Effect of gene transfection timing on TGF-ß1 expression in rabbit mandibular distraction gap.

Transforming growth factor-ß1 (TGF-ß1) is a member of the TGF-ß superfamily, and plays an important role in promoting various stages of intramembranous and endochondral bone formation. It is one of the major growth factors that influence new bone formation in the distraction gap during distraction osteogenesis (DO). The major problem of DO is the time required for the treatment. Reports show that gene therapy accelerates osteogenesis, which can significantly benefit patients with DO. However, the optimal timing of gene transfection has not yet been reported. In this study, we used the New Zealand rabbit mandibular DO model for transfecting recombinant plasmid pIRES-hVEGF165-hBMP2 during the latency, distraction, and consolidation periods of DO. The TGF-ß1 levels in the distraction gap were detected at different time-points by immunohistochemistry and analyzed semi-quantitatively with the CMIAS-2001A computerized image analyzer. The TGF-ß1 levels peaked after 7 days and decreased after 14 days of consolidation in each group. In contrast, the TGF-ß1 levels in the transfected distraction period group were significantly higher than those in the other groups. After 28 days of consolidation, TGF-ß1 levels decreased and there was no significant difference among the groups. These results indicated that the genes transfected in the distraction period up-regulated the expression of TGF-ß1 more than in the latency and consolidation periods, which promoted bone formation in the distraction gap through a series of biological effects. Thus, we obtained a remarkable effect on new bone formation, and showed that the distraction period is optimal for gene therapy.

Oral health disparities of children among Southeast Asian immigrant women in arranged transnational marriages in Taiwan.

This study assessed the oral health disparities and oral health care needs of children whose parents are Southeast Asian immigrant women in arranged transnational marriages. We used the baseline data of the Lay Health Advisor Approach to Promote Oral Health Program (LHA-POHP) to explore the disparities in oral health between immigrant and native children, and the factors associated with their oral health. A cross-sectional community-based study was conducted to collect data from mothers and their preschool children in Southern Taiwan in 2011. A total of 590 (440 natives, 150 immigrants) children aged 4-6 years and their mothers completed the questionnaire and oral examination. Multiple regression models were used to analyze the association between children's oral health and their related factors. The caries index was 6.05 in immigrant children and 3.88 in native children (p < 0.001). The caries prevalence of maxillary anterior teeth in the labial surfaces was higher among immigrants, ranging from 14.7 to 22%. The factor associated with children's caries index was maternal tooth brushing frequency (adjusted odds ratio [aOR] = 8.95, 95% confidence interval [CI] 1.95-41.05). When the mothers did not direct children to brush teeth after eating sweets, their children were more likely to have decayed teeth (aOR = 3.54, 95% CI 1.04-12.03). Children's filled teeth were related to their dental regular check-ups (aOR = 2.28, 95% CI 1.26-4.10). Disparities in oral health among immigrant and native children were observed. The findings suggest that culturally adequate oral health promotion intervention programs should be implemented for immigrants.

Abnormal cell wall structure caused by boron nutrient imbalance in orchards could affect psyllid feeding behaviour, resulting in epidemic variation of Asian citrus psyllid.

The control of Huanglongbing (HLB), one of the most destructive pests of citrus, relies heavily on the reduction of Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. An in-depth understanding of ACP feeding behaviours among citrus plants is urgent for comprehensive management of orchards. An investigation was conducted in 37 citrus orchards in HLB epidemic areas, sampling shoots in the area with aggregation feeding of ACP (ACPf) and shoots in a neighbouring area without ACP feeding (CK), to study the interaction between leaf chemical composition and ACP psyllid feeding behaviours. Results of FTIR showed a strong absorption peak intensity, mainly representing functional groups originating from cell wall components in the leaf with ACP feeding. As compared with the control, cell wall components, such as alkali-soluble pectin, water-soluble pectin, total soluble pectin, cellulose, and hemicellulose, of the cell wall of ACPf increased by 134.0%, 14.0%, 18.0%, 12.5%, and 20.35%, respectively. These results suggest that cell wall mechanical properties significantly decreased in the term of decreases in pectin performance and cellulose mechanical properties. In addition, there was a remarkably lower boron (B) content in leaves and cell wall components with ACP feeding. Further analysis indicated that leaf B content significantly affected leaf cell wall components. Taken together, we provide evidence to demonstrate that the regional distribution of nutrient imbalance in orchards could affect psyllid feeding behaviour by weakening the cell wall structure, resulting in epidemic variation in ACP. This could help us to understand the management of psyllid infections in orchards with unbalanced nutrition.

