Activity of bone morphogenetic protein-7 after treatment at various temperatures: freezing vs. pasteurization vs. allograft

Insufficient bone union is the occasional complication of biomechanical reconstruction after malignant bone tumor resection using temperature treated tumor bearing bone; freezing, pasteurization, and autoclaving. Since bone morphogenetic protein (BMP) plays an important role in bone formation, we assessed the amount and activity of BMP preserved after several temperature treatments, including −196 and −73 °C for 20 min, 60 and 100 °C for 30 min, 60 °C for 10 h following −80 °C for 12 h as an allograft model, and 4 °C as the control. The material extracted from the human femoral bone was treated, and the amount of BMP-7 was analyzed using an enzyme-linked immunosorbent assay. Then, the activity of recombinant human BMP-7 after the treatment was assessed using a bioassay with NIH3T3 cells and immunoblotting analysis to measure the amount of phospho-Smad, one of the signaling substrates that reflect the intracellular reaction of BMPs. Both experiments revealed that BMP-7 was significantly better preserved in the hypothermia groups. The percentages of the amount of BMP-7 in which the control group was set at 100% were 114%, 108%, 70%, 49%, and 53% in the −196, −73, 60, 100 °C, and the allograft-model group, respectively. The percentages of the amount of phospho-Smad were 89%, 87%, 24%, 4.9%, and 14% in the −196, −73, 60, 100 °C, and the allograft-model group, respectively. These results suggested that freezing possibly preserves osteoinductive ability than hyperthermia treatment.     

Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes

MII mouse oocytes in 1 and 1.5M ethylene glycol(EG)/phosphate buffered saline have been subjected to rapid freezing at 50 degrees C/min to -70 degrees C. When this rapid freezing is preceded by a variable hold time of 0-3 min after the initial extracellular ice formation (EIF), the duration of the hold time has a substantial effect on the temperature at which the oocytes subsequently undergo intracellular ice formation (IIF). For example, in 1M EG, the IIF temperatures are -23.7 and -39.2 degrees C with 0 and 2 min hold times; in 1.5M EG, the corresponding IIF temperatures are -29.1 and -40.8 degrees C.     

Comparison of frozen and freeze-dried particulate bone allografts

Freeze-dried and frozen particulate bone allografts are used interchangeably on the assumption that the biologic behavior of these grafts is similar. Dissimilarities in biologic behavior and differences in the rate and extent of bone incorporation of freeze-dried and frozen particulate grafts were demonstrated in a comparative study using a non-human primate model. Freeze-dried particulate allografts induced new bone formation and healing of the osseous defects much faster than the frozen allografts.     

Effects of bone marrow stromal cell injection in an experimental glaucoma model

We investigated if bone marrow stromal cells (BMSCs) transplanted into the vitreous body of a glaucoma model eye could be integrated in the host retina and also whether they could rescue the retinal ganglion cells (RGCs) from death induced by the elevated intraocular pressure. Glaucoma was induced in the right eye of adult Wistar rats by ligating the episcleral veins. The GFP-expressing BMSCs (GFP-BMSCs) were injected into the vitreous body of both the control and the glaucomatous eyes. After transplantation, GFP-BMSCs were mostly present along with the inner limiting membrane and only a few cells were integrated into the ganglion cell layer. At 2 or 4 weeks after transplantation, GFP-BMSCs were observed to express various trophic factors. The BMSCs injected glaucoma model eyes showed less reduction in the number of RGCs compared to the glaucomatous eyes with PBS injection. This study suggests that BMSC transplantation may be worthy as a neuroprotective tool to treat glaucoma.     

Effects of freezing on isolated plant mitochondria

Mitochondria isolated from spinach leaves (Spinacia oleracea L.) and potato tubers (Solanum tuberosum L.) were partly injured when subjected to freezing for 2 to 4 h at-25°C in salt solutions in the absence of cryoprotectants. Damage was manifested by the inactivation of respiratory properties and increase in the permeability of the mitochondrial membranes. Decrease in respiratory control indicated that the control mechanism of the electron transport chain was influenced by freezing. Oxidative phosphorylation was only slightly more affected than electron transport. The inactivation of the membrane systems was caused by an increase in the concentration of membrane-toxic solutes. This was confirmed by treatment of the organelles at 0°C in solutions of high salt concentrations. When sugar was present in the course of freezing, mitochondria were partly or completely protected. On a molar basis, sucrose was more effective in membrane protection than glucose. Under certain conditions amino acids, e.g., proline and hydroxyproline, also stabilized isolated mitochondria during freezing.Abbreviations BSA bovine albumin - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MOPS 2-N-morpholinopropane sulfonic acid - PVP polyvinyl pyrrolidone - RC respiratory control - Tris tris (hydroxymethyl) aminomethane     

