Vitrification of porcine articular cartilage

The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. A vitrification solution containing a 55% cryoprotectant formulation, VS55, previously studied using rabbit articular cartilage, was evaluated using porcine articular cartilage. Specimens ranging from 2 to 6 mm in thickness were obtained from 6 mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The results of post-rewarming viability assessments employing alamarBlue demonstrated a large decrease (p < 0.001) in viability in all three sizes of cartilage specimen vitrified with VS55. This is in marked contrast with prior experience with full thickness, 0.6 mm rabbit cartilage. Microscopic examination following cryosubstitution confirmed ice formation in the chondrocytes of porcine cartilage vitrified using VS55. Experiments using a more concentrated vitrification formulation (83%), VS83, showed a significant treatment benefit for larger segments of articular cartilage. Differences between the VS55 and the VS83 treatment groups were significant at p < 0.001 for 2 mm and 4 mm plugs, and at p < 0.01 for full thickness, 6 mm plugs. The percentage viability in fresh controls, compared to VS55 and VS83, was 24.7% and 80.7% in the 2 mm size group, 18.2% and 55.5% in the 4 mm size group, and 5.2% and 43.6% in the 6 mm group, respectively. The results of this study continue to indicate that vitrification is superior to conventional cryopreservation with low concentrations of dimethyl sulfoxide by freezing for cartilage. The vitrification technology presented here may, with further process development, enable the long-term storage and transportation of living cartilage for repair of human articular surfaces.     

Effects of cryopreservation on the immunogenicity of porcine arterial allografts in early stages of transplant vasculopathy

BACKGROUND: The number of revascularization procedures including coronary and lower extremity bypass, have increased greatly in the last decade. It suggests a growing need for vascular grafts. Cryopreserved allografts could represent a viable alternative but their immunologic reactivity remains controversial. METHODS: 71 pigs (40 recipients and 31 donors) were used. Two femoral grafts per recipient animal were implanted for 3, 7, and 30 days. Types of grafts: fresh autograft as a control graft (n=19), fresh allograft (n=31) and cryopreserved allograft (n=30). Histological and immunohistochemical studies were performed. RESULTS: Fresh allografts compared to autografts showed intimal inflammatory infiltration at 3 days (328 vs. 0 macrophages/mm2; P<0.05) and 7 days (962 vs. 139 T lymphocytes/mm2; P<0.05) post-transplantation. At 30 days, there was a loss of endothelial cells, presence of luminal thrombus and aneurismal lesions (total area=15.8 vs. 8.4 mm2; P<0.05). Cryopreservation did not reduce these lesions nor modify endothelial nitric oxide synthase (eNOS) expression nor modify the number of animals that developed anti-SLA antibodies. Moreover, at 7 days, cryopreserved allografts compared to fresh allografts showed a higher expression of P-selectin (5 out of 5 vs. 1 out of 5; P<0.05) and, at 30 days, a greater inflammatory reactivity (2692 vs. 1107 T lymphocytes/mm2 in media; P<0.05) with a trend towards a higher presence of multinucleated giant cells than in the fresh ones. CONCLUSIONS: The cryopreservation method used maintained immunogenicity of allografts and increased the inflammatory reactivity found in fresh allografts up to 30 days of vascular transplantation.     

改良皮片4℃低温保存方法在创面二次植皮治疗中的应用研究

目的:探讨改良皮片4℃低温保存方法在创面二次植皮治疗中的应用效果。方法:①动物实验:健康成年豚鼠3只,处死后,取背部皮肤做成32个1 cm×1 cm小皮样,随机分为新鲜皮组、庆大盐水保存组、RPMI保存组,改良RPMI保存组进行4℃保存;1周后测定皮肤活力;②临床实验:观察自2018年10月至2018年12月,二期植皮患者33例,应用改良RPMI 4℃低温保存皮肤二期回植的患者16例,与重新取皮植皮患者17例比较其皮片成活率。结果:动物实验证实:改良RPMI保存组4℃保存组在皮肤储存1周时皮肤平均活力较庆大盐水保存组、RPMI保存组高(P0.05);临床实验证实:改良RPMI保存组与重新植皮的皮片平均成活率没有显著性差别(P0.05)。结论:改良4℃断层皮片低温保存方法可短期内保存皮肤较高的活力,是皮片再利用的一种有效方法。     

Pre-grafting histological studies of skin grafts cryopreserved in α helix antarctic yeast oriented antifreeze peptide (Afp1m)

A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing antifreeze proteins (AFP). Antifreeze peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of −10 °C and −20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (−10 and −20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p < 0.01) among the different concentrations at −10 and −20 °C. In conclusion, the integrity of cryopreserved skin grafts with lower concentrations of Afp1m (0.5, 1 and 2 mg/mL) or at −20 °C was not maintained. The present study attested that Afp1m is a good cryoprotective agent for the cryopreservation of skin graft. Higher Afp1m concentrations (5 and 10 mg/mL) at −10 °C found to be suitable for the future in vivo study using (SD) rat skin grafts.     

