Chick Tendon Fibroblasts Research Articles

The influence of tantalum on human cell lineages important for healing in soft-tissue reattachment surgery: an in-vitro analysis

BackgroundPorous tantalum is currently used in orthopaedic surgery for a variety of indications including soft tissue re-attachment. However, the clinical results have been variable and a previous laboratory study has suggested that tantalum may actually inhibit chick tendon fibroblasts. The influence of tantalum on human cell-types involved in soft tissue re-attachment has not been defined.MethodsHuman fibroblasts, human osteoblasts and human mesenchymal stem cells were plated on glass cover slips, half of which were coated with tantalum. Cell numbers were assessed at 1, 2, 7 and 14 days using Cyquant® assay. Cell adhesion and morphology were assessed using light microscopy at 7, 14 and 28 days. To reduce the effect of an expected rate of error, n = 4 was utilised for each cell type and the experiment was repeated twice.ResultsStatistically similar numbers of human osteoblasts and human mesenchymal stem cells were present at 14 days on tantalum-coated and uncoated glass cover slips, revealing no inhibitory effect on cell proliferation. More than double the number of human fibroblasts was seen on tantalum-coated cover slips at that time point (compared to controls), which was statistically significant (p < 0.0001). Morphological assessment revealed normal cell spreading and adhesion on both substrates at all time points.ConclusionsIn vitro study demonstrates that Tantalum causes a significant increase in the proliferation of human fibroblasts with no quantifiable negative effects seen on fibroblast behaviour after 28 days culture. Furthermore, tantalum does not exert any inhibitory effects on the proliferation or behaviour of human osteoblasts or human mesenchymal stem cells. Tantalum could be an appropriate biomaterial for use in situations where soft tissue requires direct reattachment to implants and may stimulate soft tissue healing.

Chick tendon fibroblast transcriptome and shape depend on whether the cell has made its own collagen matrix.

Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D–3D and collagen-fibrin comparisons of gene expression, cell shape and mechanotransduction, with an in vivo reference, have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs). CTFs synthesised their own collagen matrix in fibrin-based TECs and better recapitulated the gene expression, collagen fibril alignment and cell shape seen in vivo. In contrast, cells in 3D collagen gels exhibited a 2D-like morphology and expressed fewer of the genes expressed in vivo. Analysis of YAP/TAZ target genes showed that collagen gels desensitise mechanotransduction pathways. In conclusion, gene expression and cell shape are similar on plastic and 3D collagen whereas cells in 3D fibrin have a shape and transcriptome better resembling the in vivo situation. Implications for wound healing are discussed.

Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ

Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)–Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell–matrix interface.

Triple Helix Formation of Procollagen Type I Can Occur at the Rough Endoplasmic Reticulum Membrane

One key problem in understanding the biosynthesis of collagens remains the assembly of the three alpha-chains. How and when are the different gene products selected, aligned, and folded into a triple helix? As the spatial arrangement during biosynthesis might be important, we concentrated on whether the rough endoplasmic reticular membrane is involved in this process. Microsomes were prepared from biosynthetically labeled chick tendon fibroblasts. Vesicles were spread as a monomolecular film which was then transferred over several compartments of a filmbalance containing fresh subphase. Fluorograms of the surface film showed that the monolayer contains procollagen chains. When the monolayer was transferred onto a chymotrypsin/trypsin-containing subphase, the gel bands of the proalpha-chains were shifted into the position of mature alpha-chains, indicating that only the propeptides were digested and the collagenous regions were protected due to triple helix formation. Our results suggest that newly synthesized proalpha-chains can associate as trimers and fold into a triple helical conformation while they are still associated with the membranes of the rough endoplasmic reticulum. These processes also occur when interchain disulfide linkage is inhibited, indicating that chain selection and registration is not dependent on formation of covalent bonds among the carboxyl propeptides.

Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine

We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids ( K m for lysine: 1.6 μM; K i for S-2-aminoethylcysteine: 1.4 μM). The analogue also profoundly inhibited the synthesis and secretion of [ 14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [ 14C]proline-labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25°C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis.

