Effects of gamma irradiation and repetitive freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

An increasing number of tissue banks have begun to focus on gamma irradiation and freeze-thaw in the reconstruction of anterior cruciate ligaments using allografts. The purpose of this study was to evaluate the biomechanical properties of human tendons after exposure to gamma radiation and repeated freeze-thaw cycles and to compare them with fresh specimens. Forty flexor digitorum superficialis tendons were surgically procured from five fresh cadavers and divided into four groups: fresh tendon, gamma irradiation, freeze-thaw and gamma irradiation+freeze-thaw. The dose of gamma irradiation was 25 kGy. Each freeze-thaw cycle consisted of freezing at -80 °C for 7 day and thawing at 25 °C for 6 h. These tendons underwent 4 freeze-thaw cycles. Biomechanical properties were analyzed during load-to-failure testing. The fresh tendons were found to be significantly different in ultimate load, stiffness and ultimate stress relative to the other three groups. The tendons of the gamma+freeze-thaw group showed a significant decrease in ultimate load, ultimate stress and stiffness compared with the other three groups. Gamma irradiation and repeated freezing-thawing (4 cycles) can change the biomechanical properties. However, no significant difference was found between these two processes on the effect of biomechanical properties. It is recommended that gamma irradiation (25 kGy) and repetitive freeze-thaw cycles (4 cycles) should not be adopted in the processing of the allograft tendons.     

Effects of refrigeration and freezing on the electromechanical and biomechanical properties of articular cartilage

In vitro electromechanical and biomechanical testing of articular cartilage provide critical information about the structure and function of this tissue. Difficulties obtaining fresh tissue and lengthy experimental testing procedures often necessitate a storage protocol, which may adversely affect the functional properties of cartilage. The effects of storage at either 4°C for periods of 6 days and 12 days, or during a single freeze-thaw cycle at -20°C were examined in young bovine cartilage. Non-destructive electromechanical measurements and unconfined compression testing on 3 mm diameter disks were used to assess cartilage properties, including the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Cartilage disks were also examined histologically. Compared with controls, significant decreases in SPI (to 32.3±5.5% of control values, p<0.001), Ef (to 31.3±41.3% [corrected] of control values, p=0.046), Em (to 6.4±8.5% of control values, p<0.0001), and an increase in k (to 2676.7±2562.0% of control values, p=0.004) were observed at day 12 of refrigeration at 4°C, but no significant changes were detected at day 6. A trend toward detecting a decrease in SPI (to 94.2±6.2% of control values, p=0.083) was identified following a single freeze-thaw cycle, but no detectable changes were observed for any biomechanical parameters. All numbers are mean±95% confidence interval. These results indicate that fresh cartilage can be stored in a humid chamber at 4°C for a maximum of 6 days with no detrimental effects to cartilage electromechanical and biomechanical properties, while one freeze-thaw cycle produces minimal deterioration of biomechanical and electromechanical properties. A comparison to literature suggested that particular attention should be paid to the manner in which specimens are thawed after freezing, specifically by minimizing thawing time at higher temperatures.     

Biomechanical response of human spleen in tensile loading

Blunt splenic injuries are most frequently caused as a result of motor vehicle collisions and are associated with high mortality rates. In order to accurately assess the risk of automotive related spleen injuries using tools such as finite element models, tissue level tolerance values and suitable material models must be developed and validated based on appropriate biomechanical data. This study presents a total of 41 tension tests performed on spleen parenchyma coupons and 29 tension tests performed on spleen capsule/parenchyma coupons. Standard dog-bone coupons were obtained from fresh human spleen and tested within 48 h of death. Each coupon was tested once to failure at one of the four loading rates to investigate the effects of rate dependence. Load and acceleration data were obtained at each of the specimen grips. High-speed video and optical markers placed on the specimens were used to measure local displacement. Failure stress and strain were calculated at the location of failure in the gage length of the coupon. The results of the study showed that both the spleen parenchyma and the capsule are rate dependent, with higher loading rates yielding higher failure stresses and lower failure strains. The results also show that the failure stress of the splenic capsule is significantly greater than that of the underlying parenchyma. Overall, this study provides novel biomechanical data that demonstrate the rate dependent tissue level tolerance values of human spleen tissue in tensile loading, which can aid in the improvement of finite element models used to assess injury risk in blunt trauma.     

