A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles

The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from − 8 to − 10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Influence of salt on the structure of DMPG studied by SAXS and optical microscopy

Aqueous dispersions of 50 mM dimyristoylphosphatidylglycerol (DMPG) in the presence of increasing salt concentrations (2-500 mM NaCl) were studied by small angle X-ray scattering (SAXS) and optical microscopy between 15 and 35 °C. SAXS data show the presence of a broad peak around q ∼ 0.12 Å^− 1 at all temperatures and conditions, arising from the electron density contrasts within the bilayer. Up to 100 mM NaCl, this broad peak is the main feature observed in the gel and fluid phases. At higher ionic strength (250-500 mM NaCl), an incipient lamellar repeat distance around d = 90-100 Å is detected superimposed to the bilayer form factor. The data with high salt were fit and showed that the emergent Bragg peak is due to loose multilamellar structures, with the local order vanishing after ∼ 4d. Optical microscopy revealed that up to 20 mM NaCl, DMPG is arranged in submicroscopic vesicles. Giant (loose) multilamellar vesicles (MLVs) start to appear with 50 mM NaCl, although most lipids are arranged in small vesicles. As the ionic strength increases, more and denser MLVs are seen, up to 500 mM NaCl, when MLVs are the prevailing structure. The DLVO theory could account for the experimentally found interbilayer distances.     

Interactions of inorganic phosphate and sulfate anions with collagen

We use direct infrared measurements to determine the number of binding sites, their dissociation constants, and preferential interaction parameters for inorganic phosphate and sulfate anions in collagen fibrils from rat tail tendons. In contrast to previous reports of up to 150 bound phosphates per collagen molecule, we find only 1-2 binding sites for sulfate and divalent phosphate under physiological conditions and approximately 10 binding sites at low ionic strength. The corresponding dissociation constants depend on NaCl concentration and pH and vary from approximately 50 microM to approximately 1-5 mM in the physiological range of pH. In fibrils, bound anions appear to form salt bridges between positively charged amino acid residues within regions of high excess positive charge. In solution, we found no evidence of appreciable sulfate or phosphate binding to isolated collagen molecules. Although sulfate and divalent phosphate bind to fibrillar collagen at physiological concentrations, our X-ray diffraction and in vitro fibrillogenesis experiments suggest that this binding plays little role in the formation, stability and structure of fibrils. In particular, we demonstrate that the previously reported increase in the critical fibrillogenesis concentration of collagen is caused by preferential exclusion of "free" (not bound to specific sites) sulfate and divalent phosphate from interstitial water in fibrils rather than by anion binding. Contrary to divalent phosphate, monovalent phosphate does not bind to collagen. It is preferentially excluded from interstitial water in fibrils, but it has no apparent effect on critical fibrillogenesis concentration at physiological NaCl and pH.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Structural characteristics of alpha-synuclein oligomers stabilized by the flavonoid baicalein

The flavonoid baicalein inhibits fibrillation of α-synuclein, which is a major component of Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of α-synuclein oligomers and consequently prevents their fibrillation. In order to evaluate the structural properties of baicalein-stabilized oligomers, we purified oligomer species by HPLC and examined their stability and structure by CD, Fourier transform infrared spectroscopy, size exclusion chromatography HPLC, small-angle X-ray scattering, and atomic force microscopy. Baicalein-stabilized oligomers are β-sheet-enriched according to CD and Fourier transform infrared spectroscopy analyses. They did not form fibrils even after very prolonged incubation. From small-angle X-ray scattering data and atomic force microscopy images, the oligomers were characterized as quite compact globular species. Oligomers were extremely stable, with a GdmCl C_m = 3.3 M. This high stability explains the previously observed inhibition properties of baicalein against α-synuclein fibrillation. These baicalein-stabilized oligomers, added to the solution of aggregating α-synuclein, were able to noticeably inhibit its fibrillation. After prolonged coincubation, short fibrils were formed, suggesting an effective interaction of oligomers with monomeric α-synuclein. Membrane permeability tests suggested that the baicalein-stabilized oligomers had a mild effect on the integrity of the membrane surface. This effect was rather similar to that of the monomeric protein, suggesting that targeted stabilization of certain α-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.     