Effect of electromagnetic stimulation with different waveforms on cultured chick tendon fibroblasts

An energy efficient electromagnetic stimulator device for fracture healing was compared to a commercially available device in stimulating cell growth in tissue cultures. The energy efficient device, which conserves energy by using a bidirectional time-dependent magnetic wave form, and the commercially available stimulator, which uses a unidirectional time-dependent magnetic wave form, were tested on chick tendon fibroblasts in primary culture. Comparing non-stimulated control and cells electromagnetically stimulated with unidirectional and bidirectional waveforms showed that at the growth phase between days 2 and 3, both electrical stimulation techniques increased cell division as measured by DNA synthesis. When cells were dividing rapidly, collagen synthesis was reduced. When the cells reached the confluence there was no difference among the groups (control, unidirectionally stimulated, and bidirectionally stimulated) in terms of number of cells or collagen produced.

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

The first intron of the alpha 1(I) collagen gene contains several transcriptional regulatory elements.

The first intron of the human alpha 1(I) collagen gene contains a negatively acting element that inhibits transcription of the chloramphenicol acetyltransferase gene driven by either a collagen or an SV40 basal promoter (Bornstein, P., McKay, J., Morishima, J., Devarayalu, S., and Gelinas, R. E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, in press). We now find that this element is flanked by sequences that both neutralize the inhibitory effect and impart a net positive effect on transcription. A collagen-human growth hormone minigene was constructed in which varying lengths of the collagen intron were retained. Plasmids were transfected into chick tendon fibroblasts, and transcriptional activity was measured by solution hybridization with an antisense RNA probe. The presence of the intact intronic sequence stimulated transcription by a factor of 2-3-fold in comparison with intron-deleted plasmids. However, the isolated negatively acting element inhibited transcription by a factor of 15-20-fold. Surprisingly, this effect was markedly orientation-dependent. Intronic segments flanking the negatively acting element stimulated transcription both when cloned 5' to the collagen promoter in chloramphenicol acetyltransferase-based plasmids and 3' in collagen-human growth hormone constructions. We conclude that expression of the alpha 1(I) collagen gene is controlled by several intronic elements that function coordinately with 5'-flanking and promoter elements.

Extracellular matrix morphogenesis: HVEM analysis

Matrix morphogenesis requires the synthesis, assembly and deposition of collagen molecules in a tissue specific manner. Collagen fibrillogenesis is a multistep process involving both intracellular and extracellular assembly reactions. Collagen synthesis, molecular assembly and formation of supramolecular aggregates occurs within a series of well defined cytoplasmic compartments while the assembly and deposition of the newly synthesized collagen fibrils, fibril bundles and tissue specific collagen macroaggregates occurs within a series of extracellular compartments defined by the fibroblast.In the studies described here we will focus our attention on the cellular influences involved in the assembly of collagen molecules into fibrils, fibril bundles and tissue specific macroaggregates. The chick tendon and corneal fibroblasts partition the extracellular space during morphogenesis, forming at least three compartments. These extracytoplasmic compartments have been described using high voltage electron microscopy of thick sections and serial thick sections as well as by conventional transmission electron microscopy.

A monoclonal antibody to the carboxyterminal domain of procollagen type I visualizes collagen-synthesizing fibroblasts. Detection of an altered fibroblast phenotype in lungs of patients with pulmonary fibrosis.

