Quantification of growth factors in allogenic bone grafts extracted with three different methods

Background Bony allografts are used for defect filling. A reliable sterilization method is the peracetic acid–ethanol sterilization procedure (PES). Several studies showed the antimicrobiological efficacy of this method. Aim of this study was the quantification of growth factors necessary for bone formation in PES sterilized allografts (n = 9). Methods To extract the growth factors from the tissue three different methods were used: (a) use of collagenase 1 for extraction, (b) incubation of the material in a proteinase inhibitor cocktail (Complete), and (c) extraction with guanidine HCl. The supernatants from the different methods were analyzed for the total protein concentration and different growth factors. Results The extraction with guanidine HCl resulted in the highest amount of protein measurable in the supernatants of the samples. For comparison of the individual growth factor values the results were normalized to the protein content. The highest growth factor amount/protein was detectable for BMP-2 using the GndHCL method followed by FGFa, IGF-I, TGF-β1, VEGF, and PDGF. Comparing the three extraction methods, significant differences were measured for the individual growth factor content. Conclusion PES sterilized bony allografts contain several growth factors. Depending on the extraction method, the quantity of the analyzed growth factors varies.     

Biomechanical comparison of human bone-patellar tendon-bone grafts after sterilization with peracetic acid ethanol

Recent reports of disease transmission following ACL reconstruction with fresh-frozen non-sterilized allografts have highlighted the need for new sterilization techniques that do not impair the mechanical properties as it was shown for most of the current sterilization techniques. In this in-vitro biomechanical study, it was investigated if peracetic acid ethanol sterilization (PES) has any adverse effects on the mechanical properties of human bone-patellar tendon-bone grafts (BPTB). Paired human BPTB grafts either underwent PES or were used as fresh-frozen non-sterilized grafts. Viscoelastic properties (strain, creep) were analyzed during cyclic submaximal loading and mechanical properties were investigated during load-to-failure (LTF) testing. It was found that there were no differences in viscoelastic and mechanical properties between both groups. The findings of this study provide baseline data for future in vitro and in vivo analyses of this promising new sterilization technique for soft-tissue allografts.     

Preservation and sterilization methods of the meniscal allografts: literature review

Nowadays, there are four types of meniscal allografts known: fresh, cryopreserved, deep-frozen and lyophilized ones but only two of them are widely used in clinical practice. Use of different types of meniscal allografts still remains controversial due to preparation method, their biomechanical properties as well as cost which is connected with processing and storage. The main aim of this review is to present the current status of knowledge concerning meniscal allograft preservation and sterilization, especially the advantages and disadvantages of each method. Authors wanted to show a broad spectrum of methods used and conceptions presented by other authors. The second aim is to gather available information about meniscal preservation and sterilization methods in one paper. Deep-frozen and cryopreserved meniscal allografts are the most frequently used ones in the clinical practice. The use of fresh grafts stays controversial but also has many followers. Lyophilized grafts in turn are not applied at present due to some serious drawbacks including reduction of tensile strength, poor rehydration, graft shrinkage and post-transplantation joint effusion as well as increased risk of meniscal size reduction. An application of sterilizing agents make the meniscal allograft free from the bacteria and viruses, but also it may cause serious structure changes. Therefore, choosing just one ideal method of meniscal allograft preservation and sterilization is complicated and should be based on broad knowledge and experience of surgeon performing the transplantation.     

