Development of larval and transformed teeth in Ambystoma mexicanum (Urodela, Amphibia): an ultrastructural study

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.     

Comparison of tryptophan-labeled constituents of developing rodent molar enamel matrix, non-enamel occlusal cusp, Hertwig's epithelial root sheath, and presumptive root furcation regions: light microscopic autoradiography

During the process of organogenesis involving the developing rodent molar and incisor tooth organs, novel gene products termed enamel proteins are expressed by ectodermally-derived enamel organ epithelia at precise times and positions within the course of morphogenesis. The present studies were designed to identify the relative distribution of tryptophan-labeled, non-collagenous, epithelial-derived proteins associated with rat maxillary first molar crown (M') and initial root formation. Our experimental strategy was to utilize semi-quantitative autoradiography methods to compare and contrast the distribution of silver grains resulting from tryptophan incorporation into developing postnatal pups associated with enamel matrix, non-enamel occlusal cusp, Hertwig's Epithelial Root Sheath (HERS), and presumptive root furcation regions of M'. Five-day-old Wistar rats were injected with 14C-labeled tryptophan. Four animals were sacrificed at 15 minutes and then at 1, 2, 4, and 24 hour intervals following the administration of this essential aromatic amino acid. Following fixation and subsequent processing for autoradiography, semiquantitative analyses were performed of the silver grain distribution localized within selected regions of the developing M' tooth organs. All enamel organ epithelia were found to incorporate tryptophan and silver grains were identified (above background) in the extracellular matrices (ECM) of the enamel matrix, non-enamel occlusal cusp adjacent to the inner enamel epithelia, and the ECM (2-4, micron) adjacent to presumptive root furcation and HERS regions. Tryptophan incorporation was not significant in the odontoblasts or dentine ECM of the crown or forming presumptive root regions. These results support the hypothesis that inner enamel epithelia associated with rat molar crown formation, as well as HERS, synthesize tryptophan-labeled, non-collagenous, ECM molecules. We speculate that HERS participates in root development by possibly producing non-collagenous proteins for intermediate cementum formation.     

Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig.

During tooth formation nearly all of the protein matrix of enamel is removed before final mineralization. To study this process, enamel proteins and proteinases were extracted from pig enamel at different stages of tooth development. In the enamel maturation zones, the major enamel matrix proteins, the amelogenins, were rapidly processed and removed. Possibly associated with this process in vivo are two groups of proteinases which were identified in the enamel extracts by enzymography using amelogenin-substrate and gelatin-substrate polyacrylamide gels and by the degradation in vitro of guanidinium chloride-extracted amelogenins. One group of proteinases with gelatinolytic activity consisted of several neutral metalloendoproteinases having Mr values from 62,000 to 130,000. These proteinases were inactive against amelogenins, casein and albumin, and were present in approximately equal proportions in enamel at all developmental stages. In the other group, two serine proteinases, with apparent non-reduced Mr of 31,000 and 36,000 exhibited amelogeninolytic activity. The substrate preference of the enamel serine proteinases was indicated by their limited degradation of casein and their inability to degrade gelatin and albumin. Contrasting with the distribution of the metalloendoproteinase enzymes, the serine proteinases were found only in the enamel scrapings taken from late-maturing enamel. The amelogenin degradation patterns in vivo, observed in the enamel scrapings, were similar to those produced in assays in vitro using partially purified fractions of enamel proteinases and amelogenin substrate. Together, these data strongly indicate an important role for the serine proteinases, and possibly the gelatinolytic proteinases, in the organized processing of the enamel protein matrix during enamel formation.     

Amelogenesis in vitro: a model for studies of epithelial postsecretory processing during tissue-specific extracellular matrix biomineralization

The extracellular matrix (ECM) of developing mammalian enamel comprises a complex of unusual epithelial-derived proteins, which appear to function in concert to initiate and propagate tissue-specific biomineralization. Following enamel protein synthesis by ameloblast cells within the enamel organ, the subsequent steps of posttranslational modification, secretion, postsecretory processing and eventual removal of these proteins from forming enamel are largely unknown. To address this issue we have designed studies to investigate the hypothesis that enamel proteins are removed from enamel and translocated into the vasculature as relatively high-molecular-weight components. We examined enamel proteins recovered from serumless medium during prolonged organ culture of mouse capstage mandibular first molars. By 21 days in vitro the tooth crown formed and dentine and enamel biomineralization were apparent. At 31 days, explants retained metabolic activity and the enamel matrix showed extensive transformation. Immunologically identified enamel proteins of 26-18 k Da were produced by cultured tooth organs, translocated from tooth explants to the culture medium, recovered from the medium and then compared to control enamel protein from in vivo preparations. Comparable postsecretory processing of the 26-k Da amelogenin protein was observed in vitro and in vivo. We speculate that the pathway reported in the present studies is comparable to the processing of the enamel protein polypeptides of the maturing enamel which occurs in vivo. The in vitro organ culture model described in this report provides an approach with which to investigate the molecular events associated with epithelial-derived postsecretory processing of ECM molecules associated with tissue-specific biomineralization.     

