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Denaturation of Collagen Via Heating: An Irreversible Rate Process
Abstract
Heating therapies are increasingly used in cardiology, dermatology, gynecology, neurosurgery, oncology, ophthalmology, orthopedics, and urology, among other medical specialties. This widespread use of heating is driven primarily by the availability of new technology, not by a detailed understanding of the biothermomechanics. Without basic quantification of the underlying physical and chemical processes in terms of parameters that can be controlled clinically, identification of preferred interventions will continue to be based primarily on trial and error, thus necessitating large clinical studies and years of accumulative experience. Perusal of the literature reveals that much has been learned over the past century about the response of cells, proteins, and tissues to supra-physiologic temperatures; yet, the associated findings are reported in diverse journals and the underlying basic processes remain unidentified. In this review, we seek to contrast various findings on the kinetics of the thermal denaturation of collagen and to encourage investigators to consider the many open problems in part via a synthesis of results from the diverse literatures.
... Therefore, there is a significant gap in research involving the quantitative characterization of the effects of wide-ranging temperatures on tissues under realistic conditions [3,8]. Such data would enable the development of comprehensive computational predictive models for skin burn tissue response that could help achieve positive patient outcomes while minimizing complications due to damage to the surrounding tissue [7,[9][10][11][12]. ...
... A needle-point K-type thermocouple with a Digi-Sense 20250-44 temperature data logger was used to measure the temperature of skin platforms during experiments, whereas the temperature of platforms during contact/local burn testing could not be measured with sufficient spatial and temporal resolution. Reductions in compression modulus, measured as described above, were also determined as an indicator of collagen thermal degradation [12] because of the relative simplicity of the degradation mechanism compared to cell death and expression [13,58]. Due to the slow rate of heating and the minimal thickness of the samples, the temperature throughout the platforms can be assumed to be uniform, as confirmed by calculating the Biot number (Bi) of the platforms at less than 0.01. ...
... Therefore, the correlation of experimentally observed injury and the computationally modeled temperature is valid if the thermal characteristics are known for the platforms [4,5]. Tissue stiffness, as represented by the compression modulus, influences cell growth and affects how hightemperature, short-duration burns injure tissue due to the thermally induced deformation of tissue [10,12]. The average compression moduli of multilayer skin samples recorded in the present study were found to be 20.01 kPa. ...
This study presents a multilayer in vitro human skin platform to quantitatively relate predicted spatial time–temperature history with measured tissue injury response. This information is needed to elucidate high-temperature, short-duration burn injury kinetics and enables determination of relevant input parameters for computational models to facilitate treatment planning. Multilayer in vitro skin platforms were constructed using human dermal keratinocytes and fibroblasts embedded in collagen I hydrogels. After three seconds of contact with a 50–100 °C burn tip, ablation, cell death, apoptosis, and HSP70 expression were spatially measured using immunofluorescence confocal microscopy. Finite element modeling was performed using the measured thermal characteristics of skin platforms to determine the temperature distribution within platforms over time. The process coefficients for the Arrhenius thermal injury model describing tissue ablation and cell death were determined such that the predictions calculated from the time–temperature histories fit the experimental burn results. The activation energy for thermal collagen ablation and cell death was found to be significantly lower for short-duration, high-temperature burns than those found for long-duration, low-temperature burns. Analysis of results suggests that different injury mechanisms dominate at higher temperatures, necessitating burn research in the temperature ranges of interest and demonstrating the practicality of the proposed skin platform for this purpose.
... They considered that shrinkage of collagen is governed by an irreversible rate process in which native collagen is transformed to a denatured state via a highly temperature dependent rate constant, not by equilibrium thermodynamics. The same results have also been reported by subsequent works [11][12][13]. ...
... At the beginning of last century, researchers have long proposed that hydrothermal shrinkage would be generally to a cumulative effect in which the polypeptide chains, by the rupture of certain cross-links of various strengths, assume a more compact space arrangement than their hitherto extended forms [14,15]. Further studies have shown that hydrothermal shrinkage of collagen occurs by the rupture of inter-and intra-molecular hydrogen bonds and hydrophobic bonds, resulting in the irreversible transformation of the native triple helical structure into a more random (coiled) structure [13]. Both hydrogen bonds and hydrophobic bonds contribute to the stabilization of collagen triple helix [16,17]. ...
... It shows that the TS value of pigskin is gradually decreased with the increasing hydrothermal treatment temperature. Previous studies have shown that the inter-and intra-molecular hydrogen bonds and hydrophobic bonds of collagen molecules would be broken under hydrothermal treatment [13]. The ordered structure of collagen fibrils in pigskin is destroyed continuously with the increasing hydrothermal treatment temperature. ...
The present work focuses on in situ studying the transformation process of collagen molecules and fibrils in pigskin along with hydrothermal shrinkage. The results indicate that the triple helix of collagen molecules is firstly destabilized by hydrothermal treatment and more water molecules enter the space of three polypeptide chains of collagen molecules to slight increase the diameter of collagen fibrils. Then, the thermally labile domain of collagen molecules unfolds first to form tiny nodules. The nodules are shrinkage foci, which enlarge further and finally coalesce resulting in the shrinkage of pigskin. During the shrinkage process, the diameter of fibrils increases sharply. The spaces between collagen fibrils and fibers are compressed to form a more compact space arrangement than original extended forms, resulting in extrusion of water molecules. After shrinkage, partial fibrils structure was still observed and retained their characteristic banding patterns. Subsequently, the residual fibrils structure gradually disappeared and degraded.
... The thermal stability of keratin, gelatin, and collagen solutions was investigated using μDSC, and the parameters of denaturation (peak temperature and denaturation enthalpy change) are presented in Table 3. The μDSC signal of all samples presented a broad endothermic peak attributed to the helix to random coil state transition [36,38,39]. ...
... The thermal stability of keratin, gelatin, and collagen solutions was investigated using µDSC, and the parameters of denaturation (peak temperature and denaturation enthalpy change) are presented in Table 3. The µDSC signal of all samples presented a broad endothermic peak attributed to the helix to random coil state transition [36,38,39]. The µDSC measurements for the aqueous solution of keratin ( Figure 8) indicate a higher thermal stability of keratin after UV irradiation, while the denaturation enthalpy change slightly decreases as a result of the conformational changes observed in the CD spectra. ...
In this study, the effect of gamma irradiation (10 kGy) on proteins extracted from animal hide, scales, and wool was evidenced by calorimetric (μDSC) and spectroscopic (IR, circular dichroism, and EPR) methods. Keratin was obtained from sheep wool, collagen and bovine gelatin from bovine hide, and fish gelatin from fish scales. The μDSC experiments evidenced that gamma irradiation influences the thermal stability of these proteins differently. The thermal stability of keratin decreases, while a resistance to thermal denaturation was noticed for collagen and gelatins after gamma irradiation. The analysis of the IR spectra demonstrated that gamma irradiation determines changes in the vibrational modes of the amide groups that are associated with protein denaturation, most meaningfully in the case of keratin. As evidenced by circular dichroism for all proteins considered, exposure to gamma radiation produces changes in the secondary structure that are more significant than those produced by UV irradiation. Riboflavin has different effects on the secondary structure of the investigated proteins, a stabilizing effect for keratin and fish gelatin and a destabilizing effect for bovine gelatin, observed in both irradiated and non-irradiated samples. The EPR spectroscopy evidences the presence, in the gamma-irradiated samples, of free radicals centered on oxygen, and the increase in their EPR signals over time due to the presence of riboflavin.
... In these conditions, degraded collagen molecules usually result in a local unfolding or unwinding of the collagen triple helix. This unfolding might be caused by the breaking of a small number of consecutive hydrogen bonds [28][29][30] . Collagen damage increases the risk of fracture in these populations and reduces bone's resistance to crack formation and crack growth 14 . ...
... Samples were heat-treated for two hours at temperatures in the range known to denature mineralized collagen (100°C to 190°C). Heat treatment can either be reversible when the collagen molecule regains its original structure at normal temperature, or permanent with irreversible unfolding or damage of triple helical structure into an uncoiled structure 28 . ...
When studying bone fragility diseases, it is difficult to identify which factors reduce bone’s resistance to fracture because these diseases alter bone at many length scales. Here, we investigate the contribution of nanoscale collagen behavior on macroscale toughness and microscale toughening mechanisms using a bovine heat-treatment fragility model. This model is assessed by developing an in situ toughness testing technique for synchrotron radiation micro-computed tomography to study the evolution of microscale crack growth in 3D. Low-dose imaging is employed with deep learning to denoise images while maintaining bone’s innate mechanical properties. We show that collagen damage significantly reduces macroscale toughness and post-yield properties. We also find that bone samples with a compromised collagen network have reduced amounts of crack deflection, the main microscale mechanism of fracture resistance. This research demonstrates that collagen damage at the nanoscale adversely affects bone’s toughening mechanisms at the microscale and reduces the overall toughness of bone.
... Severe heating results in a time-dependent irreversible transformation of the native triple helical structure into a more random (coiled) structure. The transformation resulting from severe heating occurs primarily via the breaking of longer sequences of hydrogen bonds that stabilize the triple helix; particular subdomains along the molecule (Wright and Humphrey, 2002). The stability of collagen has been linked to the hydroxyproline content, which according to Ramachandran et al. (1973) is due to the intermolecular hydrogen-bonding involving the hydroxyl group of hydroxyproline. ...
... Therefore, the electrostatic interaction between collagen and CMC, which led to the aggregation of collagen fibres, also leads to increased thermal stability. The decrease in G' and the corresponding increase in tan δ indicates a softening of the paste structure, caused by the denaturation of the collagen triple helical structure, more specifically the disruption of the hydrogen bonds stabilising the collagen structure (Wright and Humphrey, 2002). ...
Collagen film (casings) obtained from acid-swollen collagen fibres is widely used as an alternative to natural casings for sausage production. However, collagen casings possess weak properties such as low mechanical properties (tensile strength and stiffness) and thermal stability compared to natural casings. Therefore, there is a need industrially to improve these properties. The main purpose of this work was to study the effects of polysaccharides on the properties of acid-swollen collagen pastes and films as a function of collagen paste concentrations (2.5 %, 3.5 % and 4 %wt/wt). In this work, polysaccharides dispersions: cellulose fibres of different length and waxy (WS) and high amylose (HAS) maize starch granules and molecular solutions: Hydroxypropylmethylcellulose (HPMC), Methylcellulose (MC), high molecular weight (GH), low molecular weight guar gum (GM) and Carboxymethylcellulose (CMC) were blended with acid-swollen collagen paste to fabricate collagen films with improved properties such as mechanical properties (tensile strength, stiffness and flexibility) and thermal stability. The viscoelastic of the blend pastes and denaturation of collagen was studied by rheological and thermal techniques. The pure and composite films were studied by sorption, mechanical, spectroscopic, structural, and thermal techniques. The focus of the first part of this study is to investigate the effect of uncharged and negatively charged molecular solutions at comparable low-shear viscosity on the viscoelastic and thermal properties of acid-swollen collagen paste. Dynamic rheological data indicated that the addition of non-charged hydrocolloids: HPMC, MC, GH and GM increased the storage modulus (G’) and loss modulus (G’’) of the acid-swollen collagen paste. By contrast, negatively charged CMC decreased the G’ and G” of the collagen pastes. At the level of addition of non-charged solutions (HPMC, MC, GH and GM) considered in this study, the denaturation temperature of collagen as determined by DSC was not affected while negatively charged CMC increased the denaturation temperature. Composite films containing blends of collagen paste with the individual molecular solutions were formed. Films were characterised for their mechanical, thermal, sorption and structural properties. Collagen/CMC films were not tested due to the difficulty in analysing the films. The thickness of the films increased and was dependent on the collagen concentration as well as the hydrocolloid concentration in the film network. Mechanical data revealed that the addition of hydrocolloids increased the tensile strength (TS), stiffness (YM), and elongation at break (EAB) of the films. Derivatised cellulose showed higher enhancement than the guars. Consistent with the mechanical data, DSC revealed an increase in peak temperature and a decrease in enthalpy of the films with the addition of the polymers. An increase in TS, YM, and EAB and an increase in peak temperatures were dependent on the collagen concentration. XRD data of the composite film showed a reduction in the intensity of the crystalline peak of collagen. FTIR spectra of the films helped to understand the structural changes and the interaction between the collagen and hydrocolloids. The thermal degradation temperature of collagen was not affected, as evidenced by the TGA curves. Furthermore, the composite films showed lower moisture uptake than the pure collagen films. The next study focused on investigating the effect of polysaccharide dispersions, cellulose with different fibre length, waxy and high amylose maize starches at comparable dispersed phase volume on the rheological and thermal properties of acid-swollen collagen paste. The dynamic rheological measurement revealed the dominant elastic behaviour (G’ > G’’) of the blend and control pastes. Cellulose fibres, waxy and high amylose starch granules increased the storage and loss modulus, and values increased with increasing collagen content. The starches exhibited a higher value due to the high concentration used. According to the DSC data, the denaturation of collagen and enthalpy of melting was not affected by the addition of the dispersions. On the other hand, on reheating the blend pastes, the starches lowered the enthalpy of the denatured collagen. Films were made from the blend pastes and were characterised for their mechanical, thermal, sorption and structural properties. The surface of composite films appeared rough because of the protrusion of the cellulose fibres and starch granules. The thickness of the films increased with the addition of the cellulose fibres and starch granules. Values increased with increasing levels of collagen and dispersions concentration in the film-forming paste. Reinforcing collagen films with cellulose fibres increased the mechanical properties (TS, YM and EAB) of the films. The mechanical properties of collagen with starch granules films could not be tested due to the brittleness of the films. DSC data showed that cellulose fibres increased the peak temperature of the films. By contrast, starch granules decreased the peak temperature. The enthalpy of the films was significantly reduced with the addition of cellulose fibres and starch granules. Collagen with starch granules films had the lowest enthalpy values. XRD data showed a decrease in the intensity of the crystalline peak of collagen in the blend films. The thermal stability of collagen was reduced, as evidenced by the TGA data. Additionally, the water uptake of the films decreased with the addition of cellulose fibres and starch granules. For the final study, the effect of collagen pastes (2.5%, 3.5% and 4 % wt) on the pasting properties of waxy (WS), high amylose (HAS) and normal (NS) maize starches were studied using Rapid Viscous Analyser (RVA) at conventional (up to 95 °C) and high-temperature (up to 140 °C) heating modes. Results showed that collagen pastes modified the pasting properties of the starches. At conventional heating mode, high amylose did not show a noticeable pasting profile. The pasting temperature of waxy starch was unaffected by the addition of collagen paste. By contrast, the addition of collagen paste lowered the pasting temperature of normal starches. The viscosities (peak, setback, and breakdown) of NS and WS increased. The final viscosity of WS decreased while that of NS increased with the increase in collagen paste concentration. When the samples are heated to temperatures 140 °C higher, HAS showed a noticeable pasting profile. The pasting temperature of HAS decreased with increasing levels of collagen paste addition. Peak and breakdown viscosities of NS, WS, and HAS increased with increasing collagen paste levels. In contrast, setback and final viscosities reduced.
