Papain‐gel degrades intact nonmineralized type I collagen fibrils

Papain‐gel has been utilized as a chemo‐mechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papain‐gel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain‐gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM‐based nanoindentation. AFM images showed that the papain‐gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two‐fold drop from the values of normal dentin (E=20±1.9, H=0.8±0.08 GPa) was found after four applications (E=9.7±3.2, H=0.24±0.1 GPa) (P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain‐gel. SCANNING 31: 253–258, 2009. © 2010 Wiley Periodicals, Inc.     

Calcium hypochlorite as a dentin deproteinization agent: Microleakage,scanning electron microscopy and elemental analysis

Purpose: This study investigated the influence of collagen removal with calcium hypochlorite on the surface morphology of acid‐etched dentin and on the microleakage of composite restorations. In addition, the elemental composition (EC) of dentin after removal of the collagen fibrils was analyzed. Materials and Methods: Forty third molars received two cavities and were divided into four groups according to dentin treatment: CTRL—no pre‐treatment; Na10—10% NaOCl for 30 s; Ca10—10% CaOCl for 30 s, and Ca15—15% CaOCl for 30 s. The cavities were filled using an acetone‐based adhesive system and a resin composite; they were then subjected to thermal cycling for 5,000 cycles, immersed in methylene blue for 4 h and sectioned into 1‐mm thick slabs. Two examiners evaluated two slices per tooth using a stereomicroscope and assigned the degree of infiltration (scores 0–3). The data were analyzed using the Kruskal–Wallis (α = 0.05). Four teeth received surface treatment according to the groups and were submitted to SEM and EDS to carry at the EC. Results: There was no significant difference between the experimental groups (P = 0.533). CaOCl alters the morphology and surface composition of the dentin, resulting in an increase in the amount of calcium in the interface. Conclusions: When used prior to an acetone‐based adhesive system, CaOCl did not produce any differences in microleakage when compared to the CTRL group or to the Na10 group. Microsc. Res. Tech. 78:676–681, 2015. © 2015 Wiley Periodicals, Inc.     

The effect varied scanning electron microscopy desiccation techniques has on demineralized dentin

The study objective was to assess (a) the effect of a rubbing‐application of ethylenediaminetetraacetic acid (EDTA) or citric acid (CA) has on the ultrastructure of surface dentin and (b) the effect of two scanning electron microscopy (SEM) desiccation preparation techniques have on the collagen surface produced. Treatment regions on proximal root surfaces of extracted human teeth were root planned to expose dentin. Cotton pellets soaked in either 30% CA or 24% EDTA solution were rubbed on the treatment region then processed for SEM using one of two desiccation techniques, that is, (a) critically point dried from liquid CO₂ (control) or (b) air‐dried from tetramethylsilane (experimental). Specimens were coated with gold/palladium and viewed/photographed with an SEM. Specimens of the control groups displayed tufted fibrils (CA > EDTA) with many dentin tubules being partially obscured by overhanging fibrils. Air‐dried specimens of both treatment groups displayed a flat intact monolayer devoid of a matted meshwork of fibrous collagen. Discrete fibril “sprigs,” emanating from the surface monolayer, were characteristic of the EDTA group only. The rubbing‐application of EDTA on dentin produces a tufted fibril surface somewhat similar to that produced by CA. Air‐drying desiccation of both resulted in marked distortion with fibril collapse/coalescence of the tufted collagen matrix.     

Dentin bond strength and nanoleakage of the adhesive interface after intracoronal bleaching

