The effects of dipalmitoyl phosphatidyl choline on the precipitation of native fibrils and segment-long-spacing aggregates from collagen solution

The effect of dipalmitoyl phosphatidyl choline (DPPC), the major phospholipid component of pulmonary surfactant, on the precipitation of collagen in the form of native fibrils and segment-long-spacing (SLS) aggregates was studied in vitro. The effects of DPPC on both phases of collagen fibrillogenesis were analyzed spectrophotometrically, and alterations in the morphology of precipitated fibrils and SLS aggregates were ascertained by transmission electron microscopy (TEM). Low concentrations of DPPC inhibited the growth phase of fibrillogenesis, while higher concentrations were required to inhibit nucleation. Both the meshwork density and mean width of precipitated fibrils were altered by DPPC, as was the size of SLS aggregates. Segment-long-spacing aggregates prepared from pepsin-treated collagen were inhibited to a greater degree than SLS aggregates prepared from untreated collagen, indicating that the pepsin-susceptible residues of the telopeptide extensions of tropocollagen molecules stabilize SLS aggregates against the effects of DPPC. Based on these results and the inhibition of the growth phase at lower concentrations than those which inhibited the nucleation phase of fibrillogenesis, it was concluded that the primary mechanism of DPPC inhibition is electrostatic interference between the positively charged phospholipid molecules and the net positive charge of collagen. It is proposed that pathological conditions involving the pulmonary epithelium may allow interaction between surfactant and collagen, which could further weaken the interstitial connective tissue.     

D-periodic distribution of collagen type IX along cartilage fibrils

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.     

Release of plasminogen activator and a calcium-dependent metalloprotease from cultured sympathetic and sensory neurons

Cultures of neurons from neonatal rat superior cervical, dorsal root, and trigeminal ganglia were grown in the absence of nonneuronal cells in serum-free defined medium. Proteins metabolically labeled with radioactive amino acids and spontaneously released into the culture medium were studied using two-dimensional gel electrophoresis and photofluorography. All three populations of neurons released 12-15 major proteins into the culture medium. Four proteins were released selectively by sympathetic neurons and two proteins were consistently released by both populations of sensory neurons but not by sympathetic neurons. Enzymatic activities are associated with at least two of the released proteins. One is a calcium-dependent metalloprotease, and the other a plasminogen activator. The calcium-dependent metalloprotease has a MW of 62 kDa, requires millimolar calcium for maximum activity, and has a restricted substrate specificity. It degraded native and denatured collagen more readily than casein, albumin, or fibronectin and denatured collagen (gelatin) was a better substrate than native collagen. The plasminogen activator released by neurons has a MW of 51 kDa and is converted to an active 32 kDa form. Its physiochemical properties are similar to urokinase and it was precipitated by a rabbit antiserum produced against human urokinase. A large fraction of both proteases was released by distal processes and/or growth cones suggesting that these proteases could be involved in growth cone functions.     

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.     

Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation.

Types I and III collagens were solubilized from fetal human skin by limited digestion with pepsin and precipitated by dialysis against 0.02 M Na₂HPO₄. Heat denaturation of the collagens in 2 M guanidine-HCl, pH 7.5, resulted in the precipitation of the contaminant pepsin which could be removed by centrifugation. Renaturation of the denatured collagens by dialysis against deionized water at 22° for 2 hours selectively precipitated the type III collagen fibrils. Type I collagen remained in solution. The simplicity and high recovery (77%) make this a suitable approach for the rapid estimation of type III collagen in small tissue samples.     

A role for disulphide bridges in the protein core in the interaction of proteodermatan sulphate and collagen

Proteodermatan sulphate from bovine skin retarded precipitation of fibrils from solutions of purified acid-soluble bovine skin collagen. The isolated protein core was as effective as the intact proteoglycan. Thermal denaturation leading to almost complete loss of the native secondary structure, (determined by circular dichroism spectroscopy to consist of about 60% beta structure) did not diminish the effect unless accompanied by reduction of disulphides, of which there were shown to be three per molecule. The reduced and alkylated protein core was totally ineffective. Electron-microscopy revealed a D-periodic arrangement of glycosaminoglycan on the surfaces of collagen fibrils precipitated in the presence of proteodermatan sulphate. Dermatan sulphate (with attached small peptide) prepared from the proteoglycan, had no effect on the rate of fibrillogenesis and was apparently not bound to the fibrils.     

