Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering

AIM:To investigate the interaction between mesenchymal stem cells(MSCs) and bone grafts using two different cultivation methods:static and dynamic.METHODS:MSCs were isolated from rat bone marrow.MSC culture was analyzed according to the morphology,cell differentiation potential,and surface molecular markers.Before cell culture,freeze-dried bone(FDB) was maintained in culture for 3 d in order to verify culture medium pH.MSCs were co-cultured with FDB using two different cultivation methods:static co-culture(two-dimensional) and dynamic co-culture(threedimensional).After 24 h of cultivation by dynamic or static methods,histological analysis of Cell adhesion on FDB was performed.Cell viability was assessed by the Trypan Blue exclusion method on days 0,3 and 6 after dynamic or static culture.Adherent cells were detached from FDB surface,stained with Trypan Blue,and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture.Statistical analyses were performed with SPSS and a P < 0.05 was considered significant.RESULTS:The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures.Rat MSCs were positive for CD44,CD90 and CD29 and negative for CD34,CD45 and CD11bc.FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH(P > 0.05).In histological analysis,there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods(P < 0.05).The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method.On day 0,the cell viability in the dynamic system was significantly higher than in the static system(P < 0.05).There was a statistical difference in cell viability between days 0,3 and 6 after dynamic culture(P < 0.05).In static culture,cell viability on day 6 was significantly lower than on day 3 and 0(P < 0.05).CONCLUSION:An alternative cultivation method was developed to improve the MSCs adhesion on FDB,demonstrating that dynamic co-culture provides a superior environment over static conditions.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Screening of APOB gene mutations in subjects with clinical diagnosis of familial hypercholesterolemia

Monogenic hypercholesterolemia is a group of lipid disorders, most of which have autosomal dominant transmission. Familial defective apoB (FDB) resulting from mutations in the APOB gene is a well-recognized cause of autosomal dominant monogenic hypercholesterolemia (ADMH). However, the frequency of FDB among patients with ADMH is not well established. The aim of our research was to screen for mutations responsible for FDB in subjects with a clinical diagnosis of familial hypercholesterolemia. We studied 408 patients from the Spanish Register of Familial Hypercholesterolemia, proportionally distributed among all Spanish regions. Abnormal SSCP patterns of the APOB gene were checked by DNA sequencing and restriction analysis. Three out of the 408 patients were carriers of the R3500Q mutation, and 2 subjects were carriers of the silent T3552T mutation; in both of these patients functional mutations in the LDL receptor gene were found. We conclude that FDB is not a common cause of ADMH in Spain; the R3500Q mutation is the only mutation in APOB causing FDB, and the LDL receptor binding domain of APOB is highly conserved in the studied sample.     

Evidence that plumage bacteria influence feather coloration and body condition of eastern bluebirds Sialia sialis

Parasites influence the expression of secondary sexual traits and the health of infected individuals. We set out to test the influence of reputed exogenous parasites, plumage bacteria, including feather-degrading bacteria (FDB), on secondary sexual characteristics and body condition of wild adult eastern bluebirds Sialia sialis . Previous work has shown that FDB alter the coloration of structurally-colored bluebird feathers in vitro ( Shawkey et al. 2007 ). In a correlational study of how bacteria affect birds in the wild, we found that female plumage got duller with increasing FDB intensity. Males tended to get brighter with increasing FDB intensity, but the relationship was not significant. We also found significant associations between plumage bacteria intensity and body condition, but, again, with gender-based differences. Female body condition was negatively associated with plumage bacteria intensity, while male body condition was positively associated with plumage bacteria intensity. Interestingly, plumage bacteria intensity of males and females in nesting pairs was significantly positively correlated. We also report the highest prevalence of FDB measured in a wild bird population, with FDB detected on 67/68 (99%) of individuals. Further work is needed to fully understand the relationships between plumage bacteria and birds, but our data indicate that plumage bacteria may have sex-dependent effects on multiple phenotypic traits.     