High-resolution human cervical spinal cord imaging at 7 T.

We present high-resolution anatomical imaging of the cervical spinal cord in healthy volunteers at the ultrahigh field of 7 T with a prototype four-channel radiofrequency coil array, in comparison with 3-T imaging of the same subjects. Signal-to-noise ratios at both field strengths were estimated using the rigorous Kellman method. Spinal cord cross-sectional area measurements were performed, including whole-cord measurements at both fields and gray matter segmentation at 7 T. The 7-T array coil showed reduced sagittal coverage, comparable axial coverage and the expected significantly higher signal-to-noise ratio compared with equivalent 3-T protocols. In the cervical spinal cord, the signal-to-noise ratio was found by the Kellman method to be higher by a factor of 3.5 with the 7-T coil than with standard 3-T coils. Cervical spine imaging in healthy volunteers at 7 T revealed not only detailed white/gray matter differentiation, but also structures not visualized at lower fields, such as denticulate ligaments, nerve roots and rostral-caudal blood vessels. Whole-cord cross-sectional area measurements showed good agreement at both field strengths. The measurable gray/white matter cross-sectional areas at 7 T were found to be comparable with reports from histology. These pilot data demonstrate the use of higher signal-to-noise ratios at the ultrahigh field of 7 T for significant improvement in anatomical resolution of the cervical spinal cord, allowing the visualization of structures not seen at lower field strength, particularly for axial imaging.

High-Performance Dental Composites Based on Hierarchical Reinforcements.

Use of high-performance fibers such as poly(p-phenylene-2,6-benzobisoxazole) (PBO) improves the mechanical properties of dental fiber-reinforced composites (FRCs). However, the surfaces of high-performance fibers are relatively inert, and the interface with the resin matrix is poor. This has become a limitation restricting the performance of PBO FRCs in dentistry. Nanomaterials were introduced onto PBO fibers to construct various hierarchical reinforcements to obtain a dental FRC with higher flexural performance and optimized interface bonding. Four hierarchical reinforcements were constructed: PBO-ZnO nanoparticles (NPs), PBO-ZnO nanowires (NWs), PBO-ZnO NPs-cage silsesquioxane (POSS), and PBO-ZnO NWs-POSS. Performance following this optimized method was evaluated at macroscale and microscale levels, including measurement of the interfacial properties and mechanical properties of FRCs. The physicochemical characteristics of PBO fibers before and after modification were measured to determine the interfacial bonding mechanisms and to verify the connection between the microinterface and macromechanical properties. The cytotoxicity of the preferred PBO FRC was evaluated using the CCK8 assay. In comparison to other designs, the interfacial shear strength (IFSS) of PBO-ZnO NWs-POSS was the highest (29.31 ± 2.40 MPa). The corresponding FRC had the highest flexural strength under a static load (925.0 ± 39.2 MPa), the flexural modulus (39.39 ± 1.41 GPa) was equivalent to that of human dentin, and in vitro cytotoxicity was acceptable. The interfacial bonding mechanisms of PBO-ZnO NWs-POSS resulted from mechanical interlocking, chemical bonds, hydrogen bonds, and van der Waals forces. In summary, the PBO-ZnO NWs-POSS hierarchical reinforcement was introduced in dental FRCs and showed remarkable enhancement of the IFSS and flexural properties. We verified that the PBO-ZnO NWs-POSS hierarchical reinforcement was successful. This PBO FRC may be applied in dentistry as a new option for endodontic posts. Our study provides an interface design strategy for developing high-performance FRCs reinforced with high-performance fibers for dental applications.

The synergistic effect of grain boundary and grain orientation on micro-mechanical properties of austenitic stainless steel.

Micro/nano-scale deformation behavior including hardness, elastic modulus, and pop-ins, was studied in a medical austenitic stainless steel followed by post-mortem EBSD characterization. Relatively higher hardness and modulus was observed near {101} and more pop-ins occurred in this orientation at high loading rate. The activation volume (v) obtained from nanoindentation had weak dependence on grain orientation and was ~10-20 b3, indicating that neither diffusional creep processes nor conventional dislocation segments passing through dislocation forests controls plastic deformation in our study. The plastic zone radius (c) and the distance of the indent from the grain boundary (d) were used to describe the effect of grain boundary on the pop-in effect. The ratio of c/d meets amplitude version of Gaussian peak function distribution for a given orientation, whose peak value remains nearly constant for all the orientations.

