Bioactive nanofibrous scaffolds for regenerative endodontics.

Here we report the synthesis, materials characterization, antimicrobial capacity, and cytocompatibility of novel antibiotic-containing scaffolds. Metronidazole (MET) or Ciprofloxacin/(CIP) was mixed with a polydioxanone (PDS)polymer solution at 5 and 25 wt% and processed into fibers. PDS fibers served as a control. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), tensile testing, and high-performance liquid chromatography (HPLC) were used to assess fiber morphology, chemical structure, mechanical properties, and drug release, respectively. Antimicrobial properties were evaluated against those of Porphyromonas gingivalis/Pg and Enterococcus faecalis/Ef. Cytotoxicity was assessed in human dental pulp stem cells (hDPSCs). Statistics were performed, and significance was set at the 5% level. SEM imaging revealed a submicron fiber diameter. FTIR confirmed antibiotic incorporation. The tensile values of hydrated 25 wt% CIP scaffold were significantly lower than those of all other groups. Analysis of HPLC data confirmed gradual, sustained drug release from the scaffolds over 48 hrs. CIP-containing scaffolds significantly (p < .00001) inhibited biofilm growth of both bacteria. Conversely, MET-containing scaffolds inhibited only Pg growth. Agar diffusion confirmed the antimicrobial properties against specific bacteria for the antibiotic-containing scaffolds. Only the 25 wt% CIP-containing scaffolds were cytotoxic. Collectively, this study suggests that polymer-based antibiotic-containing electrospun scaffolds could function as a biologically safe antimicrobial drug delivery system for regenerative endodontics.

The effect of medicaments used in endodontic regeneration on root fracture and microhardness of radicular dentine.

AIM: To investigate the effect of medicaments used in endodontic regeneration on root fracture resistance and microhardness of radicular dentine. METHODOLOGY: The root canals of mandibular premolars (n = 180) were instrumented and randomized into three treatment groups and an untreated control group. Each treatment group received either triple antibiotic paste (TAP), double antibiotic paste (DAP) or calcium hydroxide paste [Ca(OH)2] intracanal medicament. Teeth were kept in saline for 1 week, 1 month or 3 months. After each time-point, 15 teeth were randomly selected from each group and two root cylinders were obtained from each tooth. One cylinder was subjected to a fracture resistance test, and the other cylinder was used for a microhardness test. Two-way ANOVA and Tukey's pairwise comparisons were used for statistical analysis. RESULTS: For the microhardness test, the two-way interaction between group and time was significant (P < 0.001). The intracanal application of TAP and DAP caused significant and continuous decrease in root dentine microhardness after one (P < 0.05) and 3 months (P < 0.001), respectively. The three-month intracanal application of Ca(OH)2 significantly increased the microhardness of root dentine (P < 0.05). The time factor had a significant effect on fracture resistance (P < 0.001). The three intracanal medicaments caused significant decreases in fracture resistance ranging between 19% and 30% after 3-month application compared to 1-week application. CONCLUSION: In this laboratory study, the 3-month application of triple antibiotic paste, double antibiotic paste or calcium hydroxide paste medicaments significantly reduced the root fracture resistance of extracted teeth compared to a 1-week application.

A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors.

C57BL/6J-c(2J) (c2J) albino mice showed much less damage to their photoreceptors after exposure to prolonged light than BALB/c mice and seven other albino strains tested. There were no gender differences, and preliminary studies suggested that the c2J relative protective effect was a complex trait. A genome-wide scan using dinucleotide repeat markers was carried out for the analysis of 194 progeny of the backcross (c2J x BALB/c)F(1) x c2J and the thickness of the outer nuclear layer (ONL) of the retina was the quantitative trait reflecting retinal damage. Our results revealed a strong and highly significant quantitative trait locus (QTL) on mouse Chromosome (Chr) 3 that contributes almost 50% of the c2J protective effect, and three other very weak but significant QTLs on Chrs 9, 12, and 14. Interestingly, the Chrs 9 and 12 QTLs corresponded to relative susceptibility alleles in c2J (or relative protection alleles in BALB/c), the opposite of the relative protective effect of the QTLs on Chrs 3 and 14. We mapped the Rpe65 gene to the apex of the Chr 3 QTL (LOD score = 19.3). Northern analysis showed no difference in retinal expression of Rpe65 message between c2J and BALB/c mice. However, sequencing of the Rpe65 message revealed a single base change in codon 450, predicting a methionine in c2J and a leucine in BALB/c. When the retinas of aging BALB/c and c2J mice reared in normal cyclic light were compared, the BALB/c retinas showed a small but significant loss of photoreceptor cells, while the c2J retinas did not. Finding light damage-modifying genes in the mouse may open avenues of study for understanding age-related macular degeneration and other retinal degenerations, since light exposures may contribute to the course of these diseases.

