A new longitudinal variation in the structure of collagen fibrils and its relationship to locations of mechanical damage susceptibility.

The hierarchical architecture of the collagen fibril is well understood, involving non-integer staggering of collagen molecules which results in a 67 nm periodic molecular density variation termed D-banding. Other than this variation, collagen fibrils are considered to be homogeneous at the micro-scale and beyond. Interestingly, serial kink structures have been shown to form at discrete locations along the length of collagen fibrils from some mechanically overloaded tendons. The formation of these kinks at discrete locations along the length of fibrils (discrete plasticity) may indicate pre-existing structural variations at a length scale greater than that of the D-banding. Using a high velocity nanomechanical mapping technique, 25 tendon collagen fibrils, were mechanically and structurally mapped along 10 µm of their length in dehydrated and hydrated states with resolutions of 20 nm and 8 nm respectively. Analysis of the variation in hydrated indentation modulus along individual collagen fibrils revealed a micro-scale structural variation not observed in the hydrated or dehydrated structural maps. The spacing distribution of this variation was similar to that observed for inter-kink distances seen in SEM images of discrete plasticity type damage. We propose that longitudinal variation in collagen fibril structure leads to localized mechanical susceptibility to damage under overload. Furthermore, we suggest that this variation has its origins in heterogeneous crosslink density along the length of collagen fibrils. The presence of pre-existing sites of mechanical vulnerability along the length of collagen fibrils may be important to biological remodeling of tendon, with mechanically-activated sites having distinct protein binding capabilities and enzyme susceptibility.

MMP-9 selectively cleaves non-D-banded material on collagen fibrils with discrete plasticity damage in mechanically-overloaded tendon.

The mechanical properties of tendon are due to the properties and arrangement of its collagen fibril content. Collagen fibrils are highly-organized supermolecular structures with a periodic banding pattern (D-band) indicative of the geometry of molecular organization. Following mechanical overload of whole tendon, collagen fibrils may plastically deform at discrete sites along their length, forming kinks, and acquiring a fuzzy, non-D-banded, outer layer (shell). Termed discrete plasticity, such non-uniform damage to collagen fibrils suggests localized cellular response at the fibril level during subsequent repair/replacement. Matrix metallo-proteinases (MMPs) are enzymes which act upon the extracellular matrix, facilitating cell mobility and playing important roles in wound healing. A sub-group within this family are the gelatinases, MMP-2 and MMP-9, which selectively cleave denatured collagen molecules. Of these two, MMP-9 is specifically upregulated during the initial stages of tendon repair. This suggests a singular function in damage debridement. Using atomic force microscopy (AFM), a novel fibril-level enzymatic assay was employed to assess enzymatic removal of material by trypsin and MMP-9 from individual fibrils which were: (i) untreated, (ii) partially heat denatured, (iii) or displaying discrete plasticity damaged after repeated mechanical overload. Both enzymes removed material from heat denatured and discrete plasticity-damaged fibrils; however, only MMP-9 demonstrated the selective removal of non-D-banded material, with greater removal from more damaged fibrils. The selectivity of MMP-9, coupled with documented upregulation, suggests a likely mechanism for the in vivo debridement of individual collagen fibrils, following tendon overload injury, and prior to deposition of new collagen.

Advanced glycation end-product cross-linking inhibits biomechanical plasticity and characteristic failure morphology of native tendon.