似鲇高原鳅皮肤与鳍的组织学观察

采用石蜡切片技术及H.E和AB-PAS染色方法,对似鲇高原鳅(Triplophysa siluroides)头部、腹部、背部、侧线部和尾部皮肤结构及胸鳍、腹鳍、背鳍、臀鳍和尾鳍的组织结构进行观察。各部位皮肤均由表皮和真皮构成,真皮包括疏松层和致密层,不同部位皮肤厚度不同。表皮层腹部最厚,为(84.62±10.82)μm,侧线部最薄,为(14.97±3.95)μm,各部位表皮厚度差异显著。表皮层分布着黏液细胞、棒状细胞及味蕾。疏松层头部最厚,为(282.71±70.56)μm,尾部最薄,为(29.07±4.88)μm,该层分布有黑色素细胞、空泡状细胞和颗粒腺,而黏液腺分布于致密层。各部位的鳍均由表皮层、胶原纤维层、胶原下层及鳍条构成,表皮层与皮肤表皮层组成相似,鳍条是矿化的结缔组织。     

Cryopreservation of fetal skin is improved by extracellular trehalose

In this study, we tested a non-permeating cryoprotectant, trehalose, in combination with dimethyl sulfoxide (Me(2)SO) in the cryopreservation of human fetal skin and compared it to Me(2)SO and glycerol, protocols that are routinely used by skin banks. The viability of fetal skin from four groups (fresh, and cryopreserved with glycerol, Me(2)SO, or trehalose/Me(2)SO) were evaluated using an in vitro membrane integrity assay and by transplantation to immunodeficient mice. The membrane integrity assay showed a 90% integrity in fresh, unfrozen fetal skin. The number of intact cells dropped to 23 and 44% in fetal skin cryopreserved with glycerol and Me(2)SO, respectively. When trehalose was added to the cryopreservation medium containing Me(2)SO, the membrane integrity rose to 65%. When transplanted to immunodeficient mice, fetal skin cryopreserved with trehalose/Me(2)SO showed a graft performance indistinguishable from fresh unfrozen fetal skin and strikingly better graft take than that of fetal skin cryopreserved with Me(2)SO or glycerol only. These results suggest that cryopreservation protocols routinely used the skin banks can be improved by combining sugars such as trehalose with a permeating cryoprotectant.     

SD大鼠与巴马小型猪的皮肤组织学比较

目的研究及观察SD大鼠和巴马小型猪皮肤的正常比较组织学。方法取SD大鼠和巴马小型猪不同部位的皮肤进行石蜡切片、HE染色,光学显微镜观察。结果两种动物的皮肤组织学结构在以下方面存在着显著差异：1.SD大鼠的毛囊成簇分布,平均3～9成群,而巴马小型猪的毛囊较稀少;2.SD大鼠表皮较薄,没有透明层,基底细胞缺乏异质性,真皮与表皮连接面平坦,没有皮钉;而在巴马小型猪皮肤表皮和真皮连接区,有上下交错的表皮皮钉和真皮乳头;3.SD大鼠的真皮结构相对松散,真皮血管系统不发达,而巴马小型猪皮肤的真皮网织层和乳头层交界的地方,水平分布着很多的浅表小静脉和小动脉丛,这种血管分布的方式与人类皮肤中的血管分布极为类似;4.SD大鼠的汗腺只局限于足垫的皮肤,汗腺上皮只有一种细胞类型,腺细胞呈立方形或矮柱状,胞核圆形,导管短而弯曲,由两层上皮细胞组成。而巴马小型猪皮肤的汗腺是顶泌汗腺,分布于真皮和脂肪相接的真皮深层,分泌部为粗管,管腔大,盘曲成团。腺细胞呈立方形或扁平,胞核圆形或长梭形。腺细胞与基膜之间也有肌上皮细胞。导管较细而直,开口于毛囊上段。     