FAM83H and Autosomal Dominant Hypocalcified Amelogenesis Imperfecta.

Autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI; OMIM #130900) is a genetic disorder exhibiting severe hardness defects and reduced fracture toughness of dental enamel. While the condition is nonsyndromic, it can be associated with other craniofacial anomalies, such as malocclusions and delayed or failed tooth eruption. Truncation mutations in FAM83H (OMIM *611927) are hitherto the sole cause of ADHCAI. With human genetic studies, Fam83h knockout and mutation-knock-in mouse models indicated that FAM83H does not serve a critical physiologic function during enamel formation and suggested a neomorphic mutation mechanism causing ADHCAI. The function of FAM83H remains obscure. FAM83H has been shown to interact with various isoforms of casein kinase 1 (CK1) and keratins and to mediate organization of keratin cytoskeletons and desmosomes. By considering FAM83H a scaffold protein to anchor CK1s, further molecular characterization of the protein could gain insight into its functions. In this study, we characterized 9 kindreds with ADHCAI and identified 3 novel FAM83H truncation mutations: p.His437*, p.Gln459*, and p.Glu610*. Some affected individuals exhibited hypoplastic phenotypes, in addition to the characteristic hypocalcification enamel defects, which have never been well documented. Failed eruption of canines or second molars in affected persons was observed in 4 of the families. The p.Glu610* mutation was located in a gap area (amino acids 470 to 625) within the zone of previously reported pathogenic variants (amino acids 287 to 694). In vitro pull-down studies with overexpressed FAM83H proteins in HEK293 cells demonstrated an interaction between FAM83H and SEC16A, a protein component of the COP II complex at endoplasmic reticulum exit sites. The interaction was mediated by the middle part (amino acids 287 to 657) of mouse FAM83H protein. Results of this study significantly extended the phenotypic and genotypic spectrums of FAM83H-associated ADHCAI and suggested a role for FAM83H in endoplasmic reticulum-to-Golgi vesicle trafficking and protein secretion (dbGaP phs001491.v1.p1).

Study of the physicochemical properties and oral bioavailability of the solid dispersion of cantharidin with polyethylene glycol 4000.

Cantharidin (CA) is partially water-soluble. Solid dispersion of CA (CA-SD) in polyethylene glycol 4000 (PEG 4000) was carried out by a solvent-fusion method to increase its dissolution rate and oral bioavailability. The physicochemical properties of this solid dispersion (SD) were evaluated immediately after preparation by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM), and the oral in vivo bioavailability was studied. In in vitro experiments CA was analyzed by high-pressure liquid chromatography (HPLC) and by gas chromatography-mass spectrometry (GC-MS) in in vivo experiments. The solubility and dissolution rate of CA were improved by the SD technique. A comparison of the pharmacokinetics between CA-SD and free CA was performed in rats. The results showed that CA-SD had a higher bioavailability than free CA after oral dosing. By comparing the AUC(0-t) of CA and CA-SD, the relative bioavailability of CA-SD to free CA was 295.4%. From these observations it could be concluded that the CA-SD has a higher absorption than pure CA and this corresponds with the dissolution result in vitro.

The significance of phase reversion-induced nanograined/ultrafine-grained structure on the load-controlled deformation response and related mechanism in copper-bearing austenitic stainless steel.

The ingenious concept of phase reversion annealing involving cold deformation of parent austenite to strain-induced martensite, followed by annealing was used to obtain nano-grained/ultrafine-grained (NG/UFG) structure in a Cu-bearing biomedical austenitic stainless steel resulting in high strength-high ductility combination. Having employed the concept effectively, the primary objective of this study is to critically analyze the interplay between the load-controlled deformation response, strain-rate sensitivity and deformation mechanism of NG/UFG austenitic stainless steel via nanoscale deformation experiments and compare with its coarse-grained (CG) counterpart. The study demonstrated that the strain-rate sensitivity of NG/UFG was ~1.5 times that of the CG structure. Post-mortem electron microscopy of plastic zone surrounding the indents indicated that the active deformation mechanism was nanoscale twinning with typical characteristics of a network of intersecting twins in the NG/UFG structure, while strain-induced martensite transformation was the effective deformation mechanism for the CG structure. The fracture morphology was also different for the two steels, essentially ductile in nature, and was characterized by striations marking the line-up of voids in NG/UFG steel and microvoid coalescence in CG counterpart. The differences in deformation mechanisms between the NG/UFG and CG structure are attributed to the austenite stability - strain energy relationship. Furthermore, the presence of ~3 wt % Cu in austenitic stainless steel had somewhat moderate effect on strain-rate sensitivity and activation volume at similar level of grain size in its Cu-free counterpart. Specifically, in the NG/UFG structure, the nanoscale twin density was noticeably higher in Cu-bearing austenitic stainless steel as compared to Cu-free counterpart, as Cu is known to increase the stacking fault energy.