Advanced glycation end-products (AGEs) are formed in vivo from the nonenzymatic reaction between sugars and proteins. AGEs accumulate in long-lived tissues like tendons, cross-linking neighboring collagen molecules, and are in part complicit in connective tissue pathologies experienced in aging and with diabetes. We have previously described discrete plasticity: a characteristic form of nanoscale collagen fibril damage consisting of serial fibril kinking and collagen denaturation that occurs in some mechanically overloaded tendons. We suspect that this failure mechanism may be an adaptive trait of collagen fibrils and have published evidence that inflammatory cells may be able to recognize and digest the denatured collagen produced by overload. In this study, we treated bovine tail tendons with ribose to simulate long-term AGE cross-linking in vitro. We hypothesized that a high degree of cross-linking would inhibit the intermolecular sliding thought to be necessary for discrete plasticity to occur. Tendons were mechanically overloaded, and properties were investigated by differential scanning calorimetry and scanning election microscopy. Ribose cross-linking treatment altered the mechanical response of tendons after the yield point, significantly decreasing postyield extensibility and strain energy capacity before rupture. Coincident with altered mechanics, ribose cross-linking completely inhibited the discrete plasticity failure mechanism of tendon. Our results suggest that discrete plasticity, which may be an important physiological mechanism, becomes pathologically disabled by the formation of AGE cross-links in aging and diabetes. NEW & NOTEWORTHY We have previously shown that mechanically overloaded collagen fibrils in mammalian tendons accrue nanoscaled damage. This includes development of a characteristic kinking morphology within a shell of denatured collagen: discrete plasticity. Here, using a ribose-incubation model, we show that advanced glycation end-product cross-linking associated with aging and diabetes completely inhibits this mechanism. Since discrete plasticity appears to cue cellular remodeling, this result has important implications for diabetic tendinopathy.

High spatial resolution (1.1 µm and 20 nm) FTIR polarization contrast imaging reveals pre-rupture disorder in damaged tendon.

Collagen is a major constituent in many life forms; in mammals, collagen appears as a component of skin, bone, tendon and cartilage, where it performs critical functions. Vibrational spectroscopy methods are excellent for studying the structure and function of collagen-containing tissues, as they provide molecular insight into composition and organization. The latter is particularly important for collagenous materials, given that a key feature is their hierarchical, oriented structure, organized from molecular to macroscopic length scales. Here, we present the first results of high-resolution FTIR polarization contrast imaging, at 1.1 µm and 20 nm scales, on control and mechanically damaged tendon. The spectroscopic data are supported with parallel SEM and correlated AFM imaging. Our goal is to explore the changes induced in tendon after the application of damaging mechanical stress, and the consequences for the healing processes. The results and possibilities for the application of these high-spatial-resolution FTIR techniques in spectral pathology, and eventually in clinical applications, are discussed.

Characterization via atomic force microscopy of discrete plasticity in collagen fibrils from mechanically overloaded tendons: Nano-scale structural changes mimic rope failure.

Tendons exposed to tensile overload show a structural alteration at the fibril scale termed discrete plasticity. Serial kinks appear along individual collagen fibrils that are susceptible to enzymatic digestion and are thermally unstable. Using atomic force microscopy we mapped the topography and mechanical properties in dehydrated and hydrated states of 25 control fibrils and 25 fibrils displaying periodic kinks, extracted from overloaded bovine tail tendons. Using the measured modulus of the hydrated fibrils as a probe of molecular density, we observed a non-linear negative correlation between molecular density and kink density of individual fibrils. This is accompanied by an increase in water uptake with kink density and a doubling of the coefficient of variation of the modulus between kinked, and control fibrils. The mechanical property maps of kinked collagen fibrils show radial heterogeneity that can be modeled as a high-density core surrounded by a low-density shell. The core of the fibril contains the kink structures characteristic of discrete plasticity; separated by inter-kink regions, which often retain the D-banding structure. We propose that the shell and kink structures mimic characteristic damage motifs observed in laid rope strands.

Pregnancy-induced remodeling of heart valves.

