Alteration of the thermodynamic characteristics of corneal collagen denaturation as a result of nonenzymatic glycation

The thermal stability of the scleral and corneal tissues after in vitro treatment with ribose, threose, and glyceraldehyde was investigated. The thermal transition temperature and the enthalpy of collagen fiber crosslinking were determined by differential scanning calorimetry (DSC). The resistance of the tissues toward trypsin was also determined after heating tissue samples in the DSC furnace. It was shown that the denaturation temperature of scleral and corneal samples treated by crosslinking agents increased, but the enthalpy of denaturation decreased. It is suggested that crosslinking in the collagen matrix of the cornea and sclera prevents complete collagen denaturation if the temperature does not rise up to 110 °C.     

The effect of deep-freezing on the structure of patellar and Achilles tendon allografts used for ACL reconstruction

According to the literature and to our recent studies, utilization of different types of allografts is a useful method for the reconstruction of the injured anterior crucial ligament. Of the different kind of allografts, in our practice, the patellar and Achilles tendons are the most commonly used types. Following our protocol, these allografts are subjected to cryopreservation at ?80 °C until utilization. The purpose of this study was to analyse the effects of cryopreservation at ?80 °C on the histological and structural properties of the human patellar and Achilles tendons. Total of four patellar and five Achilles tendons with human origin were collected for this study. The samples were subjected to fresh freezing and cryopreservation at ?80 °C. After 1, 3, 6, 12 and 24 weeks, the structure of the frozen tendons was compared to freshly collected samples by standard histological examination and thermal analyses, using differential scanning calorimetry (DSC). The thermal denaturation of different parts of human samples was monitored by a SETARAM Micro DSC-II calorimeter. The data treatment after ASCII conversion was done by Origin 6.0. DSC scans clearly demonstrated significant differences between the fresh and frozen samples. The decrease in collagen fibril density was supported by calorimetric and histological analyses in a time-dependent fashion. Our data suggest that application of allografts requires special attention paid to the proper storage time. The selection of grafts with possibly shorter storage time is recommended.     

Differential Scanning Calorimetry of Native Silk Feedstock

Native silk proteins, extracted directly from the silk gland prior to spinning, offer access to a naturally hydrated protein that has undergone little to no processing. Combined with differential scanning calorimetry (DSC), it is possible to probe the thermal stability and hydration status of silk and thus investigate its denaturation and solidification, echoing that of the natural spinning process. It is found that native silk is stable between ?10 °C and 55 °C, and both the high‐temperature enthalpy of denaturation (measured via modulated temperature DSC) and a newly reported low‐temperature ice‐melting transition may serve as useful quality indicators in the future for artificial silks. Finally, compared to albumin, silk's denaturation enthalpy is much lower than expected, which is interpreted within a recently proposed entropic desolvation framework which can serve to unveil the low‐energy aquamelt processing pathway.     

The influence of sodium chloride on the melting temperature of collagen crystalline region in parchments

In order to observe the influence of sodium chloride on the melting temperature of collagen crystalline region in three new parchments, samples were soaked in water (blanks) and NaCl solutions of different concentrations, then removed, dried in air and measured by means of differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The melting temperature of crystalline region of collagen, T _m, was determined as the minimum of the endothermal peak in the range 200–250 °C and as the inflection point of the decrease of storage modulus, respectively. There was observed a decrease in melting temperature of the salt-treated parchments compared to the samples soaked in water, sometimes significant (~20 °C) at certain concentrations of NaCl. Simultaneous TG/DTG/DSC thermal analysis (STA) was also applied for the determination of the amount of sodium chloride in salt-treated parchments, by calculating the mass loss due to the vaporization of NaCl, which occurs above 800 °C. By plotting T _m determined by DSC and DMA versus the NaCl content of the samples, an apparent minimum is observed. Additional information regarding the structural features was also obtained through X-ray diffraction (XRD) and attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR). XRD data put in evidence the preservation of collagen crystalline region in all salt-treated samples, while FTIR measurements did not showed significant modification of collagen. By removing the sodium chloride from the salt-treated parchments through washing with water, there is a return of the melting temperatures to the values of blank samples, demonstrating the reversibility of this phenomenon.     