On the mechanical behavior of austenitic stainless steel with nano/ultrafine grains and comparison with micrometer austenitic grains counterpart and their biological functions.

Conventional coarse-grained (CG) biomedical austenitic stainless steel with grain size in the micrometer range was subjected to a novel phase reversion concept involving severe cold deformation, followed by annealing, when the cold deformed martensite reverts to austenite with grain size in the nanometer/ultrafine (NG/UFG) regime (~200-400 nm). The mechanical behavior of CG and NG/UFG steels was studied via load-controlled and displacement-controlled experiments using a nanoindentation technique with the aim to simulate micromotion. The plastic zone associated with the indentation-induced deformed region was characterized by post-mortem electron microscopy of the deformed region to elucidate the deformation mechanism. Nanoscale twinning was the deformation mechanism in steel with grain size in the NG/UFG regime, and contributed to the ductility of high strength steel. In contrast, strain-induced martensite contributed to the ductility of low strength CG steel with micrometer grain size. Interestingly, besides the differences in the mechanical behavior, the biological functions of the two steels were remarkably different. Higher cell attachment, proliferation and higher expression level of prominent proteins, fibronection, actin and vinculin were favored by a surface with grain size in the nanometer regime and was in striking contrast with the surface with micrometer grain size. This behavior is attributed to the differences in the fraction of grain boundaries that are high energy two-dimensional defects. The study advances our understanding of the mechanical behavior of biomaterials and their cellular functions.

microRNA-21 Contributes to Orthodontic Tooth Movement.

microRNAs could be mechanosensitive and emerge as critical posttranscriptional regulators in the bone-remodeling process. During orthodontic tooth movement (OTM), the application of mechanical force induces alveolar bone remodeling, but whether microRNAs respond to orthodontic force and contribute to OTM is unknown. microRNA-21 (miR-21) has been previously reported in vitro to mediate stretch-induced osteogenic differentiation of periodontal ligament stem cells and support osteoclast differentiation. In this study, the authors show that miR-21 responded to orthodontic force in periodontal tissue in a dose- and time-dependent manner and regulated the osteogenesis of human periodontal ligament stem cells following OTM. Using mmu-miR-21-deficient (miR-21-/-) mice, the authors discovered that mmu-miR-21 deficiency inhibited OTM and prevented force-induced maxillary bone loss. The authors found that miR-21-/- mice showed a normal skeletal phenotype in development and a similar alveolar bone formation rate to wild-type mice postnatally. During OTM, mmu-miR-21 regulated force-induced alveolar osteoblastogenesis in the tensile side, while no effects were detected in the compressive side. However, miR-21-/- mice showed inhibited alveolar osteoclastogenesis when compared with wild-type mice. During OTM, mmu-miR-21 deficiency blocked alveolar bone resorption in both the compressive and tensile sides. To dissect the mechanism by which miR-21 regulates alveolar bone remodeling, the authors screened the reported functional targets of miR-21 and found that periodontal expression of programmed cell death 4 ( Pdcd4) was inhibited following OTM. Furthermore, mmu-miR-21 deficiency removed the suppression of Pdcd4 at both the mRNA and protein levels in the periodontium, resulting in upregulation of the downstream effector C-fos. Further analysis of OTM under lipopolysaccharide-induced periodontal inflammation showed that mmu-miR-21 mediated lipopolysaccharide (LPS)-accelerated OTM and that mmu-miR-21 deficiency blocked lipopolysaccharide-induced maxillary bone loss. In summary, these findings reveal a previously unrecognized mechanism that a microRNA can modulate OTM and alveolar bone remodeling under both normal and inflammatory microenvironments in vivo.

The disordering effect of hyoscyamine drugs on phospholipid membranes.