Learning from Synthetic Models of Extracellular Matrix; Differential Binding of Wild Type and Amyloidogenic Human Apolipoprotein A-I to Hydrogels Formed from Molecules Having Charges Similar to Those Found in Natural GAGs

Among other components of the extracellular matrix (ECM), glycoproteins and glycosaminoglycans (GAGs) have been strongly associated to the retention or misfolding of different proteins inducing the formation of deposits in amyloid diseases. The composition of these molecules is highly diverse and a key issue seems to be the equilibrium between physiological and pathological events. In order to have a model in which the composition of the matrix could be finely controlled, we designed and synthesized crosslinked hydrophilic polymers, the so-called hydrogels varying the amounts of negative charges and hydroxyl groups that are prevalent in GAGs. We checked and compared by fluorescence techniques the binding of human apolipoprotein A-I and a natural mutant involved in amyloidosis to the hydrogel scaffolds. Our results indicate that both proteins are highly retained as long as the negative charge increases, and in addition it was shown that the mutant is more retained than the Wt, indicating that the retention of specific proteins in the ECM could be part of the pathogenicity. These results show the importance of the use of these polymers as a model to get deep insight into the studies of proteins within macromolecules.     

Developmental stages in permanent porcine enamel

Developing permanent teeth of different ages were obtained from Danish Landrace pigs. Visibly distinct zones on their enamel surfaces were shown to correspond to changes in chemical composition previously reported for other species. The time of appearance, rate of progress and duration of each stage was determined for each tooth type.     

Ameloblastic secretion and calcification of the enamel layer in shark teeth

Tooth primordia at early stages of mineralization in the sharks Negaprion brevirostris and Triaenodon obesus were examined electron microscopically for evidence of ameloblastic secretion and its relation to calcification of the enamel (enameloid) layer. Ameloblasts are polarized with most of the mitochondria and all of the Golgi dictyosomes localized in the infranuclear end of the cell toward the squamous outer cells of the enamel organ. Endoplasmic reticular membranes and ribosomes are also abundant in this region. Ameloblastic vesicles bud from the Golgi membranes and evidently move through perinuclear and supranuclear zones to accumulate at the apical end of the cell. The vesicles secrete their contents through the apical cell membrane in merocrine fashion and appear to contribute precursor material both for the basal lamina and the enameline matrix. The enamel layer consists of four zones: a juxta-laminar zone containing newly polymerized mineralizing fibrils (tubules); a pre-enamel zone of assembly of matrix constituents; palisadal zones of mineralizing fibrils (tubules); and interpalisadal zones containing granular amorphous matrix, fine unit fibrils, and giant cross-banded fibers with a periodicity of 17.9 nm. It seems probable that amorphous, non-mineralizing fibrillar and mineralizing fibrillar constituents of the matrix are all products of ameloblastic secretion. Odontoblastic processes are tightly embedded in the matrix of the palisadal zones and do not appear to be secretory at the stages investigated. The shark tooth enamel layer is considered homologous with that of other vertebrates with respect to origin of its mineralizing fibrils from the innerental epithelium. The term enameloid is appropriate to connote the histological distinction that the enamel layer contains odontoblastic processes but should not signify that shark tooth enamel is a modified type of dentine. How amelogenins and/or enamelins secreted by amelo- blasts in the shark and other vertebrates are related to nucleation and growth of enamel crystallites is still not known.     

