Morphological characterization of tendon development during chick embryogenesis: measurement of birefringence retardation

Morphological observations and physical measurement of (I) birefringence retardation, (2) mean fibre profile width, and (3) cell volume fraction were used to characterize chick hind limb extensor tendon development. Observations were made at days 7, 10, 14 and 17 embryologic and 1-1.5 post-hatching. Microanatomical observations illustrated a sequential development of tendon microanatomy consisting of (1) a uniaxial cellular framework with discontinuous collagen fibril bundles present in day 7 embryos; (2) a continuous network of birefringent collagen fibres, and early evidence of tendon fasciculation and crimp development by embryonic day 10; and (3) completion of the basic cytoarchitecture of tendon observed at day 14 of embryogenesis. These observations suggest that collagen deposition in tendon involves first a longitudinal and then a lateral organization of tendon fibroblasts. Associated with the progressive anatomical development of tendon was an increase in birefringence retardation, mean collagen fibre profile width, and a decrease in the cell volume fraction. Birefringence retardation per unit thickness, however, did not change. This suggested that the fibril packing density of the fibres remained constant, although the fibres were observed to increase in size. These results indicate that collagen fibrillogenesis in vivo can be quantitatively studied by measurement of the birefringence retardation using polarized light.     

Mechanism of in vitro collagen fibril assembly. Kinetic and morphological studies

The kinetics of in vitro fibril assembly of Type I collagen preparations that contain different amounts of covalently cross-linked oligomers was studied with turbidimetry. Fibril formation showed a lag phase with no solution turbidity and a growth phase with a sigmoidal increase in the solution turbidity. The length of the lag phase was inversely related to both the total collagen concentration and the amount of covalently cross-linked oligomers in the solution. Double logarithmic plots of t1/4, the amount of time it takes for 1/4 of the collagen to assemble into fibrils, versus the total collagen concentration were linear but the slope decreased from -0.84 to -2.3 with decreasing amounts of covalently cross-linked oligomers in the samples. Electron microscopy showed the formation of unbanded microfibrils with diameters in the range of 3-15 nm early in the lag phase and larger diameter banded fibrils coexisting with the microfibrils near the end of the lag phase. Centrifugation of the solution at the lag phase prolonged the lag time, presumably by removal of microfibrils, but subsequent growth of the fibrils was unaffected. The results suggest a cooperative nucleation-growth mechanism for the in vitro assembly of collagen fibrils which is consistent with the results of an equilibrium study of the fibril assembly reaction we reported earlier (Na, G. C., Butz, L. J., Bailey, D. G., and Carroll, R. J. (1986) Biochemistry 25, 958-966).     

Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules

Turbidity measurements have been used to study the in vitro assembly and disassembly of porcine neurotubules. All measurements were carried out with tubulin with a purity higher than 80%. Tubules formed by in vitro assembly of this protein are so long that the turbidity is insensitive to length and is a function only of the total mass of high molecular weight material. Porcine tubulin shows a critical concentration for assembly of about 0.2 mg/ml under optimal conditions, pH 6.6, 0.1m-2-(N-morpholino)ethane sulfonic acid, 26 to 37 °C. Under these conditions assembly and disassembly are essentially fully reversible in the presence of excess GTP. The kinetics of assembly show an initial lag and initial rates which are strongly temperature dependent. Our samples show a concentration dependence of no more than second order. The apparent activation enthalpy of assembly is 25 kcal/mol; the apparent reaction enthalpy of assembly for the chain propagation step is 21 kcal/mol. Disassembly kinetics show an apparent negative activation enthalpy of ?28 kcal/mol. They are independent of tubule length implying a slow activation step followed by rapid depolymerization. At 20 °C, cycles of polymerization and depolymerization show hysteresis effects in the assembly kinetics though not in disassembly rates or final states. This is most easily explained by postulating a slow reversible inactivation at 4 °C of the initiation complex for tubule assembly. Conditions are reported for producing tubulin in a state which cannot assemble in aqueous buffer unless nucleotides are added. GTP, ATP and ADP, but not GDP, are effective in promoting tubule assembly. An adenylate kinase impurity in our preparation may be the cause of this unusual effect. Whether or not it is actually associated with tubulin or tubules is unknown.     

The mechanism of nucleation for in vitro collagen fibril formation.

