tendon cells with different rates of procollagen synthesis

The relative amounts of iso-tRNAs^Gly and iso-tRNAs^Pro existing in chick embryo tendon are indicative of a specialization of the tRNA population for collagen synthesis. These amounts are not modified (i) in primary avian tendon (PAT) cells in culture for which the procollagen production varies from about 10% of total protein synthesis to 60% and (ii) in tendons from immature chicks, which show a 3-fold decrease of procollagen production with increasing age. The characteristic tRNA pattern was not maintained in cells which had lost the ability to make high levels of collagen as observed in the cases of: (i) PAT cells reaching confluency; (ii) virus-transformed PAT cells and (iii) tendon from adult chick. Our data are consistent with the idea that tendon tRNA specialization for collagen synthesis is a differentiation feature independent of the expression level of the collagenic function but related to its maintenance.     

The uncoupled regulation of fibronectin and collagen synthesis in Rous sarcoma virus transformed avian tendon cells.

We have investigated the regulation of fibronectin and procollagen synthesis in normal and Rous sarcoma virus transformed primary avian tendon cells. These two proteins interact at the cell periphery and both are reportedly lost upon transformation. We thus examined whether their synthesis was coordinately regulated in Rous sarcoma virus-infected cells. It was found that while the synthesis of both pro alpha 1 and pro alpha 2 peptides was reduced upon transformation, the synthesis of fibronectin was not altered. Nevertheless, long term radiolabeling demonstrated that fibronectin levels were reduced in transformed cells. It is concluded that the reduction in levels of these components at the surface is brought about by different mechanisms; collagen levels being regulated by procollagen synthesis and fibronectin levels by degradation and/or release into the culture medium. The possibility is discussed that fibronectin is lost from the cell periphery of primary avian tendon cells as a consequence of decreased levels of anchoring collagen molecules.     

An in vitro model of fibroplasia: simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis

Previous studies of fibroblast proliferation, migration, and collagen synthesis have been limited in their ability to define the interrelationship among these events in response to various inflammatory mediators. We have now defined an in vitro tissue culture model for the synchronous quantification of these parameters of fibroplasia. Biopsies (2 mm) of chicken flexor tendons are embedded in a fibrin matrix and exposed to various factors for 5 days in tissue culture. The availability of the fibrin matrix surrounding the tendon biopsy satisfies the need for a solid support medium for fibroblast migration. Multiple measurements of tendon fibroblast proliferation, migration into the fibrin matrix, and relative collagen synthesis are then made on these preparations. Fetal calf serum stimulated tendon fibroblast proliferation and migration in a dose responsive fashion, whereas the selective expression of collagen synthesis was decreased. Platelet lysate stimulated fibroblast proliferation at low concentration, but migration only at high concentration and was without effect on relative collagen synthesis. This model now provides a means of more clearly defining the influence of various inflammatory factors on the events of fibroplasia.     

Modulation of collagen production in cultured fibroblasts by a low-frequency, pulsed magnetic field

Primary cultures of chicken tendon fibroblasts have been exposed for various periods to a low-frequency, pulsed magnetic field, and the effects on protein and collagen synthesis have been examined by radioisotopic incorporation. Total protein synthesis was increased in confluent cells treated with a pulsed magnetic field for the last 24 h of culture as well as in cells treated for a total of 6 days. However, in 6 day-treated cultures, collagen accumulation was specifically enhanced as compared to total protein, whereas after short-term exposure, collagen production was increased only to the same extent as total protein. Levels of cyclic AMP were significantly decreased after 6-day pulsed magnetic field treatment, probably as a consequence of diminished adenylate cyclase activity. Exposure to pulsed magnetic field had no effect on cell proliferation or collagen phenotype. These results indicate that a pulsed magnetic field can specifically increase production of collagen, the major differentiated function of fibroblasts, possibly by altering cyclic-AMP metabolism.     

The effects of transforming growth factor-beta and serum on proteoglycan synthesis by tendon fibrocartilage.