... HIFU uses the characteristic that ultrasound can penetrate the human body without damage and focus on tumors in vivo causing a thermal effect (main effect), cavitation effect and mechanical effect. Since the high temperature generated by thermal effect on the focus can destroy the cell structure and damage the protein, it will lead to the coagulation necrosis of the tumor target [16][17][18] instead of burning the surrounding normal tissue. The sensor emits high-intensity ultrasound signals to the tissue target, and then the tissue absorbs ultrasound energy and converts energy into thermal energy during therapy. ...
... HIFU uses the characteristic that ultrasound can penetrate the human body without damage and focus on tumors in vivo causing a thermal effect (main effect), cavitation effect and mechanical effect. Since the high temperature generated by thermal effect on the focus can destroy the cell structure and damage the protein, it will lead to the coagulation necrosis of the tumor target [16][17][18] instead of burning the surrounding normal tissue. The sensor emits high-intensity ultrasound signals to the tissue target, and then the tissue absorbs ultrasound energy and converts energy into thermal energy during therapy. ...
Hyperthermia therapy (HT) is used to treat diseases through heating of high temperature usually in conjunction with some other medical therapeutics such as chemotherapy and radiotherapy. In this study, we propose a promising temperature-controlled hyperthermia method that uses high-intensity focused ultrasound (HIFU) for clinical tumor treatment combined with diagnostic ultrasound image guidance and non-invasive temperature monitoring through speed of sound (SOS) imaging. HIFU heating is realized by a ring ultrasound transducer array with 256 elements. In this study, tumors in the human thigh were set as heating targets. The inner structure information of thigh tissue is obtained by B-mode ultrasound imaging. Since the relationship between temperature and SOS in different human tissue is available, the temperature detection is converted to the SOS detection obtained by the full-wave inversion (FWI) method. Simulation results show that our model can achieve expected hyperthermia of constant temperature on tumor target with 0.2 °C maximum temperature fluctuation for 5 h. Through simulation, our proposed thermal therapy model achieves accurate temperature control of ±0.2 °C in human thigh tumors, which verifies the feasibility of the proposed temperature-controlled hyperthermia model. Furthermore, the temperature measurement can share the same ring ultrasound transducer array for HIFU heating and B-mode ultrasound imaging, which provides a guiding significance for clinical application.
... Regarding the thermal implications, the onset of protein denaturation after thermal coagulation usually begins to develop at a temperature of 42 °C, whereas heat-induced shrinkage occurs at temperatures > 50 °C 23 . This investigation was primarily conducted based on COMSOL simulation data to exclude thermal effects. ...
Nonthermal irreversible electroporation (NTIRE) is emerging as a promising tissue ablation technique. However, maintaining irreversible electroporation (IRE) electrodes against displacement during strong esophageal spasms remains an obstacle. The present study aimed to evaluate the efficacy and safety of newly designed balloon-type endoscopic IRE catheters. Six pigs were randomly allocated to each catheter group, and each pig was subjected to four ablations at alternating voltages of 1500 V and 2000 V. Esophagogastroscopy was performed during the IRE. The ability of balloon-type catheters to execute complete IRE with 40 pulses was assessed. The success rate was higher for the balloon-type catheter than that for the basket-type (12/12 [100%] vs. 2/12 [16.7%], p < 0.001). Following gross inspection and histologic analysis of the 1500-V vs. 2000-V balloon-type catheter revealed a larger mucosal damage area (105.3 mm² vs. 140.8 mm², p = 0.004) and greater damage depth (476 μm vs. 900 μm, p = 0.02). Histopathology of the ablated tissue revealed separated epithelium, inflamed lamina propria, congested muscularis mucosa, necrotized submucosa, and disorganized muscularis propria. Balloon-type catheters demonstrated efficacy, achieving full electrical pulse sequences under NTIRE conditions, and a safe histological profile below 2000 V (1274 V/cm). Optimal electrical conditions and electrode arrays pose ongoing challenges.
... As they are both of protein origin, they are susceptible to denaturation and a possible decrease in the communication capacity and cellular anchorage of the stem or cardiac cells in tissue regeneration therapies [138,139]. However, the preparation and application of the scaffolds containing collagen and the extracellular matrix using the techniques of sample heating, such as electrospinning in the infarcted cardiac tissue, are limited due to their possible protein denaturation, which significantly affects the biological properties of collagen [140]. Chitosan is used to develop some scaffolds through crosslinking the reactions to avoid denaturation and maintain the biological properties of collagen and the extracellular matrix [139,141]. ...
Cardiovascular diseases (CVD), such as myocardial infarction (MI), constitute one of the world’s leading causes of annual deaths. This cardiomyopathy generates a tissue scar with poor anatomical properties and cell necrosis that can lead to heart failure. Necrotic tissue repair is required through pharmaceutical or surgical treatments to avoid such loss, which has associated adverse collateral effects. However, to recover the infarcted myocardial tissue, biopolymer-based scaffolds are used as safer alternative treatments with fewer side effects due to their biocompatibility, chemical adaptability and biodegradability. For this reason, a systematic review of the literature from the last five years on the production and application of chitosan scaffolds for the reconstructive engineering of myocardial tissue was carried out. Seventy-five records were included for review using the “preferred reporting items for systematic reviews and meta-analyses” data collection strategy. It was observed that the chitosan scaffolds have a remarkable capacity for restoring the essential functions of the heart through the mimicry of its physiological environment and with a controlled porosity that allows for the exchange of nutrients, the improvement of the electrical conductivity and the stimulation of cell differentiation of the stem cells. In addition, the chitosan scaffolds can significantly improve angiogenesis in the infarcted tissue by stimulating the production of the glycoprotein receptors of the vascular endothelial growth factor (VEGF) family. Therefore, the possible mechanisms of action of the chitosan scaffolds on cardiomyocytes and stem cells were analyzed. For all the advantages observed, it is considered that the treatment of MI with the chitosan scaffolds is promising, showing multiple advantages within the regenerative therapies of CVD.
... This emphasizes the need for (50). An irreversible denaturation of the native triple helical structure triggers a shrinkage of the dermal skin layer (51,52). Loss of amino acid bonding randomizes the orientation of the collagen fibers (53). ...
Introduction
Severe burns cause unique pathophysiological alterations especially on the immune system. A murine scald model was optimized as a basis for the understanding of immunological reactions in response to heat induced injury. The understanding of the roles of neutrophil extracellular traps (NETs) and DNases will support the development of new surgical or pharmacological strategies for the therapy of severe burns.
Methods
We studied C57BL/6 mice (n=30) and employed four scalding protocols with varying exposure times to hot water. An additional scald group with a shorter observational time was generated to reduce mortality and study the very early phase of pathophysiology. At 24h or 72h, blood was drawn and tissue (wound, liver, lung, spleen) was analyzed for the presence of NETs, oxidative stress, apoptosis, bacterial translocation, and extracellular matrix re-organization. In addition, we analyzed the transcriptome from lung and liver tissues.
Results
Exposure to hot water for 7s led to significant systemic and local effects and caused considerable late mortality. Therefore, we used an observation time of 24h in this groups. To study later phases of burns (72h) an exposure time of 6s is optimal. Both conditions led to significant disorganization of collagen, increased oxidative stress, NET formation (by immunodetection of H3cit, NE, MPO), apoptosis (cC3) and alterations of the levels of DNase1 and DNase1L3. Transcriptome analysis revealed remarkable alterations in genes involved in acute phase signaling, cell cohesion, extracellular matrix organization, and immune response.
Conclusion
We identified two scald models that allow the analysis of early (24h) or late (72h) severe burn effects, thereby generating reproducible and standardized scald injuries. The study elucidated the important involvement of neutrophil activity and the role of NETs in burns. Extensive transcriptome analysis characterized the acute phase and tissue remodeling pathways involved in the process of healing and may serve as crucial basis for future in-depth studies.
... It has been well recognized that the collagen fibers are composed principally of the tropocollagen molecule as a basic unit (Fang et al., 2012). The tropocollagen molecule consists of three polypeptide chains arranged at a right-handed triple-helix structure presenting as a repetitious Gly-X-Y sequence among which G corresponds to glycine, and X and Y often corresponds to proline and hydroxyproline, respectively (Hulmes, 2002;Wright & Humphrey, 2002). ...
This study aimed to investigate the effects of acid or alkaline treatments on the textural properties of Chinese traditional stewed pig trotter in relation to the degradation of collagen fibers. Pig trotters were subjected to different pHs of 4, 5, 6, 7, 8 and then stewed at 95°C for 60 min. Textural parameters (springiness, chewiness, hardness and gumminess) of pig trotters and Raman spectroscopy, cross‐links, decorin, glycosaminoglycans contents of collagen fibers were assessed. The acid or alkaline treatments at pH 4, 5, 6, and 8 improved the textural properties evidenced by lower chewiness, hardness and gumminess, and promoted the unfolding of the secondary structure evidenced by a loss of α‐helix paralleled with an increase of random coil, as well as induced a breakage to the covalent crosslinks evidenced by the reduction of cross‐links, decorin and glycosaminoglycans. This study thus concluded positive effects of acid or alkaline treatments on the textural modification of Chinese traditional stewed pig trotter in relation to the induced degradation of the collagen fibers. This article is protected by copyright. All rights reserved.
... Regarding the thermal implications, the onset of protein denaturation after thermal coagulation usually begins to develop at a temperature of 42℃, whereas heat-induced shrinkage occurs at temperatures greater than 50℃ 15 . This investigation was primarily conducted based on the COMSOL simulation data to exclude the thermal effects. ...
Non-thermal irreversible electroporation (NTIRE) is emerging as a promising tissue ablation technique. However, maintaining irreversible electroporation (IRE) electrodes against displacement from the forces of strong esophageal spasms remains an obstacle. Therefore, the present study evaluates the efficacy and safety of newly designed balloon-type endoscopic IRE catheters. Six pigs were randomly allocated to each catheter group, and four ablations were performed on each at alternating voltages (1500 and 2000 V). Esophagogastroscopy was performed during IRE. The ability of balloon-type catheters to deliver full NTIRE sequences was assessed, followed by a histopathologic analysis. The success rate was higher for the balloon- than basket-type catheter (12/12 [100%] vs. 2/12 [16.7%], p < 0.001). Gross inspection and histologic analysis of the 2000-V vs. 1000-V balloon-type catheter revealed a larger mucosal damage area (105.3 mm ² vs. 140.8 mm ² , p = 0.004) and deeper damage depth (476 μm vs. 900 μm, p = 0.02). Histopathology of the ablated tissue revealed a separated epithelium, inflamed lamina propria, congested muscularis mucosa, necrotized submucosa, and disorganized muscularis propria. Balloon-type catheters demonstrated remarkable efficacy, achieving full electrical pulse sequences under NTIRE conditions and a safe histological profile below 2000 V (1274 V/cm).
... Collagen plays a significant role in the tensile strength of ligaments and tendons, in the elasticity of skin, and provides structural support for the cornea [4]. The right-handed triple helix collagen molecular structure is composed of three parallel polypeptide chains with 1300-1700 amino acid residues and the repeated G-X-Y sequence known as (G-X-Y)n, where G usually represents glycine, and X and Y often correspond to proline and hydroxyproline [12]. The collagen molecules were stabilized by the extensive hydrogen bonds of hydroxyproline, combined to form nanometer microfibrils, polymerized to form collagen fibrils, and further bundled into macroscopic collagen fibers [13]. ...
In order to clarify the influence of the thermal degradation of collagen fibers on the texture profile analysis (TPA) parameters of pig trotter stewed with soy sauce (PTSWSS), TPA (springiness, chewiness, hardness, and gumminess), the secondary structures, the cross-linkage, decorin (DCN) and glycosaminoglycan (GAG) levels, and the histochemical morphology of collagen fibers during the stewing process (0, 30, 60, 120 min) were assessed. The springiness and hardness increased after 30 min of stewing, along with the denaturation of collagen proteins. TPA parameters improved with the prolonged stewing times of 60 and 120 min, along with the ultra-structural dissolution of collagen fibers, and a substantial reduction in cross-linkage, DCN, and GAG levels, and the unfolded triple-helix structure. This study concluded that the TPA parameters of PTSWSS were dependent on the stewing time, and that the improvement in TPA parameters with longer stewing time could primarily be attributed to the thermal degradation of collagen fibers.
... 42,43 Their unwinding requires longer timescales. 44 The final temperature step at about 72 C fixes the network by establishing cross-links by disulfide linkage of cysteine of different proteins. [45][46][47] At such temperatures, the viscoelastic meat batter solidifies, water is bound in the process, and a strong gel is formed. ...
