The effects of prolonged deep freezing on the biomechanical properties of osteochondral allografts

Musculo-skeletal allografts sterilized and deep frozen are among the most common human tissue to be preserved and utilized in modern medicine. The effects of a long deep freezing period on cortical bone has already been evaluated and found to be insignificant. However, there are no reports about the influences of a protracted deep freezing period on osteochondral allografts. One hundred osteochondral cylinders were taken from a fresh specimen and humeral heads of 1 year, 2 years, 3 years and 4 year old bones. Twenty chips from each period, with a minimum of 3 chips per humeral head. Each was mechanically tested by 3 point compression. The fresh osteochondral allografts were significantly mechanically better than the deep frozen osteochondral allografts. There was no statistical significant time dependent difference between the deep frozen groups in relation to the freezing period. Therefore, we conclude that, from the mechanical point of view deep freezing of osteochondral allografts over a period of 4 years, is safe without further deterioration of the biomechanical properties of the osteochondral allografts.     

Activation and expression of urokinase-type plasminogen activator are modulated by freezing/thawing process through activation of redox signal pathway in primary porcine endometrial cells

Plasminogen activators (PAs) play a pivotal role in a variety of uterine physiologies, such as endometrial function, trophoblast invasion, and implantation process, but its alteration in expression or activity during cryopreservation of primary uterine cells has received little attention. In this study, we investigated whether PA expression and activity were modulated in first passage primary porcine uterus endometrial epithelium cells (PUEECs) treated with or without a freezing-thawing procedure. Western blotting and zymographic analysis showed that uPA expression and activity increased significantly in frozen-thawed PUEECs in a passage-dependent manner as compared to freshly prepared control cells. Moreover, intracellular reactive oxygen species (ROS) were increased by freezing-thawing and longer culturing, and were more prominent in frozen-thawed PUEECs than in control cells. However, the increase in both uPA expression and activity was greatly reduced or alleviated by treatment with either ROS scavenger N-acetylcysteine or extracellular signal-regulated kinase (ERK) inhibitor PD98059. These results suggest that ROS/ERK-mediated uPA activation may be an important factor in cryo-damage of primary uterine cells.     

Effect of freeze-drying and gamma irradiation on biomechanical properties of bovine pericardium

Freeze-drying and gamma irradiation are the techniques widely use in tissue banking for preservation and sterilization of tissue grafts respectively. However, the effect of these techniques on biomechanical properties of bovine pericardium is poorly known. A total of 300 strips of bovine pericardium each measured 4 cm × 1 cm were used in this study to evaluate the effect of freeze-drying on biomechanical properties of fresh bovine pericardium and the effect of gamma irradiation on biomechanical properties of freeze-dried bovine pericardium. The strips were divided into three equal groups, which consist of 100 strips each group. The three groups were fresh bovine pericardium, freeze-dried bovine pericardium and irradiated freeze-dried bovine pericardium. The biomechanical properties of the pericardial strips were measured by a computer controlled instron tensiometer while the strips thickness was measured by Mitutoyo thickness gauge. The results of the study revealed that freeze-drying has no significant (p > 0.05) effect on the tensile strength, Youngs modulus (stiffness) and elongation rate of fresh bovine pericardium. Irradiation with 25 kGy gamma rays caused significant decreased in the tensile strength, Youngs modulus and elongation rate of the freeze-dried pericardium. However, gamma irradiation has no significant effect on the thickness of freeze-dried bovine pericardium, while freeze-drying caused significant decreased in the thickness of the fresh bovine pericardium. The outcome of this study demonstrated that freeze-drying has no significant effect on the biomechanical properties of fresh bovine pericardium, and gamma irradiation caused significant effect on the biomechanical properties of freeze-dried bovine pericardium.     

Effect of freezing and thawing on the structure and function of cytochrome oxidase

The effect of freezing-thawing on cytochrome oxidase from cattle heart was studied. The enzyme was characterized for its activity, absorption spectra, temperature perturbation differential spectra and conformational transitions. A decrease in the activity and conformational changes depend on the composition of the buffer system and the presence of potassium and sodium chlorides in the solution.     