Freezing of rat lymphocytes. 2. Distribution and circulation of frozen-thawed 51Cr-labelled cells

Radioactive labelled synegenic fresh and frozen-thawed rat lymphoid cells were injected into groups of animals and their distribution in liver, spleen, lung, lymph nodes, and thoracic duct recorded. Principally the same pattern of distribution was demonstrated after injection of both fresh and frozen-thawed cells. However, more radioactivity was recovered from the liver and less from lymph nodes and thoracic ducts in recipients of frozen-thawed than of fresh cell preparations. This is caused by some damage to the cells in the freeze-thaw process.     

Biomechanical properties of abdominal organs in vivo and postmortem under compression loads

Accurate knowledge of biomechanical characteristics of tissues is essential for developing realistic computer-based surgical simulators incorporating haptic feedback, as well as for the design of surgical robots and tools. As simulation technologies continue to be capable of modeling more complex behavior, an in vivo tissue property database is needed. Most past and current biomechanical research is focused on soft and hard anatomical structures that are subject to physiological loading, testing the organs in situ. Internal organs are different in that respect since they are not subject to extensive loads as part of their regular physiological function. However, during surgery, a different set of loading conditions are imposed on these organs as a result of the interaction with the surgical tools. Following previous research studying the kinematics and dynamics of tool/tissue interaction in real surgical procedures, the focus of the current study was to obtain the structural biomechanical properties (engineering stress-strain and stress relaxation) of seven abdominal organs, including bladder, gallbladder, large and small intestines, liver, spleen, and stomach, using a porcine animal model. The organs were tested in vivo, in situ, and ex corpus (the latter two conditions being postmortem) under cyclical and step strain compressions using a motorized endoscopic grasper and a universal-testing machine. The tissues were tested with the same loading conditions commonly applied by surgeons during minimally invasive surgical procedures. Phenomenological models were developed for the various organs, testing conditions, and experimental devices. A property database-unique to the literature-has been created that contains the average elastic and relaxation model parameters measured for these tissues in vivo and postmortem. The results quantitatively indicate the significant differences between tissue properties measured in vivo and postmortem. A quantitative understanding of how the unconditioned tissue properties and model parameters are influenced by time postmortem and loading condition has been obtained. The results provide the material property foundations for developing science-based haptic surgical simulators, as well as surgical tools for manual and robotic systems.     

Biomechanical and histological characteristics of passive esophagus: Experimental investigation and comparative constitutive modeling

Information on the passive biomechanical properties of two-layered esophagus is still limited, although this would enhance our understanding of its physiology/pathophysiology and help to address problems in surgery, medical-device applications, and for the optimal design of prostheses. In this study, rabbit esophagi were excised and dissected into mucosa–submucosa and muscle layers that were submitted to histological quantification of elastin and collagen content and orientation, as well as to inflation-extension testing and geometrical analysis, i.e. delineation of the zero-stress state serving as a reference configuration for biomechanical analysis. The pressure–radius data of both layers displayed a monotonically rising slope with inflating pressure, unlike the sigma shape characterizing elastin-rich tissues, for which biphasic constitutive models were initially postulated. Three phenomenological expressions of strain-energy function (SEF), commonly appearing in soft-tissue biomechanics literature, were used in an attempt to model the pseudoelastic response of esophageal tissue, namely the exponential Fung-type SEF, and the combined neo-Hookean (isotropic) or quadratic (anisotropic) and exponential Fung-type SEF. Accurate fits were attained for the pressure–radius–force data, spanning a wide range of longitudinal stretch ratios, when using the exponential form; the biphasic SEFs failed to generate improved fits, being also over-parameterized. According to the calculated material parameters, mucosa–submucosa was stiffer than muscle in both directions, justified by our histological observation of increased collagen content in that layer, and tissue was stiffer longitudinally, substantiated by the increased elastin and collagen contents and their preferential alignment towards that direction. Our results demonstrate that the passive response of esophagus is best modeled with an exponential Fung-type SEF.     