Familial defective apolipoprotein B-100: a lesson from homozygous and heterozygous patients

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder caused by a substitution of glutamine for arginine at residue 3500 of the apolipoprotein B-100 molecule. We have identified 23 heterozygotes and one homozygote for FDB (frequency 1:20) in a group of 510 patients with hypercholesterolemia. Mean age of the patients (18 females and 6 males) was 46 years. The diagnosis of FDB was based on point mutation PCR analysis of exon 26 of the apo B gene. Plasma lipids in heterozygous patients were: total cholesterol 8.76+/-1.2 mmol/l, triglycerides 1.42+/-0.5 mmol/l, HDL-cholesterol 1.43+/-0.3 mmol/l, LDL-cholesterol 6.69+/-1.2 mmol/l, apoB 1.69+/-0.4 g/l, Lp(a) 0.26+/-0.2 g/l. The most frequent apoE genotype was 3/3 (19 patients), apoE 3/4 genotype was found in 3 patients and one person had apoE 2/3. Xanthelasma palpebrarum was present in 4 patients and tendon xanthomas in 3 patients including the homozygote. Premature manifestation of coronary heart disease was revealed in 3 patients. Sixteen patients were treated with statins, a combination of statin and resin was used in 2 patients (including the homozygote), whereas six patients were treated with the diet only. We conclude that although the plasma lipid levels of total and LDL cholesterol in FDB patients are lower than in patients with familial hypercholesterolemia, the patients with FDB suffer from premature atherosclerosis. The therapeutic approach to FDB individuals and patients with familial hypercholesterolemia is very similar.     

Two Horizontally Transferred Xenobiotic Resistance Gene Clusters Associated with Detoxification of Benzoxazolinones by Fusarium Species

Microbes encounter a broad spectrum of antimicrobial compounds in their environments and often possess metabolic strategies to detoxify such xenobiotics. We have previously shown that Fusarium verticillioides, a fungal pathogen of maize known for its production of fumonisin mycotoxins, possesses two unlinked loci, FDB1 and FDB2, necessary for detoxification of antimicrobial compounds produced by maize, including the γ-lactam 2-benzoxazolinone (BOA). In support of these earlier studies, microarray analysis of F. verticillioides exposed to BOA identified the induction of multiple genes at FDB1 and FDB2, indicating the loci consist of gene clusters. One of the FDB1 cluster genes encoded a protein having domain homology to the metallo-β-lactamase (MBL) superfamily. Deletion of this gene (MBL1) rendered F. verticillioides incapable of metabolizing BOA and thus unable to grow on BOA-amended media. Deletion of other FDB1 cluster genes, in particular AMD1 and DLH1, did not affect BOA degradation. Phylogenetic analyses and topology testing of the FDB1 and FDB2 cluster genes suggested two horizontal transfer events among fungi, one being transfer of FDB1 from Fusarium to Colletotrichum, and the second being transfer of the FDB2 cluster from Fusarium to Aspergillus. Together, the results suggest that plant-derived xenobiotics have exerted evolutionary pressure on these fungi, leading to horizontal transfer of genes that enhance fitness or virulence.     

Lactobacillus salivarius strain FDB89 induced longevity in Caenorhabditis elegans by dietary restriction

In this study, we utilized the nematode Caenorhabditis elegans to assess potential life-expanding effect of Lactobacillus salivarius strain FDB89 (FDB89) isolated from feces of centenarians in Bama County (Guangxi, China). This study showed that feeding FDB89 extended the mean life span in C. elegans by up to 11.9% compared to that of control nematodes. The reduced reproductive capacities, pharyngeal pumping rate, growth, and increased superoxide dismutase (SOD) activity and XTT reduction capacity were also observed in FDB89 feeding worms. To probe the anti-aging mechanism further, we incorporated a food gradient feeding assay and assayed the life span of eat-2 mutant. The results demonstrated that the maximal life span of C. elegans fed on FDB89 was achieved at the concentration of 1.0 mg bacterial cells/plate, which was 10-fold greater than that of C. elegans fed on E. coli OP50 (0.1 mg bacterial cells/plate). However, feeding FDB89 could not further extend the life span of eat-2 mutant. These results indicated that FDB89 modulated the longevity of C. elegans in a dietary restriction-dependent manner and expanded the understanding of anti-aging effect of probiotics.     

从松叶蜂分离的球孢白僵菌及其致病性

从东北伊通红松林采集的自然染病死亡的帕克阿扁松叶蜂Acantholyda 虫尸上分离获得一株球孢白僵菌,定名为FDB01。用该菌株孢子悬浮液感染油松阿扁叶蜂Acantholyda posticalis和落叶松叶蜂Pristiphora erichsonii幼虫,研究该菌株的致病情况。结果表明,用浓度为2×108孢子/mL的孢子悬浮液处理16d后,该菌株对油松阿扁叶蜂和落叶松叶蜂的致死率分别达到了94.4%和100%,说明该菌株对松叶蜂具有很强的致病力。     