Plant based meat surrogates attract increasing interest. Modern methods of biotechnology, food chemistry/technology, and process engineering allow for surrogates with high optical similarity. Nowadays, targeted molecular-sensory methods taste and smell to be largely approximated to the original products. Nevertheless, the products appear completely different on a molecular scale, which is clearly noticeable in texture, oral processing, friction, and bolus formation. A consequent physical consideration of the function and effects of the proteins of different origin reveals the strengths of the respective products and offers suggestions how sensory weak points can be understood better and avoided. This is illustrated here by means of exemplary examples and experiments joined with underpinned by molecular models. Meat sausages, vegetarian, and vegan surrogates are microscopically investigated by rheology, tribology, and tensile experiments. The interpretation of the results is illustrated and supported by simple models.
... (1)] used by Henriques and Moritz and others. 4,7,[15][16][17][18][19][20][21] Classically, threshold thermal damage occurs when the integration in Eq. (6), with respect to α, generates a value of one at some time during a nonisothermal insult. The resulting equation is provided as EQ-TARGET;temp:intralink-;e007;116;652 ...
Significance:
Physics-based models supply simulated temperature rises to photothermal damage rate models and provide comprehensive risk assessments for laser-induced damage. As the physics-based models continue to be refined, the damage rate models have not advanced. This peculiar lack of improvement is counterintuitive considering the damage integral (Ω), originally derived for isothermal heating events, and fails to accurately represent the nonisothermal heating from short laser exposures.
Aim:
Derive a nonisothermal form of the damage integral and predict more accurately the damage induced by short laser exposures, as well as identify the role of heating rate in laser damage.
Approach:
From first principles, we derived a version of the damage integral specific to the shape of thermal profiles rather than the square function provided by Arrhenius plots. We used previously published threshold thermal profiles, where all nonisothermal frequency factors (Anon) solved all Ωnon values to unity. Nonisothermal correction factors correct isothermal Aiso values.
Results:
The Ea values were identical for both the isothermal and nonisothermal conventions. Correction factor values for Ωiso ranged from 0.0 (20-s exposures at thermal steady state) to -0.93 (0.05-s exposures). Based on empirical results, we have derived a two-dimensional empirical formula that predicts the heating rate as a function of exposure duration and ambient temperature. Threshold peak temperatures (Tpthr) and threshold critical temperatures are mathematically determined without thermal profiles when appropriate Ea and Anon values are established.
Conclusions:
We have identified a modified damage integral that does not rely on the Arrhenius plot and provides a value for the frequency factor (A) that accounts for the nonisothermal nature of short laser exposures. The method, validated in our in vitro retinal model, requires thermal profiles recorded under threshold conditions, such as at minimum visible lesions or the boundary of cell death. The method is a new option for laser damage modelers.
... However, occasionally the dynamic shrinking 'stain' manifested and was permanently visible after irradiation as exemplarily depicted in Fig. 7.11 (a,b,f). The origin of the observed shrinking might be found in the temperaturedependent contraction (shrinkage Wall et al. 1999;Wright and Humphrey 2002) as well as other structural alterations (Schober et al. 1986) of collagen fibers in the epineurial respectively perineurial layer due to confined laser tissue heating. Nevertheless, the frequently observed phenomenon of shrinking is contradictory to other reports that claim a nerve surface displacement in the sub-micrometer range upon pulsed irradiation of excised nerve samples (Wells et al. 2007a). ...
Infrared nerve stimulation (INS) is a novel optical neurostimulation technique using mil- lisecond near-infrared light pulses to elicit neural activation in unmodified neural targets. It has recently seen growing interest in research and was successfully demonstrated in pe- ripheral nerves, the auditory system, the central nervous system as well as other neural tissues in-vivo and in-vitro. INS features unambiguous advantages, specifically contact-less and artifact-free stimulation with superior spatial resolution, and is therefore discussed as a promising alternative to conventional electrical stimulation, which, unfortunately, faces sev- eral challenges regarding spatial selectivity, electrode invasiveness, stimulation artifacts and unnatural motor recruitment. Although the underlying neural activation mechanism dur- ing INS is not fully understood, it is currently assumed to result from thermally-mediated alteration of neural membrane capacitance.
In order to advance INS towards clinical application, deeper understanding of laser-induced neural excitation and determination of reliable yet safe irradiation parameters is required. In most reported studies on INS, infrared radiation is provided by a bare glass fiber in close distance to the target tissue, however, this widely used approach is inherently sensitive to minute distance variation. This may lead to inconsistent irradiation modalities and there- fore, previously reported stimulation thresholds respectively exposure limits specified by exclusively the radiant exposure (He) may be erroneous, ambivalent and are hence difficult to compare or reproduce.
In this work, we developed a novel experimental methodology that allows investigation of INS modalities under defined conditions, specifically in the rat sciatic nerve in vivo. Basi- cally, our approach comprised 1.) the implementation of a biophotonical simulation model that allows estimation of light distribution (Monte-Carlo), tissue heating and thermally- mediated axon recruitment in a realistic peripheral nerve under laser irradiation, in addition to 2.) the development and validation of a versatile experimental setup that facilitates well- defined, consistent and controlled pulsed infrared irradiation (focused beam, λ = 1470 nm) of neural targets in-vivo with high spatial precision, simultaneous to acquisition of evoked neuro-motor response (intramuscular EMG) and high-speed thermal monitoring, and 3.) es- tablishing histological procedures to assess potential adverse effects of INS on the irradiated peripheral nerve target.
Based on the observed spatio-temporal heating behavior of the rat sciatic nerve we iden- tified adequate irradiation parameters (He ≤ 5J/cm2, 2 Hz, 5 seconds for beam diameter 0.45...0.75 mm) regarding the laser-induced nerve surface temperature, which may be con- siderably lower than the nerve’s internal temperature (simulation) that supposedly drives motor axon depolarization. INS successfully evoked neuro-motor activity (CMAPs) in all (N=74) animals tested (rat sciatic nerve), however, at adequate irradiation levels we ob- served wide differences in excitability across the nerve surface. Given the small spot sizes used, INS seemed to facilitate spatially-mediated recruitment at the level of single motor axons (respectively clusters) in confined, presumably superficial nerve domains and thus provides superior spatial selectivity, which eventually allowed detailed investigation of INS across several nerve surface regions at high spatial resolution (functional nerve mapping). Further, at identical nominal radiant exposure (He), larger spot size (0.75 mm) significantly increased the overall stimulation yield compared to smaller spot size (0.45mm, n = 8). Evaluating CMAP latency and long-term fatigue behavior (n = 8), we found strong evi- dence that INS may achieve the elusive goal of orderly motor recruitment in the rat sciatic nerve. Ultimately, reliable stimulation of the rat sciatic nerve was achieved at radiant ex- posure He ≥ 4.4J/cm2 (n = 46, beam diameter ds = 0.45mm). The occurrence of acute laser-induced tissue damage across all experiments was unsystematic and not reproducible. Therefore no clear exposure limit for INS was obtained, which requires further investigation in future work.
Overall, in this thesis we demonstrate our consistent experimental methodology and discuss obtained findings on INS modalities in peripheral nerves.
... Collagen is the primary protein in the skin and subdermal fibroseptal network. Denaturation of collagen begins at 66.8°C [5] resulting in rapid contraction by up to 1/3 of the collagen fiber length [6]. Furthermore, thermal injury initiates a wound healing response that results in neocollagenesis which can occur over 6 months. ...
Energy based devices have been developed for the purposes of tissue contraction and skin tightening. Its application in the face and neck have been explored using lasers, temperature controlled monopolar and bipolar radiofrequency, and ultrasound. The purpose of this chapter is to explore the various applications for the face and neck using Renuvion™, a unique energy driven device based on plasma generated from the combination of helium gas and radiofrequency energy. The advantage of this technology is its ability to offer precise delivery of heat to tissue with minimal thermal spread, in part due to the rapid cooling aided by the helium gas. We will explore the options in which this technology can be incorporated to rejuvenate the face and neck, the patient selection considerations in choosing method of approach, surgical technique, anticipated outcomes, potential concerns and or complications associated with this and expected perioperative care. Applications in the face and neck include: (1) Subdermally in the neck as a stand alone procedure with or without liposuction. (2) Subdermally in a limited incision, non-excisional technique with a concomitant platysmaplasty either with an open approach or percutaneous use of suture suspension for the platysmal muscle. (3) Subdermally in conjunction with an open traditional rhytidectomy involving skin excision. (4) Ablative resurfacing—fractional or pulsed and full continuous modalities (non-FDA cleared at the time of this writing). It is the authors’ experience that with appropriate patient selection this can be a powerful tool that can deliver skin tightening and rhytid reduction not seen by other technologies available.
... Renuvion (Apyx Medical, Clearwater, FL, USA) combines radiofrequency energy and helium plasma to create subdermal tissue contraction. The device reaches the optimal temperature for subdermal tissue contraction (85°C) quickly with minimal thermal spread and cools to baseline temperatures in less than a second [40][41][42][43][44][45]. ...
This chapter will discuss the various energy-based modalities that are available to optimize circumferential trunk liposuction to enhance patient results. We will discuss multimodal liposuction using power assisted liposuction, ultrasonic energy addition, as well as radiofrequency assisted modalities to achieve adipose reduction and concurrent skin and soft tissue contraction. An in-depth look at patient selection and intraoperative technique will be presented. The postoperative management for each modality will be discussed in detail, including expected results from each modality and potential complications and their ensuing management. We will also discuss the application of different modalities alone and in combination to achieve superior results.
... Interestingly, the isolated eyes that had been scalded by immersion in hot buffer in the laboratory (group D) showed the smallest increase in central corneal thickness, which also did not reach statistical significance (p = 0.128). Perhaps this is an effect of thermally induced collagen denaturation, which has been demonstrated to harden connective tissue and even cause tissue shrinkage [14][15][16][17] . Both processes would limit or even counteract corneal swelling especially in the immersion scalded eyes, since here, the whole globes are surrounded by hot liquid. ...
The purpose of this study was to elucidate, under which conditions abattoir-acquired pig eyes are suitable for refractive excimer laser experiments. Porcine eyes from tunnel-scalded (n = 5) and tank-scalded (n = 10) pigs were compared to unscalded eyes (n = 5) and to eyes scalded in the laboratory (n = 5). The corneal epithelium was removed before an excimer laser was used to perform a − 8.0 D photoablation. Corneal thickness was measured by optical coherence topography before and after photoablation. The ablation depth was determined with a contour measuring station, the morphology of the ablated areas was characterized by scanning electron microscopy and white-light profilometry. The scalded eyes showed an increase in corneal swelling which gained statistical significance in tank-scalded eyes showing a wedge-shaped opaque stromal lesion in the nasal corneal quadrant. A measurable deterioration of photoablation was only found in tank-scalded eyes that exhibited the opaque lesion. Ablated area morphology was smooth and regular in the unscalded and tunnel-scalded eyes. The tank-scalded eyes showed conspicuous wrinkles. While unscalded eyes should always be preferred for excimer laser laboratory experiments, the data suggest that the use of tunnel-scalded eyes may also be acceptable and should be chosen over tank-scalded eyes.
... HIFU uses the characteristic that ultrasound can penetrate human body without damage and focus on vivo causing thermal effect (main effect), cavitation effect and mechanical effect. Since the high temperature can destroy the cell structure and damage the protein, the high temperature generated by thermal effect on the focus will lead to the coagulation necrosis of the tumor target [10][11][12] instead of burning out the surrounding normal tissue outside the focus. The sensor emits HIFU signals to the tissue target and then the tissue absorbs ultrasonic energy to convert it into thermal energy during tumor therapy. ...
Hyperthermia therapy (HT) is used to treat diseases through heating of high temperature usually in conjunction with some other medical therapeutics like chemotherapy and radiotherapy. In this study, we propose a promising thermostatic hyperthermia method that uses high-intensity focused ultrasound (HIFU) for clinical tumor treatment combined with diagnostic ultrasound image guidance and non-invasive temperature monitoring through the speed of sound (SOS) imaging. HIFU heating is realized by a ring ultrasound transducer array with 256 elements. The inner structure information of thigh tissue is obtained by B-mode ultrasound imaging. Since the relationship between the temperature and the SOS in the different human tissue is available, the temperature detection is converted to the SOS detection obtained by the full-wave inversion (FWI) method. Simulation results show that our model can achieve expected thermostatic hyperthermia on tumor target with 0.2 degree maximum temperature fluctuation for 5 hours. This study verifies the feasibility of the proposed thermostatic hyperthermia model. Furthermore, the temperature measurement can share the same ultrasound transducer array for HIFU heating and B-mode ultrasound imaging, which provides a guiding significance for clinical application.
... This transformation comes with a loss of load-bearing ability, i.e., stiffness and toughness [3]. Additionally, soft tissues shrink significantly during denaturation [4]. These processes are not instantaneous but follow exponential functions that demonstrate Arrheniustype temperature-dependence [5]. ...
The mechanics of collageneous soft tissues, such as skin, are sensitive to heat. Thus, quantifying and modeling thermo-mechanical coupling of skin is critical to our understanding of skin's physiology, pathophysiology, as well as its treatment. However, key gaps persist in our knowledge about skin's coupled thermo-mechanics. Among them, we haven't quantified the role of skin's microstructural organization in its response to superphysiological loading. To fill this gap, we conducted a comprehensive set of experiments in which we combined biaxial mechanical testing with histology and two-photon imaging under liquid heat treatment. Among other observations, we found that unconstrained skin, when exposed to high temperatures, shrinks anisotropically with the principle direction of shrinkage being aligned with collagen's principle orientation. Additionally, we found that when skin is isometrically constrained, it produces significant forces during denaturing that are also anisotropic. Finally, we found that denaturation significantly alters the mechanical behavior of skin. For short exposure times, this alteration is reflected in a reduction of stiffness at high strains. At long exposure times, the tissue softened to a point where it became untestable. We supplemented our findings with confirmation of collagen denaturation in skin via loss of birefringence and second harmonic generation. Finally, we captured all time-, temperature-, and direction-dependent experimental findings in a hypothetical model. Thus, this work fills a fundamental gap in our current understanding of skin thermo-mechanics and will support future developments in thermal injury prevention, thermal injury management, and thermal therapeutics of skin.