Effect of multiple freeze-thaw cycles on intervertebral dynamic motion characteristics in the porcine lumbar spine

Fresh frozen spine specimens are commonly used in biomechanical investigations of the spine. Since many study designs require staged preparation and testing, the effect of multiple freeze-thaw cycles on motion behavior should be understood. The objective of this study was to investigate the effect of multiple freeze-thaw cycles on the biomechanical parameters measured during dynamic pure moment loading. Ten porcine lumbar motion segments were harvested immediately after death and potted in acrylic fixtures. Specimens were tested in continuous pure moment flexion-extension, lateral flexion, and rotation cycles up to a limit of +/-5Nm. Moment-angular displacement data were analyzed and parameters quantified including range of motion, elastic zone, transitional zone (neutral region) size and slope, and width of the hysteresis loop. All specimens were tested at baseline and after each of three subsequent cycles of freezing and thawing. The transitional zone size decreased and the transitional zone slope increased during flexion-extension and lateral bending after the initial freeze-thaw cycle. These parameters were not altered after subsequent cycles. No significant change was observed in the elastic zone or width of hysteresis loop. Although freezing porcine spine specimens increased the stiffness in the neutral region of motion, up to three subsequent cycles of freezing and thawing did not further affect these motion characteristics. This suggests that data obtained from porcine spines which have been frozen and thawed multiple times are stable after initial freezing.     

Tropoelastin

Tropoelastin is a 60-72 kDa alternatively spliced extracellular matrix protein and a key component of elastic fibres. It is found in all vertebrates except for cyclostomes. Secreted tropoelastin is tethered to the cell surface, where it aggregates into organised spheres for cross-linking and incorporation into growing elastic fibres. Tropoelastin is characterised by alternating hydrophobic and hydrophilic domains and is highly flexible. The conserved C-terminus is an area of the molecule of particular biological importance in that it is required for both incorporation into elastin and for cellular interactions. Mature cross-linked tropoelastin gives elastin, which confers resilience and elasticity on a diverse range of tissues. Elastin gene disruptions in disease states and knockout mice emphasise the importance of proper tropoelastin production and assembly, particularly in vascular tissue. Tropoelastin constructs hold promise as biomaterials as they mimic many of elastin's physical and biological properties with the capacity to replace damaged elastin-rich tissue.     

MicroRNAs differentially expressed in postnatal aortic development downregulate elastin via 3' UTR and coding-sequence binding sites

Elastin production is characteristically turned off during the maturation of elastin-rich organs such as the aorta. MicroRNAs (miRNAs) are small regulatory RNAs that down-regulate target mRNAs by binding to miRNA regulatory elements (MREs) typically located in the 3' UTR. Here we show a striking up-regulation of miR-29 and miR-15 family miRNAs during murine aortic development with commensurate down-regulation of targets including elastin and other extracellular matrix (ECM) genes. There were a total of 14 MREs for miR-29 in the coding sequences (CDS) and 3' UTR of elastin, which was highly significant, and up to 22 miR-29 MREs were found in the CDS of multiple ECM genes including several collagens. This overrepresentation was conserved throughout mammalian evolution. Luciferase reporter assays showed synergistic effects of miR-29 and miR-15 family miRNAs on 3' UTR and coding-sequence elastin constructs. Our results demonstrate that multiple miR-29 and miR-15 family MREs are characteristic for some ECM genes and suggest that miR-29 and miR-15 family miRNAs are involved in the down-regulation of elastin in the adult aorta.     