Dissociated flexor digitorum brevis myofiber culture system--a more mature muscle culture system

Considerable knowledge regarding skeletal muscle physiology and disease has been gleaned from cultured myoblastic cell lines or isolated primary myoblasts. Such muscle cultures can be induced to differentiate into multinucleated myotubes that become striated. However they in general do not fully mature and therefore do not model mature muscle. Contrastingly, fresh and cultured dissociated adult mouse flexor digitorum brevis (FDB) myofibers have been studied for many years. We aimed to investigate the possibility of using the FDB myofiber culture system for drug screening and thus long-term cultures of enzymatically dissociated FDB myofibers were established in 96-well plates. Ca2+ handling experiments were used to investigate the functional state of the myofibers. Imaging of intracellular Ca2+ during electric field stimulation revealed that calcium handling was maintained throughout the culture period of at least 8 days. Western blot and immunostaining analysis showed that the FDB cultures maintained expression of mature proteins throughout the culture period, including alpha-sarcoglycan, dystrophin, fast myosin heavy chain and skeletal muscle alpha-actin. The high levels of the fetal proteins cardiac alpha-actin and utrophin, seen in cultured C2C12 myotubes, were absent in the FDB cultures. The expression of developmentally mature proteins and the absence of fetal proteins, in addition to the maintenance of normal calcium handling, highlights the FDB culture system as a more mature and perhaps more relevant culture system for the study of adult skeletal muscle function. Moreover, it may be a useful system for screening therapeutic agents for the treatment of skeletal muscle disorders.     

Resistance of melanized feathers to bacterial degradation: is it really so black and white?

Melanins are common feather pigments that contribute to signaling and crypsis. Melanins may also help feathers resist feather‐degrading bacteria (FDB). Two recent studies (Goldstein et al. 2004, Grande et al. 2004) tested the resistance of melanized versus unmelanized feathers to FDB using in vitro experiments, but draw opposite conclusions. Goldstein et al. (2004) concluded that melanized feathers resist FDB more than unmelanized feathers, while Grande et al. (2004) concluded that unmelanized feathers resist FDB more than melanized feathers. To resolve this conflict in the literature, we replicated previous studies but included additional tests not previously used. We inoculated melanized and unmelanized feathers of domestic geese Anser anser domesticus, with the FDB Bacillus licheniformis and measured bacterial activity every two days over two weeks. Three metrics of bacterial activity on feathers were measured: soluble protein content around feathers in solution, bacterial growth on feathers, and loss of feather mass. The latter two metrics were not considered in the aforementioned studies, which indirectly measured bacterial activity. We conducted two trials, one in which feathers were sterilized by autoclaving before inoculation (Goldstein et al. 2004, Grande et al. 2004), and a second in which feathers were sterilized by ethylene oxide gas. This allowed us to test whether autoclaving, done in previous studies, influences bacterial activity on feathers and could confound results. In both trials, unmelanized feathers degraded earlier, supported greater bacterial growth, and lost more mass than melanized feathers. These results support the findings of Goldstein et al. (2004); melanized feathers are more resistant to FDB than unmelanized feathers. Thus, using direct metrics of bacterial activity, we resolve a current conflict in the literature. We also found that autoclaving feathers influences FDB activity on them, and thus autoclaving should be avoided in future studies.     

Identification of intermediate and branch metabolites resulting from biotransformation of 2-benzoxazolinone by Fusarium verticillioides

Detoxification of the maize (Zea mays) antimicrobial compound 2-benzoxazolinone by the fungal endophyte Fusarium verticillioides involves two genetic loci, FDB1 and FDB2, and results in the formation of N-(2-hydroxyphenyl)malonamic acid. Intermediate and branch metabolites were previously suggested to be part of the biotransformation pathway. Evidence is presented here in support of 2-aminophenol as the intermediate metabolite and 2-acetamidophenol as the branch metabolite, which was previously designated as BOA-X. Overall, 2-benzoxazolinone metabolism involves hydrolysis (FDB1) to produce 2-aminophenol, which is then modified (FDB2) by addition of a malonyl group to produce N-(2-hydroxyphenyl)malonamic acid. If the modification is prevented due to genetic mutation (fbd2), then 2-acetamidophenol may accumulate as a result of addition of an acetyl group to 2-aminophenol. This study resolves the overall chemistry of the 2-benzoxazolinone detoxification pathway, and we hypothesize that biotransformation of the related antimicrobial 6-methoxy-2-benzoxazolinone to produce N-(2-hydroxy-4-methoxyphenyl)malonamic acid also occurs via the same enzymatic modifications. Detoxification of these antimicrobials by F. verticillioides apparently is not a major virulence factor but may enhance the ecological fitness of the fungus during colonization of maize stubble and field debris.     