Variation in modern human premolar enamel formation times: implications for Neandertals

A recent study demonstrated that variation in enamel cap crown formation in the anterior teeth is greater than that in the molars from two geographically distinct populations: native indigenous southern Africans and northern Europeans. Eighty southern African and 69 northern European premolars (P3 and P4) were analyzed in the present study. Cuspal, lateral, and total enamel formation times were assessed. Although cuspal enamel formation times were not consistently different between the two populations, both lateral and total enamel formation times generally were. Bonferroni-corrected t-tests showed that southern Africans had significantly shorter lateral enamel formation time for five of the six cusps, as well as significantly shorter total enamel formation time for these same cusps. An analysis of covariance performed on the lingual cusps of the upper third and fourth premolars showed that differences in enamel formation times between these populations remained when crown height was statistically controlled. A further goal of this study was to ascertain, based on perikymata counts, what Neandertal periodicities would have to be in order for their teeth to have lateral enamel formation times equivalent to either southern Africans or northern Europeans. To this end, perikymata were counted on 32 Neandertal premolars, and the counts were inserted into regression formulae relating perikymata counts to periodicity for each population and each tooth type. Neandertal enamel formation times could be equivalent to those of southern Africans or northern Europeans only if their hypothetical periodicities fall within the range of periodicities for African apes and modern humans (i.e., 6-12 days). The analysis revealed that both populations could encompass Neandertal timings, with hypothetical periodicities based on the southern African population necessitating a lower range of periodicity (6-8 days) than those based on the northern European population (8-11 days).     

硫酸糖胺聚糖(Sulfated Glycosaminoglycans)在花背蟾蜍眼早期形态发生中的合成

本文用放射自显影追踪注射入胚胎的~(35)S-硫酸盐的方法,研究了花背蟾蜍早期形态发生时眼的各部分组织和细胞外基质中的硫酸糖胺聚糖(Sulfated Glycosaminoglycans简称:硫酸GAG)的合成,并分析了其在眼形态发生中的作用。结果表明:1.在眼早期形态发生时,合成的硫酸GAG主要是硫酸软骨素。2.眼各部分组织中在即将分化时硫酸GAG合成率增高,分化开始后逐渐下降到原基形成时的水平。3.在晶状体被诱导时,在视杯和晶状体相互贴近的组织及两者间的细胞外基质中硫酸GAG的合成率明显增加,提示这是晶状体诱导的重要因素。4.角膜上皮形成时即向角膜上皮下层和细胞外基质分泌硫酸GAG;角膜上皮透明时,合成更多的硫酸角质素。     

Anchorage of collagen-tailed acetylcholinesterase to the extracellular matrix is mediated by heparan sulfate proteoglycans

Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (AChE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and approximately 80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina.     

Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure–function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress–strain curves \((p<0.05)\). However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries \((p<0.05)\). These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.     

Erythrocytes express chondroitin sulphate/dermatan sulphate, which undergoes quantitative changes during diabetes and mediate erythrocyte adhesion to extracellular matrix components

Glycosaminoglycans (GAGs) such as chondroitin sulphate/dermatan sulphate (CS/DS) are complex molecules that are widely expressed on the cell membrane and extracellular matrix (ECM). They play an important role in wide range of biological activities especially during pathological conditions. Diabetes, a metabolic disorder characterized by sustained hyperglycemia, is known to affect GAGs in different tissues and affect erythrocyte adhesion. The present investigation was aimed at exploring the nature of GAGs present in erythrocytes and its role on adhesion of erythrocytes from control and diabetic rats to major extracellular matrix components. GAGs isolated from erythrocytes were demonstrated to be CS/DS and a 2-fold increase was observed in erythrocytes from diabetic rats. Disaccharide composition analysis by HPLC after depolymerization by the enzyme, chondroitinase ABC showed the presence of 4-O sulphated disaccharide units with small amounts of non-sulphated disaccharides, in both control and diabetic erythrocytes. Erythrocytes from diabetic rats, however, showed significantly increased binding to poly-l-ornithine (P-orn), type IV collagen, laminin and fibronectin, which was abrogated on treatment with chondroitinase ABC to various degrees. This study sheds new light on CS/DS in erythrocytes and its likely biological implications in vivo.     

Matrix metalloproteinase inhibition impairs the processing, formation and mineralization of dental tissues during mouse molar development

Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.     

Purification and characterization of heparan sulfate from human primary osteoblasts

Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts—soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non‐identical by a number of parameters, and that these differences have significant ramifications for their ligand‐binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS‐dependent factors between the ECM and the cell surface. J. Cell. Biochem. 108: 1132–1142, 2009. © 2009 Wiley‐Liss, Inc.     

Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.     

