Virtual tissue alignment and cutting plane definition--a new method to obtain optimal longitudinal histological sections

Abstract

Histomorphometric evaluation of the buccal aspects of periodontal tissues in rodents requires reproducible alignment of maxillae and highly precise sections containing central sections of buccal roots; this is a cumbersome and technically sensitive process due to the small specimen size. The aim of the present report is to describe and analyze a method to transfer virtual sections of micro-computer tomographic (CT)-generated image stacks to the microtome for undecalcified histological processing and to describe the anatomy of the periodontium in rat molars. A total of 84 undecalcified sections of all buccal roots of seven untreated rats was analyzed. The accuracy of section coordinate transfer from virtual micro-CT slice to the histological slice, right-left side differences and the measurement error for linear and angular measurements on micro-CT and on histological micrographs were calculated using the Bland-Altman method, interclass correlation coefficient and the method of moments estimator. Also, manual alignment of the micro-CT-scanned rat maxilla was compared with multiplanar computer-reconstructed alignment. The supra alveolar rat anatomy is rather similar to human anatomy, whereas the alveolar bone is of compact type and the keratinized gingival epithelium bends apical to join the junctional epithelium. The high methodological standardization presented herein ensures retrieval of histological slices with excellent display of anatomical microstructures, in a reproducible manner, minimizes random errors, and thereby may contribute to the reduction of number of animals needed.

Keywords: bone; histology; morphometric measurements; orthodontics; periodontal ligament; rat.

Figure 1

Consecutive histomicrographs every 1000 μm (red lines) of an undissected control rat. Some of the buccal roots were located between the cutting planes and thus do not appear on the sections. In most of the cases, it is not possible to evaluate both sides on the same section. MPR-aligned virtual slices located at lateral root prominences normally include the pulp chamber at the buccal bone level (M1c, M1d, M2m, M2d, M3m, M3d). At these sites dental parameters were evaluated (yellow lines).

Figure 2

A custom sample-holder was used to align the samples. The maxilla was placed on the molars and the mesial aspects of the first molars were in contact with the anterior ridge (arrows), thereby creating an approximately perpendicular cut to the occlusal plane and the MPS with a diamond band-saw.

Figure 3

SH-aligned micro-CT sections, the corresponding histomicrographs, and the relocated micro-CT sections from three specimens. The virtual cutting plane was defined to include the pulp chamber at the buccal bone level and most of the root canal length of the buccal root (A, D, G). After processing, the plane of the resulting histological section (B, E, H) was visually identified within the micro-CT data, representing the relocated micro-CT section (C, F, I) by comparing hard-tissue appearance and choosing the most resembling slice (i.e. B–C or E–F or H–I). Differences in cutting plane level between the intended and relocated SH-aligned micro-CT slice, herein, were 36 μm (A–C), −204 μm (D–F) and −312 μm (G–I). Gray values in micro-CT sections (resolution: 12 μm) are displayed in a NIH color look-up table to enhance contrast.

Figure 4

MPR-aligned virtual micro-CT section (A) and the corresponding histomicrograph of the obtained section (B). The same measurements made on micro-CT and histomicrographs were compared to evaluate methodological errors and differences between the methods regarding tooth position and inclination. (C) 3D display of the interactive geometrical construction by the software DreiDEdit Archimedes Geo 3D.

Figure 5

Bland–Altman comparison between the MPR-aligned virtual micro-CT section level and the obtained histological section level, evaluating the accuracy of virtual section coordinate transfer. The obtained histological section level tended to be located closer (on average, 148 µm) to the RP than the MPR-aligned virtual micro-CT section level.

Figure 6

Bland–Altman comparison between right and left side tooth position (RC–MPS) measured on the MPR-aligned virtual micro-CT section and on the histomicrographs.

Figure 7

The histomicrograph shows the buccal aspect of the coronal periodontium of a rat molar. The periodontal ligament (1) and the supracrestal gingival fiber-network [dentoperiosteal fibers (2), apical (3a), horizontal (3b) and coronal (3c) dentogingival fibers] are clearly visible in dark blue color (basic fuchsin, toluidin blue and dinatriumtetraborat staining).

Figure 8

In rats most of the maxillary bone is compact. Some minor portions of cancellous bone are present in central aspects of the maxilla. Areas of remodeling are found on vestibular and coronal aspects of the buccal bone plate, the oral aspect of the palatal shelf, at the apex of roots (see also Fig. 1).