This study evaluated dentin bond strength (BS) and nanoleakage of non‐ and pre‐etched dentin immediately (T₀,), 7 days (T₇), and 14 days (T₁₄) after bleaching. Bovine incisors (150) were selected and half of them submitted to intrapulpal dentin etching (e). Non‐ and pre‐etched dentin were subjected to the following (n = 15): no bleaching/control (C); 35% carbamide peroxide (CP); 35% hydrogen peroxide (35% HP); 25% hydrogen peroxide (25% HP); and sodium perborate (SP). Bleaching agents were applied to the pulp chamber four times within a 72‐h interval. Afterwards, pulp chamber dentin was prepared for the BS test at different evaluation times (n = 5): T₀, T₇, and T₁₄. Composite blocks were built on pulp chamber and sectioned in slices. Slices were reduced to an hour‐glass shape with a cross‐sectional area of 0.8 mm² and submitted to microtensile BS test. Two additional specimens for each group were prepared for nanoleakage evaluation by transmission electron microscopy (TEM). Results were analyzed by ANOVA (two‐way) and Dunnett's test (p < .05). BS decreased immediately after intracoronal bleaching for both sound and pre‐etched dentin (p < .05). At T₁₄, the BS of non‐etched bleached dentin increased for all groups, whereas the pre‐etched SPe group presented BS similar to the Ce. Nanoleakage within the hybrid layer was perceptible immediately after bleaching, although a decrease in nanoleakage was observed for all groups at T₁₄. Adhesive restorations should be performed 7–14 days after bleaching, according to the bleaching agent used. Intracoronal bleaching should be performed preferably with sodium perborate if previous dentin etching is applied.     

Caries removal with Er:YAG laser followed by dentin biomodification with carbodiimide and chitosan: Wettability and surface morphology analysis

This study aimed to investigate dentin wettability and surface morphology after selective removal of carious lesion by erbium‐doped yttrium aluminum garnet (Er:YAG) laser, followed by dentin biomodification with carbodiimide (EDC) and chitosan (CHI). Seventy‐eight bovine dentin specimens were submitted to caries induction. Specimens were distributed according to methods of carious removal (n = 39): bur at low‐speed (40,000 rpm) or Er:YAG laser (noncontact mode, 250 mJ/pulse and 4Hz). All specimens were etched with 35% phosphoric acid, and subdivided according to dentin biomodification (n = 13): Control (no biomodification), EDC or CHI. The contact angle (n = 10) between adhesive system (3M ESPE) and dentin surface was measured by a goniometer. Eighteen specimens (n = 3) were analyzed by scanning electron microscopy. Data were analyzed by two‐way ANOVA and Tukey's test (α = .05). The method used to remove carious lesion did not influence the wettability of dentinal surface (p = .748). The angles produced on the remaining dentin after biomodification were influenced (p = .007). CHI promoted higher contact angles (p = .007) and EDC did not differ from the control group (p = .586). In the bur‐treated group, most tubules were open, regardless of which biomodifier was used. Laser modified the organic matrix layer. CHI promoted partially closed tubules in some areas while EDC exposed dentinal tubules. Regardless of which method was used for selective removal of carious lesion, biomodification with EDC did not affect the dentin wettability, whereas CHI changed the wettability of remaining dentin. Both biomodifiers promoted a slight change on dentin morphology.     

Short‐term effect of cryotherapy on human scleral tissue by atomic force microscopy

This study investigated the inflammatory effect of cryotherapy application on collagen matrix network in human infant sclera. Donor scleral tissues taken from three infant patients divided into five groups: control group, sham‐treated group, and three cryotreated groups. In the cryotherapy groups, the sclera was treated for 5 s, 10 s, and 20 s with ?80°C freezing by a cryosurgical system. The cryotreated reactions were examined using double histological analysis with hematoxylin‐eosin and Masson's trichrome, and atomic force microscopy analysis to quantify the diameter and D‐banding of collagen fibrils. The infant scleral tissues treated with cryotherapy showed a significantly increased collagen density associated with inflammatory response (p < 0.05), increased fibril diameter (p < 0.005) compared to the scleral tissues in the control group. The results directly suggest that the cryotherapy affects the morphology of scleral collagen. SCANNING 35:302‐307, 2013. © 2012 Wiley Periodicals, Inc.     