Effects of milrinone on left ventricular cardiac function during cooling in an intact animal model

A significant reduction in immunogenicity has been observed in some frozen-thawed tissues after cryopreservation. The objective of this study was to evaluate the effects of programmed cryopreservation on immunogenicity of rabbit bladder mucosa and on the extent of immunological rejection caused by the allograft. This study would provide theoretical support for the application of allogenic frozen-thawed bladder mucosa in the treatment of urethral stricture. Forty-two adult male New Zealand rabbits were used in this study. The immunogenicity was detected by mixed lymphocyte reaction using the allograft of bladder mucosa (fresh and frozen-thawed) and spleen lymphocytes. Twelve urethral stricture models were established in New Zealand rabbits for substitution urethroplasty using the allograft of bladder mucosa, which were divided into fresh and frozen-thawed group. Two weeks after operation, lymphocyte proliferation was detected in both blood and spleen of recipient rabbits. At the same time, immunohistochemical staining of urethral allograft was performed and the expression of CD3, CD4 and CD8 were observed. The mRNA of bladder mucosa (fresh and frozen-thawed) was extracted and the expressions of RLA-I, RLA-II and RLA-III gene were detected by real-time PCR. By mixed lymphocyte reaction, we found that allogenic lymphocyte proliferation stimulated by frozen-thawed bladder epithelial cells was significantly weaker than that of the fresh cells. The blood and spleen lymphocytes from fresh bladder mucosa group showed significantly higher proliferation rate than frozen-thawed group. Compared with the fresh group, the expression of CD3+ and CD8+ T cells infiltrated in the operation locus of bladder mucosa urethroplasty was significantly decreased in the frozen-thawed group. However, the expressions of RLA genes did not change significantly after the freeze-thaw procedure. This study demonstrates for the first time that a programmed freeze-thaw procedure of rabbit bladder mucosa could reduce its immunogenicity in allogenic bladder mucosa urethroplasty and thus restrict the extent of immunological rejection, therefore, provides theoretical support for the application of frozen-thawed bladder mucosa in the treatment of urethral stricture.     

Spinal biomechanical properties are significantly altered with a novel embalming method

In vitro tests on the biomechanical properties of human spines are often performed using fresh frozen specimens. However, this carries the risk of pathogen transfer from specimen to the worker and the specimens can only be used for a limited amount of time. Human spinal specimens embalmed with formaldehyde carry an almost absent risk of transfer of pathogens and can be stored and used for a long time, but the tissue properties are strongly affected making this method inapplicable for biomechanical testing. In this study, a new embalming technique called Fix for Life (F4L), which claims to preserve the tissue properties, was tested. The range of motion (ROM) and stiffness of six fresh human spinal specimens was measured using a spinal motion simulator before and after F4L embalming. After F4L embalming, spinal stiffness increased in flexion-extension by 230%, in lateral bending by 284% and in axial rotation by 271%. ROM decreased by 46% in flexion-extension, 56% in lateral bending and 54% in axial rotation. In conclusion, based on this study, F4L does not maintain physiological spinal biomechanical properties, and we propose that this method should not be used for biomechanical studies. Nevertheless, the method may be an alternative to formaldehyde fixation in situations such as training and education because the effect on spinal biomechanics is less detrimental than formaldehyde and tissue color is maintained.     

Course of liver regeneration after partial hepatectomy in rats treated with dialysates obtained 17 hours after partial hepatectomy