Glycosaminoglycan composition changes with MG-63 osteosarcoma osteogenesis in vitro and induces human mesenchymal stem cell aggregation

Osteogenic differentiation is coordinated by the exposure of cells to temporal changes in a combination of growth factors and elements within the extracellular matrix (ECM). Many of the key proteins that drive these changes share the property of being dependent on ECM glycosaminoglycans (GAGs) for their activity. Here, we examined whether GAGs isolated from proliferating, differentiating and mineralizing MG-63 osteosarcoma cells differed in their physical properties, and thus in their capacities to coordinate the osteogenic cascade both in human MG-63 osteosarcoma cells and primary human mesenchymal stem cells (hMSCs). Our results show that the size distribution of GAGs, the expression of GAG-carrying proteoglycan cores and the expression of enzymes involved in their modification systematically change as MG-63 cells mature in culture. When dosed back onto cells exogenously in soluble form, GAGs regulated MG-63 survival and growth in a dose-dependent manner, but not differentiation in either cell type. In contrast, hMSCs aggregated into distinct colonies when grown on GAG-coated substrates, while MG-63 cells did not. Heparin-coated substrates improved hMSC viability without inducing aggregation. These results suggest a complex role for GAGs in coordinating the emergence of the osteoblast phenotype, and provide further evidence for the use of heparans in bone tissue repair applications.     

Osteoblast-released Matrix Vesicles,Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans

Our aging population has to deal with the increasing threat of age-related diseases that impair bone healing. One promising therapeutic approach involves the coating of implants with modified glycosaminoglycans (GAGs) that mimic the native bone environment and actively facilitate skeletogenesis. In previous studies, we reported that coatings containing GAGs, such as hyaluronic acid (HA) and its synthetically sulfated derivative (sHA1) as well as the naturally low-sulfated GAG chondroitin sulfate (CS1), reduce the activity of bone-resorbing osteoclasts, but they also induce functions of the bone-forming cells, the osteoblasts. However, it remained open whether GAGs influence the osteoblasts alone or whether they also directly affect the formation, composition, activity, and distribution of osteoblast-released matrix vesicles (MV), which are supposed to be the active machinery for bone formation. Here, we studied the molecular effects of sHA1, HA, and CS1 on MV activity and on the distribution of marker proteins. Furthermore, we used comparative proteomic methods to study the relative protein compositions of isolated MVs and MV-releasing osteoblasts. The MV proteome is much more strongly regulated by GAGs than the cellular proteome. GAGs, especially sHA1, were found to severely impact vesicle-extracellular matrix interaction and matrix vesicle activity, leading to stronger extracellular matrix formation and mineralization. This study shows that the regulation of MV activity is one important mode of action of GAGs and provides information on underlying molecular mechanisms.Skeletogenesis is a complex process that involves differentiation and proliferation, but the most important step is the mineralization of the extracellular matrix (ECM)¹ to form bone to physically support body functions (1). Our aging population is facing an increase in age-related diseases (e.g. diabetes and osteoporosis) that impair bone healing and require situation-adapted solutions for bone grafts and implants (2). One promising approach is the use of glycosaminoglycans (GAGs) to modify biomaterials (3). GAGs are the major organic components of ECM and play an important regulatory role in the development and remodeling of bone tissue. GAGs are polysaccharides consisting of alternating monosaccharide residues. Their sequence varies with respect to saccharide composition, glycosidic bond, and modification of the disaccharide unit, e.g. the degree of sulfation (3). GAGs modulate water and extracellular cation homeostasis. Moreover, they interact with and modulate the function of proteins like cytokines, adhesion molecules, and enzymes and thereby regulate processes such as migration, adhesion, differentiation, and proliferation of bone cells (2, 4–13). Thus, because human bone marrow stromal cells (hBMSC) sense their microenvironment, especially the chemical composition of the ECM (14), GAGs also promote the differentiation of bone-forming osteoblasts from hBMSC, as different studies have shown for sulfated GAGs (15, 16). Additionally, GAGs are potent molecules to promote bone anabolic activities (2).Osteoblasts synthesize the majority of extracellular matrix components and control the mineralization of the organic ECM by secreting regulatory proteins such as osteocalcin, bone sialoprotein II, and osteoadherin and modulate the local concentration of phosphate ions by tissue nonspecific alkaline phosphatase. With ongoing differentiation, osteoblasts release matrix vesicles (MV) (17). MVs are extracellular membrane-limited structures with a diameter of 100–400 nm (18, 19). According their size and biogenesis, they are grouped into the category of ectosomes (20). Mineralizing osteoblasts as well as hypertrophic chondrocytes were shown to have high levels of Ca²⁺ ions in their mitochondria and inorganic phosphate (P_i) in their cytoplasm prior to mineralization. Ca²⁺ ions are released by mitochondria and in combination with P_i, amorphous calcium phosphate is formed at sites of MV formation (1). MVs are released from apical microvilli into the ECM by pinching off or budding (18, 19). They continue to accumulate Ca²⁺ ions and P_i, which promotes the formation of hydroxyapatite in their lumen (21). In the second phase of mineralization, MVs release hydroxyapatite crystals that propagate continuous mineral formation in the ECM (22). Furthermore, MVs possess proteins and lipids to execute essential functions for initiating mineral formation. This includes Ca²⁺/P_i ion homeostasis, mineral nucleation, ECM remodeling, degradation of mineralization inhibitors or the maintenance of membrane lipid composition, and the control of ECM interactions that are crucial for controlling mineral growth and localization (22–24).In previous studies we have reported that GAGs such as HA and its synthetically sulfated derivatives induce osteoblast functions, e.g. cell-matrix interaction, differentiation, mineralization, and endocytosis (25). However, it is not clear whether GAGs influence only the osteoblasts or also the formation, composition, activity, and adhesion to the ECM of secreted MVs. To delineate the molecular effects, the synthetically low-sulfated hyaluronic acid derivative (sHA1, degree of sulfation ∼1) was studied in terms of MV biogenesis, release, and composition, and the effects were compared with those caused by naturally equally low-sulfated chondroitin sulfate (CS1, degree of sulfation ∼1) as well as by non-sulfated HA. Furthermore, we isolated MVs from osteoblasts after cultivation with those GAGs and analyzed their respective protein composition in a quantitative manner using a global proteomic approach after stable isotope labeling by amino acids in cell culture (SILAC) labeling. To find out whether the alteration of the MV proteome is a reflection of the changes of the cellular proteome or whether the MV proteome is independently regulated, we compared the GAG-induced changes in both proteomes.     