... Indeed, mammals thermoregulate within a very narrow body temperature range of 35-40C. Beyond these temperatures, vital proteins, such as the ubiquitous structural protein collagen, 5 thermally denature [2]. That is, collagen's complex hierarchical organization disassociates. ...
Understanding the response of skin to superphysiological temperatures is critical to the diagnosis and prognosis of thermal injuries, and to the development of temperature-based medical therapeutics. Unfortunately, this understanding has been hindered by our incomplete knowledge about the nonlinear coupling between skin temperature and its mechanics. In Part I of this study we experimentally demonstrated a complex interdependence of time, temperature, direction, and load in skin's response to superphysiological temperatures. In Part II of our study, we test two different models of skin's thermo-mechanics to explain our observations. In both models we assume that skin's response to superphysiological temperatures is governed by the denaturation of its highly collageneous microstructure. Thus, we capture skin's native mechanics via a microstructurally-motivated strain energy function which includes probability distributions for collagen fiber orientation and waviness. In the first model, we capture skin's response to superphysiological temperatures as a transition between two states that link the kinetics of collagen fiber denaturation to fiber coiling and to the transformation of each fiber's constitutive behavior from purely elastic to viscoelastic. In the second model, we capture skin's response to superphysiological temperatures instead via three states in which a sequence of two reactions link the kinetics of collagen fiber denaturation to fiber coiling, followed by a state of fiber damage. Given the success of both models in qualitatively capturing our observations, we expect that our work will provide guidance for future experiments that could probe each model's assumptions toward a better understanding of skin's coupled thermo-mechanics and that our work will be used to guide the engineering design of heat treatment therapies.
... Collagen fibers rapidly contract approximately one third the resting length, in response to heat to an extent that is dependent upon the temperature and duration of the temperature [2]. The temperature at which collagen denaturation occurs is 66.8° C, depending upon the tissue type [3,4]. Thermally induced soft tissue contraction that follows is a product of the wound healing response that includes neo-collagenesis, restructuring of collagen fibers and tissue remodeling. ...
Body contouring with liposuction has evolved significantly from the early approach of simply debulking excess fatty tissue, to affecting the mechanical properties of different tissue types and layers. Operative paradigms have been expanded to apply energy-based technologies intraoperatively to provide more uniform aspiration, selective fatty tissue emulsification in soft and fibrous body areas, minimize trauma to nerves and vessels, expose the fibrous septal network, reduce operator fatigue and help deliver smooth shapes with less discomfort and bruising. Advanced refinements with the delivery of monopolar and bipolar radiofrequency energy for soft tissue heating have been shown to reduce the residual soft tissue laxity that often follows voluminous fat removal. The Renuvion® (Apyx™ Medical, Clearwater, FL) radiofrequency powered helium plasma technology introduces an emerging concept in which the delivery of subdermal thermal energy preferentially coagulates the fascia and fibrous septal network through a conductive helium plasma stream seeking the path of least resistance, which in turn results in collagen contraction and tissue shrinkage that permits re-draping of the skin and enhanced definition. The physics and mechanics of Renuvion® subdermal soft tissue coagulation will be presented, along with clinical applications that have provided the authors more contouring finesse and has augmented liposuction outcomes.
... Like the cellular response, collagen denaturation does not only depend on the magnitude of temperature, but also on the exposure time. Isothermal exposure tests have shown that reversible localised unfolding occurs prior to irreversible changes in conformation 31 . Chen et al. 23 investigated fibril shrinkage behaviour through isothermal temperature jump tests and observed, for example, at 70 • C , fibril shrinkage is reversible at exposure times less than ∼ 1.4 s. ...
The underlying constraint of ultrashort pulsed laser ablation in both the clinical and micromachining setting is the uncertainty regarding the impact on the composition of material surrounding the ablated region. A heat model representing the laser-tissue interaction was implemented into a finite element suite to assess the cumulative temperature response of bone during ultrashort pulsed laser ablation. As an example, we focus on the extraction of mineralised collagen fibre micropillars. Laser induced heating can cause denaturation of the collagen, resulting in ultrastructural loss which could affect mechanical testing results. Laser parameters were taken from a used micropillar extraction protocol. The laser scanning pattern consisted of 4085 pulses, with a final radial pass being 22 $$\upmu {\text {m}}$$ μ m away from the micropillar. The micropillar temperature was elevated to 70.58 $$^{\circ }{\text {C}}$$ ∘ C , remaining 79.42 $$^{\circ }{\text {C}}$$ ∘ C lower than that of which we interpret as an onset for denaturation. We verified the results by means of Raman microscopy and Energy Dispersive X-ray Microanalysis and found the laser-material interaction had no effect on the collagen molecules or mineral nanocrystals that constitute the micropillars. We, thus, show that ultrashort pulsed laser ablation is a safe and viable tool to fabricate bone specimens for mechanical testing at the micro- and nanoscale and we provide a computational model to efficiently assess this.
... The concept of wound closure via heat-induced collagen denaturation and shrinkage has been previously applied in the field of vascular medicine and cardiology. The process allows a robust, safe and effective arterial puncture wound closure in patients undergoing arterial catheterizations, with the advantage of no foreign material at the closure site [8][9][10][11][12][13]. Several studies have demonstrated that controlled heating of the collagen results in the breakage of the internal cross-links of the collagen helix, causing conformational changes and shrinkage within the fibrils [14,15]. ...
Objective
Easy and safe methods of fascia closure are needed in order to reduce the risk for trocar site hernias without affecting procedure time significantly. Here we present a method for port site closure using heat induced collagen denaturation.
Material and methods
Controlled heat-induced collagen denaturation was applied to laparoscopic trocar sites in living porcine animal models. These were compared to control trocar sites which were left open. Port sites were evaluated visually at days 14 and 28 after the procedure, and both visually and pathologically at post-procedural day 42.
Results
A total of 12 port sites were evaluated in three pigs. No incisional hernias were noted at any of the trocar sites in both groups. Histological evaluation revealed that one of the six control ports appeared to have a complete transfascial defect, whereas none of the study group trocars showed this finding. Furthermore, the study port sites showed a more robust scarring pattern.
Conclusions
Heat-induced collagen denaturation in this preliminary study was found to be safe and allowed better scarring of the healing port sites. We believe that this technology may offer a safe and efficient closure of laparoscopic trocar sites. More studies are needed to further evaluate the true effectiveness of this technology.
Abbreviations: TSH: trocar site hernia; IACUC: institutional animal care and use committee
Collagens constitute a family of triple-helical proteins with a high level of structural polymorphism and a broad diversity of structural and chemical characteristics. Collagens are designed to form supporting aggregates in the extracellular spaces of our body, but they can be isolated from animal sources and processed to become available as biomaterials with wide applications in biomedicine and bioengineering. Collagens can be conveniently modified chemically, and their propensity for participating in crosslinking reactions is an important feature. While the crosslinking promoted by a variety of agents provides a range of collagen-based products, there has been minor interest for therapies based on the crosslinking of collagen while located within living connective tissues, known as exogenous crosslinking. Currently, there is only one such treatment in ocular therapeutics (for keratoconus), and another two in development, all based on mechanical augmentation of tissues due to ultraviolet (UV)-induced crosslinking. As seen in this review, there was some interest to employ exogenous crosslinking in order to reinforce mechanically the lax tendons with an aim to arrest tear propagation, stabilize the tissue, and facilitate the healing. Here we reviewed in details both the early stages and the actual status of the experimental research dedicated to the topic. Many results have not been encouraging, however there is sufficient evidence that tendons can be mechanically reinforced by chemical or photochemical exogenous crosslinking. We also compare the exogenous crosslinking using chemical agents, which was predominant in the literature reviewed, to that promoted by UV radiation, which was rather neglected but might have some advantages.
Purpose:
Magnetic resonance - high-intensity focused ultrasound (MR-HIFU) is a noninvasive treatment option for symptomatic uterine leiomyomas. Currently, pretreatment MRI is used to assess tissue characteristics and predict the most likely therapeutic response for individual patients. However, these predictions still entail significant uncertainties. The impact of tissue properties on therapeutic outcomes remains poorly understood and detailed knowledge of the histological effects of ultrasound ablation is lacking. Investigating these aspects could aid in optimizing patient selection, enhancing treatment effects and improving treatment outcomes.
Methods and materials:
We present seven patients who underwent MR-HIFU treatment for leiomyoma followed by second-line surgical treatment. Tissue samples obtained during the surgery were stained with hematoxylin and eosin, Masson's trichrome and Herovici to evaluate general morphology, fibrosis and collagen deposition of leiomyomas. Immunohistochemical CD31, Ki-67 and MMP-2 stainings were performed to study vascularization, proliferation and matrix metalloproteinase-2 protein expression in leiomyomas, respectively.
Results:
The clinical characteristics and radiological findings of the leiomyomas prior to treatment as well as qualitative histological findings after the treatment are presented and discussed in the context of current literature. A tentative model for volume reduction is presented.
Conclusion:
These findings provide insights into potential factors contributing to suboptimal therapeutic outcomes and the variability in histological changes following treatment.
Background:
Minimally invasive procedures that deliver thermal energy to subcutaneous tissue offer a solution between excisional and non-invasive options to address face and neck, aging-related changes. A minimally invasive helium plasma device, Renuvion, was first used for subdermal tissue heating to reduce skin laxity under a general clearance for cutting, coagulation, and ablation of soft tissue.
Objectives:
The purpose of this study was to demonstrate the safety and effectiveness of the helium plasma device for improving the appearance of loose skin in the neck and submental region.
Methods:
Subjects undergoing a procedure with the helium plasma device in the neck and submentum were studied. Subjects were seen through 6 months post-procedure. The primary effectiveness endpoint was improvement in lax skin in the treatment area as determined by 2 of 3 blinded photographic reviewers. The primary safety endpoint was the level of pain after treatment.
Results:
The primary effectiveness endpoint was met; 82.5% demonstrated improvement at Day 180. The primary safety endpoint was met; 96.9% of subjects experienced no pain to moderate pain to Day 7. There were no serious adverse events reported as related to the study device or procedure.
Conclusions:
The data demonstrates benefit to subjects in improvement of the appearance of lax skin in the neck and submental region. Outcomes resulted in FDA 510k clearance in July of 2022 expanding indications for the device to include subcutaneous dermatological and aesthetic procedures to improve the appearance of loose skin in the neck and submental region.
Liposuction is a cosmetic procedure to remove unwanted fat, which may be performed under local or general anesthesia. It requires three-dimensional (3D) understanding of the anatomy, of the adipose tissue, precision, skill, and artistry that is attained only with hands-on experience. There are many terms used to refer to this procedure, such as suction-assisted lipectomy, suction lipoplasty, liposculpting, and liposculpture. The concept of liposuction, suctioning out unwanted fat, traces its origins back to the early 1900s, but it was not until the 1970s that Illouz really popularized liposuction as we know it today (Table 4.1) [1, 2]. With the constantly growing worldwide body mass index (BMI), and given the fact that aside from using implants, fat contouring is the only way to really sculpt the body, it is not surprising that liposuction continues to grow and slowly become a major component, or an add-on to most procedures cosmetic plastic surgeons perform today. It is the second most common surgical procedures performed by board-certified plastic surgeons in the USA and is performed by 92% of all plastic surgeons. Overall, 289,261 liposuction procedures were performed by ASAPS member surgeons in 2018 [10]. If we add to it liposuctions performed by dermatologists and other cosmetic physicians, liposuction is likely the most commonly performed cosmetic procedure worldwide. These numbers as evidenced by the last decade will only continue to increase every year.
Label-free quantitative proteomic analysis was utilized to determine the key proteins that affect texture properties of sea cucumber body wall (SCBW) with different boiling heating treatment. 862, 363, 315, and 258 proteins were confirmed in water-soluble fractions from fresh group, 0.5 h-, 2 h- and 4 h-heat treatment group, respectively. During boiling heating treatment, proteins with an increased abundance in water-soluble fraction primarily belong to structural proteins, such as collagens, microfibril-associated proteins, glycoproteins, and muscle proteins. It was speculated that the degradation of these structural proteins caused the progressive disintegration of network skeleton of collagen fibres and FMs as well as the gelatinization, thus resulted in the decrease of hardness and shear force. Besides, the degradation of FMs was occurred layer by layer during boiling heating treatment, and the fibrilin-1 outer layer degraded first, followed by the fibrilin-2 core component.
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) biosensors have captured more attention than the conventional methodologies for SARS‐CoV‐2 detection due to having cost‐effective platforms and fast detection. However, these reported SARS‐CoV‐2 biosensors suffer from drawbacks including issues in detection sensitivity, degradation of biomaterials on the sensor's surface, and incapability to reuse the biosensors. To overcome these shortcomings, molecularly imprinted polymer nanoparticles (nanoMIPs) incorporated conductometric biosensor for highly accurate, rapid, and selective detection of two model SARS‐CoV‐2 proteins: (i) receptor binding domain (RBD) of the spike (S) glycoprotein and (ii) full length trimeric spike protein are introduced. In addition, these biosensors successfully responded to several other SARS‐CoV‐2 RBD spike protein variants including Alpha, Beta, Gamma, and Delta. Our conductometric biosensor selectively detects the two model proteins and SARS‐CoV‐2 RBD spike protein variant samples in real‐time with sensitivity to a detection limit of 7 pg mL–1 within 10 min of sample incubation. A battery‐free, wireless near‐field communication (NFC) interface is incorporated with the biosensor for fast and contactless detection of SARS‐CoV‐2 variants. The smartphone enabled real‐time detection and on‐screen rapid result for SARS‐CoV‐2 variants can curve the outbreak due to its ability to alert the user to infection in real time. Sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) proteins is important for early identification of COVID‐19 infection. The conventional SARS‐CoV‐2 biosensors often produce false results due to poor sensitivity and sensor degradation. Novel high resistivity silicon‐based conductometric sensor modified with SARS‐CoV‐2 molecularly imprinted polymers for rapid, sensitive, selective, stable, and accurate detection of SARS‐CoV‐2 proteins in human saliva is introduced.