Characterization of nuclei in in vitro collagen fibril formation.

Heat precipitation fibril formation in collagen solutions depends upon the prior thermal history of the solution. Collagen solutions were heat precipitated to various extents at 30°C, cooled, and then brought to a second precipitation. Kinetic analysis of the secondary precipitation demonstrated that only the nucleation phase of the precipitation was affected, not the fibril growth phase. Thermal history, or memory, is thus related to the formation of low-temperature-stable nuclei. A range of nuclei sizes is evident, supporting the concept of a homogeneous nucleation process. Schiffs base formation and establishment of cross-linkages play no role in the in vitro nucleation: thiosemicarbazide treated collagen behaves identically to untreated collagen in kinetics of assembly to fibrils. Low-temperature-stable nuclei formed at neutral pH are dissociated in the cold in acetic acid at pH 4. Pronase and pepsin susceptible molecular end regions are important in establishing the low-temperature-stable nuclei. Pronase treatment completely abolishes the acquisition of memory of prior thermal history in collagen solutions. We speculate that biological control mechanisms for fibril formation in vivo relate to specific interactions between non-helical, enzyme susceptible regions on collagen molecules.     

Excess K+ and Phorbol Ester Activate Protein Kinase C and Support the Survival of Chick Sympathetic Neurons in Culture

The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum-free culture medium. The protein kinase C activator phorbol 12,13-dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester and NGF did not significantly increase the number of surviving neurons. Phorbol ester-supported sympathetic neurons possessed desipramine-sensitive [3H]-norepinephrine uptake mechanism, and therefore were noradrenegic in character. Two days after the start of cultures, if NGF was replaced by phorbol ester, or phorbol ester was replaced by NGF, the number of surviving sympathetic neurons was essentially the same in both groups, and the uptake of [3H]norepinephrine was also comparable when examined 2 days after the switchover. Interchangeability between phorbol ester and NGF in the survival of sympathetic neurons suggests that both agents act on the same subpopulation of neurons of the chick sympathetic ganglia. The protein kinase C activity of cytosol and particulate fractions of NGF-supported neurons was 0.14 and 0.09 pmol/min/mg protein, respectively. In phorbol ester-supported neurons the activity in the particulate fraction increased by about fivefold. Removal of the phorbol ester after 2 days resulted in restoration of the enzyme activity in less than 1 h, and readdition of the phorbol ester again increased the activity by fivefold. When NGF was added to these neurons (1 microgram for 15 min), there was no change in the enzyme activity. Phorbol 13-acetate was ineffective in supporting sympathetic neurons in culture, as well as in enhancing protein kinase C activity. We also compared the protein kinase C activity of sympathetic neurons supported in culture by NGF and excess potassium (35 mM K+) Neurons supported in culture by 35 mM K+ for 2 days had almost eightfold more protein kinase C activity in their particulate fraction than in cytosol fraction. In NGF-supported neurons were acutely treated with excess K+, the protein kinase C activity was increased in the particulate fraction by about sevenfold in a concentration- and time-dependent manner. Excess K+ plus phorbol ester did not produce an additive effect on protein kinase C activity. PDB and excess K+ had no effect on cyclic AMP content of sympathetic neurons. In summary, the present data suggest that the neurotrophic action of PDB and excess K+ is probably mediated through protein kinase C.     