Size-selective oviposition by parasitoids and the evolution of life-history timing in hosts: fixed preferences vs frequency-dependent host selection

The influence of size-selective oviposition behaviour by parasitoids on the evolution of life-history timing in their hosts was examined using an optimization model of a two-stage life history similar to a genetic algorithm. Host populations with varying durations of early-larval development were subjected to selection in scenarios where parasitoids had fixed preferences for oviposition on late-stage larvae, or those where parasitoid attack was dependent on the relative frequencies of the two life stages present in the population. Fixed preference for oviposition on late-stage larvae caused positive directional selection on the duration of early-larval development. Surviving individuals remained for as long as possible in the first stage of development in order to avoid parasitoid attack. Frequency-dependent parasitoid attack, in contrast, caused maintenance of variation in the duration of early-larval development. The influence of the fitness payoffs of different life stages on the plasticity of size-selective oviposition behaviour is discussed, as are possible implications of the model results for parasitoid-host population dynamics.     

Binding and detection of glycosaminoglycans immobilized on membranes treated with cationic detergents

Immobilization of molecules on surfaces is used for preparative, quantitative, and qualitative studies. Glycosaminoglycans (GAGs) are strongly hydrophilic and negatively charged molecules that do not bind well to either polystyrene surfaces or hydrophobic blotting membranes. Hydrophobic membranes were derivatized with cationic detergents to become hydrophilic and positively charged. The ability of the polyvinylidene fluoride and nitrocellulose membranes to retain GAGs increased up to 12.8 microg per spot in the dot blot assay when the membrane was treated with a cationic detergent. Immobilized GAGs were stained with alcian blue, and the staining intensity was quantitated by scanning and densitometry. The derivatized membranes were used for solid-phase extraction of GAGs in blood plasma, urine, or cerebrospinal fluid. The detection sensitivity was equal for different types of GAGs but there was a slight negative interference from fibrinogen in blood plasma. The immobilized GAGs could also be released from the membrane using a nonionic detergent at high ionic strength. Recovery of different proteoglycan populations, separated by electrophoresis and detected by reversible staining with toluidine blue, was 70-100%.     

Modelling cell migration strategies in the extracellular matrix

The extracellular matrix (ECM) is a highly organised structure with the capacity to direct cell migration through their tendency to follow matrix fibres, a process known as contact guidance. Amoeboid cell populations migrate in the ECM by making frequent shape changes and have minimal impact on its structure. Mesenchymal cells actively remodel the matrix to generate the space in which they can move. In this paper, these different types of movement are studied through simulation of a continuous transport model. It is shown that the process of contact guidance in a structured ECM can spatially organise cell populations. Furthermore, when combined with ECM remodelling, it can give rise to cellular pattern formation in the form of "cell-chains" or networks without additional environmental cues such as chemoattractants. These results are applied to a simple model for tumour invasion where it is shown that the interactions between invading cells and the ECM structure surrounding the tumour can have a profound impact on the pattern and rate of cell infiltration, including the formation of characteristic "fingering" patterns. The results are further discussed in the context of a variety of relevant processes during embryonic and adult stages.