林蛙属3物种皮肤的组织结构比较

利用石蜡切片和H.E染色技术,对蛙科(Ranidae)林蛙属(Rana)高原林蛙(R.kukunoris)、昭觉林蛙(R.chaochiaoensis)和峨眉林蛙(R.omeimontis)的皮肤组织结构进行了观察。应用SPSS 13.0统计软件,对皮肤的厚度、皮肤腺的相对数量和面积作了比较分析。3物种皮肤的基本结构相似,都由表皮和真皮组成。表皮是角质化的复层扁平上皮,由角质层、颗粒层、棘细胞层和生发层构成。真皮又分为疏松层和致密层,疏松层内分布有黏液腺、颗粒腺和脂腺3种类型的皮肤腺,黏液腺在体背和体腹皮肤内基本均匀分布,而颗粒腺主要以团块聚集形式散布在体背皮肤中。在高原林蛙皮肤中还发现了1种与以往描述不同的特殊嗜酸性腺体。皮肤厚度存在种间差异和部位差异。高原林蛙的表皮里有少量毛细血管和发达的色素细胞分布,真皮疏松层里有发达的腺体,这些可能是其对高海拔、低氧、低温和强紫外线辐射生活环境的适应策略。在峨眉林蛙和昭觉林蛙皮肤真皮的疏松层和致密层相邻处,发现有呈波浪条带状的、H.E染色呈蓝色的钙化层结构,体背部的钙化层比体腹部的发达。钙化层的功能可能包括防止体内水分散失、贮存钙离子、构成与体外环境进行物质交换的屏障等方面。     

In vitro measurement of the mechanical properties of skin by nano/microindentation methods

The elastic behaviors of stratum corneum, viable epidermis, dermis, and whole skin were investigated by nano/microindentation techniques. Insignificant differences in reduced elastic modulus of skin samples obtained from three different porcine breeds revealed breed type independent measurements. The reduced elastic modulus of stratum corneum is shown to be about three orders of magnitude higher than that of dermis. As a result, for relatively shallow and deep indentations, skin elasticity is controlled by that of stratum corneum and dermis, respectively. Skin deformation is interpreted in the context of a layered structure model consisting of a stiff and hard surface layer on a compliant and soft substrate, supported by microscopy observations and indentation measurements.     

Mechanical properties of radiation-sterilised human Bone-Tendon-Bone grafts preserved by different methods

Patellar tendon auto- and allo-grafts are commonly used in orthopedic surgery for reconstruction of the anterior cruciate ligaments (ACL). Autografts are mainly used for primary reconstruction, while allografts are useful for revision surgery. To avoid the risk of infectious disease transmission allografts should be radiation-sterilised. As radiation-sterilisation supposedly decreases the mechanical strength of tendon it is important to establish methods of allograft preservation and sterilisation assuring the best quality of grafts and their safety at the same time. Therefore, the purpose of this study was to compare the tensile strength of human patellar tendon (cut out as for ACL reconstruction), preserved by various methods (deep fresh freezing, glycerolisation, lyophilisation) and subsequently radiation-sterilised with doses of 0, 25, 50 or 100 kGy. Bone-Tendon-Bone grafts (BTB) were prepared from cadaveric human patella tendons with both patellar and tibial attachments. BTB grafts were preserved by deep freezing, glycerolisation or lyophilisation and were subsequently radiation-sterilised with doses of 0 (control), 25, 50 or 100 kGy. All samples were subjected to mechanical failure tensile tests with the use of Instron system in order to estimate their mechanical properties. All lyophilised grafts were rehydrated before performing of those tests. Obtained mechanical tests results of examined grafts suggest that deep-frozen irradiated grafts retain their initial mechanical properties to an extent which does not exclude their clinical application. All conducted experiments were approved by the Local Ethical Committee.     

Osmotic tolerance of mouse spermatozoa from various genetic backgrounds: acrosome integrity, membrane integrity, and maintenance of motility

All cells have an intrinsic biophysical property related to their ability to undergo osmotically driven volume changes. This project is of fundamental importance to our understanding of the basic cryobiology of mouse spermatozoa. The objectives of this study were to determine the osmotic tolerance limits for (1) motility, (2) acrosome integrity, and (3) membrane integrity of mouse spermatozoa from multiple genetic backgrounds including: C57BL/6, BALB/c, FVB, C3H, 129/SVS2 hsd B6C3F1, CB6F1, and ICR. The maintenance of acrosomal and plasma membrane integrity was not affected by genetic background (p=0.13), however, there was an interaction between genetic background and osmolality. In addition, acrosome and plasma membrane integrity was highly correlated within each strain (p<0.01). In contrast to acrosome and plasma membrane integrity, the motility of spermatozoa from different genetic backgrounds fell sharply on both sides of isosmolality, both with and without return to isosmotic conditions. Exposure to hyposmotic conditions caused morphological changes in the spermatozoa, which inhibited motility. However, this morphological change was not reversible in all cases when returned to isosmotic conditions. The ability to maintain motility in an anisosmotic media was affected by genetic background, osmolality as well as the interaction between genetic background and osmolality (p<0.05). In conclusion, mice with different genetic backgrounds appear to have similar tolerance to osmotic changes in terms of sperm acrosome and plasma membrane integrity; however, the ability to maintain motility differs between genetic backgrounds.     