THE FORMATION OF FIBRILS FROM COLLAGEN SOLUTIONS : IV. Effect of Mucopolysaccharides and Nucleic Acids: An Electron Microscope Study

The kinetics of collagen reprecipitation from solutions of salt-extracted calf dermis in the presence of small amounts of mucopolysaccharide and nucleic acids (0.005 per cent in the final reaction mixture) has been reported by Wood (1960). The present paper is a parallel study using the same materials, and describes the electron microscopic (EM) morphology of the collagen precipitates replicated after 24 hours at room temperature. Satisfactory, uncontaminated EM preparations were obtained which showed that all the deposits were fibrous and bore the 640 A cross-banding characteristic of collagen except some narrow, background fibrils 200 to 1000 A wide precipitated in the presence of heparin. These exhibited fine striations about 220 A apart. Chondroitin sulfate greatly increased the rate of precipitation to give a deposit of low optical density consisting of narrow, rigid, discrete fibrils resembling fresh dermis. In contrast, heparin prevented macroscopic gelation, delayed precipitation, and only produced a scanty deposit of abnormal, short, wide, striated tactoids and compound fibers of varying length. The control preparations and the deposits formed in the presence of hyaluronic acid were intermediate between these two extremes. Delayed precipitation was associated with a coarser deposit and aggregation of the fibrils. A duplicate series of deposits precipitated in the presence of RNA and DNA, together with their controls, were examined after ½, 1, 1½, 3, 9, and 24 hours. One set employed an acetic extract of whole calf dermis and the other salt-extracted dermis. The presence of 0.005 per cent DNA in the reaction mixture markedly delayed collagen precipitation with the slow formation of abnormal, short, wide tactoids and compound fibers. RNA also interfered with the quantity and quality of the deposits which contained far less collagen resembling unfixed, normal, adult human dermis, than the controls at the corresponding time intervals. Comparison of the experiments employing whole calf dermis with those employing the salt-extracted material demonstrated that at every time interval in all the experiments the deposits were retarded when salt-extracted dermis was used. This suggests that the salt-soluble components of the dermis play a part in fiber formation.     

Statistical modeling of ultrastructural features of murine dermal collagen under chronic low-dose whole body X-irradiation

The present study investigated the changes in ultrastructural features of dermal collagen fibrils of mice following exposure to different cumulative chronic low-dose X-irradiation through digital image analysis-based statistical modeling. Pubertal mice were X-irradiated and dorsal skin biopsies were collected and processed for transmission electron microscopic (TEM) analysis. TEM features of collagen fibrils showed alteration in the cross-sectional area, population density and in the axial periodic pattern of light and dark bands. The mathematical analysis of histogram data from TEM images revealed some adaptive behavior in collagen structures of the X-irradiated group. This finding indicated that exposure to chronic low-dose X-radiation induced an altered steady state with adaptive variation in dermal collagen fibrils in irradiated mice.     

Biomimetic Synthesis of Collagen/Nano-Hydroxyapitate Scaffold for Tissue Engineering

This paper reports a precipitation method for the fabrication of compositionally graded biomimetic collagen/nano-hydroxyapatite (HA) composite scaffold. The method is centrifugation based and produce the precipitation of nano-HA crystallites in situ (calcium ions (Ca²⁺) react phosphate ions (PO₄³⁻) and precipitate a non-stoichiometric hydroxyapatite). It was observed that prism needle-like nano-HA crystallites (about 2.5 nm × 3 nm× 25 nm) precipitated on collagen fibrils in the interior of collagen matrix. Chemical and microstructure analysis revealed a gradient of the Ca to P ratio across the width of the scaffold, lead to the formation of a HA-rich side and a HA-deplete side of scaffold. The HA-rich side featured low porosity and agglomerates of the nano-HA crystallites; while HA-depleted side featured higher porosity and nano-HA crystallites integrated with collagen fibrils to form a porous network structure.     

Different behavior of type I and type I-trimer collagen in neutral sodium chloride solutions

Type I and type I-trimer collagen, isolated from ductal infiltrating carcinoma of the human breast, have been tested for their behavior in neutral NaCl solutions. Evident diversities in their rate of precipitation at different saline concentrations have been found, since type I-trimer collagen precipitates at low NaCl molarity while type I collagen is mostly recovered in 2.6-3.6 M NaCl solutions. The native conformation of homotrimer collagen is proved by its ability to produce segment long-spacing crystallites and native-type fibrils.     

