Adherence,proliferation and collagen turnover by human fibroblasts seeded into different types of collagen sponges

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted non-crosslinked collagen, (2) reconstituted collagen that was chemically crosslinked with either glutaraldehyde, aluminium alginate or acetate, and (3) native collagen fibres, with or without other extracellular matrix molecules (elastin hydrolysate, hyaluronic acid or fibronectin). The non-crosslinked reconstituted collagen was degraded rapidly by human fibroblasts. Teh chemically crosslinked materials proved to be cytotoxic. Native collagen fibres were stable. In the absence of ascorbic acid, the addition of elastin hydrolysate to this type of matrix reduced the rate of collagen degradation. Both elastin hydrolysate and fibronectin partially prevented fibroblast-mediated contraction. Hyaluronic acid was only slightly effective in reducing the collagen degradation rate and more fibroblast-mediated contraction of the material was found than for the native collagen fibres with elastin hydrolysate and fibronectin. In the presence of ascorbate, collagen synthesis was enhanced in the native collagen matrix without additions and in the material containing elastin hydrolysate, but not in the material with hyaluronic acid. These results are indicative of the suitability of tissue substitutes for in vivo application.     

A simplified approach to quasi-linear viscoelastic modeling

The fitting of quasi-linear viscoelastic (QLV) constitutive models to material data often involves somewhat cumbersome numerical convolution. A new approach to treating quasi-linearity in 1-D is described and applied to characterize the behavior of reconstituted collagen. This approach is based on a new principle for including nonlinearity and requires considerably less computation than other comparable models for both model calibration and response prediction, especially for smoothly applied stretching. Additionally, the approach allows relaxation to adapt with the strain history. The modeling approach is demonstrated through tests on pure reconstituted collagen. Sequences of "ramp-and-hold" stretching tests were applied to rectangular collagen specimens. The relaxation force data from the "hold" was used to calibrate a new "adaptive QLV model" and several models from literature, and the force data from the "ramp" was used to check the accuracy of model predictions. Additionally, the ability of the models to predict the force response on a reloading of the specimen was assessed. The "adaptive QLV model" based on this new approach predicts collagen behavior comparably to or better than existing models, with much less computation.     

Thermal stability,mechanical properties and reducible cross-links of rat tail tendon in experimental diabetes

Thermal stability (measured as isometric contraction force), biomechanical properties and reducible cross-links were measured in tail tendons from streptozotocin diabetic rats, with and without insulin treatment. After 10 days of diabetes the maximum thermal contraction force was unchanged, but the relaxation following the maximal contraction was retarded. After 30 days the maximum contraction force was increased and the relaxation rate was decreased. The maximum strength and stiffness of the tendons were increased after 10 days of diabetes and even more after 30 days. There was no change in the density of reducible cross-links. However, diabetes increased the amount of glucose attached to the lysine and hydroxylysine residues of collagen. Insulin treatment prevented all changes in thermal stability and mechanical properties. The results indicate that stabilization of collagen fibres in diabetes does not follow the same pattern as that seen in normal ageing.     

VASP phosphorylation at serine239 regulates the effects of NO on smooth muscle cell invasion and contraction of collagen

Nitric oxide triggers cGMP‐dependent kinase‐mediated phosphorylation of the actin regulator vasodilator‐stimulated phosphoprotein (VASP) at residue serine239. The function of this phosphorylation for smooth muscle cell (SMC) adhesion, spreading, matrix contraction, and invasion is not well understood. We reconstituted VASP deficient SMC with wild‐type VASP (wt‐VASP) or VASP mutants that mimic “locked” serine239 phosphorylation (S239D‐VASP) or “blocked” serine239 phosphorylation (S239A‐VASP). Collagen gel contraction was reduced in S239D‐VASP compared to S239A‐VASP and wt‐VASP expressing cells and nitric oxide (NO) stimulation decreased gel contraction of wt‐VASP reconstituted SMC. Invasion of collagen was enhanced in S239D‐VASP and NO‐stimulated wild‐type SMCs compared to S239A‐VASP expressing cells. Expression of S239D‐VASP impaired SMC attachment to collagen, reduced the number of membrane protrusions, and caused cell rounding compared to expression of S239A‐VASP. Treatment of wt‐VASP reconstituted SMCs with NO exerted similar effects as expression of S239D‐VASP. As unstimulated cells were spreading on collagen S239A‐VASP and wt‐VASP localized to actin fibers whereas S239D‐VASP was enriched in the cytosol. NO interferes with SMC invasion and contraction of collagen matrices. This requires phosphorylation of VASP on serine239, which reduces VASP binding to actin fibers. These findings support the conclusion that VASP phosphorylation at serine239 regulates cytoskeleton remodeling. J. Cell. Physiol. 222:230–237, 2010. © 2009 Wiley‐Liss, Inc.     