Effects of sodium hypochlorite as dentin deproteinizing agent and aging media on bond strength of two conventional adhesives

This study evaluated the effect of 10% sodium hypochlorite (NaOCl) as deproteinizing agent and storage media on bond strength (BS) of two etch‐and‐rinse adhesive systems to dentin. Twenty‐eight sound extracted human third molars were divided in four groups (n = 7), according to dentin treatment (conventional etching or etching followed by 10% NaOCl application) and adhesive systems (GB—Gluma 2Bond and OS—One‐Step). After dentin treatments and adhesive application, a composite block was built‐up on dentin surface and teeth were serially sectioned to obtain bonded sticks specimens. The sticks were submitted to three aging conditions: (24H) 24 hr in water (immediate), (SH) 3 hr of NaOCl accelerated‐aging or (1Y) 1 year of water storage. Afterward, submitted to microtensile bond strength test (μTBS), failure modes and adhesive interfaces analyzes. Data were analyzed by two‐way analysis of variance (ANOVA) and Tukey's test (α = .05). Dentin deproteinization before bonding significantly reduced μTBS for GB‐treated group (p < .05), regardless the aging conditions. Water storage for 1 year (1Y) and NaOCl accelerated‐aging (SH) decreased μTBS for both adhesives. Yet, the groups stored in NaOCl (SH) exhibited the lowest BS results (p < .05). Bond strength of deproteinized dentin was dependent on the adhesive system composition and NaOCl accelerated‐aging promoted decreased bond strength and further degradation than water storage for 1 year.     

Long‐term chlorhexidine effect on bond strength to Er:YAG laser irradiated‐dentin

This study evaluates the bond strength of dentin prepared with Er:YAG laser or bur, after rewetting with chlorhexidine on long‐term artificial saliva storage and thermocycling. One hundred and twenty human third molars were sectioned in order to expose the dentin surface (n = 10). The specimens were randomly divided in 12 groups according to treatment and aging: Er:YAG laser rewetting with deionized water (LW) and 24 h storage in artificial saliva (WC); LW and 6 months of artificial saliva storage + 12.000 thermocycling (6M), LW and 12 months of artificial saliva storage + 24.000 thermocycling (12M), Er:YAG laser rewetting with 2% chlorhexidine (LC) and WC, LC and 6M, LC and 12M, bur on high‐speed turbine rewetting with deionized water (TW) and WC, TW6M, TW12M, bur on high‐speed turbine + 2% chlorhexidine (TC) and WC, TC and 6M, TC and12M. The specimens were etched with 35% phosphoric acid, washed, and dried with air. Single Bond 2 adhesive was applied and the samples were restored with a composite. Each tooth was sectioned in order to obtain 4 sticks, which were submitted to microtensile bond strength test (µTBS). The two‐way ANOVA, showed no significant differences for the interaction between the factors and for the aging factor. Tukey 5% showed that the LC group had the lowest µTBS. The rewetting with chlorhexidine negatively influenced the bond strength of the preparation with the Er:YAG laser. The artificial saliva aging and thermocycling did not interfere with dentin bond strength. Microsc. Res. Tech. 77:37–43, 2014. © 2013 Wiley Periodicals, Inc.     

The use of an Er:YAG laser to remove demineralized dentin and its influence on dentin permeability

The purpose of this study was to analyze, correlate, and compare the demineralization and permeability of dentin remaining after caries removal with either an Er:YAG laser, a bur, or a curette. Thirty human dentin fragments were immersed in a demineralizing solution for 20 days and were randomly divided into three groups (n = 10) for the removal of the demineralized lesion. The groups were G1—Er:YAG laser (200 mJ/6 Hz; noncontact at 12 mm; spot: 0.63 mm), G2—Bur, and G3—Curette. The specimens were then immersed in a 10% copper sulfate solution, then in a 1% dithiooxamide alcoholic solution for 30 min and kept in ammonia vapor for 7 days. Next, the specimens were examined with optical microscopy. The amount of demineralized dentin and the level of copper ion infiltration in the dentin were quantified in μm using Axion Vision software. Data were analyzed with the Kruskal‐Wallis test (p < 0.05) and Pearson's Correlation test. The analysis revealed no significant differences between the three caries removal methods in terms of their capacity to remove demineralized tissue (G1: 10.6 μm; G2: 8.4 μm; G3: 11 μm), although the laser removal generated more tissue permeability than the others methods (G1: 17.6 μm; G2: 6.6 μm; G3: 5.5 μm). The correlation between the remaining demineralized dentin and the dentin permeability was moderate for the conventional methods and higher for the Er:YAG laser. It can therefore be concluded that the laser produced an increase in permeability that was directly proportional to the amount of demineralized tissue removal. Microsc. Res. Tech. 76:225–230, 2013. © 2012 Wiley Periodicals, Inc.     

