Polymeric Collagen Fibrils Research Articles

Pre-crosslinked polymeric collagen in 3-D models of mechanically stiff tissues: Blended collagen polymer hydrogels for rapid layer fabrication

Currently one factor hindering the development of collagen hydrogel constructs for tissue engineering is the mismatch between initial cellularity and mechanical strength. The main advantage of collagen hydrogel tissue constructs is their ability to support interstitially seeded cells. However, cells are sensitive to their environment, in particular, substrate stiffness, which cannot easily be replicated within hydrogels without cytotoxic cross-linking treatment. In this study, pre-crosslinked polymeric collagen fibrils are introduced as a starting material, thereby avoiding artificial cross-linking. Shear aggregation of this material in solution results in fibril alignment, but cell addition is only possible when polymeric collagen is blended with its monomeric counterparts to slow the aggregation of collagen fibrils. The hydrogel can then be brought to physiological collagen density by plastic compression. Interstitially seeded fibroblasts were supported for 14days. Although compression of blended gels resulted in some cell death due to increased rate of fluid expulsion, not normally seen in conventional collagen hydrogels, the surviving cell population recovers during subsequent culture. Importantly, the compression process can be controlled and customized to limit cell damage. This is the first report of native polymeric collagen used in a tissue engineering context, for the rapid production of a stiff collagen-cell constructs.

Inhibition of trypsin and chymotrypsin by thiols. Biphasic kinetics of reactivation and inhibition induced by sodium periodate addition

Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.

The reversible thiol-disulphide exchange of trypsin and chymotrypsinogen with a tumour-derived inhibitor Kinetic data obtained with fluorescein-labelled polymeric collagen fibrils and casein as substrates

Ehrlich ascites cells contain a cytoplasmic inhibitor of both trypsin and the granule neutral protease and possess a reactive thiol which interacts with an important disulphide bond in trypsin, resulting in the formation of the trypsin-inhibitor complex. When a fixed quantity of trypsin was completely inhibited by addition of the cytoplasmic inhibitor, the trypsin could be re-activated by the addition of either trasylol-trypsin or chymotrypsinogen. Since trasylol-trypsin, chymotrypsinogen (and any derived chymotrypsin) has no ability to solubilise fluorescein-labelled peptides from the substrate, the appearance of trypsin activity was probably due to a non-enzymic exchange reaction, in which these inactive forms displaced trypsin from the trypsin-inhibitor complex. Kinetic data suggest that this displacement was a time-dependent equilibrium reaction controlled by the relative concentration of the reacting species.

Incremental analysis: The application to quantitation of both enzyme activity and inhibitory activity in complex subcellular fractions

Single stage conventional analysis of neutral protease activity present in certain tumour cell cytoplasmic fractions has been demonstrated to be totally misleading due to hidden errors introduced by the complex interactions of the protease with a cytoplasmic inhibitor of this enzyme. A novel system has been developed which enables the rapid simultaneous quantitative analysis of neutral protease activity and inhibitor activity. This technique employs an insoluble fluorescein-labelled substrate (polymeric collagen fibrils) and relies on the sensitive fluorimetric assay of solubilised fluorescein-labelled peptides. This technique has been termed “incremental analysis” and the advantages of incremental analysis over conventional analysis methods have been described in detail. The experimentally obtained results can readily be resolved graphically or by simple computation.

Evidence for the inhibition of trypsin by thiols. The mechanism of enzyme-inhibitor complex formation.

Ehrlich ascites tumour cells contain a neutral protease, capable of solubilising fluorescein-labelled telopeptides from fluorescein-labelled polymeric collagen fibrils. The cells also contain an inhibitor for this enzyme and for trypsin. The enzymically inactive enzyme-inhibitor complex can be dissociated with the mercurial thiol agent, mersalyl, with the consequent regain of enzymic activity. The reactivated neutral protease and also trypsin can be inhibited by addition of thiols such as cysteine, mercaptoethanol and dithiothreitol. Trypsin can be protected from inactivation by the tumor inhibitor by addition of cystine or L-1-tosylamido-2-phenylethyl chloromethyl ketone(TosPheCH2Cl)-inactivated chymotrypsin. The evidence suggests that the inhibitor contains a reactive thiol group which exchanges with one or more significant disulphide bridges in trypsin and the neutral protease, resulting in enzyme-inhibitor complex formation and loss of activity. Similarly, thiols interact with these enzymes resulting in a corresponding loss of enzymic activity. The evidence obtained with Tos-PheCH2Cl-inactivated chymotrypsin, which reactivated previously inhibited trypsin and neutral protease, demonstrates that the active site of the enzyme is not involved in the interaction with the thiol of the inhibitor but that the significant disulphide bond in the enzyme is required for the maintenance of the active site conformation. This disulphide exchange mechanism is therefore a form of reversible allosteric control of proteolytic activity and has been shown to be distinct from the mechanism by which soya bean trypsin inhibitor interacts with trypsin.