Structural mechanical properties of radiation-sterilized human Bone-Tendon-Bone grafts preserved by different methods

To avoid the risk of infectious disease transmission from donor to recipient, allografts should be terminally sterilized. In the previous paper (Kaminski et al. in Cell Tissue Bank 10:215–219, 2009) we presented the effect of various methods of preservation (deep fresh freezing, glycerolization, lyophilization), followed by irradiation with different doses of electron beam (EB), on material (intrinsic) mechanical properties of human patellar tendons cut out as for anterior cruciate ligament reconstruction, obtained in failure tensile test. As structural mechanical properties are equally important to predict the behaviour of the graft as a whole functional unit, the purpose of the present paper was to show the results for failure load and elongation, obtained in the same experiment. Paired Bone-Tendon-Bone grafts (BTB) were prepared from cadaveric human patella tendons with both patellar and tibial attachments. They were preserved by deep freezing, glycerolization or lyophilization and subsequently EB-irradiated with the doses of 25, 35, 50 or 100 kGy (fresh-frozen grafts) or a single dose of 35 kGy (glycerolized and lyophilized grafts). Each experimental (irradiated) group was provided with control (non-irradiated), donor-matched group. The specimens from all groups were subjected to mechanical failure tensile test with the use of Instron system in order to measure their structural properties (failure load and elongation). All lyophilized grafts were rehydrated before mechanical testing. In our study we did not observe significant deterioration of structural mechanical properties of BTB grafts processed by fresh-freezing and then terminal sterilized with growing doses of EB up to 100 kGy. In contrast, BTB grafts processed by glycerolization or lyophilization and irradiated with 35 kGy showed significant decrease of failure load. Obtained results suggest that deep-frozen irradiated grafts retain their initial mechanical properties to an extent which does not exclude their clinical application. However, biomechanical investigations constitute only the first step to evaluate the potential clinical usefulness of such allografts and further extensive in vivo studies are needed.     

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.     

The genotypic response of mouse embryos to multiple freezing variables

Four- and eight-cell embryos from 3 mouse genotypes were cryopreserved to study the relationship of genetics and freezing variables on post-thaw survival. Embryos from outbred N:NIH(S), N:NIH(S)-B and inbred (C57BL/6N) mice were exposed to 1 of 2 cryoprotectants (glycerol [GLYC] versus dimethyl sulfoxide [DMSO]) and stored in 1 of 2 containers (ampules [AMP] versus straws [STR]). Containerized embryos were cooled at a rate of 0.5 degrees C/min to a minimum of -35 degrees C, transferred into liquid nitrogen, and later thawed and cultured in vitro. Genotypic differences (p less than 0.05) were noted for 4 interrelated embryo characteristics including post-thaw survival (PTS), embryo degeneration rate (EDR), and quality grade (QG) and viability grade (VG) ratings. The PTS for outbred embryos was greater (p less than 0.05) in GLYC than DMSO, whereas inbred C57BL/6N embryos survived similarly (p greater than 0.05) in either cryoprotectant. Compared to DMSO counterparts, embryos from GLYC-treated outbred groups had improved QG and VG ratings and reduced EDR (p less than 0.05), but comparable results were observed between GLYC- AND DMSO-treated embryos in the C57BL/6N group. Between genotypes, type of container affected PTS and EDR (p less than 0.05) but not QG or VG ratings (p greater than 0.05). Within genotypes, PTS generally ranged 15 to 20% higher (p less than 0.01) in AMP than STR groups. Increased PTS was noted in outbred mouse x GLYC x AMP groups; however, based on the degrees of difference, the inbred C57BL/6N strain appeared less affected by this 3-way interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. Gamma irradiation is an effective method for providing terminal sterilization to biological tissue, but it is also reported to have deleterious effects on tissue mechanics in a dose-dependent manner. At irradiation ranges up to 25 kGy, a clear relationship between mechanical strength and dose has yet to be established. The aim of this study was to investigate the mechanical properties of bone and soft tissue allografts, irradiated on dry ice at a low absorbed dose (18.3–21.8 kGy) and a moderate absorbed dose (24.0–28.5 kGy), using conventional compressive and tensile testing, respectively. Bone grafts consisted of Cloward dowels and iliac crest wedges, while soft tissue grafts consisted of patellar tendons, anterior tibialis tendons, semitendinosus tendons, and fascia lata. There were no statistical differences in mechanical strength or modulus of elasticity for any graft irradiated at a low absorbed dose, compared to control groups. Also, bone allografts and two soft tissue allografts (anterior tibialis and semitendinosus tendon) that were irradiated at a moderate dose demonstrated similar strength and modulus of elasticity values to control groups. The results of this study support the use of low dose and moderate dose gamma irradiation of bone grafts. For soft tissue grafts, the results support the use of low dose irradiation.     