Recent studies have demonstrated remodeling of aortic and mitral valves leaflets under the volume loading and cardiac expansion of pregnancy. Those valves' leaflets enlarge with altered collagen fiber architecture, content, and cross-linking and biphasic changes (decreases, then increases) in extensibility during gestation. This study extends our analyses to right-sided valves, with additional compositional measurements for all valves. Valve leaflets were harvested from nonpregnant heifers and pregnant cows. Leaflet structure was characterized by leaflet dimensions, and ECM composition was determined using standard biochemical assays. Histological studies assessed changes in cellular and ECM components. Leaflet mechanical properties were assessed using equibiaxial mechanical testing. Collagen thermal stability and cross-linking were assessed using denaturation and hydrothermal isometric tension tests. Pulmonary and tricuspid leaflet areas increased during pregnancy by 35 and 55%, respectively. Leaflet thickness increased by 20% only in the pulmonary valve and largely in the fibrosa (30% thickening). Collagen crimp length was reduced in both the tricuspid (61%) and pulmonary (42%) valves, with loss of crimped area in the pulmonary valve. Thermomechanics showed decreased collagen thermal stability with surprisingly maintained cross-link maturity. The pulmonary leaflet exhibited the biphasic change in extensibility seen in left side valves, whereas the tricuspid leaflet mechanics remained largely unchanged throughout pregnancy. The tricuspid valve exhibits a remodeling response during pregnancy that is significantly diminished from the other three valves. All valves of the heart remodel in pregnancy in a manner distinct from cardiac pathology, with much similarity valve to valve, but with interesting valve-specific responses in the aortic and tricuspid valves.

Biaxial Creep Resistance and Structural Remodeling of the Aortic and Mitral Valves in Pregnancy.

Pregnancy produces rapid, dramatic volume-overload changes to the maternal circulation. This paper examines pregnancy-induced structural-mechanical changes in bovine aortic and mitral heart valve leaflets. Valve leaflets were harvested from non-pregnant heifers and pregnant cows. Dimensions, biaxial extensibility and creep resistance were assessed and related to changes in the collagen network: histological leaflet and anatomic layer thicknesses plus collagen crimp, and biochemical collagen content. Collagen stability and crosslinking were assessed thermomechanically. Pregnancy altered both aortic and mitral valve leaflets. Both valves demonstrated biphasic changes in leaflet stretch, decreasing in early pregnancy and recovering by late pregnancy. Creep in leaflets from both valves was minimal and decreased even further with pregnancy in the mitral valve. There were valve-specific changes in preconditioning areal extension with pregnancy: increasing in the aortic valve and decreasing in the mitral valve. Leaflet area increased dramatically (84% aortic, 56% mitral), with thickening mainly in the fibrosa, accompanied by increases in collagen content (8% aortic, 16% mitral): together suggesting synthesis of new collagen. Collagen crimp was almost completely lost in pregnancy, with the denaturation temperature decreased by approximately 2 °C. Mature and total crosslinking increased, curiously without a significant increase in immature crosslinking. Mature aortic and mitral heart valve leaflets in the maternal cardiovascular system remodel substantially and similarly-despite their different embryological origins.

Simulation of a Bead Placement Protocol for Follow-up of Thoracic Spinal Fusion Using Radiostereometric Analysis.

STUDY DESIGN: Computer simulation to detect intervertebral motion enabling future follow-up of spinal fusions performed on patients with multilevel thoracic scoliosis. OBJECTIVES: To propose a method using computer simulation to evaluate a radiostereometric analysis (RSA) marker placement protocol for visibility and redundancy and validate the performance of the developed RSA system in detecting intervertebral motion. SUMMARY OF BACKGROUND DATA: Radiostereometric analysis is a stereo x-ray technique in which clusters of tantalum markers are implanted to label well-defined landmarks and measure the relative motion between rigid bodies. METHODS: A model of the spine with the instrumentation and the RSA markers was developed. The vertebrae were aligned to mimic multilevel thoracic scoliosis after correction. The researchers performed virtual segment motion to validate the performance of the developed system. X-ray images were simulated and RSA was used to evaluate the proposed marker placement protocol and detect virtual motion. The authors performed a physical phantom study to evaluate marker visibility. RESULTS: All markers were located and matched between simulations and the condition numbers were well below the recommended value of 100. Based on computer simulation, average translational accuracy was 0.14, 0.01, and 0.24 mm along the x, y, and z axes, respectively, and average rotational accuracy was 0.23°, 0.12°, and 0.11° about the x, y, and z axes, respectively. The translational and rotational precision of the simulated RSA system was generally high. The physical phantom study agreed with the computer simulation and validated marker visibility. CONCLUSIONS: Computer simulation is a powerful tool that can be used to facilitate the development and refinement of an RSA system before its application in patients, particularly when the anatomy involved is complex. The proposed marker placement protocol yielded translational and rotational accuracy below the limits of clinical significance, which enables future follow-up of multilevel thoracic scoliosis with Lenke classification 1AN.