Polymeric collagen fibrils: An example of substrate-mediated steric obstruction of enzymic digestion

Polymeric collagen fibrils have been reacted with fluorescein and rhodamine isothiocyanates to produce fluorescent dye-labelled fibrils, containing seven dye substituents per molecule of tropocollagen within the polymeric collagen fibrils. Two dye-labelled peptides per molecule of tropocollagen were solubilised by trypsin (EC 3.4.21.4) from the telopeptide regions and four dye-labelled peptides were located in the helical regions solubilised by bacterial collagenase (EC 3.4.24.3). The solubilisation of dye-labelled peptides from these insoluble substrates were employed to measure the kinetics of trypsin and collagenase digestion of the telopeptide and helical regions, respectively, of the insoluble polymeric collagen fibrils. These studies demonstrated an apparent excess of enzyme for the readily available substrate under conditions when it was known that a vast excess of substrate existed in the reaction mixture calculated in terms of a molecular ratio. A point of equivalence was established for both trypsin and bacterial collagenase, approximately one enzyme molecule per 870 substrate molecules. On either side of this point the quantity of products formed was controlled by either the enzyme concentration or the substrate concentration. The results can be explained in terms of the inaccessibility of tropocollagen molecules within the molecular architecture of the polymeric collagen fibrils. The external layer of tropocollagen molecules obstruct collagenolytic enzymes penetrating to, and forming enzyme-substrate complexes with, the bulk of the substrate within the interior of the fibrils.

Human-polymorphonuclear-leucocyte neutral protease and its inhibitor. Studies with fluorescein-labelled polymeric collagen fibrils as a substrate.

Human polymorphonuclear leucocytes were obtained from the synovial fluids of patients with inflamed knee joints suffering either from Reiter's syndrome or from rheumatoid arthritis. The polymorphonuclear leucocytes were collected by gentle centrifugation followed by disruption and their subcellular fractionation by centrifugation in 0.34 M sucrose to provide a granule fraction and a post-granule supernatant fraction. 0.5 M KCl extraction of the granule fraction yielded neutral protease activity, similar to trypsin, when assayed against fluorescein-labelled polymeric collagen fibrils. The post-granule supernatant fraction contained an inhibitor towards the neutral protease and trypsin. The inhibition of the neutral protease was found to be time-dependent, this inhibition being released after 1.5-2 h. In contrast, the inhibition of trypsin was irreversible and this property was used to devise an assay procedure for the inhibitor.

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.

A neutral protease in rheumatoid synovial fluid capable of attacking the telopeptide regions of polymeric collagen fibrils

Fluorescent-labelled polymeric collagen fibrils have been prepared which contain three fluorescein residues in the telopeptide regions and four fluorescein residues in the helical region of each tropocollagen unit within the polymer. This material has been used as a substrate for the study of enzymes present in the synovial fluid of inflamed rheumatoid joints which are capable of degrading polymeric collagen fibrils. Two enzyme systems were observed, one inhibited by EDTA and having the properties of the known synovial collagenase, the other having the properties of a neutral protease. The neutral protease was found to be present in sonicates of the polymorphonuclear leucocytes in the synovial fluids of inflamed joints. This enzyme attacked the telopeptides of fluorescein-labelled polymeric collagen fibrils and was similar to trypsin in removing two residues of fluorescein-labelled peptides per tropocollagen molecule within the polymeric collagen fibrils but did not depolymerise the polymeric collagen fibrils.