Regenerative medicine requires both tissue restoration and ease of compliance for clinical application. Considering this, sticky tissue sealants have been shown to have great potentials over surgical suturing and wound treatment. However, tissue sealants currently used pose challenges such as uncontrollable adhesion formation, mechanical mismatch, and lack of tissue restoration. A new sticky sealant based on gelatinized cornea-derived extracellular matrix (GelCodE) with a visible light-activating system is firstly being introduced in this study. De novo tissue regeneration relies on the matrisome in charge of tissue-organization and development within GelCodE while visible light-based photopolymerization with ruthenium/sodium persulfate rapidly induces covalent bonds with the adjacent tissues. The ease of not only in vivo application, biocompatibility, and biointegration, but also exceptional de novo tissue formation is demonstrated in this study. Interestingly, newly regenerated tissues were shown to have normal tissue-like matrices with little scar formation. Hence, this work presents a promising strategy to meet clinical demands for scar-free tissue recovery with superior ease of clinical application.
The natural extracellular matrices of most tissues of the human body are collagen fibre reinforced structures. From a biomimetic point of view, collagen fibres are considered particularly suitable for regenerative medicine because they are a natural component of the extracellular matrix. Nevertheless, there are no textile scaffolds made of collagen fibres because there are no suitable technologies to produce collagen fibres in suitable quality and quantity.
This dissertation addresses the challenge of insufficient production of collagen yarns. The aim was therefore to enable the continuous production of collagen multifilament yarns on a large scale. In addition, a textile-technological process was to be developed with which the collagen yarns can be further processed into biomimetic scaffolds by means of electrostatic flocking technology.
A laboratory spinning plant based on wet spinning was developed for the production of collagen mono- and multifilament yarns. For this purpose, new types of spin packs were developed with which multiple collagen strands could be extruded centrally and coaxially to a coagulation medium into a long coagulation tube. In the tube, the strands solidified into filaments as the coagulation medium initiated fibrillogenesis of the collagen molecules. After leaving the coagulation tube, the filaments were passed through washing baths and a drying section, and finally wound onto a spool. With the developed wet-spinning process, non-crosslinked and crosslinked collagen yarns could be produced in sufficient quantities for scaffold fabrication.
The electrostatic flocking technique was used to fabricate biomimetic scaffolds. This technology is suitable for the production of vertically aligned fiber-based structures that can be used as scaffolds for regenerative medicine of hyaline cartilage. Wet-spun yarns were processed into short fibers of 2 mm length. Fiber-based scaffolds were then fabricated using these short fibers, a newly developed collagen adhesive, and the electrostatic flocking method. Due to their vertical fiber orientation, these scaffolds exhibited a structure similar to natural cartilage and thus a biomimetic structure. Due to the high similarity, these scaffolds have a very high potential for regenerative medicine in osteochondral defects.
So far, many researches have been done on temperature distribution due to thermal therapy. In these studies, the temperature field is obtained using the Fourier heat transfer equation. In biological tissues, because of the inhomogeneity nature of materials, temperature distribution has time delay and temperature oscillations. In this case, using the Fourier heat transfer creates an error, and for higher accuracy, using non-Fourier models for obtaining temperature distribution is necessary. In this study, the three-dimensional non-Fourier temperature distribution in liver tumor under HIFU radiation in the presence of significant blood vessel are studied. Temperature results for different distances of the ultrasonic transducer axis relative to the vessel are obtained and compared. The Helmholtz equation is used to simulate the acoustic pressure field, and the Fourier heat transfer model and non-Fourier TWMBT and DPL models are used to simulate the temperature distributions. The results show that by moving the wave axis away from the center of the vessel until the coordinates of the axis are smaller than the radius of the vessel, the temperature distribution decreases, and then as the transducer axis moves away from the vessel wall, the resulting temperature distribution values increase. The results also show the high accuracy of the non-Fourier heat transfer models, especially the DPL model compared to the Fourier heat transfer model in simulating the temperature distribution in biological tissues.
Background
Protein denaturation and collagen contraction occur when living tissue is heated to well-defined temperatures. The result is reduced volume and surface area of the heated tissue.
Objective
To evaluate the adverse events of procedures in which a helium-based plasma technology was used with and without ultrasound-assisted liposuction for the coagulation of soft tissue.
Methods
A multicenter retrospective chart review was performed in which patients (n=192) were divided into two groups: one that received only soft tissue coagulation and the other that received both soft-tissue coagulation and liposuction. Each of the two groups was subdivided into patients with and without adverse events, including seroma. Odds ratios for adverse events were calculated for both demographic and surgical subgroups. Seroma data were analyzed separately.
Results
No serious adverse events were observed. Forty-six (24.0%) patients reported 51 total adverse events. Seroma was the most frequently occurring adverse event with 13 patients (6.8%) reporting 17 (33.3%) events in 12 body areas. In these cases, all areas were treated with both liposuction and soft tissue coagulation. Seroma was not observed in patients receiving soft tissue coagulation alone. Patients aged 61 to 76 years and males were more likely to experience seroma or other adverse event than younger patients or females, respectively.
Conclusions
The use of the helium-based plasma technology for soft tissue coagulation in combination with ultrasound for liposuction is associated with non-serious adverse events. The most frequently occurring adverse event, seroma, was not observed in patients treated with helium-based plasma technology alone.
Purpose
To determine the effect of temperature on the accommodative response of non-human primate crystalline lenses during simulated accommodation.
Methods
Eight lenses from 7 cynomolgus monkeys (Macaca fascicularis, ages: 4.5–7.3 years; post-mortem time: 17.0 ± 16.4 h) were mounted in a lens stretcher. Stretching experiments were performed on each lens at 24 °C (room temperature), then the tissue was warmed to 35 °C (intraocular temperature) and the stretching experiments were repeated. The lens diameter, thickness, anterior and posterior surface radii of curvature, optical power, and the stretching force (load) were measured at each stretch position and the linear optomechanical relationships were quantified: load–lens diameter, load–thickness, power-load, load–anterior radius, and load–posterior radius. The rate of change for each parameter was quantified by performing a linear regression. The slopes of the linear regressions were compared at the two temperatures using a paired sample t-test.
Results
The average changes in the lens with stretching at 24 °C and 35 °C were: 3.07 ± 0.17 and 2.58 ± 0.15 for load–lens diameter (g/mm), −2.38 ± 0.20 and −2.00 ± 0.32 for load–thickness (g/mm), −13.35 ± 1.21 and −13.75 ± 1.26 for power-load (D/g), 0.41 ± 0.10 and 0.34 ± 0.05 for load–anterior radius of curvature (g/mm), and 1.35 ± 0.24 and 1.31 ± 0.35 for load–posterior radius of curvature (g/mm), respectively. The changes in load-diameter and load-thickness with lens stretching were significantly different for the two temperatures.
Conclusions
Temperature influences the change in lens shape observed during simulated accommodation in non-human primate lenses. These results suggest that lens stretching experiments and other optomechanical measurement techniques on ex vivo crystalline lenses be conducted at 35 °C and that the temperature of the tissue sample be documented and maintained constant to ensure repeatability.
The interactions between proteins and polysaccharides are of considerable importance in the food industry. In this study, the effect of adding non-charged methylcellulose (MC), hydroxypropylmethylcellulose (HPMC), medium (GM) and high (GH) molecular weight guar gum and negatively charged sodium carboxymethylcellulose (CMC) was investigated on the rheological and thermal properties of acid-swollen collagen pastes, as a function of collagen concentration. Dynamic frequency sweeps showed that the addition of the hydrocolloids, except for CMC, increased the storage modulus (G′) and loss modulus (G″) of collagen pastes at all collagen levels investigated. MC, HPMC, GH, and GM increased the collagen pastes' storage modulus to a similar extent. In contrast, the values of the loss moduli were found to be markedly different for each hydrocolloid. The loss factor (tan δ) showed that incorporating the hydrocolloids made the pastes less elastic than pure collagen pastes. The phase transition temperature of collagen measured during the temperature sweep tests was not affected by MC, HPMC, GH, and GM. However, CMC shifted the transition temperature of collagen to higher temperatures. Micro Differential scanning calorimeter (microDSC) results showed that the presence of MC, HPMC, and guar gums did not affect the denaturation temperature (Td) and enthalpy (ΔH). In contrast, the addition of CMC increased the denaturation temperature and denaturation enthalpy of collagen pastes. By fixing the hydrocolloids' viscosifying power and changing the collagen concentration, collagen pastes with different hydrocolloids show different viscoelastic properties.
The article describes the research of a new treatment method of lower limb varicose vein using low-frequency ultrasound. To study the effectiveness of this method we analyzed the interaction between the ultrasound instrument and the venous wall and defined the mechanisms of action during endovenous ultrasound treatment (EUT): thermal, mechanical, and chemical. We developed three models to study the impact of different combinations of mechanisms of action on varicose venous wall. The first model describes the endothelial cell layer delamination during mechanical mechanism. Other models investigate the two main modes of EUT. The EUT mode 1 models mechanical and thermal mechanisms of action, while the EUT mode 2 also studies the impregnation process of sclerosant. During the finite element analysis of both EUT modes, the instrument with an axisymmetric tip was inserted inside the vein with varying blood flow velocity. The amplitude of ultrasound oscillations required for the endothelial cell layer delamination was equal to 40 µm. The ultrasound exposure induced the impregnation enhancement of sclerosant up to 6 times. The temperature distribution and the necrotic tissue fraction of the venous wall were also evaluated. The results of the study demonstrate uniform irreversible damage to the venous wall for both EUT modes.
Understanding the response of skin to superphysiological temperatures is critical to the diagnosis and prognosis of thermal injuries, and to the development of temperature-based medical therapeutics. Unfortunately, this understanding has been hindered by our incomplete knowledge about the nonlinear coupling between skin temperature and its mechanics. In Part I of this study we experimentally demonstrated a complex interdependence of time, temperature, direction, and load in skin’s response to superphysiological temperatures. In Part II of our study, we test two different models of skin’s thermo-mechanics to explain our observations. In both models we assume that skin’s response to superphysiological temperatures is governed by the denaturation of its highly collageneous microstructure. Thus, we capture skin’s native mechanics via a microstructurally-motivated strain energy function which includes probability distributions for collagen fiber orientation and waviness. In the first model, we capture skin’s response to superphysiological temperatures as a transition between two states that link the kinetics of collagen fiber denaturation to fiber coiling and to the transformation of each fiber’s constitutive behavior from purely elastic to viscoelastic. In the second model, we capture skin’s response to superphysiological temperatures instead via three states in which a sequence of two reactions link the kinetics of collagen fiber denaturation to fiber coiling, followed by a state of fiber damage. Given the success of both models in qualitatively and quantitatively capturing our observations, we expect that our work will provide guidance for future experiments that could probe each model’s assumptions toward a better understanding of skin’s coupled thermo-mechanics and that our work will be used to guide the engineering design of heat treatment therapies.
Statement of significance
Quantifying and modeling skin thermo-mechanics is critical to our understanding of skin physiology, pathophysiology, as well as heat-based treatments. This work addresses a lack of theoretical and computational models of the coupled thermo-mechanics of skin. Our model accounts for skin microstructure through modeling the probability of fiber orientation and fiber stress-free states. Denaturing induces changes in the stress-free configuration of collagen, as well as changes in fiber stiffness and viscoelastic properties. We propose two competing models that fit all of our experimental observations. These models will enable future developments of thermal-therapeutics, prevention and management of skin thermal injuries, and set a foundation for improved mechanistic models of skin thermo-mechanics.
The mechanics of collagenous soft tissues, such as skin, are sensitive to heat. Thus, quantifying and modeling thermo-mechanical coupling of skin is critical to our understanding of skin’s physiology, pathophysiology, and its treatment. However, key gaps persist in our knowledge about skin’s coupled thermo-mechanics. Among them, we haven’t quantified the role of skin’s microstructural organization in its response to superphysiological loading. To fill this gap, we conducted a comprehensive set of experiments in which we combined biaxial mechanical testing with histology and two-photon imaging under liquid heat treatment at temperatures ranging from 37∘C to 95∘C lasting between 2 seconds and 5 minutes. Among other observations, we found that unconstrained skin, when exposed to high temperatures, shrinks anisotropically with the principal direction of shrinkage being aligned with collagen’s principal orientation. Additionally, we found that when skin is isometrically constrained, it produces significant forces during denaturation that are also anisotropic. Finally, we found that denaturation significantly alters the mechanical behavior of skin. For short exposure times, this alteration is reflected in a reduction of stiffness at high strains. At long exposure times, the tissue softened to a point where it became untestable. We supplemented our findings with confirmation of collagen denaturation in skin via loss of birefringence and second harmonic generation. Finally, we captured all time-, temperature-, and direction-dependent experimental findings in a hypothetical model. Thus, this work fills a fundamental gap in our current understanding of skin thermo-mechanics and will support future developments in thermal injury prevention, thermal injury management, and thermal therapeutics of skin.