Nanosecond fluctuations of the molecular backbone of collagen in hard and soft tissues: a carbon-13 nuclear magnetic resonance relaxation study

We have determined the amplitude of nanosecond fluctuations of the collagen azimuthal orientation in intact tissues and reconstituted fibers from an analysis of 13C NMR relaxation data. We have labeled intact rat calvaria and tibia collagen (mineralized and cross-linked), intact rat tail tendon and demineralized bone collagen (cross-linked), and reconstituted lathyritic (non-cross-linked) chick calvaria collagen with [2-13C]glycine. This label was chosen because one-third of the amino acid residues in collagen are glycine and because the 1H-13C dipolar coupling is the dominant relaxation mechanism. Spin-lattice relaxation times (T1) and nuclear Overhauser enhancements were measured at 15.09 and 62.98 MHz at 22 and -35 degrees C. The measured NMR parameters have been analyzed by using a dynamic model in which the azimuthal orientation of the molecule fluctuates as a consequence of reorientation about the axis of the triple helix. We have shown that if root mean square fluctuations in the azimuthal orientations are small, gamma rms much less than 1 rad, the correlation function decays with a single correlation time tau and T1 depends only upon tau and gamma rms and not the detailed model of motion. Our analysis shows that, at 22 degrees C, tau is in the 1-5-ns range for all samples and gamma rms is 10 degrees, 9 degrees, and 5.5 degrees for the non-cross-linked, cross-linked, and mineralized samples, respectively. At -35 degrees C, gamma rms is less than 3 degrees for all samples. These results show that mineral and low temperature significantly restrict the amplitude of nanosecond motions of the collagen backbone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional role of PKC in contraction of cultured human prostatic stromal cells

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI-17 was over-expressed, PDBu induced a large contraction, but the agonist-induced contraction did not become larger than expected. In alpha-toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI-17 over-expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI-17 and also that the PKC-CPI-17 pathway does not appear to play a major role in the agonist-induced contraction even when CPI-17 was over-expressed.     

Expression and purification of human cardiac troponin subunits and their functional incorporation into isolated cardiac mouse myofibrils

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca(2+) concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca(2+) regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca(2+)-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca(2+)-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca(2+). Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.     

Fibronectin in rat heart: a link between cardiac myocytes and collagen

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.     

The role of the NH(2)- and COOH-terminal domains of the inhibitory region of troponin I in the regulation of skeletal muscle contraction.

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Delta96-116)), 2) the COOH-terminal domain (TnI-(Delta105-115)), and 3) the NH(2)-terminal domain (TnI-(Delta95-106)). Measurements of Ca(2+)-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI.TnC complex restored the Ca(2+) sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Delta96-116).TnC and TnI-(Delta105-115).TnC. The TnI-(Delta95-106).TnC mutant retained approximately 55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca(2+) sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca(2+), and two of them (TnI-(Delta96-116) and TnI-(Delta105-115)) gave significant activation in the absence of Ca(2+). These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH(2)-terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca(2+)-sensitive molecular switch during muscle contraction.     

Continuum rheology of muscle contraction and its application to cardiac contractility.

A set of constitutive equations is proposed to describe the mechanics of contraction of skeletal and heart muscle. Fiber tension is assumed to depend on the degree of chemical activation, the stretch ratio, and the rate of stretching of the fibers. The time rate of change of activation is governed by a differential equation. The proposed constitutive equations are used to model the time courses of isotonic and isometric twitches during contraction and relaxation phases of the muscle response to stimulation. Various contractility indices of the left ventricle are considered next by using the proposed constitutive equations. The present analysis introduces a new interpretation of the index of contractility (dP/dt)/P used in cardiac literature. It is shown that this index may not be related at all to the maximum speed of shortening and that it may be dependent on both preload and afterload. The development of pressure during isovolumetric contraction of the left ventricle is shown to be governed by a differential equation describing the time rate of change of tension during isometric contraction of myocardium fibers.     

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.     

Research and comparative characteristic of the factors determining elasticity of the human skin

Changes in skin elasticity have been analyzed under different conditions (upon skin stretching, at different thickness of the skin, at different contents of collagen, intercellular and endocellular liquids, upon changes of venous pressure, and during contraction and relaxation of smooth muscles in skin vessels - vasomotions). Elasticity was defined by the acoustic method from the speed of diffusion of a superficial sheared acoustic wave in the skin, by the autoresonant method from the mechanical resonance frequency of skin, and from the vacuum pressure needed for skin site deformation of constant volume. It was shown that the major factors determining the elasticity of skin are it stretching, thickness, and the contents of collagen and liquids in it. The influence on elasticity of venous pressure and the contraction activity of smooth muscles in vessels is not essential. This suggests that the parameters of skin elasticity can be used as indicators of systemic and local lesions of the connective tissue.     

Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium,Silica and Chitosan

Using eucollagen solutions from ox hide, we cast collagen films to assess the influence of calcium and silica on the reconstitution of the fibrous structure of collagen. The tensile strength and the breaking elongation of the reconstituted collagen films were measured and analysed. Significant differences were observed between reconstituted collagen films with and without calcium and silica. The breaking elongation of the films obtained in the presence of silica was significantly greater, and the degradation was lower than other films of reconstituted collagen. Collagen and chitosan do not exist together as blends in nature, but the specific properties of each may be used to produce in biomimetic way man-made blends with biomedical applications, that confer unique structural, mechanical （detail） and in vivo properties.     

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.     

Anisotropic viscoelastic properties of cortical bone

Relaxation Young's modulus of cortical bone was investigated for two different directions with respect to the longitudinal axis of bone (bone axis, BA): the modulus parallel (P) and normal (N) to the BA. The relaxation modulus was analyzed by fitting to the empirical equation previously proposed for cortical bones, i.e., a linear combination of two Kohlraush-Williams-Watts (KWW) functions (Iyo et al., 2003. Biorheology, submitted): E(t)=E0 (A1 exp[-(t/tau1)beta]+(1-A1) exp[-(t/tau2)gamma]), [0 < A1, beta, gamma < 1], where E0 is the initial modulus value E0. Tau1 and tau2(>tau1) are characteristic times of the relaxation, A1 is the fractional contribution of the fast relaxation (KWW1 process) to the whole relaxation process, and beta and gamma are parameters describing the shape of the relaxation modulus. In both P and N samples, the relaxation modulus was described well by the empirical equation. The KWW1 process of a P sample almost completely coincided with that of an N sample. In the slow process (KWW2 process), there was a difference between the relaxation modulus of a P sample and that of an N sample. The results indicate that the KWW1 process in the empirical equation represents the relaxation in the collagen matrix in bone and that the KWW2 process is related to a higher-order structure of bone that is responsible for the anisotropic mechanical properties of bone.     

The rheological behaviour of cortical bone and cartilage of Bos taurus. The importance of maximal damping capacity in collagen

The relaxation behaviour of bone and cartilage was investigated with a free-oscillating torsional pendulum. Two relaxations (260 K, and 150 K) were found for bone an; two (at 260 K and 210 K) for cartilage at a frequency of 1 Hz. The translations were the same as those known for collagen. The translation at 260 K is consistent with the frequency of the impulses causing the contraction of skeleton muscles when extrapolated to 37 centrigrades and a frequency of 500 Hz.     

Contractile Characteristics of Mimosa pudica L

The force-velocity characteristics of the primary pulvinus of Mimosa pudica have been determined using a new polytonic measurement technique. The contractile characteristics were determined from a modified form of Hill's equation (Hill, A. V. 1938. Proc. Roy. Soc. London B126: 136-195) describing the physiological contractile behavior of animal muscle. The values of the resulting Hill's constants were found to be remarkably similar to those of intact animal muscle and reconstituted contractile collagen.     

Stress relaxation function of bone and bone collagen

Relaxation Young's and shear moduli of bovine bone and bone collagen were investigated. It was found that each relaxation process observed had two stages, which were referred to as process I and process II in order of time. Process II was described by a simple exponential decay while process I was not. The Kohlrausch-Williams-Watts (KWW) function, ψ(t) = exp[t/τ₁)^B] (0 < B < 1), was found to be suitable to describe process I. The normalized relaxation modulus, M_r(t), was expressed by the combination of the simple exponential type relaxation function and the KWW function

M_r=A₁exp[−(t/τ₁)^B]+A₂exp[(t/τ₁)](0<B1)

On the basis of this equation, the relaxation mechanism in bone and bone collagen was identified. According to the model proposed for the KWW relaxation function, the stress relaxation process in bone was considered to be governed by viscoelastic properties of matrix collagen fiber. The model for the KWW relaxation function requires the disordered glassy structure of collagen fiber, which is consistent with the results of the structural investigations.     

PACAP- and PHI-mediated sustained relaxation in circular muscle of gastric fundus: findings obtained in PACAP knockout mice

Mediators of neurogenic responses of the gastric fundus were studied in wild type and pituitary adenylate cyclase activating peptide (PACAP) knockout mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild type mouse fundus induced a tri-phasic response, rapid transient contraction and relaxation, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by N(G)-nitro-L-arginine and atropine, respectively. The sustained relaxation was completely inhibited by a PACAP receptors antagonist, PACAP(6-38). The strips prepared from PACAP knockout mice exhibited a large contraction without rapid relaxation and unexpectedly, a sustained relaxation. However, the sustained relaxation was decreased to about a half of that observed in wild type mice. Anti-peptide histidine isoleucine (PHI) serum abolished the sustained relaxation in the knockout mice. The serum partially inhibited the sustained relaxation in wild type mice and PACAP(6-38) abolished the relaxation that remained after the antiserum-treatment. PHI relaxed the strips prepared from wild type mice. The relaxation was completely inhibited by PACAP(6-38). It was concluded that PACAP and PHI separately mediate the sustained relaxation in the mouse gastric fundus, and that nitric oxide and ACh mediate transient relaxation and contraction, respectively.     

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.