Alptekin Aksan

THERMAL MODIFICATION OF CONNECTIVE TISSUES:BASIC SCIENCE CONSIDERATIONS AND CLINICAL IMPLICATIONS

&NA; Thermal modification (shrinkage) of capsular connective tissue has gained increasing popularity as an adjunctive or even a primary procedure in the arthroscopic treatment of shoulder instability. Although the physical effects of heat on collagenous tissues are well known, the long‐term biologic fate of these shrunken tissues is still a matter of debate. The temperatures required to alter the molecular bonding of collagen and thus cause tissue shrinkage (65°C to 70°C) are also known to destroy cellular viability. Therefore, thermally modified tissues are devitalized and must undergo a biologic remodeling process. During this remodeling, the mechanical properties of the treated tissues are altered (decreased stiffness) and can be at risk for elongation if the postoperative rehabilitation regimen is too aggressive. Although anecdotal reports suggest that thermal capsular shrinkage does have a beneficial effect, the exact mechanism responsible for this clinical improvement has yet to be fully defined. The reported improvement could be due to the maintenance of initial capsular shrinkage, secondary fibroplasia and resultant thickening of the joint capsule, a loss of afferent sensory stimulation due to the destruction of sensory receptors, or a combination of all three. The clinical role for thermal modification of connective tissues has not yet been defined, but it appears that it may prove most useful as a stimulant for inducing a biologic repair response.

Painting and printing living bacteria: Engineering nanoporous biocatalytic coatings to preserve microbial viability and intensify reactivity

Latex biocatalytic coatings containing ∼50% by volume of microorganisms stabilize, concentrate and preserve cell viability on surfaces at ambient temperature. Coatings can be formed on a variety of surfaces, delaminated to generate stand‐alone membranes or formulated as reactive inks for piezoelectric deposition of viable microbes. As the latex emulsion dries, cell preservation by partial desiccation occurs simultaneously with the formation of pores and adhesion to the substrate. The result is living cells permanently entrapped, surrounded by nanopores generated by partially coalesced polymer particles. Nanoporosity is essential for preserving microbial viability and coating reactivity. Cryo‐SEM methods have been developed to visualize hydrated coating microstructure, confocal microscopy and dispersible coating methods have been developed to quantify the activity of the entrapped cells, and FTIR methods are being developed to determine the structure of vitrified biomolecules within and surrounding the cells in dry coatings. Coating microstructure, stability and reactivity are investigated using small patch or strip coatings where bacteria are concentrated 102‐ to 103‐fold in 5–75 μm thick layers with pores formed by carbohydrate porogens. The carbohydrate porogens also function as osmoprotectants and are postulated to preserve microbial viability by formation of glasses inside the microbes during coat drying; however, the molecular mechanism of cell preservation by latex coatings is not known. Emerging applications include coatings for multistep oxidations, photoreactive coatings, stabilization of hyperthermophiles, environmental biosensors, microbial fuel cells, as reaction zones in microfluidic devices, or as very high intensity (>100 g·L‐1 coating volume·h‐1) industrial or environmental biocatalysts. We anticipate expanded use of nanoporous adhesive coatings for prokaryotic and eukaryotic cell preservation at ambient temperature and the design of highly reactive “living” paints and inks.

Freezing-induced phase separation and spatial microheterogeneity in protein solutions.

Amid decades of research, the basic mechanisms of lyo-/cryostabilization of proteins and more complex organisms have not yet been fully established. One major bottleneck is the inability to probe into and control the molecular level interactions. The molecular interactions are responsible for the significant differences in the outcome of the preservation processes. (1) In this communication, we have utilized confocal Raman microspectroscopy to quantify the freezing-induced microheterogeneity and phase separation (solid and liquid) in a frozen solution composed of a model protein (lysozyme) and a lyo-/cryoprotectant (trehalose), which experienced different degrees of supercooling. Detailed quantitative spectral analysis was performed across the ice, the freeze-concentrated liquid (FCL) phases, and the interface region between them. It was established that the characteristics of the microstructures observed after freezing depended not only on the concentration of trehalose in the solution but also on the degree of supercooling. It was shown that, when samples were frozen after high supercooling, small amounts of lysozyme and trehalose were occluded in the ice phase. Lysozyme preserved its native-like secondary structure in the FCL region but was denatured in the ice phase. Also, it was observed that induction of freezing after a high degree of supercooling of high trehalose concentrations resulted in aggregation of the sugar and the protein.