Copolymerization of pNcollagen III and collagen I. pNcollagen III decreases the rate of incorporation of collagen I into fibrils, the amount of collagen I incorporated, and the diameter of the fibrils formed

Previous observations suggested that pNcollagen III, the partially processed form of type III procollagen, coats fibrils of collagen I and thereby helps regulate the diameter of fibrils formed by collagen I. The previous observations, however, did not exclude the possibility that pNcollagen III was deposited on preformed collagen I fibrils after the fibrils were assembled. Here, mixtures of pNcollagen III and collagen I were generated simultaneously by enzymatic cleavage of precursor forms of the proteins. The results demonstrated that pNcollagen III forms true copolymers with collagen I. The presence of pNcollagen III both inhibited the rate at which collagen I assembled into fibrils and decreased the amount of collagen I incorporated into fibrils at steady-state equilibrium. In addition, the results demonstrated that copolymerization of pNcollagen III with collagen I generated fibrils that were thinner than fibrils generated under the same conditions from collagen I alone. Increasing the initial molar ratio of pNcollagen III to collagen I in the solution-phase increased the amount of pNcollagen III copolymerizing with collagen I and progressively decreased the diameter of the fibrils. Therefore, the copolymers were heterogeneous in that the stoichiometry of the two monomers in the fibrils varied. The results are consistent with a model in which pNcollagen III can regulate the diameter of collagen I fibrils by coating the surface of the fibrils and thereby allow tip growth but not lateral growth of the fibrils.     

Differential anion effects on thermal stability of collagen in the dispersed and aggregated states (Short Communication)

The effects of KCNS and KI on thermal transition temperatures of calf skin collagen molecules in dilute acid solution and precipitated collagen fibrils from the same source were compared as a function of salt concentration and pH. The two salts produced qualitatively similar effects on each collagen form, but the response shown by single collagen molecules in dilute solution differed from that observed for molecular aggregates present in native-type fibrils.     

A sensitive and rapid method for assaying the activity of type III procollagen amino-terminal proteinase

A rapid assay procedure was developed for measuring the rate of cleavage of the amino-terminal propeptide of type III procollagen. The method was based on the sequential precipitation of type III collagen and uncleaved pN-collagen by 30% ammonium sulfate, while the free amino-terminal propeptide remained in solution and could be further precipitated by 60% ammonium sulfate. Consistently better results were obtained than with the earlier method in which absolute ethanol was used as the precipitant, and selective precipitation was confirmed by polyacrylamide gel electrophoresis of the pellets. The high sensitivity of this method facilitates relatively rapid assays even from small amounts of cultured cells.     

Embryonic avian cornea contains layers of collagen with greater than average stability

A unique morphological feature of the embryonic avian cornea is the uniformity of its complement of striated collagen fibrils, each of which has a diameter of 25 nm. We have asked whether this apparent morphological uniformity also reflects an inherent uniformity of the structural and physical properties of these fibrils. For this we have examined the in situ thermal stability of the type I collagen within these fibrils. Corneal tissue sections were reacted at progressively higher temperatures with conformation-dependent monoclonal antibodies directed against the triple-helical domain of the type I collagen molecule. These studies show that the cornea contains layers of collagen fibrils with greater than average stability. The two most prominent of these extend uninterrupted across the entire width of the cornea, and then appear to insert into thick bundles of scleral collagen, which in turn appear to insert into the scleral ossicles, a ring of bony plates which circumscribe the sclera of the avian eye. Once formed, the bands may act to stabilize the shape of the cornea or, conversely, to alter it during accommodation.     

Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: Comparison of different substrata and tissue culture media formulations