Investigation of nanostructural changes following acute injury using atomic force microscopy in rabbit vocal folds

There continues to be a paucity of data regarding the nanostructural changes of vocal fold (VF) collagen after injury. The aim of this study is to investigate the nanostructural and morphological changes in the rabbit VF lamina propria following acute injury using atomic force microscopy (AFM). Unilateral VF injury was performed on 9 New Zealand breeder rabbits. Sacrifice and laryngeal harvest were performed at three time points: 1 day, 3 days, and 7 days after injury. Histology and immunohistochemistry data were collected to confirm extracellular matrix (ECM) changes in rabbit VF. The progressive changes in thickness and D‐spacing of VF collagen fibrils were investigated over a 7‐day postinjury period using AFM. At post‐injury day 1, a fibrin clot and inflammatory cell infiltration were observed at the injured VF. The inflammatory score at postinjury day 1 was highest in injured VF tissue, with a significant decrease at postinjury day 7. The immunoreactivity of inflammatory proteins (COX‐2, TNF‐α) was observed in VF up to day 7 after injury. AFM investigation showed clustered and disorganized collagen fibrils at the nanoscale resolution at post‐injury day 7. Collagen fibrils in injured VF at postinjury day 7 were significantly thicker than control and postinjury days 1 and 3 (P < 0.001). D‐spacing of collagen at postinjury day 7 was not studied due to loss of distinct edges resulting from immature collagen deposition. AFM investigation of VF could add valuable information to understanding micromechanical changes in VF scar tissue. Microsc. Res. Tech. 78:569–576, 2015. © 2015 Wiley Periodicals, Inc.     

TiF4 gel effects on tubular occlusion of eroded/abraded human dentin

This in situ study evaluated the tubular occlusion caused by 4% TiF₄ gel on the surface of eroded/abraded dentin. Sixty human dentin samples were eroded in vitro and assigned into six groups (n = 10) according to the in situ surface treatment and number of cycling days: 4% TiF₄ gel applied once (TiF₄1), twice (TiF₄2), or three times (TiF₄3) followed by 2, 4, and 6 days of erosive/abrasive in situ cycling, respectively. Control groups (no treatment) were subjected to 2 (C1), 4 (C2), and 6 (C3) days of erosive/abrasive in situ cycling only. A seventh group (n = 10) was comprised by in vitro uneroded samples (UN), subjected to 6 days of in situ erosive/abrasive cycling. Each cycling day consisted on six erosive (0.5% citric acid, pH 2.6) and one abrasive events. Environmental scanning electron microscopy micrographs were taken. For all groups, blinded examiners assessed dentin tubules occlusion using visual scores (0—unoccluded, 1—partially occluded by granular deposits, 2—partially occluded by reduction in tubular lumen into diamond shape, 3—completely occluded) on images captured prior and after the in situ phase. Scheirer‐Ray‐Hare test demonstrated that treatments significantly affected tubule occlusion (p < .001). Dunn's test showed that tubule occlusion in TiF₄3 was significantly higher than in C1. Tubule occlusion in remaining groups did not differ from that observed in groups TiF₄3 and C1. Tubule occlusion was significantly higher after in situ phase. It may be suggested that TiF₄, when applied three times, was able to positively change tubule occlusion of dentin samples.     

Micro‐ and nanoscale structures of mesiodens dentin: Combined study of FTIR and SAXS/WAXS techniques