Insoluble Collagen I.: The Effect of Substrate Weight on the Rate of Solubilisation of Polymeric Collagen Fibrils by Bacterial Collagenase

The enzymic solubilisation of insoluble collagen fibrils by bacterial collagenase has been examined employing unlabelled polymeric collagen fibrils (PC), rhodamine labelled fibrils (RB-PC), and fluorescein labelled fibrils (F-PC) as substrates. The presence of the label did not affect the kinetics of enzyme digestion but enabled the solubilized products to be rapidly estimated. The quantity of substrate used was found to play a major role in determining the quantity of peptides solubilised by a given enzyme concentration. It is suggested that in the PC fibrils only a limited number of TC molecules were suitably located to form an enzyme substrate complex with bacterial collagenase. Conditions were found in which the F-PC and RB-PC could be used as substrates in the assay of this enzyme.

Insoluble Collagen II.: The Use of Fluorescein Labelled Polymeric Collagen Fibrils in a Very Sensitive Assay Procedure for Enzymes Degrading Insoluble Collagen

98% of the collagen in mature connective tissue is in the form of insoluble collagen fibers, consisting of bundles of polymeric collagen (PC) fibrils. The enzymes concerned in connective tissue remodeling degrade PC rather than tropocollagen (TC). TC is the most usual substrate for collagenase assays, and we believe it is essential to employ PC in any study of the activity of collagenolytic enzymes. In order to facilitate the study of enzymic degradation of PC we have labelled PC with fluorescein iso-thiocyanate to produce F-PC fibrils, containing 5 fluorescein labelled epilson-NH2 groups of lysine per TC molecule within the PC. The fluorescent F-PC is degraded at the same rate as PC with the release of hydroxyprolyl peptides but has the great advantage that the solubilised F-peptides can be quantitated by their fluorescent emission. The technique is described in detail employing bacterial collagenase and mammalian collagenase preparations to illustrate the methodology. The advantages of the fluorescent technique over the collagenolytic assay methods currently in use are outlined.

An electron microscopic study of the effect of crude bacterial α-amylase and ethylenediaminetetraacetic acid on human tendon

Crude bacterial α -amylase and ethylenediaminetetraacetic acid act on the interfibrillar matrix of the fiber to weaken the fibril—fibril interaction and allow the constituent fibrils to disperse in dilute acetic acid. The morphology, range of diameter, amino acid composition, carbohydrate content, and stability of the polymeric collagen fibrils thus obtained is identical to that of native fibrils.

Depolymerisation of Human and Bovine Polymeric Collagen Fibrils

Bovine and human polymeric collagen fibrils were incubated at 37°C over a range of pH with a crude lysosomal enzyme preparation obtained from rat liver. Electron microscopic analysis demonstrated depolymerisation of the fibrils over the pH range 3.5 to 7.5 with the cleavage of the fibrils into short segments, followed by unwinding of the protofibrils and cleavage of the protofibrils into protofibrillar particles. The depolymerisation of polymeric collagen to soluble products was followed by hydroxyproline analysis; the pH optimum for this process was approxi- mately 4.0. The soluble depolymerisation products of bovine polymeric collagen fibrils digested at pH 4.0 were found to be native tropocollagen-like molecules which rapidly aggregated at physiological pH to form intermolecularly cross-linked native type fibrils. No soluble hydroxyproline containing products were produced by this enzyme system at physiological pH, although the cleavage of polymeric collagen fibrils could be observed at the electronmic...

The solubilization of mature, polymeric collagen fibrils by lyotropic relaxation

Insoluble bovine corium collagen was dissolved in acid following a lyotropic swelling step at 25° and neutral pH at low ionic strength. The lyotropic swelling apparently relaxes interfibrillar bonding and permits the fibrils to swell without limit in acetic acid. Yields of soluble collagen were on the order of 20% of the total. The use of purified amylase or chelating agents did not increase the yields. Electron microscopic examination and intrinsic viscosity determination showed the soluble collagen to be present as very long thin filaments. CM-cellulose chromatography and disc electrophoresis of the denatured collagen indicated that the solubilized collagen was very highly cross-linked. These data support the concept of the limiting microfibrillar structure of collagen fibers and indicate that one main cross-linking system is entirely internal to the microfibrils.