The formation of collagen fibrils from soluble monomers and aggregates by thermal gelation at neutral pH can be divided into two distinct stages: a nucleation phase and a growth phase. Turbidity studies of the kinetics of the precipitation reaction show that the lag-phase time or nucleation reaction time, t′_l, is markedly temperature dependent while the growth reaction time is temperature independent. The activation energy of the nucleation reaction is essentially constant over the temperature range studied. In monitoring the nucleation-phase reaction by various physicochemical techniques, including viscosity, sedimentation equilibrium, and light scattering, no evidence for the formation of aggregates was observed. Enrichment of the initial collagen solution with aggregates accelerates nucleation, but de novo nuclei formation is still required even in highly aggregated collagen preparations. Removal of pepsin and pronase susceptible peptides lengthens the nucleation reaction time and increases the sensitivity of the rate of nuclei formation to changes in ionic strength. Electron microscope studies show the fibrils formed from the protease-treated collagen to be less well organized. With pepsin-treated collagen, subfibrils and obliquely striated fibrils are seen, showing that while microfibrils are formed interactions between them are modulated by the enzyme susceptible peptides in the same way that these regions modulate nuclei assembly. It appears that pepsin and pronase susceptible peptide regions of collagen play a more prominent role in the in vitro assembly of collagen molecules to form D-stagger nuclei and fibrils than do ionic interactions between helical molecular regions. A mechanism of nucleation of collagen fibrillogenesis is discussed.     

Fibrin assembly after fibrinopeptide A release in model systems and human plasma studied with magnetic birefringence.

Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by the action of the snake-venom-derived enzymes reptilase and ancrod. A range of conditions was investigated. Fibrin polymerization in solutions of purified fibrinogen shows a distinct break near the gelation point. On addition of Ca2+ or albumin the lag period is shortened, fibre thickness is increased and the break in assembly almost vanishes, probably because both of these additives promote lateral aggregation. There are minor differences in the kinetics, depending on the venom enzyme used. The kinetics of fibrin assembly in model systems containing either Ca2+ or albumin and in human plasma with a largely dormant coagulation cascade are very similar. Therefore in the latter condition there is no significant alteration in the assembly process due to interaction between fibrin or the venom enzymes and any of the plasma proteins. When the cascade is activated, the polymerization progress curves have a character that resembles a combination of the reactions observed when the venom enzymes and endogenously generated thrombin separately induce coagulation, except for a region near gelation where, paradoxically, polymerization appears to be slower on activation. The low-angle neutron-diffraction patterns from oriented gels made with thrombin or reptilase are identical. Therefore at low resolution the packing of the monomers within fibres is the same when fibrinopeptide A only or both fibrinopeptides A and B are removed.     

Monomer and oligomer of type I collagen: molecular properties and fibril assembly

Type I collagen purified from calf skin was further separated into monomeric and oligomeric fractions and characterized with gel electrophoresis and measurement of solution viscosity. The thermal stabilities of the triple-helical structure of the collagen molecules of these preparations and the fibrils assembled therefrom were determined with differential UV spectroscopy and scanning microcalorimetry. The monomeric collagen was reduced with NaBH4-, and the kinetics and equilibrium of the reversible fibril assembly-disassembly were examined in detail. Fibril assembly and disassembly of the collagen induced by slow scans of temperature showed hysteresis. The assembly curve was very sharp whereas the disassembly curve was gradual. Equilibrium centrifugation showed the collagen disassembled from the fibrils to be predominantly monomers. However, unlike the unassembled collagen, the collagen disassembled from fibrils by cooling showed no lag phase in subsequent cycles of fibril assembly. The thermodynamic parameters of fibril growth were derived from a fibril disassembly curve. Fibril growth was weaker for the NaBH4-reduced monomeric collagen than the native crude collagen, perhaps due to the removal of oligomers and the changes in the molecular structure brought by the reduction. The results corroborated the strongly cooperative mechanism for the fibril assembly proposed in the preceding paper.     