A mesiodens is the most common type of supernumerary tooth present in conjunction to normal dentition. A mesiodens may commonly occur in the central region of the upper or lower jaw. A mesiodens is different from normal teeth in terms of structure and shape. The aim of this study is to evaluate the micro‐ and nanoscale structural properties of mesiodens dentin by combined small‐ and wide‐angle X‐ray scattering (SAXS/WAXS) and Fourier transform infrared (FTIR) spectroscopy. Five freshly extracted, noncarious mesiodens and five normal dentin disks prepared from human incisor teeth were compared. Using FTIR, the phosphate‐to‐amide I, carbonate‐to‐phosphate, and carbonate‐to‐amide I band area ratios and the crystallinity index were quantified. SAXS/WAXS were used to study the nanostructure of mesiodens. An increase in the mineral content in the mesiodens dentin with respect to the normal group was found. Crystallinity was also significantly increased and the protein content decreased in the mesiodens dentin compared with that of normal dentin. SAXS/WAXS results revealed that mesiodens dentin has a more calcified tissue. Further, SAXS analysis revealed a nonuniform distribution of dentin fibrils in mesiodens. Microsc. Res. Tech., 78:52–58, 2015. © 2014 Wiley Periodicals, Inc.     

Shear bond strength and ultrastructural interface analysis of different adhesive systems to bleached dentin

Background: It remains unclear as to whether or not dental bleaching affects the bond strength of dentin/resin restoration. Purpose: To evaluated the bond strength of adhesive systems to dentin submitted to bleaching with 38% hydrogen peroxide (HP) activated by LED‐laser and to assess the adhesive/dentin interfaces by means of SEM. Study design: Sixty fragments of dentin (25 mm²) were included and divided into two groups: bleached and unbleached. HP was applied for 20 s and photoactivated for 45 s. Groups were subdivided according to the adhesive systems (n = 10): (1) two‐steps conventional system (Adper Single Bond), (2) two‐steps self‐etching system (Clearfil standard error (SE) Bond), and (3) one‐step self‐etching system (Prompt L‐Pop). The specimens received the Z250 resin and, after 24 h, were submitted to the bond strength test. Additional 30 dentin fragments (n = 5) received the same surface treatments and were prepared for SEM. Data were analyzed by ANOVA and Tukey's test (α = 0.05). Results: There was significant strength reduction in bleached group when compared to unbleached group (P < 0.05). Higher bond strength was observed for Prompt. Single Bond and Clearfil presented the smallest values when used in bleached dentin. SEM analysis of the unbleached specimens revealed long tags and uniform hybrid layer for all adhesives. In bleached dentin, Single Bond provided open tubules and with few tags, Clearfil determined the absence of tags and hybrid layer, and Prompt promoted a regular hybrid layer with some tags. Conclusions: Prompt promoted higher shear bond strength, regardless of the bleaching treatment and allowed the formation of a regular and fine hybrid layer with less deep tags, when compared to Single Bond and Clearfil. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

Micro‐shear bond strength of adhesives with different degrees of acidity: Effect on sound and artificially hypermineralized dentin

This study evaluates the bond strength of four self‐etching adhesive systems with different acidity levels in normal and artificially hypermineralized dentin substrate. Healthy human molars were divided into groups: normal dentin—N (n = 36) and artificially hypermineralized dentin—H (n = 36). Self‐etching adhesive systems Clearfil S³ Bond (n = 9), Optibond All in One (n = 9), Clearfil SE Bond (n = 9), and Adhese (n = 9) were used for both the N and H groups. Transparent cylindrical matrices were positioned on the treated dentin surfaces, filled with composite resin, and light‐cured for 40 s. After the transparent cylindrical matrices were removed, the specimens were stored for 24 hr in a humid environment at 37°C and were subjected to a micro‐shear bond strength test. For each group, a specimen was prepared and evaluated in scanning electron microscope for adhesive interface observation. Normality was confirmed and the two‐way analysis of variance and Games–Howell post‐tests were conducted (α = .05). The data demonstrated an interaction between the adhesive system and type of dentin substrate (p < .01). For normal dentin, all adhesive systems assessed were adequate; however, in the hypermineralized dentin, the Clearfil SE Bond two‐step self‐etching adhesive system with mild pH presented the highest immediate bond strength. There was a predominance of adhesive failures for all adhesive systems in the different dentin substrates evaluated. It was concluded that the self‐etching adhesive systems evaluated were efficient for both substrates, and for the hypermineralized dentin, the Clearfil SE Bond presented a higher bond strength value.     