Effect of arbuscular mycorrhizal fungi, organic fertilizer and soil sterilization on maize growth

The effects of inoculation with arbuscular mycorrhizal (AM) fungi, organic fertilizer (F) applications, and soil sterilization on maize growth were evaluated in a pot experiment. The experiment was in a completely randomized factorial design (2 × 4 × 2) with six replicates for each treatment. There were two soil treatments (sterilized soil, SS and unsterilized soil, US), four organic fertilizer treatments (0.0, 0.5, 1.0 and 2.0 g kg^?1 soil), and two AM fungi treatments (inoculation with Glomus mosseae, +AM and uninoculated control, ?AM). Inoculated plants generally had greater AM colonization, plant height, dry weight and phosphorus (P) uptake than uninoculated controls, and these parameters were significantly increased as the organic fertilizer application increased up to 0.5 g kg^?1 but decreased or had no significant effect compared to the uninoculated plants at the highest fertilizer rate (2.0 g kg^?1). Plant growth, P uptake and AM colonization of root system were significantly higher in sterilized soil compared to the unsterilized control. Our results indicated that the inoculation of AM fungi in field soil with optimal organic fertilizer application greatly improved maize growth and nutrient uptake, and the effect was greater under sterilized soil condition.     

Effect of arbuscular mycorrhizal fungi, organic fertilizer and soil sterilization on maize growth ☆

The effects of inoculation with arbuscular mycorrhizal (AM) fungi, organic fertilizer (F) applications, and soil sterilization on maize growth were evaluated in a pot experiment. The experiment was in a completely randomized factorial design (2 × 4 × 2) with six replicates for each treatment. There were two soil treatments (sterilized soil, SS and unsterilized soil, US), four organic fertilizer treatments (0.0, 0.5, 1.0 and 2.0 g kg^-1 soil), and two AM fungi treatments (inoculation with Glomus mosseae, +AM and uninoculated control, －AM). Inoculated plants generally had greater AM colonization, plant height, dry weight and phosphorus (P) uptake than uninoculated controls, and these parameters were significantly increased as the organic fertilizer application increased up to 0.5 g kg^-1 but decreased or had no significant effect compared to the uninoculated plants at the highest fertilizer rate (2.0 g kg^-1). Plant growth, P uptake and AM colonization of root system were significantly higher in sterilized soil compared to the unsterilized control. Our results indicated that the inoculation of AM fungi in field soil with optimal organic fertilizer application greatly improved maize growth and nutrient uptake, and the effect was greater under sterilized soil condition.     

Substantiation of 25 kGy radiation sterilization dose for banked air dried amniotic membrane and evaluation of personnel skill in influencing finished product bioburden

Preparation of amniotic membrane (AM) by air drying method followed by radiation sterilization is simple and valuable approach; sterility and quality of the final AM product are depending on the quality management system at the tissue bank. Validation and substantiation of radiation sterilization dose (RSD) for tissue allografts is an essential step for the development and validation of the standard operating procedures (SOP). Application of SOP is perfectly relying on trained staff. Skills differences among personnel involved in AM preparation could have an effect on microbiological quality of the finished product and subsequently on the RSD required. AM were processed by four different couples of the tissue bank technicians. The AM grafts were randomly selected and subjected to bioburden test to validate and substantiate the 25 kGy RSD. Bioburden test for AM grafts were also useful to evaluate the skill of the tissue bank technicians and thus, to validate the current SOP for air dried AM. Moreover, the effect of placental source on bioburden counts on AM grafts was assessed. Substantiation of the 25 kGy RSD at a sterility assurance level of 10^?1, and sample item portion = 1, was carried out using Method VD _max ²⁵ of the International Organization for Standardization, document no. 11137-2 (ISO in Sterilization of healthcare products—radiation—part 2: establishing the sterilization dose, Method VDmax—substantiation of 25 kGy or 15 kGy as the sterilization dose, International Standard Organization, 2006). The results showed that there were no significant differences in the bioburdens of the four batches (α = 1 %), this means no significant differences in the skill of the four couples of the tissue bank technicians in terms of their ability to process AM according to the air dried AM SOP. The 25 kGy RSD was validated and substantiated as a valid sterilization dose for the AM prepared with the current established SOP at the Biotechnology Research Center experimental tissue bank. The donor’s type of delivery, normal or caesarean, showed no significant effect on the levels of microbial counts on the tested AMs (α = 1 %).     