Excessive collagen deposition plays a critical role in the development of fibrosis, and early or active fibrosis may be more susceptible to therapeutic intervention than later stages of scarring. However, at present there is no simple method for assessing the collagen-synthesizing and secreting activity of fibroblasts in human tissues. Type I procollagen carboxyterminal domains are proteolytically removed during collagen secretion. Thus, antibodies to these domains should stain fibroblasts synthesizing type I collagen but not extracellular collagen fibrils which could mask the signal from the cells. We developed and characterized a monoclonal antibody (Anti-pC) specific for the carboxyterminal propeptide of type I procollagen. To determine the relationship between Anti-pC staining and collagen synthesis, we stained embryonic and adult chicken tendon. Embryonic chick tendon fibroblasts actively synthesizing type I collagen stained heavily with Anti-pC, while quiescent adult tendon fibroblasts did not stain with Anti-pC. Wounded adult tendons developed fibroblasts that stained with Anti-pC at the wound site. Thus, Anti-pC specifically visualized fibroblasts actively synthesizing collagen. Lung biopsies from patients with fibrotic lung disease were stained with Anti-pC. Interstitial and intraalveolar fibroblasts in biopsies from patients with active fibrosis stained intensely with Anti-pC, while normal human lung was unstained. The absence of staining in normal lung supports the hypothesis that fibrosis is associated with an altered collagen-synthesizing phenotype of tissue fibroblasts. Anti-pC may provide a useful clinical tool for assessing fibrogenic activity at sites of tissue injury.

Decreased synthesis and increased intracellular degradation of newly synthesized collagen in freshly isolated chick tendon cells incubated with monensin.

The synthesis and secretion of procollagen in embryonic chick tendon fibroblasts in suspension culture were inhibited with the carboxylic ionophore monensin. The synthesis of procollagen was inhibited by 50% in a 2-h exposure to 0.1 microM monensin and was inhibited by 70% in a 6-h exposure to 0.1 microM monensin. Secretion of procollagen was inhibited by greater than 90% in the 0.1 microM monensin-treated cultures and was totally inhibited by higher doses of the reagent. A cellular pool of collagenase-digestible peptides was demonstrated in the control cells, the level of which was elevated 3-4 times in the monensin-treated cultures. In order to determine whether the secretory and synthesis block caused by monensin inhibited intracellular degradation of newly synthesized collagen, the hydroxy[14C]proline in degraded collagen fragments present in control and monensin-treated cultures was determined and compared to the total hydroxy[14C]proline synthesized in each culture. The intracellular degradation of newly synthesized, pulse-labeled collagen was shown to proceed at rates comparable to those seen in the control cultures. The monensin-treated cells degraded pulse-labeled newly synthesized collagen nearly twice as long as the controls, resulting in an overall increase in the fraction of newly synthesized collagen that was degraded. These findings suggest that force generation in the activated cross-bridge cycle may occur as a result of an actin-attached cross-bridge transition between these two orientations.

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.

Topographies of extracytoplasmic compartments in developing chick tendon fibroblasts

The complex topography of the fibroblast leading to the formation of extracellular compartments is demonstrated in the native three-dimensional state in cryofractured chick embryo tendon at different stages of development. Tendon collagen fibrils, fibril bundles, and macroaggregates are intimately associated with these extracytoplasmic domains. The localization of fibrils within narrow channels, bundles within peripheral compartments, and the coalescence of bundles into macroaggregates within larger extracytoplasmic compartments is illustrated at different stages of development. Changing patterns of matrix organization are apparent as morphogenesis proceeds. Our interpretation is that collagen fibrils, oriented along the tendon axis, appear in extracytoplasmic channels defined by the fibroblast which are added at the periphery of the cell until a row of fibrils outlines the cellular border at Day 10. By Day 14, cytoplasmic processes have partitioned the fibrils into groups and then into round bundles. New fibrils are continually added to the fibril bundles through fusion of the extracytoplasmic channels. As the bundles grow larger, the cell boundaries between the bundles retract and bundles begin to coalesce into macroaggregates. The fibroblast becomes attenuated, bundle-forming compartments disappear as processes retract, bundles coalesce, and macroaggregates predominate by Day 17. This work is consistent with the model proposed for the compartmentalization of the extracellular space by the fibroblast, providing distinct microenvironments where collagen fibrillogenesis and matrix morphogenesis occur under close cellular regulation.

Fibroblasts create compartments in the extracellular space where collagen polymerizes into fibrils and fibrils associate into bundles.