Effects of different cryoprotectants on the viability and biological characteristics of porcine preadipocyte

Effective techniques for the cryopreservation of porcine preadipocytes could increase the usefulness of these cells as a model in obesity studies. The objective of this study was to test the effects of the following cryoprotective agents (CPAs) on the cytotoxicity, post-thaw survival, proliferation and differentiation capacity of porcine preadipocytes: ethylene glycol (EG), dimethyl sulphoxide (Me2SO), polyvinylpyrrolidone (PVP), Me2SO+PVP, and no-CPA. In addition to the CPAs, the CPA medium contained 80% DMEM/F12 plus 10% FBS. Trypan blue exclusion tests showed that among the CPA treatments in this study, only EG was toxic to porcine preadipocytes. The highest survival rate (94.96%) and cell viability were obtained when preadipocytes were cryopreserved with 10% PVP. Morphologically, PVP cryopreserved preadipocytes resembled fibroblasts and most underwent attachment, proliferation, and growth arrest with subsequent accumulation of intracellular lipid droplets before becoming mature adipocytes. There were no significant differences in the GPDH activity between adipocytes in the PVP treatment and primary cells from days 3 to 10 of the culture. Analysis of RT-PCR confirmed that there was no significant difference of PPARgamma2 mRNA levels between the cells in the 10% PVP treatment and primary cells. In summary, porcine preadipocytes cryopreserved with DMEM/F12 medium containing 10% PVP and 10% FBS have high survival rate and proliferation potential. Furthermore, the cryopreserved cells synthesize a range of markers that are consistent with this cell type. We conclude that 10% PVP is a suitable CPA for porcine preadipocytes.     

Cryopreservation of cornea: a low cooling rate improves functional survival of endothelium after freezing and thawing

AIM: To investigate the influence of low cooling rates on endothelial function and morphology of corneas frozen with propane-1,2-diol (PROH). METHODS: Rabbit corneas, mounted on support rings, were exposed to 1.4mol/l (10% v/v) PROH, seeded to initiate freezing, and cooled at 0.2 or 1 degrees C/min to -80 degrees C. Corneas were frozen immersed in liquid or suspended in air. After being held overnight in liquid nitrogen, corneas were warmed at 1 or 20 degrees C/min. After stepwise removal of the cryoprotectant, the ability of the endothelium actively to control corneal hydration was monitored during normothermic perfusion. Morphology was assessed after staining with trypan blue and alizarin red S, and by specular microscopy during perfusion. RESULTS: Functional survival was achieved only after slow cooling (0.2 degrees C/min) with the cornea immersed in the cryoprotectant medium, and rapid warming (20 degrees C/min). These conditions also gave the best morphology after freezing and thawing. CONCLUSION: Cooling rates lower than those typically applied to cornea improved functional survival of the endothelium. This result is in accord with previous observations showing the benefit of low cooling rates for cell monolayers [CryoLetters 17 (1996) 213-218].     

Cryopreservation of porcine articular cartilage: MRI and biochemical results after different freezing protocols

The objective of this study was to investigate the effects of cryopreservation on the components of articular cartilage (AC) matrix by utilizing magnetic resonance imaging (MRI) and biochemical assessments. Porcine AC (10mm osteochondral dowels) was collected into four groups - (1) phosphate buffered saline (PBS) control, (2) PBS snap frozen in liquid nitrogen, (3) slow-cooled in dimethyl sulfoxide (DMSO), and (4) slow cooled in PBS (in absence of DMSO). MRI results demonstrated three distinct zones in the cartilage. After exposure to ice formation during cryopreservation procedures, alterations in MRI determined matrix fixed charged density and magnetization transfer rate were noted. In addition, biochemical assays demonstrated significant alterations in chondroitin sulfate and hydroxyproline content over time without differences in hydration or DNA content. In conclusion, MRI was able to detect some changes in the intact cartilage matrix structure consistent with biochemical assessments after ice formation during cryopreservation of intact porcine AC. Furthermore, biochemical assessments supported some of these findings and changed significantly after incubating the cartilage matrix for 36-72 h in PBS in terms of chondroitin sulfate and hydroxyproline content.     

Effect of rapid freezing and thawing on cellular integrity of honey bee sperm

Abstract. Direct observations on the effect of rapid freezing and thawing on honey bee ( Apis mellifera L.) sperm were made by light, scanning and transmission electron microscopy. Rapid freezing of honey bee ejaculated sperm, suspended in freezing diluent, in liquid nitrogen followed by rapid thawing can cause cellular injuries which lead to the death of the sperm. The frozen-thawed sperm, supravitally stained, showed a significant decrease in cell viability compared with that of the control fresh sperm ( P <0.001). Significant uptake of the stain in the dead sperm resulted from damage in the cell membrane. The scanning electron micrographs of frozen-thawed sperm further demonstrated that the injury of cell membrane can lead to the splitting of mitochondrial derivatives from the flagellar axoneme. More cellular injuries including the release of acrosomal content and membrane damage at the acrosome, nucleus and the tail regions were further revealed by transmission electron microscopy. The impact of cellular injuries on the quality of honey bee sperm cryopreserved for artificial insemination of honey bee queens is discussed.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Effect of freezing and thawing on the microcirculation and capillary endothelium of the hamster cheek pouch