Response of the cell membrane–cytoskeleton complex to osmotic and freeze/thaw stresses☆

In order to develop successful cryopreservation protocols a better understanding of the freeze- and dehydration-induced changes occurring in the cell membrane and its underlying support, the actin cytoskeleton, is required. In this study, we compared the biophysical response of model mammalian cells (human foreskin fibroblasts) to hyperosmotic stress and freeze/thaw. Transmitted light, infrared spectroscopy, fluorescence- and cryo-microscopy were used to investigate the changes in the cell membrane and the actin cytoskeleton. We observed that a purely hyperosmotic challenge at room temperature resulted in bleb formation. A decrease in temperature abrogated the blebbing behavior, but was accompanied by a decrease in viability. These results suggested that cell survival depended on the availability of the membrane material to accommodate the volumetric expansion back to the original cell volume at isotonic conditions. Our data also showed that freeze/thaw stresses altered the cell membrane morphology resulting in a loss of membrane material. There was also a significantly lower incidence of blebbing after freeze/thaw as compared to isothermal osmotic stress experiments at room temperature. Significant depolymerization of the actin cytoskeleton was seen in cells whose membranes had been compromised by freeze/thaw stresses. Actin depolymerization using cytochalasin D affected the stability of the membrane against mechanical stress at isothermal conditions. This study shows that both the membrane and cytoskeleton, as a system, are involved in the osmotic and freeze/thaw-induced responses of the mammalian cells.

Spatial Distribution of the State of Water in Frozen Mammalian Cells

We describe direct determination of the state of intracellular water, measurement of the intercellular concentration of a cryoprotectant agent (dimethylsulfoxide), and the distribution of organic material in frozen mammalian cells. Confocal Raman microspectroscopy was utilized at cryogenic temperatures with single live cells to conduct high spatial resolution measurements (350 × 350 × 700 nm), which yielded two, we believe, novel observations: 1), intracellular ice formation during fast cooling (50°C/min) causes more pronounced intracellular dehydration than slow cooling (1°C/min); and 2), intracellular dimethylsulfoxide concentration is lower (by as much as 50%) during fast cooling, decreasing the propensity for intracellular vitrification. These observations have a very significant impact for developing successful biopreservation protocols for cells used for therapeutic purposes and for cellular biofluids.

Hydrogen Bonding and Kinetic/Thermodynamic Transitions of Aqueous Trehalose Solutions at Cryogenic Temperatures

Carbohydrates play important roles in the survival of freeze-tolerant organisms. In order to understand the role of carbohydrates on hydrogen bonding (HB) and thermodynamic/kinetic transitions, aqueous trehalose solutions at cryogenic temperatures were analyzed using FTIR spectroscopy. Distinct changes in water-water and water-carbohydrate HB organization were identified during supercooling, freezing, and vitrification. FTIR spectroscopy revealed the kosmotropic effect of trehalose and the presence of two distinct water families in supercooled carbohydrate solutions, (1) water molecules directly associated with the carbohydrate, forming its hydration layer(s) and (2) water molecules that are involved in water-water HB in small clusters. The latter showed characteristics of water in hydrophilic confinement.

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery.

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T(m)), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T(m) from 8.4+/-1.7 degrees C (in undried control samples) to 16.5+/-1.3 degrees C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T(m), which began approximately 1 day after initial rehydration and plateaued at 12.5+/-1.8 degrees C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T(m) and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.

State of the Art in Preservation of Fluid Biospecimens

Fluid biospecimens (blood, serum, urine, saliva, cerebrospinal fluid and bronchial lavage fluid) contain not only cells and subcellular components, but also proteins, enzymes, lipids, metabolites, and peptides, which are utilized as biomarkers. Availability of high-quality biospecimens is a requirement for biomarker discovery. The quality of the biospecimens depends upon preanalytical variables (ie, collection and processing techniques, freeze/thaw stability, and storage stability), which account for >60%-90% of the diagnostic errors. Currently, millions of fluid biospecimens are stored in hundreds of biorepositories across the nation, and tens of thousands of new biospecimens are added to the pool daily. Specimen stabilization is imperative, because fluid biospecimens degrade quickly when kept untreated at room temperature. Achieving a high-quality fluid biospecimen requires understanding the effects of storage processing parameters (eg, freezing and thawing as well as cryo-/lyoprotectant additives) and storage conditions on biomarkers contained within the biospecimens. In this article, we will discuss the main issues related to the stabilization of specific biofluids by reviewing (a) the current preservation and storage practices applied in biobanks/biorepositories and (b) the sensitivity of certain biomarkers to current storage techniques.

Thermomechanical analysis of soft-tissue thermotherapy.

Soft-tissue thermotherapy based on sub-ablative heating of collagenous tissues finds wide-spread application in medicine such as tissue welding, thermokeratoplasty, skin resurfacing, elimination of discogenic pain in the spine and treatment of joint instability. In this paper, heat-induced thermomechanical response characteristics of collagenous tissues are quantified by means of in vitro experimentation with a representative model tissue (New Zealand white rabbit patellar tendon). Three distinct heat-induced thermomechanical response regimes (defined by the rate of deformation and the variation of material properties) are identified. Arrhenius damage integral representation of collagenous tissue thermal history is shown to be adequate in establishing the master response curves for quantification of thermomechanical response for modeling purposes. The trade-off between the improved kinematical stability and compromised mechanical stability of the heated collagenous tissue is shown to be the major challenge hindering the success of subablative thermotherapies.

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens

Geobacter sulfurreducens is a delta-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.