Macrophage-like U937 cells recognize collagen fibrils with strain-induced discrete plasticity damage.

At its essence, biomechanical injury to soft tissues or tissue products means damage to collagen fibrils. To restore function, damaged collagen must be identified, then repaired or replaced. It is unclear at present what the kernel features of fibrillar damage are, how phagocytic or synthetic cells identify that damage, and how they respond. We recently identified a nanostructural motif characteristic of overloaded collagen fibrils that we have termed discrete plasticity. In this study, we have demonstrated that U937 macrophage-like cells respond specifically to overload-damaged collagen fibrils. Tendons from steer tails were bisected, one half undergoing 15 cycles of subrupture mechanical overload and the other serving as an unloaded control. Both halves were decellularized, producing sterile collagen scaffolds that contained either undamaged collagen fibrils, or fibrils with discrete plasticity damage. Matched-pairs were cultured with U937 cells differentiated to a macrophage-like form directly on the substrate. Morphological responses of the U937 cells to the two substrates-and evidence of collagenolysis by the cells-were assessed using scanning electron microscopy. Enzyme release into medium was quantified for prototypic matrix metalloproteinase-1 (MMP-1) collagenase, and MMP-9 gelatinase. When adherent to damaged collagen fibrils, the cells clustered less, showed ruffled membranes, and frequently spread: increasing their contact area with the damaged substrate. There was clear structural evidence of pericellular enzymolysis of damaged collagen-but not of control collagen. Cells on damaged collagen also released significantly less MMP-9. These results show that U937 macrophage-like cells recognize strain-induced discrete plasticity damage in collagen fibrils: an ability that may be important to their removal or repair.

Pregnancy-induced remodeling of collagen architecture and content in the mitral valve.

Pregnancy produces rapid, non-pathological volume-overload in the maternal circulation due to the demands of the growing fetus. Using a bovine model for human pregnancy, previous work in our laboratory has shown remarkable pregnancy-induced changes in leaflet size and mechanics of the mitral valve. The present study sought to relate these changes to structural alterations in the collagenous leaflet matrix. Anterior mitral valve leaflets were harvested from non-pregnant heifers and pregnant cows (pregnancy stage estimated by fetal length). We measured changes in the thickness of the leaflet and its anatomic layers via Verhoeff-Van Gieson staining, and in collagen crimp (wavelength and percent collagen crimped) via picrosirius red staining and polarized microscopy. Collagen concentration was determined biochemically: hydroxyproline assay for total collagen and pepsin-acid extraction for uncrosslinked collagen. Small-angle light scattering (SALS) assessed changes in internal fiber architecture (characterized by degree of fiber alignment and preferred fiber direction). Pregnancy produced significant changes to collagen structure in the mitral valve. Fiber alignment decreased 17% with an 11.5° rotation of fiber orientation toward the radial axis. Collagen fiber crimp was dramatically lost, accompanied by a 53% thickening of the fibrosa, and a 16% increase in total collagen concentration, both suggesting that new collagen is being synthesized. Extractable collagen concentration was low, both in the non-pregnant and pregnant state, suggesting early crosslinking of newly-synthesized collagen. This study has shown that the mitral valve is strongly adaptive during pregnancy, with significant changes in size, collagen content and architecture in response to rapidly changing demands.

Riboflavin-sensitized photo-crosslinking of collagen using a dental curing light.