Sporicidal efficacy of genipin: a potential theoretical alternative for biomaterial and tissue graft sterilization

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study’s aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin’s sterilization potential Bacillus subtilis var. niger spore strips were treated with 0–10 % genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100–100:0), and treatment duration (24–72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63–10 % genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63 % genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100 % PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin’s effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infectious species other than spores, and to assess whether sterilant activity persists after penetrating tissues and biomaterials.     

Effects of ionizing radiation and preservation on biomechanical properties of human costal cartilage

Tissue banks around the world store human cartilage obtained from cadaveric donors for use in diverse reconstructive surgical procedures. To ensure this tissue is sterile at the time of distribution, tissues may be sterilized by ionizing radiation. In this work, we evaluate the physical changes in deep frozen costal cartilage (?70 °C) or costal cartilage preserved in high concentrations of glycerol (>98 %) followed by a terminal sterilization process using ionizing radiation, at 3 different doses (15, 25 and 50 kGy). Tension and compression tests were carried out to determine the mechanical changes related both to the different preservation methods and irradiation doses. For both methods of preservation, tension strength was increased by about 24 %, when cartilage tissue was irradiated with 15 kGy. Deep frozen samples, when irradiated with 25 or 50 kGy, had a decrease in their mechanical performance, albeit to a lesser extent than when tissues were preserved in high concentration of glycerol and equally irradiated. In conclusion, processing in high concentration of glycerol did not increase tissue protection against radiation damage; while cartilage preserved in high concentrations of glycerol withstands radiation up to 25 kGy, deep frozen human costal cartilage may be sterilized with a doses up to 50 kGy without significant mechanical impact.     

Application of a high-level peracetic acid disinfection protocol to re-process antibiotic disinfected skin allografts

Skin allografts, derived from cadaveric donors, are widely used for the treatment of burns and ulcers. Prior to use in clinical situations, these allografts are disinfected using a cocktail of antibiotics and then cryopreserved. Unfortunately, this antibiotic disinfection procedure fails to decontaminate a significant proportion and these contaminated grafts can not be used clinically. We have investigated whether it is possible to apply a second, more potent disinfection procedure to these contaminated grafts and effectively to re-process them for clinical use. Cadaveric skin grafts, treated with antibiotics and cryopreserved, were thawed and a peracetic acid (PAA) disinfection protocol applied. The grafts were then preserved in a high concentration of glycerol or propylene glycol, and properties thought to be essential for successful clinical performance assessed. The cytotoxicity of the grafts was assessed using both extract and contact assays; damage to the skin collagen was assessed using a collagenase susceptibility assay and the capacity of the grafts to elicit an inflammatory response in vitro was assessed by quantifying the production of the pro-inflammatory cytokine TNF-alpha by human peripheral blood mononuclear phagocytes. PAA disinfection, in conjunction with either glycerol or propylene glycol preservation, did not render the grafts cytotoxic, pro-inflammatory, or increase their susceptibility to collagenase digestion. The rates of penetration of glycerol and propylene glycol into the re-processed skin were comparable to those of fresh skin. This study has demonstrated that PAA disinfection combined with immersion in high concentrations of either glycerol or propylene glycol was an effective method for re-processing contaminated skin allografts, and may justify their clinical use.     