Statement of significance
Our work experimentally explores how skin reacts to being heated. That is, it measures how much skin shrinks, what forces it produces, and how its mechanical properties change; all as a function of temperature, but also of direction and time. Additionally, our work connects these measurements to changes in skin’s microscopic make-up. This knowledge is important to our understanding of skin’s function and dysfunction, especially during burn injuries or heat-dependent treatments.
Fish skin is the potential gelatin source since the increase of the demand for the halal food due to the religious consideration. The yield of gelatin obtained from connective tissue of animal skin greatly affected by the extraction process and age of the animals that used as raw material. Research about the potential of the Pangasius hypophthalmus (striped catfish) skin as halal gelatin source have been developing. However, there was no information about the correlation between the length of striped catfish that representing age with the gelatin content. Here we optimize the gelatin extraction method from striped catfish skin with different length body size (sub adult and adult size) using statistical analysis of Placket Burman Design. We performed preliminary study to determine the variables used in the main study. We used 9 variables in the extraction process and analyzed the p-value of each variable. According to this analysis, three variables with the lowest p-values were selected: temperature (p=0.000); soaking time in alkali solution (p=0.055) and soaking time in acid solution (p=0.244). Range value of selected variables were determined according to the related previous studies. Results from our study showed that the maximum yield of sub adult and adult were obtained when we used 70 °C as temperature of extraction, i.e. 37.42% and 30.31%, respectively. In general, sub adult striped catfish have higher yield than the adult one at the temperature of 40 and 70 °C. However, the gelatin yielded from extraction process temperature of ≥70 °C exhibited dark pigmentation, while the gelatin that obtained with temperature treatment of ≤ 55°C showed less pigmentation. The gelatin yielded from sub adult striped catfish showed more pigmentation than the adult one. We suggested for process of extraction both size of striped catfish should be at 55°C, since it showed higher yield extraction with less pigmentation.
The mathematical foundations of the thermal ablation procedures have a noteworthy role in understanding the destruction of the ablation zones by determining the best settings for the ablation process using a specific ablation method to treat a specific organ. Since thermal ablation is based on increasing or decreasing temperatures for tumor ablation, studying the appropriate heat transfer mathematical formulations for animal/human tissues is essential. Generally, the used applicators/probes in the thermal ablation process apply energy to cool or heat the targeted tissues. However, blood perfusion may remove some of this generated energy. Accordingly, the characteristics and location of the targeted tissues have a major effect on the ablation process outcomes. To study the correct way to control the applied temperature and power to completely ablate the targeted tissues, and maximizing the coagulation volumes, the finite element method (FEM) for modeling the ablation procedure including healthy/tumor tissues, and the applicator based on the tissues and material characteristics are designed. Finite element (FE) modeling depends on the mathematical prototype of the thermal process, which happens during the ablation process of tissues. It describes and analyzes the dynamics of the temperature distribution by considering the bioheat transfer in the tissue and/or blood vessel due to the existence of an energy source (generator). In addition, such a model is used to determine the volume of the ablated regions without destroying the nearby healthy tissues and/or blood vessels by defining the optimal input power which increases the maximum temperature in the targeted tissues. This chapter studies in detail most of the mathematical formulas and relations which govern the ablation process, starting from the governing equations of the generated wave in the different ablation systems, including radiofrequency ablation (RFA), microwave ablation (MWA), laser ablation, ultrasound (US) ablation, cryosurgical ablation, and electroporation ablation, then, the heat transfer process is introduced, including Penne’s bioheat equations, Chen and Holmes equations, Weinbaum and Jiji equation, and Weinbaum, Jiji, and Lemons model. Then tissue contraction during the thermal ablation and the first-order Arrhenius rate equation are explained. Finally, the effect of cooling due to the presence of large blood vessels is highlighted, followed by an explanation of the concept of the FEM.
Collagen fiber films were prepared with the addition of softwood cellulose fiber (SWCF) and chitosan (CHS). The modulus, viscosity and yield stress of collagen fiber dispersions (CFD) increased or decreased with the addition of SWCF or CHS, respectively. The addition of SWCF significantly improved the film flexibility at dry, wet state and decreased the swelling ratio of films. The addition of CHS significantly increased the tensile strength (TS) of films at different states and decreased the swelling ratio and shrinkage ratio. For the blending of the two polymers, the CHS has more improvement effect than SWCF in TS (wet, boiled), swelling ratio and shrinkage ratio. The thermodynamic properties of films were improved by adding SWCF and CHS. Optical microscope images showed that some of the SWCF could appeared on the film surface and the CHS could well dispersed in the collagen fiber network. The SEM observation indicated that the collagen fiber formed layered network and the addition of SWCF made the fiber network irregular, while the CHS made the fiber layer denser. Electrostatic interactions excited between CHS and collagen and the addition of SWCF and CHS changed the crystal diffraction patterns to a certain extent.
Introduction and hypothesisRadiofrequency (RF) energy has been delivered in a variety of methods to the vagina, bladder, and periurethral tissue to improve myriad genitourinary complaints. Currently, practitioners are promoting transvaginal RF treatments with a minimal understanding of the various platforms and data to support or refute their utilization. This review explores how various RF technologies create desired tissue effects, review the published literature reporting outcomes of various treatment regimes, and peer into potential future uses of this technology in urogynecology.MethodsA comprehensive literature review was performed for articles pertaining to RF energy use in women for genitourinary complaints with regard to stress urinary incontinence (SUI), genitourinary syndrome of menopause (GSM), female sexual dysfunction (FSD), and overactive bladder (OAB).ResultsRadiofrequency energy devices heat tissues via direct or micro-needling applications with the goal of stimulating collagen remodeling, neovascularization, and potentially modulation of nerve function. By altering the approach and location of energy application, many new devices have been marketed for treatment of conditions such as SUI, GSM, FSD, and OAB. Available studies demonstrate promising efficacy and favorable safety; however, interpretation of studies is greatly limited by poor study quality and reporting.Conclusions
Despite a lack of high-quality evidence for efficacy, safety, and durability in the literature, practitioners around the world continue to promote RF technology for a variety of genitourinary complaints. Currently, it appears that RF energy can potentially treat a variety of genitourinary conditions, but more robust data are needed to substantiate evidence-based use.
The effects of heat treatment on the proteome of Apostichopus japonicus have been evaluated using label-free quantitative proteomics by ultrahigh performance liquid chromatography-quadrupole/time of flight (UHPLC-Q/TOF) mass spectrometry with sequential window acquisition of all the theoretical fragment ion (SWATH) acquisition mode. Chemometric tools are integrated to reveal proteomic changes by mining the protein quantitation data from fresh and differently heat-treated samples. SWATH allows the quantitation of 548 proteins, of which 24 proteins are significantly sensitive to heat treatment and 13 proteins vary significantly responding to different heat procedures (boiling, steaming, and microwave heating), and 5 of them are sharing proteins. Gene ontology (GO) annotation of the differentiating proteins highlights most of them are relevant to molecular functions. The results can be favorable to evaluate the effects of heat treatment on the nutrition and function of processed sea cucumbers and facilitate the selection of an optimal thermal treatment.
Temperature dependence of heat capacity of native and denatured collagen samples with different content of bound water (6 divided by 27%) has been studied by DSC method in the temperature range from -50 to 150 degrees C. Heat capacity of denatured samples demonstrates a jump of 0.50 J/g.grad. at temperature Tg, which depends on humidity of the sample. It has been shown that Tg value also depends on the heating rate and thermal history. Annealing at the temperature below Tg produces an additional maximum in the temperature dependence on heat capacity. The magnitude of this maximum, as well as the Tg value increase with the annealing time. It is concluded that these properties of heat capacity reflect glass transition in the denatured collagen.
Thermal stability measured as area shrinkage without tension during heating was determined for membranes of collagen fibrils reconstituted from solutions of highly purified rat skin collagens. Shrinkage in per cent of area at 25 degrees C and shrinkage temperature were quantitated in a standardized way and determined as a function of in vitro maturation time for 11 to 104 days after aggregation for the collagen membranes. Similar to reports on intact rat skin, shrinkage temperature remained constant and shrinkage per cent declined with a rate decreasing with time during maturation. Solubility in water at 80 degrees C for 2 hours was 95-96% and remained unchanged for the maturation time (about 2 months) studied. The decreased shrinkage reflecting a lower degree of collapse is ascribed to an increasing thermal stability of the membranes during maturation. Development of heat-stable bonds in the reconstituted collagen fibrils is taken up to be amenable to this increased stability. Similarity in changes of shrinkage characteristics during in vivo and in vitro maturation indicates that maturation changes in reconstituted collagen fibrils reflect those occurring in intact collagen during in vivo aging.
Minimally invasive heat therapies are of continued clinical interest to alter the functional attributes of tissue in the treatment of a number of pathologies. The extent of heating and tissue alteration is governed by the physics of the mode of heating, the geometry of the heater, and the properties of the tissue. Analytical models are needed to optimize the design of protocols and predict clinical outcomes. Presented here are the results of a finite difference model of the heating and alteration of a hypothetical non-perfused artery adjacent to a joule-heated, catheter-mounted balloon as might be used to enhance coronary angioplasty. The calculations suggest that shorter heating times (∼20 s) at greater balloon lumen temperatures (95 °C) better localize heating-induced changes to tissue than longer heating at lower temperatures (470 s at 60 °C) for the same alteration of the target arterial tissue.
The chapter discusses the protein stability with emphasis on compact globular proteins representing a single cooperative system. All the small compact globular proteins represent cooperative systems; they exhibit an extreme cooperativity that integrates the whole of their structure into a single structural unit. The large proteins, to which fibrillar proteins are also related, do not present single cooperative systems, but are subdivided into definite cooperative subsystems—structural blocks or domains. The advances in studying the stability of complicated proteins are connected with two methodical achievements: (1) the appearance of the precise scanning microcalorimetric technique, which affords reliable information on the heat capacity function of proteins in a broad temperature range; and (2) realization of the fact that the complicated heat effect of disruption of a complex macromolecular structure can be analyzed thermodynamically. The thermodynamic specificity of collagen has been considered. The volume of globular proteins does not increase at denaturation but decreases, as seen from their ability to denature under high pressure. The results of calorimetric studies are discussed, presenting the specific melting enthalpy of various protein structures—globular proteins, double-stranded coiled coils, and triplestranded coiled coils. The practical importance of thermodynamic studies of protein stability—that is, its importance not only for understanding the principles of organization of these molecules, but just for obtaining structural information on the domain level is emphasized.
In laserthermokeratoplasty (LTK) thermal denaturation and shrinkage of corneal collagen is used to correct hyperopia and astigmatism. In order to optimize dosimetry, the temperature at which maximal shrinkage of collagen fibrils occurs is of major interest. Since the exposure time in clinical LTK-treatment is limited to a few seconds, the kinetics of collagen denaturation as a rate process has to be considered, thus the time of exposure is of critical importance for threshold and shrinkage temperatures. We investigated the time-temperature correlation for corneal collagen denaturation within different time domains by turbidimetry of scattered HeNe laser probe light using a temperature controlled water bath and pulsed IR laser irradiation. In the temperature range of 60 degree(s)C to 95 degree(s)C we found an exponential relation between the denaturation time and temperature. For the typical LTK-treatment time of 2 s, a temperature of 95 degree(s)C is needed to induce thermal damage. Use of pulsed Holmium laser radiation gave significant scattering of HeNe laser probe light at calculated temperatures of around 100 degree(s)DC. Rate parameters according to the formalism of Arrhenius were fitted to these results. Force measurements showed the simultaneous onset of light scattering and collagen shrinkage.
Thermal fusion or welding of collagen-rich tissues involves the thermal denaturation of collagen which is reflected by changes in birefringence intensity in histologic sections. The weld bond between two severed edges is formed when the apposed ends of the collagen fibrils unravel during heating then re-entwine during the cooling phase. Thermal coagulation of collagen can be described as an end point of a kinetic rate process of thermal damage which is linear with time of exposure and exponential with temperature. The kinetic rate coefficients, A (s-1) and E (J/mole), have been experimentally determined for birefringence loss in rat skin collagen heated in vitro -- A equals 1.606 X 1045 and E equals 3.06 X 105. Loss in collagen birefringence is a rare quantitative indicator of thermal damage; in this case, the structural alteration in tissue native-form collagen. The kinetic model coefficients were derived from exposure times between 600 and 6000s over the temperature range 45 to 90 degree(s)C. Room temperature control specimens were also analyzed for comparison.
Kinetic models of thermal damage in tissues can be used to describe pathologic end points obtained with laser irradiation. Many treatment end-point goals involve relatively low temperature coagulation or desiccation of tissue, and these end points can be conveniently described by rate process models. Thermal damage is exponentially dependent on temperature and linearly dependent on time of exposure. Damage processes can be modeled as first-order rate processes for which two experimentally derived coefficients are sufficient. The rate process models apply well to the prediction of damage thresholds and less well as the damage becomes complete, since several of the fundamental assumptions are violated. In order to be useful in evaluating laser dosimetry, the kinetic model must be coupled to quantitative pathological analysis. This chapter describes quantitative markers of thermal damage and experimental methods for estimating relevant kinetic coefficients in both constant-temperature and transient thermal history experiments. As expected, transient in vivo thermal history data yield a noisy kinetic plot; however, estimates of the appropriate rate coefficients often can be made.
The thermal denaturation in solution of five purified vertebrate collagens has been examined by viscometry and optical polarimetry. The intrinsic viscosity and specific optical rotation values of undenatured collagen in solution were found to be independent of pH under the conditions used. The temperature dependences of the denaturation processes have been examined for the five collagens as a function of pH and the activation energies and entropy changes involved have been calculated from the changes of intrinsic viscosity with temperature. Considerable and similar variations in the denaturation temperatures TD with change of pH have been observed for all the collagens; for three collagens this variation is examined in detail. The activation energy involved in the denaturation of a given collagen is found to be independent of pH and the entropy change on denaturation is shown to be a function of the distribution of electric charges along the collagen molecule.