The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits

The pedicle freezing procedure by liquid nitrogen is a method for the reconstruction of tumor-bearing bone after malignant tumor resection. However, the regenerative mechanism of bone after the pedicle freezing procedure is unclear. We investigated the complete process from devitalization to revitalization of bone after the pedicle freezing procedure in 13 rabbits. After osteotomy the 5 mm distal femurs were immersed in liquid nitrogen, and the specimens were divided into frozen area and sub-frozen area. The bilateral femurs were harvested for evaluation of bone regeneration by histological and immunohistochemical examination (VEGF, CD31, BMP-2 and Runx2) from 1 week to 52 weeks. The diameter of operating femurs was compared with contralateral femurs from 6 weeks to 52 weeks.No viable cells could be found from 1 to 8 weeks in the frozen area, and a mean 1.83 cm necrotic range were detected in the sub-frozen area. The periosteal reaction, massive fibrous tissue and immature bone matrix invaded from the normal area to the necrotic area from 12 weeks. Subsequently, the necrotic bone was gradually replaced by newly formed bone by creeping substitution, with endochondral and intramembrane bone formation. The diameter of frozen femurs was significantly larger than the contralateral femur at the same period from 8 weeks to 52 weeks (P < 0.01). All immunohistochemical factors were positively expressed in both areas at different time points. The active osteoblasts and microvessel migrated from marrow cavity and periosteum into dead bone. This study suggested that the frozen bone not only provides a scaffold but also possesses excellent osteoinductive properties.     

Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves

The effects of salicylic acid (SA) (0.01, 0.1 and 1 mM) and cold on freezing tolerance (freezing injury and ice nucleation activity) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) grown under control (20/18 °C for 15, 30 and 45-day) and cold (15/10 °C for 15-day, 10/5 °C for 30-day and 5/3 °C for 45-day) conditions. Cold acclimatisation caused a decrease of injury to leaf segments removed from the plants and subjected to freezing conditions. Exogenous SA also decreased freezing injury in the leaves grown under cold (15/10 °C) and control (15 and 30-day) conditions. Cold conditions (10/5 and 5/3 °C) caused an increase in ice nucleation activity by apoplastic proteins, which were isolated from the leaves. For the first time, it was shown that exogenous SA caused an increase in ice nucleation activity under cold (15/10 and 10/5 °C) and control conditions. These results show that salicylic acid can increase freezing tolerance in winter wheat leaves by affecting apoplastic proteins.     

Effects of thirty elements on bone metabolism

The human skeleton, made of 206 bones, plays vital roles including supporting the body, protecting organs, enabling movement, and storing minerals. Bones are made of organic structures, intimately connected with an inorganic matrix produced by bone cells. Many elements are ubiquitous in our environment, and many impact bone metabolism. Most elements have antagonistic actions depending on concentration. Indeed, some elements are essential, others are deleterious, and many can be both. Several pathways mediate effects of element deficiencies or excesses on bone metabolism. This paper aims to identify all elements that impact bone health and explore the mechanisms by which they act. To date, this is the first time that the effects of thirty minerals on bone metabolism have been summarized.     

Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells

The morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen-thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three-step and one-step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co-cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three-step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one-step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three-step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method. © 1994 Wiley-Liss, Inc.     

Collection,processing and freezing of equine bone marrow cells

There is no consensus on aspects of equine bone marrow collection and processing. The study aimed to describe the collection of large volumes of bone marrow from horses of advanced age, with emphasis on bone marrow mononuclear cells (BMMCs) recovery and viability after cryopreservation. Fourteen horses, aged 3–24 years, were divided into three experiments. E1 studied the feasibility of collecting 200 mL from the sternums of horses of advanced age; E2 examined the number of cells obtained from the first and last syringe of each puncture; and E3 investigated the influence of heparin concentration on the prevention of cell aggregation, and cell viability after freezing in liquid nitrogen. Bone marrow aspirations were done with syringes pre-filled with Iscove's modified Dulbecco's medium and different concentrations of sodium heparin. BMMCs were counted, cell viability was determined, and samples were frozen. Bone marrow collection from the sternum is safe, even at large volumes and from horses of advanced age, and the number of cells recovered decreases with successive aspirations (p < 0.0001). Heparin concentration influenced cell aggregation, and recovered cells continued to be commercially viable after 150 days in frozen storage.     