BACKGROUND: Photo-crosslinking of biomolecules such as collagen and fibrinogen is an emerging area of research interest. The use of a small dental curing light with a non-toxic photosensitizer represents a novel, practical approach to periodontal wound treatment. OBJECTIVE: This study evaluated the effects of riboflavin-sensitized photo-oxidation using a dental curing light on two collagenous biomaterials, as a preliminary step towards developing a medical technology for wound closure/healing. METHODS: A collagenous biomaterial (DBP) and type I collagen gels were treated by this photo-oxidative technique and characterized by hydrothermal isometric tension (HIT) analysis, amino acid analysis, SDS-PAGE, and rheology. RESULTS: HIT analysis suggested that dental curing light exposure for 300 s with riboflavin produced heavily crosslinked DBP. Dental curing light exposure for 300 s with riboflavin also showed a reduction in lysine concentration of DBP. SDS-PAGE showed that dental curing light exposure for 30 or 300 s with riboflavin resulted in crosslinked collagen gels. Dental curing light exposure for 30 s with riboflavin yielded a collagen gel with the strongest rheological characteristics. CONCLUSIONS: This novel approach to wound treatment has potential for wide adoption and clinical use, particularly because dental curing lights, riboflavin, and collagen biomaterials are all used clinically, but not yet combined together as one technology for broad application.

Mechanically overloading collagen fibrils uncoils collagen molecules, placing them in a stable, denatured state.

Due to the high occurrence rate of overextension injuries to tendons and ligaments, it is important to understand the fundamental mechanisms of damage to these tissues' primary load-bearing elements: collagen fibrils and their constituent molecules. Based on our recent observations of a new subrupture, overload-induced mode of fibril disruption that we call discrete plasticity, we have sought in the current study to re-explore whether the tensile overload of collagen fibrils can alter the helical conformation of collagen molecules. In order to accomplish this, we have analyzed the conformation of collagen molecules within repeatedly overloaded tendons in relation to their undamaged matched-pair controls using both differential scanning calorimetry and variable temperature trypsin digestion susceptibility. We find that tensile overload reduces the specific enthalpy of denaturation of tendons, and increases their susceptibility to trypsin digestion, even when the digestion is carried out at temperatures as low as 4 °C. Our results indicate that the tensile overload of collagen fibrils can uncoil the helix of collagen molecules, placing them in a stable, denatured state.

Cross-link stabilization does not affect the response of collagen molecules, fibrils, or tendons to tensile overload.

We investigated whether immature allysine-derived cross-links provide mechanically labile linkages by exploring the effects of immature cross-link stabilization at three levels of collagen hierarchy: damaged fibril morphology, whole tendon mechanics, and molecular stability. Tendons from the tails of young adult steers were either treated with sodium borohydride (NaBH4) to stabilize labile cross-links, exposed only to the buffer used during stabilization treatment, or maintained as untreated controls. One-half of each tendon was then subjected to five cycles of subrupture overload. Morphologic changes to collagen fibrils resulting from overload were investigated using scanning electron microscopy, and changes in the hydrothermal stability of collagen molecules were assessed using hydrothermal isometric tension testing. NaBH4 cross-link stabilization did not affect the response of tendon collagen to tensile overload at any of the three levels of hierarchy studied. Cross-link stabilization did not prevent the characteristic overload-induced mode of fibril damage that we term discrete plasticity. Similarly, stabilization did not alter the mechanical response of whole tendons to overload, and did not prevent an overload-induced thermal destabilization of collagen molecules. Our results indicate that hydrothermally labile cross-links may not be as mechanically labile as was previously thought.

Repeated subrupture overload causes progression of nanoscaled discrete plasticity damage in tendon collagen fibrils.

A critical feature of tendons and ligaments is their ability to resist rupture when overloaded, resulting in strains or sprains instead of ruptures. To treat these injuries more effectively, it is necessary to understand how overload affects the primary load-bearing elements of these tissues: collagen fibrils. We have investigated how repeated subrupture overload alters the collagen of tendons at the nanoscale. Using scanning electron microscopy to examine fibril morphology and hydrothermal isometric tension testing to look at molecular stability, we demonstrated that tendon collagen undergoes a progressive cascade of discrete plasticity damage when repeatedly overloaded. With successive overload cycles, fibrils develop an increasing number of kinks along their length. These kinks-discrete zones of plastic deformation known to contain denatured collagen molecules-are accompanied by a progressive and eventual total loss of D-banding along the surface of fibrils, indicating a loss of native molecular packing and further molecular denaturation. Thermal analysis of molecular stability showed that the destabilization of collagen molecules within fibrils is strongly related to the amount of strain energy dissipated by the tendon after yielding during tensile overload. These novel findings raise new questions about load transmission within tendons and their fibrils and about the interplay between crosslinking, strain-energy dissipation ability, and molecular denaturation within these structures.