The TD values for the collagens at a given pH are separately correlated with the contents of hydroxyproline, proline, and total pyrrolidine residues. It is considered that the correlation between TD and total pyrrolidine content is the most fundamental and that the variation in thermal stability from collagen to collagen may be explained entirely on stereochemical grounds. The results afford no direct evidence to allow a decision between the two structures collagen I and collagen II proposed to explain the collagen high-angle X-ray diagram but put both structures on an equal basis. Since collagen II is more stereochemically satisfactory than collagen I this structure would appear to be the more likely.
A sensitive instrument has been constructed to measure the development and subsequent relaxation of the isometric tension during the heating of collagenous tissues.The curves of the tension developed by rat skin plotted against temperature exhibited different shapes according to the age of the animal. In skin from new born rats the maintenance of the maximum tension up to temperatures around 95°C appeared to be correlated with the presence of the thermally stable “keto” cross-link. In adult skin the dramatic relaxation of the tension that occurred above 75°C correlated with the increased proportion of the heat labile “aldimine” cross-link. With increasing age the extent of this relaxation decreased due to the increasing proportion of thermally stable cross-links of unknown origin.The curves obtained with granulation tissues of acute inflammation during the development phase and subsequent resorption phase of the granuloma were similar to those obtained during the growth of young animals from birth to 1 month. On the other hand granulation tissues of chronic inflammation retained an isometric tension curve similar to that of the new born rat skin, even after a long period of inflammation (8 months).Determination of the isometric tension developed in heated tissues provides a rapid assay useful for obtaining preliminary information on the cross-linking of collagen fibres in normal and pathological situations.
A number of physical methods have been used to show that soluble collagen prepared by citrate extraction of carp swim bladder tunics (ichthyocol) consists of rigid, rod-shaped molecules having a very narrow size distribution. The diameter is 13.6 A., and the weight average molecular length and weight are 3000 Å. and 345,000, respectively. This very asymmetric molecule undergoes denaturation in dilute salt solution at about 30° with an activation energy of 81 kcal, to form a product known as parent gelatin. However, the tendency toward reaggregation below this temperature and the significant rate of hydrolysis above this temperature (energy of activation 24 kcal.) creates unusual difficulty in characterizing the denatured state. The demonstration that 2 M KCNS lowers the denaturation temperature below 4° makes possible the avoidance of reaggregation by the use of this solvent. The number and weight average molecular weights were then shown to be 125,000 ± 10,000 and 138,000 ± 8000, respectively. The ratio of the molecular weight of collagen to the denatured form (parent gelatin) was found to be 2.5 ± 0.15 in separate experiments involving heating directly in the light scattering cell. The interpretation of these data shows that three molecules are formed from each collagen molecule upon denaturation, but the ratio of 2.5 requires that the molecules be of substantially different molecular weights. The dimensions of the collagen molecule are so much larger than the main long spaced periodicity (700 Å.) in collagen fibrils that several, probably four, of these must be contained within each molecule. Moreover, the size of the average parent gelatin molecule is such that it can traverse the whole length of the collagen molecule. A comparison with the three-stranded collagen structures recently deduced from X-ray studies shows that the observed mass-to-length ratio of 110 is sufficiently close to the required value and that the observed diameter is in excellent agreement. The conclusion therefore follows that the soluble collagen molecule is composed of three polypeptide strands running lengthwise and that it does serve without serious modification as the structural element of collagen fibrils. Moreover, this molecule has the additional characteristics of a hypothetical particle, tropocollagen, which Schmitt, Gross and Highberger concluded must exist as the common building unit of the three forms of collagen observed electron microscopically. Finally, suggestions are made of ways in which the three chains may be mutually arranged in the molecule so as to provide for a dangling chain at one end and a double chain at the other. These rather unique arrangements permit a closer fit of our data and a basis of constructing polarized protofibrils without the need of a continuous primary bonded framework.
The transformation of native beef Achilles tendon (BAT) and rat tail tendon (RTT) collagen in admixture with ethylene glycol to the amorphous, shrunken state has been investigated over the range of volume fraction v2 of collagen from 0 to 0.83. Precision dilatometric methods employed for v2 > 0.08 reveal small but unmistakable latent volume changes (ca. 5 × 10-3 cc. per g. of collagen). The transformation is partially reversible, the latent volume change being slowly recovered in part upon cooling. Return to the previous liquidus occurs within ±2° of the original transformation temperature Tm. These observations show conclusively that the transformation is properly represented as a phase transition, involving melting of crystalline regions, rather than as a rate process. Melting temperatures Tm also have been ascertained by polarizing microscopy and, at high dilution, from the decrease in viscosity. The dependence of Tm on v2 conforms rather well with polymer melting theory, which yields 24 ± 5 cal. g.-1 for the heat of fusion of collagen. A glass-type transition observed both in native collagen-diluent mixtures below Tm and in shrunken dry collagen at 95° probably is associated with freezing-in of side chain disorder. Melting of gelatin gels resembles the re-melting of collagen allowed to re-crystallize by cooling after the initial transformation. The identity of the processes involved is indicated by the observation that Tm for gelatin-glycol is within 2° of that for collagen-glycol of the same composition.
Recent developments in protein structure make one model for globular proteins especially attractive. This model consists of polypeptide chains folded on themselves to give hydrogen-bonded secondary structures which are in turn folded in a rigid tertiary arrangement through the interaction of amino acid side chains. The implications of the model are examined in terms of possible energy states available to proteins and possible denaturation reactions. Denaturation is defined as change in conformation. The folding process is considered to be spontaneous and directed by the composition and order of peptide amino acid residues. Reversible thermal denaturation processes are presented as changes in tertiary structure, irreversible processes as changes in secondary structure. The activated complex for denaturation and for enzymic function is discussed and the possibility presented that enzymic activity, reversible denaturation and ion-binding are all aspects of the same protein property.
A theoretical analysis of several protein denaturation models (Lumry-Eyring models) that include a rate-limited step leading to an irreversibly denatured state of the protein (the final state) has been carried out. The differential scanning calorimetry transitions predicted for these models can be broadly classified into four groups: situations A, B, C, and C'. (A) The transition is calorimetrically irreversible but the rate-limited, irreversible step takes place with significant rate only at temperatures slightly above those corresponding to the transition. Equilibrium thermodynamics analysis is permissible. (B) The transition is distorted by the occurrence of the rate-limited step; nevertheless, it contains thermodynamic information about the reversible unfolding of the protein, which could be obtained upon the appropriate data treatment. (C) The heat absorption is entirely determined by the kinetics of formation of the final state and no thermodynamic information can be extracted from the calorimetric transition; the rate-determining step is the irreversible process itself. (C') same as C, but, in this case, the rate-determining step is a previous step in the unfolding pathway. It is shown that ligand and protein concentration effects on transitions corresponding to situation C (strongly rate-limited transitions) are similar to those predicted by equilibrium thermodynamics for simple reversible unfolding models. It has been widely held in recent literature that experimentally observed ligand and protein concentration effects support the applicability of equilibrium thermodynamics to irreversible protein denaturation. The theoretical analysis reported here disfavors this claim.
The two principal rationales for applying hyperthermia in cancer therapy are that: (a) the S phase, which is relatively radioresistant, is the most sensitive phase to hyperthermia, and can be selectively radiosensitized by combining hyperthermia with x-irradiation; the cycling tumor cells in S phase which would normally survive an x-ray dose could thus be killed by subjecting these cells to hyperthermia; and (b) the relatively radioresistant hypoxic cells in the tumor may be selectively destroyed by combinations of hyperthermia and x-irradiation. Both of these rationales have been mentioned as reasons for using high LET irradiation in cancer therapy; therefore where such irradiation may be of use, hyperthermia may also be advantageous.
The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Testing environment is an important factor in the outcome of mechanical tests on connective tissue. The purpose of this investigation was to determine the effect of ligament water content on ligament mechanical behaviour by altering the test environment. Water content of medial collateral ligament (MCLs) from 19 three-month-old New Zealand White rabbits was varied in subsets of ligaments pairs by means of immersion in 2, 10 or 25% sucrose or 0.9% phosphate-buffered saline (PBS) solutions for 1 h. One knee joint was cycled 50 times in the designated solution (experimental), while the contralateral knee (uncycled control) was simultaneously soaked in the same tank. Following cycling, the water contents of both test and control ligaments were determined. Water contents of 22 normal MCLs were determined immediately post-sacrifice and served as 'normal water content' controls. Normalized peak cyclic load changes were used as a measure of the viscoelastic behaviour of each MCL. Results demonstrated that only ligaments soaked (but not cycled) in a 10% sucrose solution had water contents (60.5 +/- 2.5%) which were statistically similar to the 22 fresh normal MCLs (63.9 +/- 6.0%). Ligaments soaked in PBS (74.0 +/- 1.3%) or 2% sucrose (69.2 +/- 2.3%) had significantly higher water contents compared to fresh normal MCLs. Ligaments with higher water contents (e.g. soaked in PBS or 2% sucrose) demonstrated greater cyclic load relaxation compared to ligaments with lower contents (e.g. soaked in 25 or 10% sucrose). Different fluid test environments can significantly alter ligament water content and, in turn, significantly affect ligament viscoelastic behaviour.
Differential scanning calorimetry transitions for the irreversible thermal denaturation of yeast phosphoglycerate kinase at pH 7.0 are strongly scanning-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. To test this possibility, we have carried out a kinetic study on the thermal inactivation of the enzyme. The inactivation kinetics are comparatively fast within the temperature range of the calorimetric transitions and can be described phenomenologically by the equation dC/dt = -alpha C2/(beta + C), where C is the concentration of active enzyme at a given time, t, and alpha and beta are rate coefficients that depend on temperature. This equation, together with the values of alpha and beta (within the temperature range 50-59 degrees C) have allowed us to calculate the fraction of irreversibly denatured protein versus temperature profiles corresponding to the calorimetric experiments. We have found that (a) irreversible denaturation takes place during the time the protein spends in the transition region and (b) there is an excellent correlation between the temperatures of the maximum of the calorimetric transitions (Tm) and the temperatures (Th) at which half of the protein is irreversibly denatured. These results show that the differential scanning calorimetry transitions for the denaturation of phosphoglycerate kinase are highly distorted by the rate-limited irreversible process. Finally, some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible protein denaturation.
Light microscopy using polarized transmission illumination of routinely stained histologic sections shows changes of the native birefringence of certain tissue constituents when heated by laser irradiation or electrosurgical current. The naturally occurring birefringence of cardiac muscle disappears permanently when the muscle is frozen, thawed, and heated to temperatures in excess of 42 degrees C in vitro. This loss of birefringence is produced with temperatures at which other morphologic thermal changes are hard to detect; thus, it is a low-temperature tissue marker which can be used to observe the extent of thermal damage in tissues. Partial loss of the native birefringence of collagen occurs in canine urinary bladder coagulated by laser irradiation and pericardium heated with electrodes. In addition, thermally coagulated collagens have variable birefringence color shifts when compared to the adjacent unaffected collagens in stained histologic sections. The gradual birefringence color changes are seen at tissue temperatures higher than those at which the thermally induced hyalinization (coagulation) of collagen usually occurs (about 60-70 degrees C), but below those at which carbonization is seen (200+ degrees C). Birefringence changes can be measured to test mathematical models of thermal damage necessary for development of dosimetry models in medical applications of laser irradiation.
The endotherm enthalpy changes ΔHD and temperatures TD of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g−1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔHD decreases by 2 cal−1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds.The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F− and I−), on the hydration dependence of ΔHD and TD was also studied. It was shown that the primary hydration is not influenced by these ions, but that TD and ΔHD are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.
The degree of denaturation of heat-shrunk rattail tendon collagen was determined by dissolving the tendons with pronase and then calculating the native portion in the resultant solution from optical rotation values. Re-formation of triple-helical structures during the dissolution process was largely prevented when the tendons were dissolved at +4°. In this way, it was possible to detect the reversibly denatured portion together with the more severely denatured collagen. The dependence of the entire denaturation process on temperature is shown in a sigmoid curve with a point of inflection coinciding with that of the curve for the temperature dependence of the shrinkage process of collagen fibers. Both methods of measurement thus indicated the same denaturation temperature. This method also permits measurement of the renaturation processes in denatured insoluble collagen. Considerable renaturation was observed up to 3 hr after denaturation. The degree of renaturation was dependent on the degree of previous denaturation. The optimal renaturation temperature was found to be 30°. Stretching the denatured fiber promoted the re-formation of native material considerably.
The curves obtained for skin samples of different ages and species by hydrothermal isometric tension ("HIT") measurement are compared to those obtained by differential scanning calorimetry (DSC) under the same thermal conditions (for a rise in temperature at a rate of 1.0 degrees C/min). Collagen denaturation, observed by DSC, directly affects the kinetics of the tension variations in the first part of the "HIT" curves, including the early peak due to the presence and destruction of the heat-labile cross-links in the collagen network. The presence of cross-links is in term shown to delay collagen denaturation to an extent which depends in part on their heat-stability. The final part of the "HIT" curves reflecting the effects of heat in the stable polymeric collagen network is no longer affected by collagen denaturation. Thus, both "HIT" and DSC are useful methods to evaluate collagen reticulation in connective tissues.