Effects of whole-body vibration on bone properties in aged rats

Objective:This study aimed to explore optimal conditions of whole-body vibration (WBV) for improving bone properties in aged rats.Methods:Eighty-week-old rats were divided into baseline control (BC), age-matched control (CON) and experimental groups, which underwent WBV (0.5 g) at various frequencies (15, 30, 45, 60 or 90 Hz) or WBV (45 Hz) with various magnitudes (0.3, 0.5, 0.7 or 1.0 g) for 7 weeks. After interventions, femur bone size, bone mechanical strength and circulating bone formation/resorption markers were measured, and trabecular bone microstructure (TBMS) and cortical bone geometry (CBG) of femurs were analyzed by micro-CT.Results:Several TBMS parameters and trabecular bone mineral content were significantly lower in the 15 Hz WBV (0.5 g) group than in the CON group, suggesting damage to trabecular bone. On the other hand, although frequency/magnitude of WBV did not influence any CBG parameters, the 0.7 g and 1.0 g WBV (45 Hz) group showed an increase in tissue mineral density of cortical bone compared with the BC and CON groups, suggesting the possibility of improving cortical bone properties.Conclusion:Based on these findings, it should be noted that WBV conditions are carefully considered when applied to elderly people.     

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。     

镉染毒对雄性大鼠骨生物力学性能的影响

目的探讨镉（Cd）对大鼠股骨和腰椎生物力学性能的影响。方法 24只8周龄雄性SD大鼠随机分成4组：对照组,皮下注射0.5 mL生理盐水;实验组,染毒剂量分别为0.1 mg Cd/（kg.bw）（低剂量组）,0.5 mgCd/（kg.bw）（中剂量组）和1.5 mg Cd/（kg.bw）（高剂量组）,每周根据体重调整注射量。染毒后第12周,收集全血、腰椎及股骨,分别用于血镉测定、骨镉测定、骨密度测定及生物力学测定。结果染毒组大鼠体内血镉及骨镉水平明显高于对照组,差异有统计学意义（P〈0.05）;中、高剂量染毒组大鼠骨密度较对照组显著下降（P〈0.05）;染毒组大鼠股骨和腰椎生物力学性能较对照组有不同程度的的降低,其中高剂量染毒组大鼠股骨生物力学性能的下降和对照组相比差异有显著性（P〈0.01）;中、高剂量组大鼠腰椎生物力学性能和对照组相比有显著下降,差异有统计学意义（P〈0.01）。结论镉影响大鼠股骨和腰椎的生物力学性能,并且腰椎较股骨更为敏感。     

Non-lethal freezing effects on seed degreening in Brassica napus

The effects of a non-lethal freezing stress on chlorophyll content, moisture level and distribution, and abscisic acid (ABA) levels were examined in siliques and seeds of Brassica napus (canola). A non-lethal freezing stress resulted in the retention of chlorophyll in seed at harvest that was most pronounced for seeds 28, 32 and 36 days after flowering (DAF). This increase was primarily due to an increased retention of chlorophyll a relative to chlorophyll b. Chlorophyll retention in seeds exposed to a non-lethal freezing stress correlated with an increased ABA catabolism, as measured 1, 3 or 7 days after the stress treatment. Although the non-lethal freezing stress had no significant effect on moisture content in seeds of siliques stressed at 28–44 DAF, moisture distribution, as viewed by magnetic resonance imaging, showed an uneven drying of 32 and 40 DAF siliques after exposure to the non-lethal freezing stress. Moisture was initially lost more rapidly from the silique wall between seeds, than in control non-stressed siliques. Increased moisture loss was not due to structural changes in the vasculature of the silique/seed of stressed tissues. These results are consistent with the hypothesis that a non-lethal freezing stress-induced decrease in ABA level, during seed maturation, effects an inhibition of normal chlorophyll a catabolism resulting in mature but green B. napus seed.     