The effects of transforming growth factor-beta (TGF-beta) and serum on proteoglycan synthesis by tissue explants from the fibrocartilaginous region of adult bovine tendon and by cells in culture from this region were assessed. The most characteristic effect of added TGF-beta on both explant tissue and cells in culture was enhanced synthesis of one small proteoglycan-biglycan. Lowered serum concentration diminished incorporation of Na2 35SO4 into proteoglycans. Added TGF-beta (1 ng/ml) stimulated cell proliferation, increased overall proteoglycan synthesis, and increased the length of glycosaminoglycan chains on all secreted proteoglycans. The effect of TGF-beta on cells in culture was highly consistent whereas explants from different animals showed greater variability in the response. It was concluded that TGF-beta did not specifically promote or maintain the cartilaginous nature of this tissue because supplementing medium with TGF-beta did not significantly alter the ratio of large/small proteoglycans synthesized by tissue explants. However, the observation of enhanced biglycan synthesis by TGF-beta suggests that TGF-beta could be involved in differentiation of regions of tendon subjected to compression, because compressed tendon contains both decorin and biglycan small proteoglycans whereas tensional tendon contains primarily decorin. Excess decorin added to cell culture medium did not affect the ability of TGF-beta to enhance synthesis of biglycan.     

Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate

Whole blood serum is widely recognized as essential for the growth of diploid cells in culture. Dermal fibroblasts and arterial smooth muscle cells fail to proliferate in culture in the presence of serum derived from platelet-poor plasma. Platelet-poor plasma serum is capable of maintaining monkey arterial smooth muscle cells quiescent in culture at either low (1.5 x 10(3)) or high (2.0 x 10(4)) population densities. The proportion of cell traversing the cell cycle under these conditions was approximately 3%. Equal numbers of quiescent smooth muscle cells initiated DNA synthesis and cell division when treated with whole blood serum or with an equivalent quantity of platelet-poor plasma serum supplemented with a factor(s) derived from a supernate obtained after exposure of human platelets to purified thrombin in vitro.     

Composition of cell-polymer cartilage implants

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.     

Growth of cultured mammalian cells on secondary glucose sources

Mammalian cells can grow in culture at very low glucose concentrations. They can also grow using starch or maltose as secondary sources of glucose if hydrolytic enzymes (amylase and/or maltase) are available to release the glucose. The serum supplement in the culture medium provides these enzymes in amount adequate to permit growth at as rapid a rate as when free glucose is added. Owing to the relatively slow liberation of glucose from the secondary sources, the cells produce less lactic acid, and the culture medium does not become acidic.If the amount of hydrolytic enzyme in the serum supplement is reduced by heat inactivation, the rate of glucose liberation is further reduced. As a result, glucose continues to be released into the medium even at high cell densities, when all glucose added directly to control cultures has been consumed at a time. For this reason, the cells survive longer at high density on secondary glucose sources than on free glucose. Use of such a culture system should have important practical advantages in maintaining dense cultures of any mammalian cell type.Medium containing secondary glucose sources and serum whose hydrolytic enzymes have been completely inactivated should be a selective medium for the corresponding cellular enzymes. Attempts to select for cell lines able to grow using their own amylase or maltase were not successful, but calculations based on embryonic pancreatic cells, known to synthesize amylase, showed that the amount of enzyme required should be quite low in comparison with that present in the differentiated state. The possibilities of selection for a differentiated function in cell culture have been very little explored, and such an approach may be fruitful if applied to the right cell types.     

Attachment and extracellular matrix differences between tendon and synovial fibroblastic cells

Fibroblasts of the synovium of sheathed tendons were isolated, and their biochemical properties were compared with those of the fibroblasts of the remaining tendon. The synovial cells had a lower attachment efficiency than did the tendon cells. On the day of cell isolation the synovial cells synthesized collagen as 10% of their total protein, whereas the tendon cells synthesized 30% collagen. After growth in fetal bovine serum (FBS), the percentage of collagen synthesized by both populations decreased; however, the synovial cells still made less collagen than did the tendon cells (5 versus 11%). On the basis of cyanogen bromide peptide analysis, the synovial cells were found to synthesize Types I and III collagen in primary culture, whereas the tendon cells synthesized only Type I. The synovial cells also synthesized two to three times less sulfated glycosaminoglycans in culture than did the tendon cells. Thus, the two cell populations differed in attachment efficiency and in their biosynthesis of collagen and sulfated glycosaminoglycans. These differences reflect extracellular matrix differences that have been observed in the tendon in vivo. In addition, the results augment existing data showing that not all fibroblasts have identical phenotypes.     