The effect of hyoscyamine drugs on the fluidity of dipalmitoylphosphatidylcholine liposomes has been studied by differential scanning calorimetry (DSC), electron spin resonance spectroscopy (ESR), fluorescence polarization and freeze-fracture electron microscopic techniques. DSC results indicate that anisodamine, anisodine, atropine and scopolamine all increase the fluidity of dipalmitoylphosphatidylcholine liposomes but with different degrees of efficiency. The increasing of fluidity of dipalmitoylphosphatidylcholine liposomes by hyoscyamine drugs is in a dose-dependent way. Increase of the fluidity of phosphatidylcholine liposomes by anisodamine was also shown by the other three methods. The possible mechanism of hyoscyamine-membrane interaction is discussed.

Porcine amelogenin is expressed from the X and Y chromosomes.

Amelogenin is the major enamel matrix component in developing teeth. In eutherian mammals, amelogenin is expressed from the X chromosome only, or from both the X and Y chromosomes. Two classes of porcine amelogenin cDNA clones have been characterized, but the chromosomal localization of the gene(s) encoding them is unknown. To determine if there are sex-based differences in the expression of porcine amelogenin, we paired PCR primers for exons 1a, 1b, 7a, and 7b, and amplified enamel organ-derived cDNA separately from porcine males and females. The results show that exons 1a/2a and 7a are always together and can be amplified from both males (XY) and females (XX). Exons 1b/2b and 7b are also always paired, but can be amplified only from females. We conclude that porcine amelogenin is expressed from separate genes on the X and Y chromosomes, and not, as previously proposed, from a single gene with two promoters.

Treatments of oil-refinery and steel-mill wastewaters by mesocosm constructed wetland systems.

In this study, two types of industrial wastewater, oil-refining and steel-milling, were selected for investigating their feasibility of treatment by mesocosm constructed wetland systems. The secondly treated effluents from the wastewater treatment plants were directly discharged into the systems controlled at different flow rates. Three wetland mesocosms were installed in the two industries: mesocosms A and B were in the oil refinery, and mesocosm C was in the steel mill. The substratum media used in wetland systems were sand (mesocosm A) and gravel (mesocosms B and C), while the vegetation types selected were reeds (mesocosms A and B) and mixed species of reeds and cattails (mesocosm C). The flow regimes were controlled as free water surface (FWS) and subsurface flow (SSF) for the sand- and gravel-beds, respectively. According to the experimental results, we found that the system treating oil-refining wastewater performed better than that treating steel-milling wastewater learned by comparing the removal efficiencies of COD, total N and total P. In addition, it was found that for oil-refining wastewater treatments, the SSF wetland system (mesocosm B) performed better than FWS (mesocosm A) wetland system when comparing both of their removal of pollutants and growth of vegetation. Besides, the effluents from these two industrial wetland treatment systems might be reclaimed and reused for boiler water, cooling, cleaning and miscellaneous purposes in industries. Further treatments are required if the constructed wetland effluents are thought about being reused for processing in industries.

Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis.

The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.

Effects of cello-oligosaccharide on intestinal microbiota and epithelial barrier function of weanling pigs.

A total of 144 piglets (Duroc × Landrace × Yorkshire; average initial weight of 6.13 kg weaned at 21 ± 1 d age) were allotted to 4 treatments for 2 wk, each of which had 6 pens with 6 pigs per pen. After the feeding experiment, 6 pigs per treatment were slaughtered to investigate the effects of cello-oligosaccharide (COS) on intestinal microbiota and epithelial barrier function. The COS was added to the basal diet at 0, 1.5, 3.0, and 4.5 g/kg diet at the expense of corn, respectively. Plasma -lactate, diamine oxidase (DAO), and the Ussing chamber technique were used to determine the intestinal barrier function. 16S rRNA-based methods were used for intestinal microbiota analysis. The results showed that incremental levels of COS had no effect ( > 0.05) on growth performance. Incremental levels of COS increased lactobacilli in jejunal and colonic contents ( < 0.05); decreased in jejunal contents ( < 0.05) and and in colonic contents ( < 0.05); reduced plasma DAO (linear, = 0.013, and quadratic, = 0.037); increased jejunal mucosa DAO (linear, = 0.003, and quadratic, = 0.008); decreased fluorescein isothiocyanate dextran 4 kDa flux of jejunum and colon ( < 0.05); and increased transepithelial electrical resistance (TER) in colon ( < 0.05), claudin-1 protein expression in jejunal mucosa (linear, = 0.001, and quadratic, = 0.003), and protein expressions of claudin-1 and zonula occludens-1 (ZO-1) in colonic mucosa linearly ( = 0.001 and = 0.001, respectively) and quadratically ( = 0.001 and = 0.002, respectively). The results indicated that the improved microbial ecosystem in the presence of COS might contribute to improvement in intestinal barrier function and tight junction proteins. Results also showed that the appropriate dietary COS supplementation level was 3.0 g/kg in weaned pig diets under our trial conditions.