Designed to fail: a novel mode of collagen fibril disruption and its relevance to tissue toughness.

Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules-meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils-were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils.

Macrophage differentiation and polarization on a decellularized pericardial biomaterial.

The monocyte-derived macrophage (MDM), present at biomaterial implantations, can increase, decrease or redirect the inflammatory and subsequent wound healing process associated with the presence of a biomaterial. Understanding MDM responses to biomaterials is important for improved prediction and design of biomaterials for tissue engineering. This study analyzed the direct differentiation of monocytes on intact, native collagen. Human monocytes were differentiated on decellularized bovine pericardium (DBP), polydimethylsiloxane (PDMS) or polystyrene (TCPS) for 14 d. MDMs on all surfaces released high amounts of MMP-9 compared to MMP-2 and relatively little MMP-1. MDMs differentiated on DBP released more MMP-2, but less acid phosphatase activity. MDMs on all three surfaces released low amounts of cytokines, although substrate differences were found: MDMs on DBP released higher amounts of IL-6, IL-8, and MCP-1 but lower amounts of IL-10 and IL-1ra. This research provides evidence that MDMs on decellularized matrices may not be stimulated towards an activated, inflammatory phenotype, supporting the potential of decellularized matrices for tissue engineering. This study also demonstrated that the differentiation surface affects MDM phenotype and therefore study design of macrophage interactions with biomaterials should scrutinize the specific macrophage culture method utilized and its effects on macrophage phenotype.

Three-dimensional morphologic nasal surface characteristics that predict the extremes of esthetics in patients with repaired cleft lip and palate.

OBJECTIVE: The purposes of this study were (1) to develop imaging methods and objective numeric parameters to describe nose morphology, and (2) to correlate those parameters with nasal esthetics for patients with clefts. METHODS: A total of 28 patients with repaired complete unilateral cleft lip and palate (CUCLP) and 20 age- and gender-matched individuals without clefts were identified. A panel of orthodontists rated and ranked nasal esthetics from nose casts for the cleft group. Best and worst esthetic cleft groups were established from the cast assessments. Three-dimensional surface coordinates of the casts were digitally mapped with an electromagnetic tracking device. Digitized nasal images were oriented, voxelated, sliced, and mathematically curve-fitted. Maximum difference, percent area difference, and maximum and minimum derivative differences between cleft and noncleft and between right and left nose sides were calculated. Differences in parameters between groups were assessed with the use of analysis of variance (ANOVA) and t tests, and correlations with esthetics were assessed with the Spearman rank correlation test. RESULTS: Differences were seen between cleft and noncleft and best and worst esthetic groups for all four parameters (p < .05). The best esthetic cleft group had (1) lower percent area difference (p < .0001), (2) lower maximum difference (p < .001), and (3) smaller differences in slope of the nose in the coronal plane (p < .0001) than the worst esthetic cleft group. CONCLUSIONS: Maximum difference and maximum derivative difference and, to a lesser degree, percent area difference can be used to identify differences between cleft and noncleft nasal morphology and to assess levels of nasal esthetics for patients with CUCLP.

Differential changes in the molecular stability of collagen from the pulmonary and aortic valves during the fetal-to-neonatal transition.

During the fetal-to-neonatal transition, transvalvular pressures (TVPs) on the aortic and pulmonary valves change dramatically-but differently for each valve. We have examined changes in the molecular stability and crosslinking of collagen during this transition. Aortic and pulmonary valves were harvested from fetal and neonatal cattle. Using differential scanning calorimetry (DSC), denaturation of valvular collagen was examined and, using HPLC, the types and quantities of enzymatic crosslinks were examined. No difference in hydrothermal stability was found between the collagens in the fetal aortic and pulmonary valves; this was expected since the TVP is approximately the same across both valves before birth. Only in the neonatal samples was the collagen from aortic valves (higher TVP) less stable than that from pulmonary valves (lower TVP). Surprisingly, the enthalpy of denaturation did not differ either between valve type or with age, suggesting an entropic mechanism of altered molecular stability. A significant difference in immature-to-mature crosslink ratio was found between neonatal aortic and pulmonary valves: a difference absent in fetal valves. This ratio-indicative of remodeling rate-parallels (and may be a function of) the changing in vivo load. This study highlights the relationship between in vivo load and both (i) molecular stability and (ii) collagen remodeling in heart valves.