Effects of static magnetic fields on bone microstructure and mechanical properties in mice

All the living organisms originate, evolve and live under geomagnetic field (GMF, 20–70 µT). With rapid development in science and technology, exposure to various static magnetic fields (SMFs) from natural and man-made sources remains a public environmental topic in consideration of its probable health risk for humans. Many animal studies related to health effect have demonstrated that SMF could improve bone formation and enhance bone healing. Moreover, most of the studies focused on local SMF generated by rod-type magnet. It was difficult to come to a conclusion that how SMF affected bone metabolism in mice. The present study employed hypomagnetic field (HyMF, 500 nT), and moderate SMF (MMF, 0.2 T) to systematically investigate the effects of SMF with continuous exposure on microstructure and mechanical properties of bone. Our results clearly indicated that 4-week MMF exposure did not affect bone biomechanical properties or bone microarchitecture, while HyMF significantly inhibited the growth of mice and elasticity of bone. Furthermore, mineral elements might mediate the biological effect of SMF.     

Temperature Effects on Survival of Xiphinema bakeri in Fallow Soil

Numbers of Xiphinema bakeri increased during the first month of storage at temperatures 5-30 C in naturally-infested soil sealed in polyethylene bags. From 1 to 6 months, populations trended toward later developmental stages, and total numbers declined, especially at the higher temperatures. Similarly-packaged X. bakeri eggs, larvae and adults were killed by -18 C for 48 hr or -34 C for 12 hr.     

Effects of bone morphogenetic proteins on epithelial repair

Epithelial tissue has important functions such as protection, secretion, and sensation. Epithelial damage is involved in various pathological processes. Bone morphogenetic proteins (BMPs) are a class of growth factors with multiple functions. They play important roles in epithelial cells, including in differentiation, proliferation, and migration during the repair of the epithelium. This article reviews the functions and mechanisms of the most profoundly studied BMPs in the process of epithelial damage repair and their clinical significance.     

Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections

To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization of type I collagen, bone sialoprotein, and osteopontin was investigated during different stages of fixation and decalcification. Four-week-old rat femurs were rapidly frozen, and were sectioned without fixation or decalcification. Thereafter, following or bypassing fixation in 4% paraformaldehyde, these sections were decalcified in 5% EDTA for 0-5 min. Before decalcification, marked radiopacity of bone matrix was observed in contact microradiography (CMR) images, and electron probe microanalysis (EPMA) demonstrated intense localization for phosphorus and calcium. In fixed and unfixed sections without decalcification, immunolocalization of bone matrix proteins were almost restricted to osteoid. After 1 min of decalcification, reduced radiopacity was apparent in the CMR images, and less phosphorus and calcium was observed by EPMA, which completely disappeared by 5 min decalcification. After 3-5 min of decalcification, unfixed sections showed that these proteins were immunolocalized in bone matrix, but were not detectable in osteoid. However, fixed sections demonstrated that these were found in both bone matrix and osteoid. The present findings suggest that bone matrix proteins are embedded in calcified matrix which is separated from the aqueous environment and that they hardly move, probably due to firm bonding with each other. In contrast, matrix proteins in osteoid are subject to loss after decalcification because they may be bound to scattered apatite crystals, not to each other.     

Effects of reproduction on sexual dimorphisms in rat bone mechanics

Osteoporosis most commonly affects postmenopausal women. Although men are also affected, women over 65 are 6 times more likely to develop osteoporosis than men of the same age. This is largely due to accelerated bone remodeling after menopause; however, the peak bone mass attained during young adulthood also plays an important role in osteoporosis risk. Multiple studies have demonstrated sexual dimorphisms in peak bone mass, and additionally, the female skeleton is significantly altered during pregnancy/lactation. Although clinical studies suggest that a reproductive history does not increase the risk of developing postmenopausal osteoporosis, reproduction has been shown to induce long-lasting alterations in maternal bone structure and mechanics, and the effects of pregnancy and lactation on maternal peak bone quality are not well understood. This study compared the structural and mechanical properties of male, virgin female, and post-reproductive female rat bone at multiple skeletal sites and at three different ages. We found that virgin females had a larger quantity of trabecular bone with greater trabecular number and more plate-like morphology, and, relative to their body weight, had a greater cortical bone size and greater bone strength than males. Post-reproductive females had altered trabecular microarchitecture relative to virgins, which was highly similar to that of male rats, and showed similar cortical bone size and bone mechanics to virgin females. This suggests that, to compensate for future reproductive bone losses, females may start off with more trabecular bone than is mechanically necessary, which may explain the paradox that reproduction induces long-lasting changes in maternal bone without increasing postmenopausal fracture risk.