Morphogenesis in the animal kingdom is closely coupled with the intracellular synthesis and the extracellular deposition of collagen. These morphogenetically important events involve multiple steps which begin in compartments inside the cell and continue in compartments outside the cell. The movement of matrix components from the intracellular compartments to the extracellular compartments is a functional continuum. Studies of embryonic chick tendon and cornea fibroblasts with the high-voltage (1000 kV) transmission electron microscope have provided the beginnings of an understanding of how this functional continuum is related to the structural compartments which the cells create. This series of compartments, both inside and outside the cell, is involved in collagen fibrillogenesis, collagen fibril bundle formation, and tissue morphogenesis.

Coated vesicles purified from chick tendon fibroblasts contain newly synthesized type I procollagen

Coated vesicles were purified from embryonic chick tendon fibroblasts pulsed with [ 3H]proline. They were morphologically and biochemically similar to coated vesicles purified from other sources. Furthermore, they contained newly synthesized Type I procollagen which was protected from bacterial collagenase digestion unless detergent was present. The procollagen remained associated with coated vesicles during immune precipitation and agarose gel electrophoresis. Data from pulse-chase experiments demonstrated that the specific activity of the coated vesicle preparations was approx. 5-fold higher at the 10 min chase point than at either the 0 or 40 min chase points. These data are consistent with the hypothesis that coated vesicles are intermediates in the intracellular transport of newly synthesized Type I procollagen in chick tendon fibroblasts.

The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture

The production of [ 14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH 4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitors N-p-tosyl- l-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 ± 1.4% to 10.5 ± 2.4% with 10 μ m TLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 μ m TLCK. The incorporation of [ 14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 μg/ml leupeptin or 60 μg/ml pepstatin A. However, the production of collagen was reduced to 10.9 ± 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [ 14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 μ m TLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.

Lysosomotropic agents ammonium chloride and chloroquine inhibit both the synthesis and secretion of procollagen by freshly isolated embryonic chick tendon cells

Freshly isolated embryonic chick tendon fibroblasts were incubated in suspension culture for six hrs in the presence or absence of ammonium chloride or chloroquine. Both agents caused a marked reduction in collagen production without inhibiting non-collagen protein synthesis. The small amounts of collagen synthesized in the presence of each agent were secreted into the culture medium at rates significantly lower than exhibited by control cells. Since ammonium chloride and chloroquine have been shown to inhibit lysosomal degradation of proteins, and interfere with the fusion of membranous vesicles and receptor recycling, these results suggest a requirement for at least one of these processes in the synthesis and secretion of procollagen.

The effect of cis-4-hydroxy-L-proline on intracellular degradation of newly synthesized collagen by freshly isolated chick tendon fibroblasts.

Freshly isolated embryonic chick tendon cells were incubated for 6 hrs in suspension culture in the presence of the proline analogue, cis-4-hydroxyproline to cause the cells to synthesize collagen which was incapable of becoming triple helical. The cells were evaluated for the percentage of total protein synthesis devoted to collagen and for the percentage of newly synthesized collagen which was rapidly degraded. Collagen production in the presence of cis-4-hydroxyproline was reduced from 25% to 7% of total protein synthesis. Under normal conditions the cells degraded 8% of their newly synthesized collagen but when the cells were incubated in the presence of cis-4-hydroxyproline, 25% of their total collagen synthesized was degraded to dialyzable peptides. The enhanced degradation of nonhelical, analogue containing collagen was inhibited by inhibitors of lysosome function. These observations provide support for the concept that fibroblasts are able to recognize and degrade a portion of their newly synthesized collagen, and that defective collagen may be selectively degraded by an intracellular lysosomal process. Enhanced intracellular degradation can in part explain the decrease in collagen production by freshly isolated tendon fibroblasts incubated with cis-4-hydroxyproline.

Biosynthesis of procollagen [(pro alpha 1 V)2 (pro alpha 2 V)] by chick tendon fibroblasts and procollagen (pro alpha 1 V)3 by hamster lung cell cultures.

Biosynthesis of procollagen [(pro alpha 1 V)2 (pro alpha 2 V)] by chick tendon fibroblasts and procollagen (pro alpha 1 V)3 by hamster lung cell cultures.