Light-induced retinal damage in mice carrying a mutated SOD I gene.

Transgenic mice expressing mutated mouse Cu/Zn superoxide dismutase (SOD I), corresponding to a mutation associated with familial amyotrophic lateral sclerosis, develop a fatal motorneuron degeneration that resembles the human disease. The biochemical properties of some mutant SOD I enzymes indicate that a gain of catalytic functions, (such as increased peroxidase activity) may be the pathologic factor(s). However, at the present time there is little in vivo evidence that a mutation-induced change in the catalytic activity of SOD I is directly involved in neuronal cell death or that vulnerability to cell death is related to the level of functional/metabolic activity of cells carrying mutated SOD I. In pigmented mice carrying the G86R mutation of mouse SOD I, exposure to constant bright light for 20 days caused a diminution of electroretinographic activity and specific degeneration of photoreceptor cells, while no pathological effects were seen in transgenic littermates not exposed to bright light or in light exposed non-transgenic littermates. These findings are the first to indicate that one mechanism for neuronal cell death by mutated SOD I is use-dependent and/or related to metabolic activity, and therefore may be due to a gain in function of catalytic activities involving superoxide/hydrogen peroxide. The light-exposure pathology in this transgenic mouse model indicates an essential role for SOD I in the protection of photoreceptors from light-damage.

Cytogenesis in the monkey retina.

Time of cell origin in the retina of the rhesus monkey (Macaca mulatta) was studied by plotting the number of heavily radiolabeled nuclei in autoradiograms prepared from 2- to 6-month-old animals, each of which was exposed to a pulse of 3H-thymidine (3H-TdR) on a single embryonic (E) or postnatal (P) day. Cell birth in the monkey retina begins just after E27, and approximately 96% of cells are generated by E120. The remaining cells are produced during the last (approximately 45) prenatal days and into the first several weeks after birth. Cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in the foveal and perifoveal regions by E56. Despite extensive overlap, a class-specific sequence of cell birth was observed. Ganglion and horizontal cells, which are born first, have largely congruent periods of cell genesis with the peak between E38 and E43, and termination around E70. The first labeled cones were apparent by E33, and their highest density was achieved between E43 and E56, tapering to low values at E70, although some cones are generated in the far periphery as late as E110. Amacrine cells are next in the cell birth sequence and begin genesis at E43, reach a peak production between E56 and E85, and cease by E110. Bipolar cell birth begins at the same time as amacrines, but appears to be separate from them temporally since their production reaches a peak between E56 and E102, and persists beyond the day of birth. Müller cells and rod photoreceptors, which begin to be generated at E45, achieve a peak, and decrease in density at the same time as bipolar cells, but continue genesis at low density on the day of birth. Thus, bipolar, Müller, and rod cells have a similar time of origin. The maximal temporal separation of cell birth is between cones and amacrine cells so that cell generation exhibits two relatively distinct phases: the first phase gives rise to ganglion, horizontal, and cone cells, and the second phase to amacrine, bipolar, rod, and Müller cells. In addition, cells of the first phase are generated faster than the second phase cells, and there are differences in the topography of spread of labeled cells between the two phases. Each cell class displays a central-to-peripheral gradient in genesis, although the spatiotemporal characteristics of the gradients differ between the classes.(ABSTRACT TRUNCATED AT 400 WORDS)

[Effects of fixation and light conditions on distribution of interphotoreceptor matrix].

By using albino, adult rats, the effects of different light conditions during enucleation and fixation on the staining of the interphotoreceptor matrix (IPM) with colloidal iron were examined. When the eyes were enucleated in the dark or under a fluorescent lamp, followed by immersion-fixing in the dark, the IPM around the photoreceptor outer segments (OS) and apical inner segments (IS) was uniformly stained. When the eyes were enucleated in the light, and were immersion-fixed in the light, the staining pattern of the IPM was dependent on the light conditions during the fixation. By increasing the intensity and the exposure-time of the light, the intensity of the interstitial IPM-staining around the OS decreased except for the apical- and basal-regions, whereas that of the IPM staining around the IS increased from the basal- to the apical-region. When the rat was perfused with the fixative in the light, the diffuse IPM staining around the IS was especially remarkable. In cases in which it is only possible to apply immersion-fixation, it is necessary to remove the light effects during the fixation in order to obtain consistent results with IPM-histochemistry. For this purpose, it may be effective to cover the bottle for fixation with aluminum foil immediately after the enucleation.

[Classification of light-evoked distribution of interphotoreceptor matrix].