Darcy permeability of agarose-glycosaminoglycan gels analyzed using fiber-mixture and donnan models

Agarose-glycosaminoglycan (GAG) membranes were synthesized to provide a model system in which the factors controlling the Darcy (or hydraulic) permeability could be assessed in composite gels of biological relevance. The membranes contained a GAG (chondroitin sulfate) that was covalently bound to agarose via terminal amine groups, and the variables examined were GAG concentration and solution ionic strength. The addition of even small amounts of GAG (0.4 vol/vol %) resulted in a twofold reduction in the Darcy permeability of 3 vol/vol % agarose gels. Electrokinetic coupling, caused by the negative charge of the GAG, resulted in an additional twofold reduction in the open-circuit permeability when the ionic strength was decreased from 1 M to 0.01 M. A microstructural hydrodynamic model was developed, based on a mixture of neutral, coarse fibers (agarose fibrils), and fine, charged fibers (GAG chains). Heterogeneity within agarose gels was modeled by assuming that fiber-rich, spherical inclusions were distributed throughout a fiber-poor matrix. That model accurately predicted the Darcy permeability when the ionic strength was high enough to suppress the effects of charge, but underestimated the influence of ionic strength. A more macroscopic approach, based on Donnan equilibria, better captured the reductions in Darcy permeability caused by GAG charge.     

Chymotrypsin–poly vinyl sulfonate interaction studied by dynamic light scattering and turbidimetric approaches

The formation of non-soluble complexes between a positively charged protein and a strong anionic polyelectrolyte, chymotrypsin, and poly vinyl sulfonate, respectively, was studied under different experimental conditions such as pH (1–3.5), protein concentration, temperature, ionic strength, and the presence of anions that modifies the water structure. Turbidimetric titration and dynamic light scattering approaches were used as study methods. When low protein–polyelectrolyte ratio was used, the formation of a soluble complex was observed. The increase in poly vinyl sulfonate concentration produced the interaction between the soluble complex particules, thus inducing macro-aggregate formation and precipitation. Stoichiometry ratios of 500 to 780 protein molecules were found in the precipitate per polyelectrolyte molecule when the medium pH varied from 1.0 to 3.5. The kinetic of the aggregation process showed to be of first order with a low activation energy value of 4.2 ± 0.2 kcal/mol. Electrostatic forces were found in the primary formation of the soluble complex, while the formation of the insoluble macro aggregate was a process driven by the disorder of the ordered water around the hydrophobic chain of the polymer.     

Structural Insights into Yeast DNA Polymerase δ by Small Angle X-ray Scattering

DNA polymerase δ (Polδ) is a multisubunit polymerase that plays an indispensable role in replication from yeast to humans. Polδ from Saccharomyces cerevisiae is composed of three subunits: Pol3, Pol31, and Pol32. Despite the elucidation of the structures and models of the individual subunits (or portions, thereof), the nature of their assembly remains unclear. We present here a small-angle X-ray scattering analysis of a yeast Polδ complex (Polδ_T) composed of Pol3, Pol31, and Pol32N (amino acids 1-103 of Pol32). From the small angle X-ray scattering global parameters and reconstructed envelopes, we show that Polδ_T adopts an elongated conformation with a radius of gyration (R_g) of ∼ 52 Å and a maximal dimension of ∼ 190 Å. We also propose an orientation for the accessory Pol31-Pol32N subunits relative to the Pol3 catalytic core that best agrees with the experimental scattering profile. The analysis also points to significant conformational variability that may allow Polδ to better coordinate its action with other proteins at the replication fork.     

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

The aggregation process of wild-type human lysozyme at pH 3.0 and 60 °C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the β-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.     

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.     

Thermodynamic and structural characteristics of collagen fibrils formed in vitro at different temperatures and concentrations

Analysis of the results of calorimetric study of reconstituted collagen (type I) fibrils, in particular, the half-width of the temperature transition, shows that the collagen packing density in the fibrils and the size of cooperative blocks therein depend on the assembly temperature and on the initial collagen concentration. The least dense fibrils are formed at subphysiological temperatures (25° or 30°C) and low concentration (0.3 mg/ml). The extent of ordering does not change upon doubling the concentration but increases upon quadrupling it. At physiological temperature (35°C) the fibrils are densely packed regardless of collagen concentration. The enthalpy of fibril assembly is minimal at 35°C, 1.2 mg/ml, and ionic strength of 0.17 M. The influence of temperature on particular steps of fibrillogenesis and the role of water in these processes are discussed.     

