Freeze–Thaw Induced Biomechanical Changes in Arteries: Role of Collagen Matrix and Smooth Muscle Cells

Abstract

Applications involving freeze-thaw, such as cryoplasty or cryopreservation can significantly alter artery biomechanics including an increase in physiological elastic modulus. Since artery biomechanics plays a significant role in hemodynamics, it is important to understand the mechanisms underlying these changes to be able to help control the biomechanical outcome post-treatments. Understanding of these mechanisms requires investigation of the freeze-thaw effect on arterial components (collagen, smooth muscle cells or SMCs), as well as the components' contribution to the overall artery biomechanics. To do this, isolated fresh swine arteries were subjected to thermal (freeze-thaw to -20 degrees C for 2 min or hyperthermia to 43 degrees C for 2 h) and osmotic (0.1-0.2 M mannitol) treatments; these treatments preferentially altered either the collagen matrix (hydration/stability) or smooth muscle cells (SMCs), respectively. Tissue dehydration, thermal stability and SMC functional changes were assessed from bulk weight measurements, analyses of the thermal denaturation profiles using Fourier transform infrared (FTIR) spectroscopy and in vitro arterial contraction/relaxation responses to norepinephrine (NE) and acetylcholine (AC), respectively. Additionally, Second Harmonic Generation (SHG) microscopy was performed on fresh and frozen-thawed arteries to directly visualize the changes in collagen matrix following freeze-thaw. Finally, the overall artery biomechanics was studied by assessing responses to uniaxial tensile testing. Freeze-thaw of arteries caused: (a) tissue dehydration (15% weight reduction), (b) increase in thermal stability (approximately 6.4 degrees C increase in denaturation onset temperature), (c) altered matrix arrangement observed using SHG and d) complete SMC destruction. While hyperthermia treatment also caused complete SMC destruction, no tissue dehydration was observed. On the other hand, while 0.2 M mannitol treatment significantly increased the thermal stability (approximately 4.8 degrees C increase in denaturation onset), 0.1 M mannitol treatment did not result in any significant change. Both 0.1 and 0.2 M treatments caused no change in SMC function. Finally, freeze-thaw (506+/-159 kPa), hyperthermia (268+/-132 kPa) and 0.2 M mannitol (304+/-125 kPa) treatments all caused significant increase in the physiological elastic modulus (Eartery) compared to control (185+/-92 kPa) with the freeze-thaw resulting in the highest modulus. These studies suggest that changes in collagen matrix arrangement due to dehydration as well as SMC destruction occurring during freeze-thaw are important mechanisms of freeze-thaw induced biomechanical changes.

The effect of a thermal renal denervation cycle on the mechanical properties of the arterial wall

“…the modulus) of the tissue is present, whereby the average collagen stiffness is only 16% of the control specimens highlighting that the thermal cycle significantly degrades the collagen structure in these specimens. This potentially is an anticipated result, as the temperature at which collagen degrades is reported as being in the range of 50-60 1C (Gross, 1964;Venkatasubramanian et al, 2010). The degradation in the collagen stiffness is not as pronounced in the native specimens, whereby the collagen retains 60% of it mechanical stiffness in comparison to the unheated controls.…”

“…Any disruption to this extracellular matrix architecture may cause vessel wall dysfunction. The denaturation temperature of collagen has been reported in a number of studies as being in the range of 50-60 1C (Gross, 1964;Venkatasubramanian et al, 2010). These results would imply that some form of reduction in mechanical stability of the arterial wall would occur post a renal denervation thermal cycle, as it contains significant amounts of collagen-the main structural element.…”

Cryoplasty Versus Conventional Balloon Angioplasty of the Femoropopliteal Artery in Diabetic Patients: Long-Term Results from a Prospective Randomized Single-Center Controlled Trial

“…We speculate that the lower pressure (B8 Atm) and the compliant nature of the cryoplasty catheter, which can be attributed to the compressible gas used for the inflation, compared with the uniformly liquid-inflated noncompliant or semicompliant conventional balloons, may result in more immediate technical failures, especially in the setting of resistant, eccentric, fibrotic, or fibrocalcific lesions. It should be stressed that although many operators may anecdotally perform multiple repeated cryoplasty sessions in the same lesion, i.e., during the same procedure, because of an initial suboptimal angioplasty result or in case of tandem lesions, there are no published data about the actual biomechanical effects as well as the induction of apoptosis after consecutive applications of freezing-thawing to the vessel wall [33,34].…”

Measurement of Spatiotemporal Intracellular Deformation of Cells Adhered to Collagen Matrix During Freezing of Biomaterials

“…This tissue-level microstructural damage has been investigated using multiphoton-induced autofluorescence and second harmonic generation microscopy [13], magnetic resonance imaging [14], and histological analysis [10,15]. Though successful preservation of microstructures was reported for aortic and pulmonary valves [8] and articular cartilages [16], significant change in tissue functionality, as well as in structural and mechanical properties was also observed in other types of tissues [17][18][19].…”

“…Although post-thaw cell viability [6] has been the primary target of these applications, it has recently been recognized that for tissues, where cells are embedded in a complex three-dimensional extracellular matrix (ECM), other features beyond viability are also important to the functionality of biomaterials. This includes the microstructure of the ECM, state of the cell-matrix adhesion, and the cytoskeletal structure and organization [7][8][9][10].…”