Gene expression, signal transduction and tissue-specific biomineralization during mammalian tooth development.

Tooth development provides a paradigm for intrinsic molecular controls for cell- and extracellular matrix (ECM)-mediated biomineralization. The intent of this review is to evaluate the sequential timing and positional information prerequisite for tissue-specific biomineralization. Recent investigations suggest that 1,25-dihydroxyvitamin D3 functions to up-regulate VDR (vitamin D receptor) that in turn could induce structural gene products, including calcium-binding proteins and several ECM proteins (e.g., enamelins, amelogenins, dentine sialoglycoproteins (DSP) and dentine phosphoproteins (DPP)), resulting in dentine and enamel formation. Inhibition of regulatory gene products and/or their receptors likely results in hypoplastic and/or hypomineralized ECM as a direct consequence of down-regulated (1) transcription and/or translation of structural and regulatory genes, (2) posttranslational modifications, (3) and/or decreased calcium transport to the forming dentine and enamel matrices. Advances in serumless in vitro culture methodology; computer-assisted access to nucleic acid sequences for probes to define when, where, and how many specific regulatory and structural gene products are expressed; antisense oligodeoxynucleotides to inhibit specific translation; and microtechniques to analyze biomineralization all provide additional avenues to investigate tissue-specific biomineralization.

Peripheral nerve regeneration using silicone rubber chambers filled with collagen, laminin and fibronectin.

A 10 mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with an extracellular gel containing collagen, laminin and fibronectin. Empty silicone rubber tubes were used as controls. Six weeks after implantation, all extracellular elements were completely degraded and absorbed, and 90% of the animals from the extracellular gel group exhibited regeneration across the nerve gaps, whereas only 60% in the control group. Both qualitative and quantitative histology of the regenerated nerves revealed a more mature ultrastructural organization with 28% larger cross-sectional area and 28% higher number of myelinated axons in the extracellular gel group than the controls. These results showed that the gel mixture of collagen, laminin and fibronectin could offer a suitable growth medium for the regeneration of axons.

Fungal mycelia as the source of chitin and polysaccharides and their applications as skin substitutes.

A wovenable skin substitute (Sacchachitin) made from the residue of the fruiting body of Ganoderma tsugae was developed in this study. Chemical analysis revealed that the treated residue was a copolymer of beta-1,3-glucan (ca 60%) and N-acetylglucosamine (ca 40%) with a filamental structure of mycelia form, as demonstrated by both optical and scanning electron microscopy. The pulp-like white residue was then woven into thin, porous sheets 7.0 cm in diameter and 0.1-0.2 mm in thickness by filtration and lyophilized for use as a skin substitute. The wound area produced by dissecting rat skin of full thickness was found to almost completely heal on the side covered with Sacchachitin, whereas the control side covered with cotton gauge was around 6.0 cm2 on the 28th day. Furthermore, the wound healing effects of the chitin sheet from crab shell (Beschitin) and Sacchachitin were not found to be significantly different.

Development of fungal mycelia as skin substitutes: effects on wound healing and fibroblast.

In this study, Sacchachitin membrane, prepared from the residue of the fruiting body of Ganoderma tsugae, was estimated for its effects on wound healing and the proliferation and migration of fibroblast cells. Two mirror-image wounds were made on the back of female guinea pigs by dissecting a 1.5 x 1.5 cm2 skin surface of full thickness. Sacchachitin membrane was placed randomly on one of the wounds and gauze or Beschitin on the other. Changes in the wound area were measured and photographed after a predetermined amount of time postoperatively. Histological examination of the wound and surrounding tissue was also performed to reveal any interaction of tissue with the dressing. The results showed that the wound area covered with Sacchachitin membrane was statistically smaller than that covering with gauze on day 10, whereas there was no significant difference in the wound size compared to that with Beschitin. Fibroblast cells from the dermis layer of guinea pigs were used. The number of fibroblast cells were counted on the predetermined days in the culture suspended with or without 0.01% w/v dressing materials. By layering on DMEM plates, the number of fibroblast cells migrating across the center line or outside of the central hole were counted after five days. All the results indicated that both 0.01% w/v of Sacchachitin and chitin significantly enhanced the proliferation and migration of fibroblast cells.