Effects of deep-freezing and storage time on human femoral cartilage

Surgical techniques including new, possible resources to repair injured joints and damaged cartilage are still evolving. The exact effects of cryopreservation on the collected cartilage samples require accurate determination prior to utilization. The aim of our study was to analyze the impact of cryopreservation at ?80 °C on the structural properties of the human cartilage. The effects of storage time were also evaluated in conjunction with optimal utilization. The human cartilage samples were derived during operation and considered to be waste material. Samples were fresh frozen and stored at ?80 °C. Cryopreservation times were: 0, 1, 3, 6, and 12 weeks. To assess the biological and structural properties of the frozen human cartilage, we performed calorimetric examinations using differential scanning calorimetry (DSC). During the first 3 weeks, the calorimetric enthalpy (ΔH _cal) showed an increasing tendency compared to controls, parallel with the denaturation temperature (T _m): ΔH _cal (J g^?1) = 1.60 versus 2.49, T _m1 (°C) = 61.73 versus 63.64. After the sixth week, both the enthalpy and the transition temperature decreased, compared to the control samples. The decrease in both the calorimetric enthalpy and T _m could be explained by the decrease in bound water and the time-related degeneration in the structure of the cartilage. Here we found that the duration of cryopreservation interferes with the morphology of human cartilage samples only after 6 weeks of storage time. The thermal analyzes of human cartilage by DSC could be a useful method to follow the morphological changes in the clinical practice.     

Reversible Melting of Thermally Fractionated Polyethylene

Different grades of linear low density polyethylenes (LLDPEs) have been quenched cooled step-wise and crystallised isothermally at (a series of increasing) temperatures in a DSC (thermal fractionated samples). These samples have been investigated by temperature modulated DSC (MDSC). The heat flow curves of the thermal fractionated materials were compared with those obtained from samples crystallised at a relatively slow cooling rate of 2 K min^-1(standard samples). The melting enthalpy obtained from the total heat flow of the thermal fractionated samples was 0-10 J g^-1higher than those of standard samples. The melting enthalpy obtained from the reversing heat flows was 13-31 J g^-1lower in the thermal fractionated samples than in the standard samples. The ratio of the reversing melting enthalpy to the total melting enthalpy increased with decreasing density of the PE. The melting temperature of the endotherms formed by the step-wise cooling was 9 K higher than the crystallisation temperature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heating effect on the DSC melting curve of flaxseed oil

Flaxseed oil is rich in the alpha-linolenic acid. The effect of heating on the thermal properties of flaxseed oil extracted from flax seeds has been investigated. The flaxseed oils were heated at a certain temperature (75, 105, and 135 °C, respectively) for 48 h. The melting curve (from ?75 to 100 °C) of flaxseed oil was determined by differential scanning calorimetry (DSC) at intervals of 4 h. Three DSC parameters of exothermic event and endothermic event, namely, peak temperature (T _peak), enthalpy, and temperature range were determined. The initial flaxseed oil exhibited an exothermic peak, two endothermic peaks, and two endothermic shoulders between ?68 and ?5 °C in the melting profile. Heating temperature had a significant influence on the oxidative deterioration of flaxseed oil. The melting curve and parameters of flaxseed oil were almost not changed when flaxseed oil was heated at 75 °C. However, the endothermic peaks of melting curve decreased dramatically with the increasing of heating time when heating temperature was above 105 °C. There is almost no change of melting heat flow of flaxseed oil when heating time exceeded 32 h at 135 °C. The preliminary results suggest that the DSC melting profile can be used as a fast and direct way to assess the deterioration degree of flaxseed oil.     

Measurement of protein denaturation in human synovial fluid and its analogs using differential scanning calorimetry

Differential scanning calorimetry (DSC) was used to evaluate the thermal transitions associated with protein constituents of synovial fluid samples from three individuals with osteoarthritis. Analysis of the multi-component DSC curves revealed that major endothermic transitions of synovial fluid occur between 60 and 80 °C and can be resolved into three peaks, likely due to the unfolding of human serum albumin and immunoglobulins, and that the enthalpies of these transitions can be quantified in terms of their relative contribution to the total system enthalpy. DSC was also used to analyze a solution of bovine calf serum, a lubricant used in simulator wear testing of joint replacement implants, and the resulting endothermic transitions occurred in a temperature range relevant to that produced by frictional heat during such wear simulator testing. Results of this study indicate a new application for DSC as a direct method for studying thermal stabilities of both bovine calf serum and synovial fluid. The use of DSC is proposed as a diagnostic tool to detect altered thermal properties or protein concentrations indicative of a diseased or injured state, and as a development tool to test the efficacy of additives in controlling protein denaturation associated with increased wear in joint replacement implants.     