Effect of pig faecal donor and of pig diet composition on in vitro fermentation of sugar beet pulp

Two experiments were undertaken to investigate the influence of (1) pig bodyweight and (2) dietary fibre content of the diet on the in vitro gas production of sugar beet pulp fibre using faecal inoculum.In the first experiment, inocula prepared from young pigs (Y; 16–50 kg), growing pigs (G; 62–93 kg) and sows (S; 216–240 kg) were compared. Sugar beet pulp, hydrolysed in vitro with pepsin and then pancreatin, was used as the fermentation substrate. The cumulated gas productions over 144 h were modelled and the kinetics parameters compared. Lag times (Y: 4.6 h; G: 6.4 h; S: 9.2 h) and half-times to asymptote (Y: 14.7 h; G: 15.9 h; S: 20.8 h) increased with pig bodyweight (P<0.001) and the fractional degradation rates of the substrate differed between the pig categories (Y: 0.110 h⁻¹; G: 0.115 h⁻¹; S: 0.100 h⁻¹; P<0.001). The final gas production was not affected (P=0.10) by the inoculum source.In the second experiment hydrolysed sugar beet pulp was fermented with four inocula prepared from pigs fed diets differing in their total and soluble dietary fibre contents, i.e. low fibre diet rich in soluble fibre (LOW-S) or in insoluble fibre (LOW-I) or high fibre diet rich in soluble fibre (HIGH-S) or in insoluble fibre (HIGH-I). The total and the soluble dietary fibres influenced the kinetics of gas production. The presence of soluble fibres decreased the lag times, whatever the total dietary fibre content (2.7 h for LOW-S versus 3.5 h for LOW-I, 4.0 h for HIGH-S versus 4.4 h for HIGH-I; P<0.001). The half-times to asymptote were higher with the low fibre diets (P<0.001) and, for similar total dietary fibre contents, they were lower when the proportion of soluble fibres increased (LOW-S: 9.9 h; LOW-I: 11.4 h; HIGH-S: 8.9 h; HIGH-I: 10.1 h; P<0.001). The fractional degradation rates of the substrate were the highest with the fibre-rich diet containing a high proportion of soluble fibres (0.158 h⁻¹; P<0.001).In conclusion, the bodyweight of the faeces donors and the dietary fibre composition of the pig diet influence the in vitro fermentation kinetics of hydrolysed sugar beet pulp, but not the final gas production.     

3D Non-Woven Polyvinylidene Fluoride Scaffolds: Fibre Cross Section and Texturizing Patterns Have Impact on Growth of Mesenchymal Stromal Cells

Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds.     

Assembly of type I collagen fibrils de novo. Between 37 and 41 degrees C the process is limited by micro-unfolding of monomers

The effects of temperature on the assembly of collagen fibrils were examined in a system in which collagen monomers are generated de novo and in a physiological buffer by specific enzymic cleavage of type I pC-collagen, an intermediate in the normal processing of type I procollagen to type I collagen. Increasing the temperature of the reaction in the range of 29-35 degrees C decreased the turbidity lag and increased the rate of propagation as assayed by turbidity. The effect of temperature on the turbidity propagation rate gave a linear Arrhenius plot with a negative slope. The predicted value of the activation energy of propagation was 113 kJ/mol. However, the effects of temperature on the rate of assembly above 37 degrees C were opposite to the effects seen at temperatures below 37 degrees C. In the range of 37-41 degrees C, the turbidity propagation rate decreased markedly with temperature. Also, the turbidity lag increased. Therefore, much longer times were required for monomers to reach equilibrium with fibrils. A large fraction of the collagen monomers remaining in solution at temperatures above 37 degrees C was sensitive to rapid digestion by trypsin and alpha-chymotrypsin. Cooling the solutions to 25 degrees C made the monomers resistant to protease digestion. The results are consistent with the conclusion that, although formation of collagen fibrils is a classical example of an entropy-driven process of self-assembly, the rate of assembly between 37 and 41 degrees C is limited by reversible micro-unfolding of the monomer.     

Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases

A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.     

The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae,Nematomorpha): observations by TEM,SEM and AFM

The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cuticle is composed of 30-50 compact layers. The number of the layers is higher in the central part of the animal's body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately 640 nm in diameter and of indefinite length. The fibres run strictly parallel within each layer, while in adjoining layers they run at a variable angle from 45 degrees in the central body to 90 degrees in the extremities. Each fibre shows a barely detectable filamentous inner structure and is enveloped in a thin highly regular net formed by hexagonal meshes. Our results suggested that these fibres should be proteinaceous although non-collagenous. Thinner radial fibres run among the large fibres and across all the layers and span the whole thickness of the cuticle from the epithelial layer located deep underneath the large fibres up to the epicuticle on the external surface of the animal.     