Changes in collagen with aging maintain molecular stability after overload: evidence from an in vitro tendon model.

Soft tissue injuries are poorly understood at the molecular level. Previous work using differential scanning calorimetry (DSC) has shown that tendon collagen becomes less thermally stable with rupture. However, most soft tissue injuries do not result in complete tissue rupture but in damaging fiber overextension. Covalent crosslinking, which increases with animal maturity and age, plays an important role in collagenous fiber mechanics. It is also a determinant of tissue strength and is hypothesized to inhibit the loss of thermal stability of collagen due to mechanical damage. Controlled overextension without rupture was investigated to determine if overextension was sufficient to reduce the thermal stability of collagen in the bovine tail tendon (BTT) model and to examine the effects of aging on the phenomenon. Baseline data from DSC and hydrothermal isometric tension (HIT) techniques were compared between two groups: steers aged 24-30 months (young group), and skeletally mature bulls and oxen aged greater than five years (old group). Covalent crosslinks were quantified by ion exchange chromatography. Overextension resulted in reduced collagen thermal stability in the BTT model. The Young specimens, showing detectably lower tissue thermomechanical competence, lost more thermal stability with overextension than did the old specimens. The effect on old specimens, while smaller, was detectable. Multiple overextension cycles increased the loss of stability in the young group. Compositional differences in covalent crosslinking corresponded with tissue thermomechanical competence and therefore inversely with the loss of thermal stability. HIT testing gave thermal denaturation temperatures similar to those measured with DSC. The thermal stability of collagen was reduced by overextension of the tendon--without tissue rupture--and this effect was amplified by increased cycles of overextension. Increased tissue thermomechanical competence with aging seemed to mitigate the loss of collagen stability due to mechanical overextension. Surprisingly, the higher tissue thermomechanical competence did not directly correlate with the concentration of endogenous enzymatically derived covalent crosslinking on a mole per mole of collagen basis. It did, however, correlate with the percentage of mature and thermally stable crosslinks. Compositional changes in fibrous collagens that occur with aging affect fibrous collagen mechanics and partially determine the nature of mechanical damage at the intermolecular level. As techniques develop and improve, this new information may lead to important future studies concerning improved detection, prediction, and modeling of mechanical damage at much finer levels of tissue hierarchy than currently possible.

Reduction in postsurgical adhesion formation after cardiac surgery by application of N,O-carboxymethyl chitosan.

OBJECTIVE: The study objectives were to assess the efficacy of N,O carboxymethyl chitosan film in reducing postsurgical adhesion in a rabbit cardiac injury model and to confirm the efficacy of N,O carboxymethyl chitosan gel and film in reducing postsurgical adhesion formation in a pig cardiac injury model. METHODS: (1) Rabbit cardiac injury model: Cardiac injury was generated by abrading the anterior surface of the heart and desiccation with oxygen. N,O carboxymethyl chitosan solution and film were administered to the injured surface. (2) Pig cardiac injury model: Cardiac injury was generated as described above. N,O carboxymethyl chitosan solution and gel (or film) were administered to the injured surface. The severity and area of adhesion between the heart and the sternum were evaluated at 14 days postcardiac surgery. RESULTS: (1) Rabbits treated with N,O carboxymethyl chitosan film plus solution showed significantly reduced severity and area of adhesion formation. (2) Both N,O carboxymethyl chitosan gel plus solution and N,O carboxymethyl chitosan film plus solution significantly reduced adhesion formation in the pig model. CONCLUSIONS: Application of N,O carboxymethyl chitosan products significantly reduces severity of postsurgical adhesion formation after cardiac surgery in the rabbit and pig models. N,O carboxymethyl chitosan products may act as a biophysical barrier.