Digit flexor muscles in the cat: their action and motor units

The relation between muscle action and the mechanical properties of motor units has been explored in the main digit flexors of the cat hind limb: plantaris (PL); flexor digitorum brevis (FDB); flexor hallucis longus (FHL); and, flexor digitorum longus (FDL). General observations on muscle action revealed that PL is an ankle extensor as well as a digit flexor. PL and FHL were shown to be the major force contributors to digit flexion with FDL playing a lesser but still significant role. The mechanical properties of PL, FHL and FDB motor units were studied by noting twitch and tetanic tensions produced by electrical stimulation of single alpha axons, functionally isolated from the ventral root filaments. These data were compared to similar data reported by Olson and Swett (1966) for flexor digitorum longus (FDL). Our sample (114 PL, 60 FDB and 124 FHL units) disclosed that PL, FDB and FHL have units of uniformly fast contraction times (means 22, 27 and 27 msec respectively). PL units developed the most tetanic tension (3 to 160, mean 62 gm-wt) followed by FHL (2 to 87, mean 31 gm-wt) with FDB units producing very little tension (1 to 20, mean 6 gm-wt). Swett and Olson's FDL sample (108 units) showed tensions ranging from 0.3 to 100 gm-wt (mean 10 gm-wt). A division of labor among the four muscles is proposed. The large PL units are advantageous for forceful phasic inputs to the digits during the locomotion and in keeping with PL's additional role as an ankle exstensor. The low output forces of FDB units are optimal for discrete input to the digits during subtle adjustments of posture. We propose that the larger fast contracting units of FHL are used primarily for forceful digit flexions required in locomotion and for phasic protrusion of the claws while the predominately small and slow contracting units of FDL are used for sustained claw protrusion.     

New insights into the epigenetics of osteoporosis

Osteoporosis is characterized by reduced bone formation and accumulation of adipocytes in the bone marrow compartment. The decrease in bone mass results from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The deficiency of bone cells to replace the resorpted bone can be due to a preferential differentiation of bone marrow stromal cells into adipocytes at the expense of osteoblasts. Consequently, the processes that control the differentiation of osteoclasts, osteoblasts and adipocytes play a crucial role in bone metabolism. It is known that epigenetic mechanisms are critical regulator of the differentiation programs for cell fate and moreover are subject to changes during aging. Here, we summarize recent findings on the role of epigenetics in the modulation of mechanisms that may be associated with osteoporosis. In particular, we focus on disturbances in the bone remodeling process described in human studies that address the epigenetic regulation of the osteoblast/adipocyte balance.     

The significance of the heel process in anthropoids

The plantar process of the tuber calcanei among pongids and hominids serves as the origin for the superficial head of the flexor digitorum brevis muscle (FDB). In a survey of the soft tissues and osteology of the foot in a diverse number of anthropoid genera, it was found that a large superficial head of the FDB is associated with a large inflated plantar process (heel process). A large FDB with a separate calcaneal origin allows toe flexion independent of foot position. This type of movement allows an animal to reach and grasp or hang by its feet during slow deliberate climbing. The presence of á heel process in anthropoids and in nonprimate mammals is correlated with those animals which are slow climbers or are likely descended from slow-climbing forms. The presence of a heel process in humans and in pongids implies that the common pongid-hominid ancestor was most likely a slow climber. In fossil catarrhines the presence of a heel process can be interpreted either as evidence of slow-climbing behavior or as a heritage feature from a slow-climbing ancestor.     

Enzymatic synthesis and characterization of novel feruloylated lipids in selected organic media

A facile enzymatic synthesis approach to prepare novel feruloylated lipids through the lipase-catalyzed transesterification reaction of ethyl ferulate (EF) with tributyrin (TB) in toluene was investigated. The nuclear magnetic resonance (NMR) and electrospray ionization-mass spectroscopy (ESI-MS) analysis confirmed the formation of two major products, 1(3)-feruloyl-monobutyryl-glycerol (FMB) and 1(3)-feruloyl-dibutyryl-glycerol (FDB). The influences of different enzyme source, organic solvent, molar ratio, reaction temperature, agitation speed and water activity on the total conversion of reaction, distribution of FMB and FDB and selectivity of these two novel derivatives of FA were analyzed systematically. Under the optimal conditions, the highest conversion of feruloylated lipids achieved was 73.6%, which was composed of FMB 58.3% and FDB 13.1%, respectively. The enzyme can be reused for 14 runs without evident loss in activity and stability.     