Comparative ultrastructural study of normal and grafted skin in the frog,Rana pipiens,with special reference to neuroepithelial connections

Summary Recent investigations have suggested specific differences in back and belly skin in anurans which appear to influence the quality of reflex responses obtained from various areas of the animals body. The present investigation represents a comparative morphological study of back and belly skin in control and skin-graftedRana pipiens, with special regard to the neuroepithelial relationships. A distinct difference in pigmentation of back and belly skin was observed. Intra-epithelial Merkel cells were present in all skin samples studied. The origins of the numerous unclassifiable cells in the Merkel cell region are discussed in relation to a presumed coordinating function of the Merkel cell during epithelial differentiation. Epitheliomesenchymal interactions were observed in the richly innvervated dermal regions. Two types of morphologically different intra-epithelial nerve endings were observed. These observations are discussed in relation to earlier observations on vertebrate skin and in relation to misdirected reflex responses obtained in skin-grafted anurans.     

Effect of strain on human dermal fibroblasts in a three-dimensional collagen sponge

Our aim was to design a simple compression system and investigate the influence of mechanical stress on skin-like structures. Many mechanical compression studies have employed intricate culture systems, so the relationship between extracellular matrix material and the response of skin cells to mechanical stress remains unknown. Our approach uses only glass vials, 6-well plates and standard laboratory equipment. We examined the influence of mechanical stress on human skin fibroblasts embedded within a collagen sponge. The results show that mechanical compression increases MMP-1 and MMP-2 release by the cells into the the cell culture. Our results suggest that pressure on the skin may affect extracellular matrix degradation through some as yet unidentified pathways and that IL-6 mRNA expression may be involved in this effect. Using our approach, the effects of static mechanical stress on protein expression by cells in the culture medium and in sponges can be easily examined, and therefore this system will be useful for further analyses of skin responses to mechanical stress.     

The biomechanical and histological sequelae of common skin banking methods

Human skin allografts are used worldwide as an adjunct for the healing of burns when autograft skin is not available or not indicated. Allograft skin comes from human cadaveric donors, and so must be preserved until use. This study forms the first investigation to compare the mechanical and histological integrity of human split-thickness skin grafts preserved by either glycerolisation or cryopreservation (with or without the cryoprotectant DMSO). Stress relaxation was used to assess mechanical properties, whilst histological analysis allowed for evaluation of structural integrity.     

In vitro study of the impact of mechanical tension on the dermal fibroblast phenotype in the context of skin wound healing

Skin wound healing is finely regulated by both matrix synthesis and degradation which are governed by dermal fibroblast activity. Actually, fibroblasts synthesize numerous extracellular matrix proteins (i.e., collagens), remodeling enzymes and their inhibitors. Moreover, they differentiate into myofibroblasts and are able to develop endogenous forces at the wound site. Such forces are crucial during skin wound healing and have been widely investigated. However, few studies have focused on the effect of exogenous mechanical tension on the dermal fibroblast phenotype, which is the objective of the present paper. To this end, an exogenous, defined, cyclic and uniaxial mechanical strain was applied to fibroblasts cultured as scratch-wounded monolayers. Results showed that fibroblasts? response was characterized by both an increase in procollagen type-I and TIMP-1 synthesis, and a decrease in MMP-1 synthesis. The monitoring of scratch-wounded monolayers did not show any decrease in kinetics of the filling up when mechanical tension was applied. Additional results obtained with proliferating fibroblasts and confluent monolayer indicated that mechanical tension-induced response of fibroblasts depends on their culture conditions. In conclusion, mechanical tension leads to the differentiation of dermal fibroblasts and may increase their wound-healing capacities. So, the exogenous uniaxial and cyclic mechanical tension reported in the present study may be considered in order to improve skin wound healing.     

蜘蛛丝的分子结构与力学性能研究

蜘蛛丝尤其是蜘蛛大囊状腺产生的拖丝,具有独特的机械性能,是自然界颇具应用潜力的生物材料。现代分子生物学技术使蜘蛛丝蛋白基因得以克隆,通过高分子物理化学手段方法的利用,有利于揭示蜘蛛丝蛋白质序列、分子结构、以及分子结构和力学性能之间的关系。对不同种类蜘蛛丝蛋白的深入研究,将为基因工程方法人工合成并改造蜘蛛丝成为可能。