Effects of etchant on the nanostructure of dentin: an atomic force microscope study

The objective of this study is to investigate effects of etchant on dentin surface as a function of etching time by atomic force microscopy (AFM). Twenty intact, freshly extracted noncarious human teeth were used to make 40 dentin discs and the discs were randomly divided into 4 groups. A commercial etchant was applied on these dentin discs. The main component of the etchant is 32% phosphoric acid and the etching time for the four dentin disc groups was 0, 20, 40, and 60 s. The AFM results show progressive changes of the surface morphology as the etching time increases. Significant difference of average roughness (R_a) exists in the dentin surface among all four groups (p<0.05). The statistic difference of diameters of dentinal tubule orifice (D_t) exists between the control group and all other groups (p<0.05), whereas the D_ts for the 40‐s group and 60‐s group are not statistically different (p>0.05). Our results showed that acid treatment has a significant influence on dentin demineralization and the effective etching time of the dentin surface appears to be 60 s. We provide a new nanoscale insight into the dentin surface treatment and this can help us to select the optimal etching time in clinic. SCANNING 31: 28–34, 2009. © 2009 Wiley Periodicals, Inc.     

Chemical and morphological changes in human dentin after Er:YAGlaser irradiation: EDS and SEM analysis

Sixty samples of human dentin were divided into six groups (n = 10) and were irradiated with Er:YAG laser at 100 mJ–19.9 J/cm², 150 mJ–29.8 J/cm², 100 mJ–35.3 J/cm², 150 mJ–53.0 J/cm², 200 mJ–70.7 J/cm², and 250 mJ–88.5 J/cm², respectively, at 7 Hz under a water spray. The atomic percentages of carbon, oxygen, magnesium, calcium, and phosphorus and the Ca‐to‐P molar ratio on the dentin were determined by energy dispersive X‐ray spectroscopy. The morphological changes were observed using scanning electron microscopy. A paired t‐test was used in statistical analysis before and after irradiation, and a one‐way ANOVA was performed (P ≤ 0.05). The atomic percent of C tended to decrease in all of the groups after irradiation with statistically significant differences, O and Mg increased with significant differences in all of the groups, and the Ca‐to‐P molar ratio increased in groups IV, V, and VI, with statistically significant differences between groups II and VI. All the irradiated samples showed morphological changes. Major changes in the chemical composition of dentin were observed in trace elements. A significant increase in the Ca‐to‐P ratio was observed in the higher energy density groups. Morphological changes included loss of smear layer with exposed dentinal tubules. The changes produced by the different energy densities employed could have clinical implications, additional studies are required to clarify them. Microsc. Res. Tech. 78:1019–1025, 2015. © 2015 Wiley Periodicals, Inc.     

Erosion effects on chemical composition and morphology of dental materials and root dentin

Purpose: This work aims to study the erosion on restorative materials and on surrounding dentin. Fifty root dentin samples were obtained from bovine incisors. Methods: Twenty samples were not restored and thirty received cavity preparations. Samples were assigned to five groups: G1, G2: sound dentin (D); G3: composite resin (CR); G4: resin‐modified glass‐ionomer cement (RMGIC); G5: glass‐ionomer cement (GIC). The samples of groups 2–5 were submitted to six cycles (demineralization–remineralization). Samples were analyzed by micro energy‐dispersive X‐ray fluorescence spectrometry (μ‐EDXRF) and by scanning electron microscopy (SEM). Results: Mineral loss was greater in G2 samples than in RMGI > CR > GIC > D (control). SEM images showed pronounced dentin demineralization in groups 2 and 4. The acid erosion has a significant effect on mineral loss (Ca and P) of root dentin without restoration. Conclusions: Composite resin had the best chemical resistance to erosion among all the materials. Fluoride contained in GIC seemed to cause some protection, however, with material degradation. Chemical interaction of tooth‐colored dental materials with root dentin could be assessed by μ‐EDXRF. Microsc. Res. Tech. 75:703–710, 2012. © 2011 Wiley Periodicals, Inc.     

Assessment of collagen fibril spacing in relation to selected region of interest (ROI) on electron micrographs--application to the mammalian corneal stroma

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center‐to‐center spacing) were measured within different ROIs of the same nominal area (0.25 μm²) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / μm² and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.