Thermal analysis of the cruciate ligaments of the human knee

Summary Cruciate ligaments of the knee joint are important structures very often affected by the degenerative process in case of osteoarthritis. One of the most controversial issues in knee arthroplasty practice nowadays is the role of the posterior cruciate ligament. With foregoing studies authors have demonstrated the feasibility of DSC in the investigation of the musculoskeletal system. With current study authors established the thermal behaviour of healthy cruciate ligaments and detected the alterations in case of osteoarthritis. By establishing the DSC scans of the normal ligaments authors demonstrated clear differences between the posterior and anterior ligament. In addition alterations between normal and arthritic samples could be detected both in terms of changes in total enthalpy and heat capacity. Calorimetric findings have been verified by histological examinations as well.     

DSC analysis of human synovial fluid samples in the diagnostics of non-septic and septic arthritis

The synovial fluid analysis is an important method in diagnosing and managing septic arthritis. To reach a quick diagnosis, preferably faster than the microbiological cultures, could be a great advantage in the therapy. The differential scanning calorimetry (DSC) has recently been found useful in the differential diagnosis of septic and non-septic periprosthetic conditions. The aim of this study was to evaluate whether there is a specific pattern in the different grades of arthritis, and the effect of three different bacterial strains inoculated in synovial fluid. The sensitivity of our method was also determined by using synovial fluid samples with the different bacterial concentrations. Authors developed a standardized, experimental model to assess the denaturation characteristics of non-septic and inoculated synovial fluid, infected by different bacterial strains. The thermal characteristics [maximal denaturation temperature (T _m) and calorimetric enthalpy change (ΔH _cal)] were monitored by SETARAM Micro DSC-II calorimeter. The denaturation scans clearly demonstrated specific, representative curves in the case of different grades of arthritis, as well as with each individual bacterial strain. Therefore, thermoanalyses of human synovial fluid samples by DSC could be a useful tool in the staging of osteoarthritis and the diagnostics of septic arthritis.     

Evaluation of blood plasma changes by differential scanning calorimetry in psoriatic patients treated with drugs

Psoriasis is a chronic inflammatory skin disease, most commonly resulting in the occurrence of red and silver scaly plaques. Application of differential scanning calorimetry (DSC) should be used as a new method to detect the different stages of the disease and to monitor medications with different anti-psoriatic drugs using patient’s blood plasma. The study included 72 white adults (35 men and 37 women; median age 56 years) with diagnosed psoriasis. According to the psoriasis area severity index (PASI) patients were selected into three groups: symptomless (PASI: 0), mild (PASI: 1–15), and serious symptoms (PASI: >15). According to medication patients were divided into untreated (n = 39) and treated (n = 33) groups. For systemic drug treatment cytostatic therapy (methotrexate, n = 12), retinoid treatment (acitretin, n = 10), and biologic response modifiers (adalimumab, n = 5; infliximab, n = 5; ustekinumab, n = 1) were applied. Denaturation of human plasma components were detected in Setaram Micro DSC II calorimeter. The patients had no third denaturation peak in the untreated mild and serious symptoms groups. In mild symptoms all the thermal parameters altered significantly, while in serious symptoms only the first melting and the calorimetric enthalpy altered significantly compared with symptoms-free states. In case of systematic cytostatic and retinoid drug treatment (methotrexate, n = 12; acitretin, n = 10) cases the DSC scans of patients with symptoms exhibited significant differences (p < 0.05) in melting temperatures and in calorimetric enthalpy compared with the untreated symptoms-free patients. Using biologic response modifier agents (adalimumab, infliximab, and ustekinumab) we had no enough samples for a statistical evaluation for each one, but after the intervention a stronger effect can be seen as in case of systematic drug treatment. In this study blood plasma measurement in psoriatic patients by DSC showed differences between untreated, conventional systemic drug treatment, and application of biologic response modifier agents, but further studies are needed to elucidate these relationships (supported by grant OTKA CO-272).     

Assessment of interactions between four proteins and benzothiazole derivatives by DSC and CD

The thermal denaturation of ovalbumin, lysozyme, myoglobin and fibrinogen at different BTS concentrations have been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Thermodynamic parameters: melting temperatures (T_m), calorimetric enthalpy (ΔH), van’t Hoff enthalpy (ΔH_v) were obtained for all the systems under study. Thermal denaturation of the four proteins was completely irreversible. Changes in the protein conformation due to the adsorption of BTS molecules have been monitored by using UV-CD spectra. Greater changes in α-helical contents correspond with the BTS higher concentrations. The lysozyme denaturation temperature increases at low concentrations BTS indicating that BTS acts as a structure stabilizer; meanwhile it acts as a destabilizer at higher concentrations in all the proteins studied. The major effect is observed in the case of myoglobin, the protein with the highest α-helical secondary structure (75%).     