Various theories have been put forward to explain the regenerative capacity of liver tissue induce by partial hepatectomy (PH). One of them presumes the existence of humoral factors stimulating proliferation of the liver tissue. We evaluated the course of liver regeneration after 65-70% PH as influenced by dialysates (DIA) of the organs of a rat killed 17 h after PH. In addition to kidney DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on rats weighting 310-370 g. Kidney, liver or spleen dialysate was administered subcutaneously and the rats were killed 12 or 24 h later by exsanguination from the abdominal aorta. In further rats, PH was performed 24 h after administering DIA and the rat were killed 18, 24, 30, 48 and 72 h after the operation. The initiation of liver regeneration was stimulated by all the given DIA, but especially by liver DIA. The faster onset of liver regeneration 18 h after PH in rats given spleen DIA is interesting. DIA did not greatly affect the hepatocytes of intact liver, but accelerated the initiation of liver regeneration after PH by synchronizing the cell cycle of proliferating hepatocytes. DIA obtained 17 h after PH contained substances which primarily stimulated liver DNA synthesis. From the changes in inhibition of the migration of spleen macrophages in the medium containing liver antigens, and from the circulating immunocomplex values, we conclude that DIA activation of the immune system, a well as the hepatic stimulator substance contained in the DIA, participates in acceleration of the liver regeneration process.     

Fluid endocytosis by rat liver and spleen. Experiments with 125I-labelled poly(vinylpyrrolidone) in vivo.

1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.     

Systemic changes in hydrolytic enzyme activities in mice affected with murine leprosy

In order to investigate the behavior of hydrolytic enzymes in chronic infections, the activities of 17 hydrolytic enzymes were tested in limb muscles, heart muscle, spleen, liver, and kidney of lepromatous mice infected with Mycobacterium lepraemurium (M. lepraemurium) and their controls. Typical increases in those enzymatic activities were seen in spleen and liver, where pathological changes were the most pronounced, especially at the 11th week after the inoculation of the bacilli. At the 16th week, the enzymatic changes became less remarkable probably because of the decreased viability of tissues in these organs. The enzymatic changes observed could not be explained as due to bacterial enzymes. These findings are compatible with the notion that the increases in hydrolytic enzyme activities are related to tissue damage caused by murine leprosy.     

Changes in the structure-function relationship of elastin and its impact on the proximal pulmonary arterial mechanics of hypertensive calves

Extracellular matrix remodeling has been proposed as one mechanism by which proximal pulmonary arteries stiffen during pulmonary arterial hypertension (PAH). Although some attention has been paid to the role of collagen and metallomatrix proteins in affecting vascular stiffness, much less work has been performed on changes in elastin structure-function relationships in PAH. Such work is warranted, given the importance of elastin as the structural protein primarily responsible for the passive elastic behavior of these conduit arteries. Here, we study structure-function relationships of fresh arterial tissue and purified arterial elastin from the main, left, and right pulmonary artery branches of normotensive and hypoxia-induced pulmonary hypertensive neonatal calves. PAH resulted in an average 81 and 72% increase in stiffness of fresh and digested tissue, respectively. Increase in stiffness appears most attributable to elevated elastic modulus, which increased 46 and 65%, respectively, for fresh and digested tissue. Comparison between fresh and digested tissues shows that, at 35% strain, a minimum of 48% of the arterial load is carried by elastin, and a minimum of 43% of the change in stiffness of arterial tissue is due to the change in elastin stiffness. Analysis of the stress-strain behavior revealed that PAH causes an increase in the strains associated with the physiological pressure range but had no effect on the strain of transition from elastin-dominant to collagen-dominant behavior. These results indicate that mechanobiological adaptations of the continuum and geometric properties of elastin, in response to PAH, significantly elevate the circumferential stiffness of proximal pulmonary arterial tissue.     