Distributional patterns of the interphotoreceptor matrix (IPM) under several different light conditions were examined in adult rats using colloidal iron staining. In the dark, the IPM distributed uniformly throughout photoreceptor outer segments including apical halves of inner segments. After exposure to light for 1 min, the colloidal iron-bound IPM concentrated in bands at apical and basal regions of outer segments and at apical inner segments. After exposure to light for 5-30 min, the IPM distributed throughout inner segments, in addition to the distribution at apical and basal outer segments. In excessive light, diffuse IPM staining was observed throughout the photoreceptor layer. These findings suggest that the light response of the IPM around outer segments precedes that around basal inner segments. It is also remarkable that the excessive light-induced distributional pattern of the IPM is similar to patterns in inherited retinal dystrophy and light-induced retinal degenerations.

[Lectin binding in the interphotoreceptor matrix in neuraminidase-induced retinal detachment].

Binding sites of fluorescence isocyanate-labeled lectins, peanut agglutinin (PNA) and Ricinus communis agglutinin-1 (RCA-1), were studied in the interphotoreceptor matrix (IPM) of adult rat retinas that were pretreated with an intravitreous injection of neuroaminidase. The localization of binding sites to the subretinal IPM differed significantly between the two lectins. PNA bound to the subretinal IPM components in a cord-like fashion that formed a bridge between the apical photoreceptor outer segments and the surface of the RPE. On the other hand, RCA-1 showed homogeneously prominent binding to the subretinal space IPM, the intensity of which appeared to inversely correlate with the extent of neuraminidase-induced retinal separation. The results suggest that the IPM may consist of mucin-type glycoconjugates as recognized by PNA and serum-type ones as bound by RCA-1, and that these IPM components may play different roles in the maintenance and organization of photoreceptor-RPE complex, e.g. the mucin-type for the retinal adhesion and the serum-type for the transport of metabolites.

The interphotoreceptor matrix in RCS rats: histochemical analysis and correlation with the rate of retinal degeneration.

We have previously demonstrated with histochemical digestions that a spatial heterogeneity exists in the interphotoreceptor matrix (IPM) of the normal rat retina. The apical outer segment zone adjacent to the pigment epithelium is mostly sialoglycoconjugates, and the basal outer segment zone appears to consist largely of chondroitin sulfates A and C, with sialogylcoconjugates in low concentration (Porrello and LaVail, in press). Although the RCS rat with inherited retinal dystrophy is missing the apical zone of IPM in the posterior retina, it has an abnormally high concentration of IPM in the basal zone and a concentrated apical zone in the far peripheral retina (LaVail, Pinto and Yasumura, 1981). We have attempted to characterize the IPM histochemically in these two regions in the RCS rat. Using the same digestive procedures which remove hyaluronic acid, chondroitin sulfates A and C, and sialoglycoconjugates, we find that the excessive basal IPM accumulation in the RCS retina appears to be composed in large part of chondroitin sulfates A and C, whereas the peripheral apical zone contains little or no chondroitin sulfates A and C. Furthermore, little or no sialoglycoconjugates are present in the basal zone, and the peripheral apical zone is composed predominantly of sialoglyconjugates. Thus, the IPM in the RCS rat possesses the same regional heterogeneity as that found in the normal rat IPM. Changes in IPM staining during the progression of retinal degeneration were examined in the eyes of both pink-eyed RCS and pigmented RCS-p+ rats. IPM staining was characterized by progressive condensation of the initially abundant basal stain into a thin band adjacent to the outer limiting membrane. The loss was concomitant with the reduction in photoreceptor inner-segment length and integrity, precisely matching the central to peripheral gradient and hemispheric differences in the rate of photoreceptor degeneration in these animals. This suggests that photoreceptor cells produce some or all of the chondroitin sulfates of the IPM. The peripheral apical staining was lost rapidly between days 30-40, which correlates closely with the loss of interstitial retinol-binding protein as described by others.

Survival of some photoreceptor cells in albino rats following long-term exposure to continuous light.

Fischer albino rats, seven weeks of age, were exposed to continuous light at 65 foot-candle incident illuminance for up to 264 days. Other Fischer rats, seven months of age, were exposed to continuous light at 140 foot-candle incident illuminance for up to 147 days. In all cases, a small percentage of the photoreceptors survived. The identification of the surviving cells as photoreceptors was made by light microscopy on the basis of nuclear heterochromatin pattern and staining and by electron microscopy by the presence of ribbon synapses and ciliary basal bodies with ciliary filaments. No outer segment membranes were observed. The percentage of cones progressively increased from the normal 1.5 per cent to about 60 per cent with increasing exposure time, indicating that cone cells are more resistant than rods to destruction by constant light.