Differential scanning calorimetric examination of ruptured lower limb tendons in human

The tendon ruptures are serious injuries of the lover limb in middle age and physically active population. While the Achilles tendon rupture is common, the patellar ligament and quadriceps ligament ruptures are an absolutely rare injury. Usually there is no correlation between the velocity of the trauma and the supervening of the rupture. The aetiology of the degenerative changes in the collagen structures of the tendons and ligaments which could be disposed for the rupture are still not clear. Our hypothesis was that before the injury there are clear pathological abnormalities in the tissues of the tendons, which are predisposed for the rupture, and could be monitored besides the classical histological methods by differential scanning calorimetry. The thermal denaturation of human samples was monitored by a SETARAM Micro DSC-II calorimeter. All the experiments were performed between 0 and 100 °C. The heating rate was 0.3 K/min. DSC scans clearly demonstrated significant differences between the control and ruptured samples (control: T _m = 59.7 °C, T _1/2 = 1.4 °C and ΔH _cal = 8.54 J/g; ruptured Achilles tendon: T _m = 62.75 °C, T _1/2 = 2.6 °C and ΔH _cal = 1.54 J/g, ruptured Quadriceps tendon: T _m = 64.8 °C, T _1/2 = 1.6 °C and ΔH _cal = 1.53 J/g, ruptured Patellar tendon: T _m = 63.9 °C, T _1/2 = 1.41 °C and ΔH _cal = 0.97 J/g). These observations could be explained with the structural alterations caused by the biochemical processes. With our investigations we could demonstrate that DSC is a useful and well applicable method for the investigation of collagen tissue of the degenerated human tendons and ligaments. We can prove with this method that the degenerative changes of the tissue elements increase the thermal stability of collagen tissues of the tendons which could be disposed for the rupture.     

Calorimetric investigation of normal and pathological human meniscus

In this study, human meniscus tissue of normal origin in young adults and in patients with early and late stages of osteoarthritic degeneration were analyzed for their thermoanalytical properties with differential scanning calorimetry. Degenerative changes of knee menisci frequently result from trauma to the joint or are associated with joint diseases. Meniscus damage may play an important role in osteoarthritis pathophysiology. The purpose was to further characterize the altered metabolism in matrix composition during different stages of meniscus degeneration that promotes disease progression. The human knee joint menisectomy specimens were received after surgical removal. The calorimetric properties of samples were determined by DSC method, samples were heated from 0 to 80 °C. The heating rate was 0.3 °C min^?1. Conventional Hastelloy batch vessels were used with 40 μL sample volume. Change in the enthalpy was observed in normal cartilage as 1632.48 J g^?1 (SD = 50.55). In case of early degeneration a greater change at 1707.83 J g^?1 (SD = 112.46), while in the severely degenerated samples at 1677.30 J g^?1 (SD = 182.48) was measured. This method proved to be suitable for compositional thermoanalytical study of normal and degenerative human meniscus samples. All samples that were extracted for this study were obtained during live surgeries. With the rise of temperature an endothermic reaction was observed in all cases. The enthalpy change of the process initiated by the temperature change showed difference between the normal and pathological groups.     

Effect of venous wall immobilization on the thermal degradation of collagen

The results from a comparative study of the thermal denaturation of collagen in the venous walls of reference samples and samples with varicose disease are presented. Changes in the organization of collagen network of the tissue matrix are detected via thermal analysis and multiphoton microscopy with recording of the second harmonic generation (SHG). It is established that the collagen network of venous walls degrades in varicose disease. It is shown that the disordering of the tertiary structure of collagen molecules is reflected in a 40% drop in the enthalpy of protein denaturation compared to reference (ΔH _D = 12.4 ± 4.9 J/g dry residue). The disorganization of fiber structures is recorded on SHG images. It is shown that upon the hydrothermal heating of sequestered samples of venous walls, the complete degradation of the tissue network occurs at 75°C. However, it is noted that upon the mechanical immobilization of samples of both types, the stability of collagen increases and complete denaturation is observed at temperatures above 84°C. It is suggested that the number of available conformations of polypeptide chains in the random coil state falls under tension, lowering ΔS _D and raising the temperature of the denaturation of protein.     