Fibrillogenesis in ADan peptides is inhibited by biphenyl ethers

In this study, biphenyl ethers of diverse functionality were used to assess their effect on fibrillogenesis of both the oxidized and reduced ADan peptides, in vitro. It was noted that these compounds not only stalled fibrillogenesis but were also able to disrupt pre-formed fibers. The EC₅₀ values for the inhibition of this process lie in the nanomolar range for 50 μM of peptide concentration, indicating the high potency of these compounds as inhibitors. It was found that these compounds impart to the peptides, an α-helical conformation which does not allow them to aggregate and form fibrils. These studies also point out that the transition of peptides through α-helical conformation may be a prelude to the onset of fibrillogenesis for oxADan peptides.     

Hinge stiffness is a barrier to RNA folding

Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg^2 +-mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4-P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4-P6 folding was followed by global and local measures as a function of Mg^2 + concentration. While all concentrations of Mg^2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg^2 + increases; collapse is greatly accelerated by Mg^2 +, while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg^2 +, a stiff hinge limits the rate of P4-P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formation.     

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D_phys, and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R_g) of the guanidine unfolded state (U) is 26.9 ± 0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R_g value extrapolated from kinetic R_g data to time zero, R_g,0, is 24.3 ± 0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R_g² was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R_g of the minor component of D_phys containing the non-native cis proline isomer (D_phys,cis) to be 25.7 ± 0.6 Å. Moreover, R_g of the major component of D_phys containing the native proline isomer (D_phys,tra) was estimated as 23.9 ± 0.2 Å based on R_g,0. Consequently, both components of the burst-phase intermediate of barnase (D_phys,tra and D_phys,cis) are still largely expanded. It was inferred that D_phys possesses the N-terminal helix and the center of the β-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.     

The ionic strength effect on the DNA complexation by DOPC - gemini surfactants liposomes

Liposome dispersions obtained from the mixture of gemini surfactants of the type alkane-α,ω-diyl-bis(alkyldimethylammonium bromide) and helper lipid DOPC create complexes with DNA showing a regular inner microstructure, identified by small angle X-ray diffraction as condensed lamellar phase (L_α^c). In addition to the L_α^c phase, a coexisting lamellar phase L_B was also identified in the complexes formed, with periodicities in the range ~ 8.8-5.7 nm, at ionic strengths corresponding to 50-200 mM NaCl. The periodicities of L_B phase did not correspond to those identified in liposome dispersion without DNA using small angle neutron scattering. The observed phase separation is shown to depend on the interplay between the surface charge density of cationic liposomes, ionic strength and method of complex preparation. The effect of ionic strength on complex formation was studied by isothermal titration calorimetry and zeta potential measurements. High ionic strength reduces the fraction of bound DNA in the complexes, and the isoelectric point is attained at a ratio of DNA/gemini surfactant which is lower than the one that can be estimated by calculation based on nominal charges of CLs and DNA.     

Detection of intermediates and kinetic control during assembly of bacteriophage P22 procapsid

Bacteriophage P22 serves as a model for the assembly and maturation of other icosahedral double-stranded DNA viruses. P22 coat and scaffolding proteins assemble in vitro into an icosahedral procapsid, which then expands during DNA packaging (maturation). Efficient in vitro assembly makes this system suitable for design and production of monodisperse spherical nanoparticles (diameter ≈ 50 nm). In this work, we explore the possibility of controlling the outcome of assembly by scaffolding protein engineering. The scaffolding protein exists in monomer-dimer-tetramer equilibrium. We address the role of monomers and dimers in assembly by using three different scaffolding proteins with altered monomer-dimer equilibrium (weak dimer, covalent dimer, monomer). The progress and outcome of assembly was monitored by time-resolved X-ray scattering, which allowed us to distinguish between closed shells and incomplete assembly intermediates. Binding of scaffolding monomer activates the coat protein for assembly. Excess dimeric scaffolding protein resulted in rapid nucleation and kinetic trapping yielding incomplete shells. Addition of monomeric wild-type scaffold with excess coat protein completed these metastable shells. Thus, the monomeric scaffolding protein plays an essential role in the elongation phase by activating the coat and effectively lowering its critical concentration for assembly.     

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg²⁺, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s^− 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I_C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.     

Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7 days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7 days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7 days.