Identification of Intermediate and Branch Metabolites Resulting from Biotransformation of 2-Benzoxazolinone by Fusarium verticillioides

Detoxification of the maize (Zea mays) antimicrobial compound 2-benzoxazolinone by the fungal endophyte Fusarium verticillioides involves two genetic loci, FDB1 and FDB2, and results in the formation of N-(2-hydroxyphenyl)malonamic acid. Intermediate and branch metabolites were previously suggested to be part of the biotransformation pathway. Evidence is presented here in support of 2-aminophenol as the intermediate metabolite and 2-acetamidophenol as the branch metabolite, which was previously designated as BOA-X. Overall, 2-benzoxazolinone metabolism involves hydrolysis (FDB1) to produce 2-aminophenol, which is then modified (FDB2) by addition of a malonyl group to produce N-(2-hydroxyphenyl)malonamic acid. If the modification is prevented due to genetic mutation (fbd2), then 2-acetamidophenol may accumulate as a result of addition of an acetyl group to 2-aminophenol. This study resolves the overall chemistry of the 2-benzoxazolinone detoxification pathway, and we hypothesize that biotransformation of the related antimicrobial 6-methoxy-2-benzoxazolinone to produce N-(2-hydroxy-4-methoxyphenyl)malonamic acid also occurs via the same enzymatic modifications. Detoxification of these antimicrobials by F. verticillioides apparently is not a major virulence factor but may enhance the ecological fitness of the fungus during colonization of maize stubble and field debris.     

A fast-twitch oxidative-glycolytic muscle with a robust inward calcium current

This report describes the histochemical and physiological properties of a rat skeletal muscle with a robust activity-dependent slow inward Ca2+ current. The muscle, the flexor digitorum brevis (FDB), is a small plantar flexor from the hindfoot. It is a homogeneous muscle consisting of approximately 90% fast-twitch oxidative-glycolytic (type IIA) fibers. Stimulation of the FDB with repetitive stimulus trains (30 or 50 Hz for 330 ms, 1 train/s for 2-5 min) produced a slow increase in the base-line or resting tension of the muscle between trains. This progressive increase in resting tension appears to be due to the activation of voltage-dependent slow Ca2+ channels, since it could be eliminated (i) by stimulating the muscle in a medium containing 2 mM EGTA and without Ca2+, and (ii) by the addition of either Co2+ or verapamil. The presence of a slow current may be associated with an increase in K+ efflux as stimulation continues, and with a prolongation of relaxation time. We also propose that the slow Ca2+ current may contribute to the allosteric activation of phosphorylase kinase during muscle activity. The FDB provides an excellent preparation to investigate the regulation of muscle metabolism by intra- and extra-cellular Ca2+ during exercise.     

Myokine Irisin promotes osteogenesis by activating BMP/SMAD signaling via αV integrin and regulates bone mass in mice

Irisin is well-known to contribute to bone homeostasis due to its bidirectional regulation on osteogenesis and osteoclastogenesis. However, the mechanisms of irisin involved in mesenchymal stem/stromal cells (MSCs)-derived osteogenesis are still under investigated. Fibronectin type III domain-containing protein 5 (FNDC5) is the precursor protein of irisin, compare with wild type (WT) littermates, FNDC5^-/- mice lost bone mass significantly, collectively evidenced by the decrease of bone mineral density (BMD), impaired bone formation and reduced N-terminal propertied of type I procollagen (P1NP) in sera. Meanwhile, the bone resorbing of FNDC5^-/- mice has enhanced accompanied by increased tartrate phosphatase (TRAP) staining cells morphologically and cross-Linked C-telopeptide of type 1 collagen (CTX) level in sera. In vitro study showed that lack of irisin impeded the MSC-derived osteogenesis of FNDC5^-/- mice. The addition of irisin promote the osteogenesis of WT and irisin-deficient MSCs, by activating αV integrin-induced ERK/STAT pathway, subsequently enhancing bone morphogenetic protein 2 (BMP2) expression and BMP/SMAD signaling activation. Taken together, these findings further indicate that irisin regulates bone homeostasis. Moreover, irisin promotes MSC-derived osteogenesis by binding to αV integrin and activating BMP/SMAD signaling consequently. Thus, irisin may be a promising therapeutic target for osteoporosis and bone defects.