Summary Long-term monolayer cultures of adult rat hepatocytes were tested for their ability to glucuronize phenol red and to maintain initial levels of cell proteins, glucose consumption, and lactic acid production. Lactate dehydrogenase leakage served as an index of culture status because a high value indicates cell death. Three tissue culture (TC) media formulations were the main variables introduced to determine ideal conditions for cell survival in vitro. Investigations of long-term cultures were preceded by studies of hepatocyte attachment to polystyrene surfaces. This attachment was influenced by the amount of substrate deposited and the number of cells seeded, but not by the uniformity of the substrate coating. A statistical analysis of our data revealed that in the absence of fetal bovine serum (FBS), air dried collagen (ADC) and Biomatrix (BMX) were superior to saline precipitated collagen and fibronectin as attachment substrates. In the presence of 10% FBS, all of the substrates performed equally. Chee's Medium (CEM) proved to be the best for preserving cell proteins over a time course of 28 d and Williams' E medium also performed adequately up to 14 d. The glucuronization of phenol red was at 50% of initial values at Day 7 in CEM-ADC hepatocytes in contrast to 30% for cells in Williams' E medium and 5% for cells grown in Waymouth's. At 14 d glucuronization was still present at 40% of original values in CEM-ADC cells but had ceased in the other two media. When BMX was used, none of the TC media supported glucuronization levels comparable to ADC cells. This research was supported in part by grant 1R01-AM-26520 from the National Institute of Arthritis, Diabetes and Digestive Kidney Diseases, NIH, Bethesda, Maryland.     

The formation and thermal stability of in vitro assembled fibrils from acid-soluble and pepsin-treated collagens.

The role of the non-helical regions of the collagen molecule in fibrillogenesis has been investigated by comparing the kinetics of fibril formation of pepsin-treated acid-soluble collagen, acid-soluble collagen and mixtures of the two and by comparison of the thermal stabilities of the fibrils formed. The acid-soluble collagen was found to aggregate more rapidly than the pepsin-treated collagen under physiological conditions of pH and ionic strength. Variations in ionic strength, at physiological pH, were found to have differing effects on the aggregation of these two forms of soluble collagen. Fibrils formed from the pepsinized-collagen had a lower thermal stability tha n those formed from the intact collagen. The behavior observed with mixtures of acid-soluble and pepsin-treated collagens was found to be quantitatively consistent with the pepsinized collagen being able to utilize the nuclei formed by the acid-soluble collagen for subsequent growth. However, the use of the acid-soluble nuclei by the pepsinized collagen for growth did not enhance its rate of precipitation during the growth phase, nor did it enhance the thermal stability of the fibrils formed from the pepsinized collagen.     

Oblique banding pattern in collagen fibrils reconstituted in vitro after trypsin treatment.

Collagen fibres from rat tail tendon suspended in small pieces in a solution (pH 7.8) containing 0.5 M CaCl2 were treated with purified bovine trypsin at 20 degrees C for 20 h. After the enzyme treatment collagen from this solution was precipitated out and reconstituted in vitro into native-type fibrils. The banding pattern in these reconstituted fibrils was found to be oblique. This is comparable to that observed recently in fibrils reconstituted from cartilage collagen. On the other hand, normal transverse banding pattern was observed in the fibrils reconstituted in vitro from collagen solution of rat tail tendon which was not pre-treated with trypsin. No significant change was, however, observed in the segment long spacing fibrils precipitated from the enzyme-treated collagen solution. It is possible that the enzyme might affect the mode of organization of tropocollagen molecules during in vitro fibrillogenesis into native-type fibrils either by interacting with the "telopeptide" regions or with the non-collagenous components associated with the native protein and this could probably result into the formation of fibrils with oblique banding pattern.     

A study of the growth of normal and iodinated collagen fibrils in vitro using electron microscope autoradiography

Electron microscopic autoradiographic observations on collagen fibrils grown in vitro allow growth rates in the N- and C-terminal directions to be measured on individual fibrils. Such observations, made on normal and iodinated collagen, show that normal fibrils grow at both ends (although rather more rapidly at the N-terminal end), whereas fully-iodinated collagen fibrils grow only at the N-terminal end. Measurements of growth rates at different temperatures provide estimates of the activation enthalpy (ΔH^≠) and entropy (ΔS^≠) of precipitation for the two types of collagen. Solubility measurements have also yielded values for the thermodynamic enthalpy (ΔH) and entropy (ΔS) of precipitation. Results show that the activated (rate-limiting) state is characterized by a large positive ΔH^≠ and ΔS^≠ similar in magnitude to the ΔH and ΔS of transition from solution to fibril. It is also concluded that the different rates of precipitation of normal and iodinated collagen cannot be explained in terms of fibril formation requiring ionization of the tyrosine residues.