Golden Syrian hamster cheek pouches were prepared in vivo for observation of microvasculature under the light microscope. On an apparatus designed especially for this purpose, pouches were cooled from plus 10 °C to minus 30 °C at 1 °C/min, held at minus 30 °C for 1 min and warmed at 1 °C/min to plus 10 °C whence spontaneous rewarming to ambient was permitted. Pouches were observed under the light microscope throughout freezing and for 1 hr after thaw. At 0, 1, 5, 15, 30, and 60 min after thaw pouch tissue was taken for electron microscopy. After thaw hemostasis evolved in a biphasic pattern. In the early phase in which the degree of stasis reached its peak at about 20 min after thaw, cessation of circulation was caused by obstructive embolic platelet aggregates and a second uncharacterized factor, possibly humoral in nature. After 30 min, blood flow spontaneously resumed in at least one portion of each pouch. In 6 specimens, by 45–50 min, blood flow had returned to normal in about 90% of the microvasculature. After 50 min hemostasis began again and by 60 min blood flow had ceased in virtually all areas of the pouch, this time in the absence of vascular obstruction by platelet aggregates. Ultrastructural damage to endothelial cells occurring immediately at thaw and progressing through 1 hr thereafter included the rupture of cell membranes, early thinning, and later condensation of ground substance, decrease in the number and concentration of pinocytotic vesicles, swelling of rough endoplasmic reticulum, and the swelling and loss of cristal structure of mitochondria. This endothelial destruction can account for the permanent hemostasis of frostbite.     

Artificial Cornea and the Future of Eye Banking

A report on the construction of functional human corneal equivalent reflects a step towards creation of an artificial cornea. We discuss the current status of supply and demand for corneas for transplanation, and wonder if the creation of an artificial cornea will have an effect on corneal transplantation in the near future     

Effect of freezing and thawing on cell membranes of Lentinus edodes,the shiitake mushroom

When cell membranes of Lentinus edodes mycelium were rapidly frozen at either 50 or 160°C/min, viability was lost and this correlated with rupture of the plasmalemma and residual membrane material and with alterations in the organelles. Although with slow cooling (1°C/min) 80% of the samples recovered viability, some cells still showed similar changes to those cooled rapidly, indicating that individual cells of the mycelium do not respond in the same way.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.     

Effect of hypertonicity on survival of unprotected human cultured cells following freezing and thawing

An investigation was carried out on the post-thaw survival of unprotected human heteroploid EUE cells, either maintained in isotonic medium (0.137 M NaCl) or adapted to hypertonicity (0.356 M NaCl) and frozen in medium with an increased concentration of NaCl. A fivefold increase in the survival fraction of the adapted cells in comparison with the unadapted ones was observed when cells were frozen in isotonic medium. When cells were frozen in hypertonic medium (0.356 M NaCl), the two cell types exhibit comparable survival values. The results are discussed, with special attention to cell defense mechanisms against freezing injury.     

石楠和蜡梅茎结冰动力学过程的差热扫描分析

在（0.66 ±0.2）℃/min（0℃~-20℃）的降温速度下,采用高分辨率差热分析法分别对石楠（Photinia serrulata Lindl.）和蜡梅（Chimonanthus praecox（L.）Link）活体幼茎和经过10 min高温煮沸的幼茎在结冰过程中的热力学行为进行分析,并根据茎的形态解剖结构对他们的结冰特征进行研究。结果显示：石楠和蜡梅的活体幼茎在结冰过程中的差热扫描曲线均出现3个放热峰;而经过高温杀死后的茎仅出现1个单放热峰。分析结果表明,2种植物活体幼茎的3个放热峰可能与其木质部、质外体、韧皮部、形成层的结冰、脱水以及髓组织的结冰、脱水过程有关。进一步采用生理盐水浸湿的滤纸进行模拟实验,结果发现差热扫描曲线出现与高温杀死的茎类似的放热峰。实验结果表明,采用高分辨率差热分析法可以探测植物组织结冰过程中的放热强度、结冰温度及其与结冰动力学过程相关的大量细节,适用于植物的结冰动力学分析。