Microstructural properties of sintered Al–Cu–Mg–Sn alloys

A non-commercial Al4Cu0.5Mg alloy has been used for investigating the effects of the elemental Sn additions. Uniaxial die compaction response of the alloys in terms of green density was examined, and the results showed that Sn addition has no effect when compacting conducted under high pressures. In total, 93–95% green density was achieved with an applied pressure of 400 MPa. Thermal events occurring during the sintering of the emerging alloys were studied by using differential scanning calorimetry (DSC). First thermal event on the DSC analysis of the Al4Cu0.5Mg1Sn alloy is the melting of elemental Sn, whereas for Al4Cu0.5Mg alloy, it is the formation of Al–Mg liquid nearly at 450 °C. Also it is clearly seen on the DSC analysis that Sn addition led to an increase in the formation enthalpy of Al–Mg liquid phase. High Sn content and high sintering temperature (620 °C), therefore high liquid-phase content, caused decrease on the mechanical properties due to thick intergranular phases and grain coarsening. Highest transverse rupture strength and hardness values were obtained from Al4Cu0.5Mg0.1Sn alloy sintered at 600 °C and measured as 390 MPa and 73 HB, respectively.     

Effect of accelerated thermal ageing on the thermal behaviour of the recently made parchments

To reveal the fire injuring of parchment, the changes in the thermal behaviour of some goat parchments, obtained from skins originating from different animals, as a result of thermal aging were determined by thermal analysis methods (DSC; simultaneous TG/DTG, DSC; micro hot table (MHT)). Thermal aging of parchments was revealed to bring about the decrease in shrinkage temperature, absolute value of enthalpy of denaturation in water and some changes in non-isothermal parameters characteristic for dehydration process in static air atmosphere. The results obtained by DSC analysis performed in N₂ and O₂ flows as well as those obtained by simultaneous TG/DTG, DSC analyses have shown that both softening (melting) process parameters and parameters of thermo-oxidative processes have not been changed by thermal ageing. The results obtained by thermal analysis methods were correlated with those obtained by microscopic investigation of parchment samples immersed in water and scanning electron microscopy (SEM). The application of these microscopic techniques has revealed the morphology changes in the investigated parchments as a result of thermal degradation.     

Laser-induced modification of collagen in nucleus pulpose gel

The thermal behavior of collagen in intact and IR laser irradiated nucleus pulpose tissues was studied. The enthalpy of denaturation of the intact tissue was determined (53.1 ± 1.2 J/g of collagen). The denaturation of collagen during laser heating at temperatures below the characteristic melting point occurred under the influence of the photomechanical component of laser irradiation. Original Russian Text ? I.V. Andreeva, N.Yu. Ignat’eva, O.L. Zakharkina, V.V. Lunin, 2009, published in Zhurnal Fizicheskoi Khimii, 2009, Vol. 83, No. 3, pp. 590–592.     

Bird muscles under hydrostatic high-pressure/temperature combinations

Breast muscles from three different birds were subjected to hydrostatic high-pressure (400 MPa)/temperature (10–75°C) combinations, and the denaturation-induced effects on the pressurized proteins monitored by DSC. Comparisons with parallel results from heating-alone processes were established. Actin was the most labile moiety to pressurization and myosin together with sarcoplasmic proteins were next in observing pressure-induced denaturation at low temperatures. Some myosin derivatives (fragments or aggregates) and collagen remained native-like under pressure at any temperature. As previously reported, pressure and temperature showed interdependent and antagonistic-like effects. Hydrostatic high-pressure caused severe proteins denaturation at non thermal denaturing temperatures. At thermally active conditions, pressure preserved proteins from subsequent thermal denaturation. This last effect was lower than in similar but destructured myosystems (batters) because of the absence of functional salts but presumably also by steric hindrance.     

Cyclophosphamide-induced changes in plasma and red blood cells detected by differential scanning calorimetry (DSC) in guinea pigs

Cyclophosphamide is one of the most widely used drugs, but it has severe side effects. Considering the variability of kinetics and metabolism of cyclophosphamide in the daily use, it is an important question how could we predict these undesirable consequences. The purpose of this study was to introduce calorimetry for the first time in the detection of cyclophosphamide-induced changes in plasma and red blood cells in an experimental animal model. Adult guinea pigs (n = 55, in 11 different groups) were injected intraperitoneally with the dose of cyclophosphamide that is comparable to the human dosage. Animals were euthanized; plasma and formed elements of blood were analyzed by a SETARAM Micro DSC-II calorimeter. The denaturation temperatures were measured, and the calorimetric enthalpies were calculated based on the areas under thermal absorption curves. The results show a definite dose dependence in both denaturation temperature and calorimetric enthalpy, that is, the DSC seems to be a suitable technique to detect the damage caused by chemotherapy.