The temperature at which collagen denatures from a triple helix to a random coil structure is a useful measure of the degree of crosslinking. A new multi-sample denaturation temperature tester (DTT) has been constructed for rapid determination of the collagen denaturation temperature of natural tissues and collagenous biomaterials. To validate the system, the denaturation temperatures measured for the DTT are compared with results from differential scanning calorimetry (DSC). Data are presented for bovine pericardium in three states with denaturation temperatures ranging from 68 to 85 degrees C: fresh, or crosslinked with glutaraldehyde or the epoxide reagent Denacol EX-512 poly (glycidyl ether). Denaturation temperatures measured by DTT were not significantly different from those measured by differential scanning calorimetry (DSC); however, DSC onset systematically occurred at a slightly lower temperature than that measured by DTT. This result, seen only for fresh tissue is in agreement with earlier experiments using hydrothermal isometric tension (HIT) testing. By contrast, DTT and DSC onset were identical for the exogenously crosslinked materials. Since the measured transition temperature was independent of initial load, this variable may be chosen to yield sharper force-temperature transitions with a given sample geometry. This instrument allows accurate assessment of collagen denaturation temperatures for multiple samples in a fraction of the time required by other methods.
This paper shows that the position and shape of the denaturation endothem of collagen fibrils are governed by the kinetics of an irreversible rate process. This was proved by measuring the rate of denaturation in rat tail tendons held isothermally at different temperatures, thereby determining rate constant characteristics such as the activation enthalpy and entropy and predicting endotherm position and shape therefrom. Comparison with actual scanning results showed good correspondence. Isothermal measurements of the rate of collagen denaturation, measured continuously using a calorimetric method, were used to determine rate constants for collagen denaturation in tendons immersed in water and 0.5 M acetic acid. The temperature dependence of the rate constants were fitted to the three rate process models, previously examined theoretically: the D and z formulation, the Arrhenius equation and the absolute rate theory. For example, in water the activation enthalpy was 0.518 (+/- 0.016) Mj mol-1 and the activation entropy 1.485 (+/- 0.049) kj mol-1 K-1, while in acetic acid the corresponding figures were 1.306 (+/- 0.099) Mj mol-1 and 4.142 (+/- 0.323) kj mol-1 K-1. These characteristics are discussed in terms of the thermal activation of a region of the molecule, the co-operative unit. The ratio of the activation enthalpy to the calorimetry enthalpy of denaturation indicated a co-operative unit that was 66 (+/- 5) residues long when fibrils were swollen in acetic and the collagen molecules acted essentially independently. On the other hand the intact fibrils in water gave a co-operative unit of 26 (+/- 1) residues long. The reason for the reduction in size of the co-operative unit is that it is surrounded, and therefore stabilized by other molecules in the fibre. It is interesting to note that the suggested co-operative unit lies almost entirely within the "gap" zone of the collagen fibril in its quarter-staggered arrangement of molecules. We believe that the co-operative unit would be represented by a domain that is free of stabilising hydroxyproline residues. Indeed such a domain exists near the C terminus of the triple helix from Gly877 to Pro941, i.e. 65 residues. In acetic acid, activation is similar to that of collagen molecules in solution. All the inter alpha-chain hydrogen bonds in the co-operative unit are broken and the separate chains in this short region are free to flail around under the action of thermal collisions relatively unimpeded by intermolecular interactions.(ABSTRACT TRUNCATED AT 400 WORDS)
Calorimetry was used to measure the rates of irreversible denaturation of collagen in intact mammalian lens capsules, isothermally stored in water at different temperatures, and the kinetic characteristics of the process determined. At 47.4 degrees C, the number of native collagen molecules declined by a factor of 10 in 21.4 +/- 2.9 min (this is called the D-value) and increasing the storage temperature by 2.32 +/- 0.12 degrees C reduced the D-value by a factor of 10. The activation energy of the process was 860 +/- 44 kJ mol-1, and the activation enthalpy and entropy were respectively 858 +/- 45 kJ mol-1 and 2.38 +/- 0.14 kJ mol-1 K-1. Using equations derived in the paper, these kinetic characteristics were used to predict the position and shape of the denaturation endotherms over a range of scanning rates. Comparison of the predicted and actual positions of peak maxima showed a close correspondence, demonstrating that the principal phenomenon determining the position of the peak maxima was an irreversible rate process. While there was fairly good agreement between the predicted and actual shape of the endotherm at low scanning rates, measurements at high rates were distorted by the finite response time of the calorimeter, and the agreement was poor.
Based upon the analysis of enzyme-catalyzed reactions occurring in living tissue, a model of thermal injury process is presented in which the fraction of denatured enzyme protein was taken as the indicator of thermal damage degrees. The results from this model describe the dependence of thermal damage on exposure time and temperature elevation.
The purpose of this study was to investigate the effect of tissue hydration on the structural properties of human patellar tendon. Specimens were subjected to a load relaxation experiment prior to being stretched to failure. The experiments indicated that tendons relaxed faster in hypotonic solutions when compared to hypertonic solutions. At a strain rate of 50% s-1 the structural stiffness was significantly higher while immersed in a hypotonic versus hypertonic solution. No difference in tensile stiffness was documented between baths for 0.5% s-1. Tendons immersed in the hypotonic solution were significantly stiffer for 50% s-1 against 0.5% s-1. The results indicate that the structural properties of human patellar tendon are more sensitive to time when the tissues are fully hydrated.
Linear birefringence is an anisotropic property of rat tail tendon, which is largely composed of collagen. Our goal is to show that the dynamic range and sensitivity of the linear birefringence loss of collagen during heating are sufficient for kinetic modeling of the reaction.
The linear birefringence loss was quantified for tendon denatured via both a heated-isotonic-saline bath and a heated stage. All measurements were made with a polarizing transmission microscope equipped with a Berek compensator.
The data show that the loss of linear birefringence is a first-order kinetic reaction. The native rat tail tendon birefringence, delta n = 3.0 +/- 0.6 x 10(-3) (mean +/- std. err.), is lost after denaturation occurs (delta n = 0). Application of the Arrhenius equation to the linear birefringence data yields the activation energy (Ea = 89 +/- 1 kcal/mole), pre-exponential coefficient (A = e130 +/- 1 s-1), enthalpy (delta H = 88 +/- 1 kcal/mole) and entropy (delta S = 197 +/- 2 cal/degree K.mole).
This study shows that dynamic changes in linear birefringence can be used to monitor thermally induced changes in collagen.
Thermal damage in heated bovine myocardial tissue is assessed from measured changes in total reflection and transmission of light. Mathematical expressions, based on random walk analysis of light propagation within tissue slabs, are used to relate the diffuse reflection and transmittance to the absorption coefficient, mu a, and effective scattering coefficient, mu's, for samples of myocardial tissue which were subjected to rapid step changes in temperature. Time-dependent changes in mu's, indicate two processes, one with a fast and temperature-dependent rate the other with a slow and apparently temperature-independent rate. For final temperatures above 56.8 degrees C and for the first 500 s after the temperature change, the optical parameters are well fit by exponential forms that exhibit temperature-dependent time constants as predicted by Arrhenius reaction rate theory of thermal damage. The scattering changes are associated with an apparent activation energy, delta E, of 162 kJ/mole and a frequency constant, A, of 3 x 10(23) s-1. This method provides a means for estimating optical coefficients which are needed to assess laser tissue dosimetry.
The outcome of many clinical heat therapies depends on the post-treatment structural integrity of the tissue. Unfortunately, there are few data on heat-induced changes in the mechanical properties of tissues due to temperature levels commonly achieved in laser, microwave, and radio-frequency-based modalities. Without such information, one cannot design optimal clinical protocols. Hence, we present new findings on the uniaxial stress strain behavior at 37 degrees C of a model collagenous tissue (chordae tendineae) both before and after thermal damage. This damage was induced via a variety of different thermo-mechanical loads: isothermal heatings at temperatures from 65 degrees C to 90 degrees C, durations of heating from 120 to 3600 s, and isotonic loads during heating from 0 to 0.65 MPa. Our data reveal that chordae exhibit pseudoelastic responses both before and after heating, but the extensibility, hysteresis, and compliance all increase with increased thermal damage. Fortunately, these complex heat-induced changes in behavior can be parameterized using a single measure of the prior thermal damage. This will clearly simplify the requisite constitutive formulations.
Isothermal data for heat-induced damage to four cells and the denaturation of two proteins are re-examined by scaling the actual heating time by τ50, a characteristic time common to biology, chemistry and physics. The data are from chordae tendineae, yeast phosphoglycerate kinase, 3T3 fibroblasts and skeletal muscle cells. The results appear to be independent of the mechanism of heat-induced damage. By conducting appropriate measurements at a minimum number of temperatures, heat-induced damage for a given material might be correlated, and predictions of cell death or protein denaturation are quickly found.
Optimization of clinical heat treatments for various pathologies requires accurate numerical modeling of the heat transfer, evolution of thermal damage, and associated changes in the material properties of the tissues. This paper presents two phenomenological equations that quantify time-dependent thermal damage in a uniaxial collagenous tissue. Specifically, an empirical rule-of-mixtures model is shown to describe well heat-induced axial shrinkage (a measure of underlying denaturation) in chordae tendineae which results from a spectrum of thermomechanical loading histories. Likewise an exponential decay model is shown to describe well the partial recovery (e.g., renaturation) of chordae when it is returned to body temperature following heating. Together these models provide the first quantitative descriptors of the evolution of heat-induced damage and subsequent recovery in collagen. Such descriptors are fundamental to numerical analyses of many heat treatments because of the prevalence of collagen in many tissues and organs.
Collagen molecules in solution unfold close to the maximum body temperature of the species of animal from which the molecules are extracted. It is therefore vital that collagen is stabilized during fiber formation. In this paper, our concept that the collagen molecule is thermally stabilized by loss of configurational entropy of the molecule in the fiber lattice, is refined by examining the process theoretically. Combining an equation for the entropy of a polymer-in-a-box with our previously published rate theory analysis of collagen denaturation, we have derived a hyperbolic relationship between the denaturation temperature, Tm, and the volume fraction, epsilon, of water in the fiber. DSC data were consistent with the model for water volume fractions greater than 0.2. At a water volume fraction of about 0.2, there was an abrupt change in the slope of the linear relationship between 1/Tm and epsilon. This may have been caused by a collapse of the gap-overlap fiber structure at low hydrations. At more than 6 moles water per tripeptide, the enthalpy of denaturation on a dry tendon basis was independent of hydration at 58.55 +/- 0.59 J g-1. Between about 6 and 1 moles water per tripeptide, dehydration caused a substantial loss of enthalpy of denaturation, caused by a loss of water bridges from the hydration network surrounding the triple helix. At very low hydrations (less than 1 mole of water per tripeptide), where there was not enough water to form bridges and only sufficient to hydrogen bond to primary binding sites on the peptide chains, the enthalpy was approximately constant at 11.6 +/- 0.69 J g-1. This was assigned mainly to the breaking of the direct hydrogen bonds between the alpha chains.
We present data from isothermal, isotonic-shrinkage tests wherein bovine chordae tendineae were subjected to well-defined constant temperatures (from 65 to 90 degrees C), durations of heating (from 180 to 3600 s), and isotonic uniaxial stresses during heating (from 100 to 650 kPa). Tissue response during heating and "recovery" at 37 degrees C following heating was evaluated in terms of the axial shrinkage, a gross indicator of underlying heat-induced denaturation. There were three key findings. First, scaling the heating time via temperature and load-dependent characteristic times for the denaturation process collapsed all shrinkage data to a single curve, and thereby revealed a time-temperature-load equivalency. Second, the characteristic times exhibited an Arrhenius-type behavior with temperature wherein the slopes were nearly independent of applied load--this suggested that applied loads during heating affect the activation entropy, not energy. Third, all specimens exhibited a time-dependent, partial recovery when returned to 37 degrees C following heating, but the degree of recovery decreased with increases in the load imposed during heating. These new findings on heat-induced changes in tissue behavior will aid in the design of improved clinical heating protocols and provide guidance for the requisite constitutive formulations.
Shoulder capsular shrinkage has recently been proposed as a therapeutic modality in a select group of patients with instability. Basic science research studying the mechanism of collagen shrinkage and the effect of shrinkage on the tissue's mechanical properties is essential to define the ideal process by which to achieve optimal tissue shrinkage. Tissue shrinkage is a function of both time and temperature. This relationship was studied, and a model was derived to describe the relationship mathematically. Tissue shrinkage rate was extremely sensitive to temperature changes. The purpose of this study, was to shrink collagenous tissue thermally and then to measure the mechanical property changes as a function of tissue shrinkage. Uniaxial tensile testing of normal and heat-shrunken bovine tendon was carried out, and a model was developed to express the relationship between shrinkage and mechanical properties. We found that the mechanical properties decreased with increasing shrinkage, and that the maximal allowable shrinkage before significant material property changes occurred was between 15% to 20%. Ultrastructural analysis with transmission electron microscopy showed denaturation of the collagen fibrillar structure and provided direct support for the observed material changes.
Thermal shrinkage of collagen currently is being used in orthopaedic surgery to treat ligamentous laxity. Understanding the kinetics of collagen shrinkage is key to revealing the events that take place during application of thermal energy. To elucidate the thermokinetic properties of collagen, punch biopsies of bovine joint capsule were immersed in a heated saline bath at temperatures between 20 degrees and 90 degrees C for periods up to 60 minutes. The resulting tissue thermal shrinkage was measured by the change in the cross-sectional area of the specimens. Only a small amount of shrinkage occurred at temperatures below 63 degrees C, and increasing amounts and rates of shrinkage were seen at temperatures between 63 degrees and 72 degrees C. The denaturation kinetics of bovine knee collagen, which could be described by a first order reaction rate, had an activation energy of 2.3 x 10(5) kJ/mol.
The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.
A detailed thermodynamic study of isolated strips of dog aorta is basic to the interpretation of the nonlinear and apparently rubber-like elastic behavior of such specimens. Experimental data indicate that the retractile force in isolated aortic strips of the dog is entropic. Volume changes with elongation in such strips are negligible. However, a large negative internal energy contribution to the total force is present at small elongations. Studies of aortic strips extracted with alkali suggest that the latter force arises from the elastic tissue itself.
Conformational transitions of proteins in water and in aqueous mixtures
- J F Brandts