Increase in Survival of Subcultured Fibroblasts mediated by Serotonin

WE have already reported a cytospecific effect of serotonin in enhancing the growth of fibroblasts in vitro¹, apparently by shortening the lag phase. We have now investigated this effect and the kinetics of cell growth in order to provide information about the mechanism of action of serotonin.     

Early Aggregation Steps in α-Synuclein as Measured by FCS and FRET: Evidence for a Contagious Conformational Change

The kinetics of aggregation of α-synuclein are usually studied by turbidity or Thio-T fluorescence. Here we follow the disappearance of monomers and the formation of early oligomers using fluorescence correlation spectroscopy. Alexa488-labeled A140C-synuclein was used as a fluorescent probe in trace amounts in the presence of excess unlabeled α-synuclein. Repeated short measurements produce a distribution of diffusion coefficients. Initially, a sharp peak is obtained corresponding to monomers, followed by a distinct transient population and the gradual formation of broader-sized distributions of higher oligomers. The kinetics of aggregation can be followed by the decreasing number of fast-diffusing species. Both the disappearance of fast-diffusing species and the appearance of turbidity can be fitted to the Finke-Watzky equation, but the apparent rate constants obtained are different. This reflects the fact that the disappearance of fast species occurs largely during the lag phase of turbidity development, due to the limited sensitivity of turbidity to the early aggregation process. The nucleation of the early oligomers is concentration-dependent and accompanied by a conformational change that precedes β-structure formation, and can be visualized using fluorescence resonance energy transfer between the donor-labeled N-terminus and the acceptor-labeled cysteine in the mutant A140C.     

Collagen fibrillogenesis in vitro: an investigation of the thermal memory effect and of the early events occurring during fibril assembly using dynamic light scattering

Dynamic light scattering has been used to characterize a variety of lathyritic rat skin collagen solutions. The technique was used to monitor the onset of fibril assembly in vitro and to investigate the thermal memory effect. Although the incorporation of thermal memory was demonstrated by reheating the sample and subsequently observing a shortened turbidimetric lag phase, no significant differences between naive solutions and ones exhibiting thermal memory could be detected using photon correlation spectroscopy. This suggests that subtle changes in the state of the collagen molecules rather than extensive changes in the degree of aggregation are responsible for the thermal memory effect. During fibrillogenesis, no large-scale changes in the distribution of monomers or aggregates occur until near the end of the lag phase.     

AN ANALYSIS OF COLLAGEN SECRETION BY ESTABLISHED MOUSE FIBROBLAST LINES

In vitro synthesis of collagen by established mouse fibroblast lines has been examined by electron microscopy. During rapid growth (log phase), when collagen could not be detected in the cultures, the cells lacked a well developed granular ergastoplasm and Golgi system. Upon cessation of growth (stationary phase), collagen accumulated in the cultures and the cells demonstrated highly developed granular and smooth ergastoplasm. Collagen appeared to be synthesized in the rough-surfaced endoplasmic reticulum and to be transported as a soluble protein to the cell surface by vesicular elements of the agranular ergastoplasm. Fusion of the limiting membranes of these vesicles with the cell membrane permitted the discharge of the soluble collagen into the extracellular space, where fibrils of two diameter distributions formed. The secretion of collagen is concluded to be of the merocrine type. Alternative theories of collagen secretion are discussed and the data for established lines compared with the results of other in vitro and in vivo studies of collagen fibrillogenesis.     

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.     

Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy

To establish an optimal method for analysis of the collagen structures from unstained tissue sections, a computerized image analysis system using a charge coupled device camera coupled to a polarizing light microscope was used. Retardation values of birefringence, which are proportional to the content and fibril orientation of collagen in the extracellular matrix of articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular cartilage, the highest retardation values were recorded from sections cut parallel to the so-called split lines indicating the anisotropic arrangement of collagen. Complete digestion of glycosaminoglycans reduced the retardation value by approximately 6.0%, suggesting a minor, but not insignificant, contribution of glycosaminoglycans to the birefringence of the matrix. The use of a mounting medium with a refractive index close to that of the collagen (e.g. DPX) increased the specificity of the method, since the optical anisotropy of collagen derives predominantly from the intrinsic (structural) birefringence. In conclusion, analysis of unstained sections after careful removal of paraffin and glycosaminoglycans from the tissues provides a sensitive and rapid quantitative assessment of oriented collagen structures in articular cartilage     

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.     

Retroviral Capsid Assembly: A Role for the CA Dimer in Initiation

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.