食物蛋白含量和限食对雌性东方田鼠生理特性的影响

为了研究食物蛋白含量和限食对雌性东方田鼠生理特性的影响,实验以成年雌性东方田鼠为对象,在控制其他因子一致的条件下,依据食物条件设置4个处理组:20％蛋白质+自由取食、20％蛋白质+70％限食、10％蛋白质+自由取食、10％蛋白质+70％限食.经过4周的食物驯化,测定了动物体重、动情周期、雌激素(雌二醇、孕酮、卵泡刺激素)和内脏器官(脾脏、肝脏、肾脏、子宫)湿重等指标.结果发现:(1)限食70％使东方田鼠体重减少,动情期缩短,脾脏、肝脏、肾脏及子宫湿重均显著减轻.(2)10％蛋白含量降低了脾脏湿重和肝脏湿重,但未显著影响体重、肾脏湿重和子宫湿重.(3)蛋白和限食均未影响雌二醇、孕酮、卵泡刺激素的含量及动情周期的长短.实验结果表明,在食物资源不理想的情况下,成年雌性东方田鼠通过降低体重来维持基本生存,内脏器官重量也相应地减轻,但是与繁殖相关的生理特性变化不显著.相对与食物蛋白含量,可获得的食物量的多少是引起东方田鼠生理变化的主要因素.实验结果反映出食物资源对成年雌性东方田鼠的生理特性产生了影响,但这些影响对其繁殖行为的调控强度及季节性的种群波动的作用还需结合其他相关方面做进一步探究.     

Expression of luciferase in selected organs following delivery of naked and formulated DNA to rainbow trout (Oncorhynchus mykiss) by different routes of administration

In the present work, the expression of luciferase in selected organs following administration of DNA delivered as naked, liposome-formulated or chitosan-formulated by different routes of administration (intramuscular, intraperitoneal and intravenous injection, immersion and anal intubation) was studied in rainbow trout (Oncorhynchus mykiss). The different formulations and routes of administration both influenced in which organs luciferase was expressed and the magnitude of expression. The highest expression levels of luciferase in the head kidney and liver were found after an intraperitoneal injection of lipoplex 2. In the spleen, the highest levels were detected after injection of naked DNA (intraperitonal or intramuscular) and lipoplex 2 (intraperitoneal). Following intravenous injection, naked DNA gave higher expression levels in the organs than the formulated plasmids and immersion and anal intubation were not effective routes of delivery as no expression of luciferase could be detected in any of the organs tested. Additionally, PCR using a primer specific for a 600 bp region of the luciferase gene pcDNA3-luc was used to assess the distribution of the plasmid itself after intramuscular and intraperitoneal injection. Positive amplification was obtained in spleen, head kidney, liver and muscle at the injection site following injection of formulated plasmids, while only muscle tissue from the injection site was positive when naked DNA was used.     

Quantity of DNA in the organs of adult Swiss male mice after AET and MEA treatment

Adult Swiss male mice were injected intraperitoneally with 2-aminoethylesothiouronium bromide hydrobromide (AET) or cysteamine hydrochloride (MEA) in a dose of 400 mg/kg body weight. In the thirtieth, sixtieth or ninetieth minute after the injection, the animals were killed and the deoxyribonuclein acid content in 100 mg of fresh tissue of testes, spleen and liver, was measured. DNA was extracted from the organs by means of Burton's method, which is based on the estimation of deoxiribose content in the colour reaction with diphenylamine. The injection of AET and MEA did not distinctly influence the DNA content in the organs of mice. Statistically significant differences among the groups of mice were not observed compared to the controls, in mice treated with the compound, a decreasing tendency in the quantity of the DNA in the organs was found only.     

Differential expression of hepatocyte growth factor in liver, kidney, lung, and spleen following burn in rats

Hepatocyte growth factor (HGF) plays a role as an organotropic factor for regeneration of injured organs. HGF is synthesized as an inactive single-chain precursor which is then converted to a biologically active heterodimeric form by proteolytic processing. Burn is the insult that results in hypovolemia which causes systemic organ injury. In this study, we investigated the induction and activation of HGF in various rat organs following burn trauma. Tissue HGF content determined as the total amount of the single-chain and heterodimeric form increased significantly in liver, lung, spleen, and kidney 12 h after burn. Molecular analysis revealed that HGF in these four organs of control rats was the single-chain precursor. In the burned rats, HGF was the single-chain form in the liver and lung, whereas heterodimeric HGF was detected in the spleen and kidney. Tissue protein content, an index of tissue injury, decreased significantly in the spleen and kidney, indicating that tissue damage was severe in these two organs. These results suggest that burn induces the production of HGF in various organs, and that the induced HGF is activated according to the severity of tissue damage caused by burn.