Cultivation of human tenocytes in high-density culture

Limited supplies of tendon tissue for use in reconstructive surgery require development of phenotypically stable tenocytes cultivated in vitro. Tenocytes in monolayer culture display an unstable phenotype and tend to dedifferentiate, but those in three-dimensional culture may remain phenotypically and functionally differentiated. In this study we established a three-dimensional high-density culture system for cultivation of human tenocytes for tissue engineering. Human tenocytes were expanded in monolayer culture before transfer to high-density culture. The synthesis of major extracellular matrix proteins and the ultrastructural morphology of the three-dimensional cultures were investigated for up to 2 weeks by electron microscopy, immunohistochemistry, immunoblotting and quantitative, real-time PCR. Differentiated tenocytes were able to survive over a period of 14 days in high-density culture. During the culture period tenocytes exhibited a typical tenocyte morphology embedded in an extensive extracellular matrix containing cross-striated collagen type I fibrils and proteoglycans. Moreover, expression of the tendon-specific marker scleraxis underlined the tenocytic identity of these cells. Taken together, we conclude that the three-dimensional high-density cultures may be useful as a new approach for obtaining differentiated tenocytes for autologous tenocyte transplantation to support tendon and ligament healing and to investigate the effect of tendon-affecting agents on tendon in vitro.     

Fibroblastic cell cycling in collagen gels

Abstract. Quiescent C3H10T1/2 mouse fibroblasts resume DNA synthesis and proliferation following incubation in medium containing fresh serum both when grown in monolayer and when grown in a collagen matrix. We observed that the rate of DNA synthesis is reduced at high initial cell densities and low initial collagen concentrations. In a collagen matrix, fibroblasts contract the matrix causing an increase in cell density and collagen concentration. We studied the chronological relationship between the kinetics of DNA synthesis and the collagen matrix contraction. The rate of collagen collection per cell changes in time, dependent on initial cell and collagen concentration. The kinetics of the collagen collection showed a positive correlation with the kinetics of DNA synthesis, 16 h later.     

Attachment and extracellular matrix differences between tendon and synovial fibroblastic cells

Summary Fibroblasts of the synovium of sheathed tendons were isolated, and their biochemical properties were compared with those of the fibroblasts of the remaining tendon. The synovial cells had a lower attachment efficiency than did the tendon cells. On the day of cell isolation the synovial cells synthesized collagen as 10% of their total protein, whereas the tendon cells synthesized 30% collagen. After growth in fetal bovine serum (FBS), the percentage of collagen synthesized by both populations decreased; however, the synovial cells still made less collagen than did the tendon cells (5 versus 11%). On the basis of cyanogen bromide peptide analysis, the synovial cells were found to synthesize Types I and III collagen in primary culture, whereas the tendon cells synthesized only Type I. The synovial cells aslo synthesized two to three times less sulfated glycosaminoglycans in culture than did the tendon cells. Thus, the two cell, populations differed in attachment efficiency and in their biosynthesis of collagen and sulfated glycosaminoglycans. These differences reflect extracellular matrix differences that have been observed in the tendon in vivo. In addition, the results augment existing data showing that not all fibroblasts have identical phenotypes. This investigation was supported by National Institutes of Health Grant AM 25749.     

Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.     

Regulation of collagen production and collagen mRNA amounts in fibroblasts in response to culture conditions.

Collagen synthesis and mRNA amounts for the alpha 1 and alpha 2 polypeptide chains of Type I collagen were measured in embryonic-chick tendons and in tendon cells both in suspension and in primary cultures. The percentage of protein production represented by collagen in suspension-cultured cells was initially the same as in the intact tendon; however, on an hourly basis, there was actually a steady decline in collagen production by suspended cells. Collagen production in primary cultures of chick tendon fibroblasts was decreased when compared with intact tendon, even though ascorbate-supplemented primary cultures were able to maintain higher rates of collagen production than were non-supplemented cultures. The amounts of mRNA for alpha 1(I) and alpha 2(I) polypeptide chains of collagen responded in similar fashions to different culture conditions and were compared with the amounts of mRNA for beta-actin. In primary cultures the available alpha 1 and alpha 2 collagen mRNAs support proportionately higher collagen production than in the intact tendon. However, the ratio of alpha 1/alpha 2 mRNA and polypeptide-chain synthesis did not remain 2:1, but increased with the concomitant production of Type I trimers composed of three alpha 1 chains. Removal of fibroblasts from their environment in vivo appears to alter the amounts of mRNA for alpha 1 and alpha 2 chains and to alter the utilization of those mRNAs for polypeptide synthesis.     

Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.