去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

植物内生放线菌的选择性分离

[目的]更好地发掘内生菌资源,建立有效的植物内生放线菌分离方法.[方法]比较不同消毒剂和消毒程序、样品预处理、选择性分离培养基等分离内生放线菌的效果,通过形态及16S rRNA基因序列分析进行菌种鉴定.[结果]用5％的次氯酸钠处理样品4-7 min消毒效果最好;100℃处理样品15 min能较好地减少真菌和细菌的干扰.丙酸钠、琥珀酸钠等培养基分离放线菌出菌率较高且类群多样性丰富.[结论]植物样品表面消毒干燥后,100℃处理15 min,用无菌搅拌杯打碎,直接撒植物于分离培养基中的分离方法效果较好.     

Biomechanical properties of sterilized human auditory ossicles.

Bone allograft material is treated with sterilization methods to prevent the transmission of diseases from the donor to the recipient. The effect of some of these treatments on the integrity of the bone is unknown. This study was performed to evaluate the effect of several sterilization methods on the mechanical behaviour of human middle ear bones. Due to the size and composition of the bones (approximately 1.5 mm diameter by 4 mm long), mechanical testing options were limited to the traditional platens compression test. Experiments were first performed with synthetic bone to evaluate the precision of this test applied to small specimens. Following this, fresh frozen human ossicles were thawed and sterilized with (i) 1 N NaOH (n = 12); (ii) 0.9% LpH, a phenolic solution (n = 12); or (iii) steam at 134 degrees C (n = 18). A group of 26 control specimens did not receive any sterilization treatment. Material and structural properties were determined from axial compression testing. Results from the synthetic bone showed that the test was reproducible, with standard deviations less than 20% of the means. Significant differences occurred in stiffness and ultimate force values between NaOH-treated and autoclaved bones when compared to normals (p<0.05), but not for LpH-treated bones. LpH is not approved for medical use, so NaOH is the most appropriate of the treatments studied for the sterilization of ossicle allografts.     

攀枝花市麻疯树根际土壤内生菌根菌分布特征

【目的】研究天然麻疯树根际土壤中内生菌根菌孢子果密度,揭示麻疯树根际土壤中内生菌根菌的分布特征,为麻疯树育苗繁殖和栽植推广提供科学依据。【方法】采用随机采样的方法对攀枝花市仁和区天然麻疯树根际土壤中内生菌根菌孢子果数量进行调查,确定了20个采样点,采集了20个土根混合样,利用解剖镜测定其孢子果密度,再根据1999年林业行标测定样品中的水分含量,养分含量等。【结果】天然麻疯树根际土壤采样从海拔1025m开始,直到1500m。土壤样品中均含有AM菌根菌孢子果,且数量丰富的,含量最高的土壤样品中AM菌根菌孢子果密度为236个/g干土,最小的密度为9个/g干土,平均密度为80个/g干土。土样含水量在4.01%-13.39%之间,平均值为6.79%,与AM菌根菌孢子果含量正相关。有机质平均含量为3.80g/kg,与AM菌根菌孢子果含量正相关。【结论】天然麻疯树根际土壤中均能检测出AM菌根菌孢子果,且含量高,但分布不均。它的密度随着海拔高度的升高逐渐降低,随土壤含水量的增加而增加,随土壤有机质含量的增加而增加。     

The Influence of Different Preservation and Sterilisation Steps on the Histological Properties of Amnion Allografts — Light and Scanning Electron Microscopic Studies

Despite thorough donor screening and preparation under aseptic conditions, conventional methods of preservation do not exclude the probability of a contamination with pathogenic germs. The purpose of this study was to investigate the changes of histological parameters of amnion transplants (ATs) through different methods of sterilisation and preservation. Therefore 10 different procedures for sterilisation and preservation of ATs were described. Specimens of each group were studied using different histological procedures such as light microscopy and scanning electron microscopy. General staining (Haematoxylin-eosin stain, periodic-acid-Schiff, Domack) and immunohistochemical methods have been applied in order to gain additional information concerning the structure of the amniotic epithelium and the basement membrane but also the distribution of collagens and intermediate filaments. Furthermore, the measurement of the ATs thickness was included in order to study the influence of the manufacturing procedures to this property.As a result we found that the histological appearance of the ATs is closely related to the applied sterilisation and preservation procedures.Although the basement membrane remained intact, especially the amniotic epithelium was partially destroyed by irradiation sterilisation. Further, the dissolution of the connective tissue layers into single fibre bundles was clearly visible. Procedures with and without peracetic acid sterilisation (PAA) preserved the tissue structure.Our results showed a significant variation in the tissue's thickness after different preservation procedures. Air- and freeze-dried ATs were found to be the thinnest tissues varying from 20 to 30 microm, the thickest ATs preserved in glycerol varied from 45 to 50 microm. Because ATs showed a preserved tissue structure after PAA sterilisation it can be recommended as an alternative for methods previously described in literature. Depending on the specific use of the AT one may choose from thinner or thicker allografts.     

Effects of preservation media on in vitro maturation of porcine oocytes

The effects of preservation media for ovaries on in vitro maturation of porcine oocytes was studied. The cumulus-oocyte complexes (COCs) obtained from ovaries that had been preserved in three different media at various temperatures for different time intervals were cultured in the M199 maturation medium. The preservation media used were 0.9% saline solution, BCS (Braun-Collins solution) and Dulbecco's phosphate buffered saline solution (PBS). Mature oocytes obtained from the ovaries preserved in three preservation media for 8 h were electrically activated. The activated oocytes were then cultured in the NCSU23 embryo culture medium for 16 h to observe activation, or for 144 h to observe embryo development. It was found that the preservation temperature significantly affected maturation of the porcine oocytes. A preservation temperature of about 25 degrees C showed an optimal maturation rate for a preservation time of 8 h for the three preservation media. Although the preservation temperature was a major factor influencing the maturation rate, different preservation media at 25 degrees C for 8 h also significantly affected the maturation rate, activation rate and embryo development. Among these three preservation media, PBS exhibited the highest cleavage rate indicating that PBS should be a better preservation medium for porcine ovaries at 25 degrees C for 8 h or longer periods.     

Contamination control of microbe Ziziphus spina [christti] seed in vitro culture

Ziziphus spina [christti] is a naturally distributed tree in subtropical, arid and semi-arid parts of Iran. It is ecologically and economically important due to its tolerance to drought and salinity. Most tree seeds are infected with parasitic and saprophytic microorganisms which decrease the seed germination and seedling establishment. The goal of this paper was to evaluate the ability of selected chemical solutions to inhibit the growth of variety of microbial contaminants in Z. spina [christti] seeds and to enhance the seed germination. Different chemical treatments were used in surface sterilization of seeds: (Treatment 1) sodium hypochlorite (NaOCl) in concentrations of 1, 2 and 4% for 20 min. (Treatment 2) hydrogen peroxide (H₂O₂) in concentration of 4, 8, 12% treated for 10 min. (Treatment 3) 1% mercuric chloride (HgCl₂) at duration 10, 15, 20, 25 min. Seeds were scarified and aseptically, planted on agar Murashige and Skoog (MS) medium. Contaminants were identified according to their morphological and cultural characteristics. Bacterial contaminants included Xanthomonas sp. While Fungal isolates were Fusarium, Penicillium, Alternaria, Rhizopus, and Aspergillus. Our experiment reveals that 4% NaOCl followed by benomyl is the best sterilization treatment for Z. spina [christti] seeds, since the highest number of germination and highest number of sterilized seeds was observed after this treatment.