K. A. Grötz

Treatment of injuries to the inferior alveolar nerve after endodontic procedures

Abstract Overextension of filling material into the mandibular canal after root treatment in the lower jaw is a rare but serious complication. Mechanical compression, chemical neurotoxicity and local infection may cause irreversible nerve damage. A report on 11 patients with neurological complaints of the inferior alveolar nerve after endodontic treatment is summarised. The neurological findings are dominated by hypaesthesia and dysaesthesia. Half of the patients reported pain. Hyperaesthesia is found much more rarely. Nearly all the patients had a combination of one or more symptoms. Initial X-rays showed root filling material in the area of the mandibular canal. Nine cases were treated with apicectomy and decompression of the nerve; in two cases, extraction of the tooth was necessary. Only one patient reported persistent pain after surgery. If neurological complaints appear after root filling in the lower jaw, a nerve injury due to root filling material should be ruled out. In cases of overfilling, immediate apicectomy and decompression of the nerve with conservation of the tooth is often the treatment of choice; the tooth may be preserved and the best chance of avoiding permanent nerve damage is provided.

Using ultrasound transmission velocity to analyse the mechanical properties of teeth after in vitro, in situ, and in vivo irradiation.

Abstractu2002Ultrasound transmission velocity (UTV) in isotropic material as a measure for the modulus of elasticity was correlated to mechanical properties. Changes in micromechanical properties of radiated teeth and influence of the oral cavity were to be evaluated nondestructively. UTV was measured in extracted teeth after 36 Gy and 62 Gy of in situ (enorally, with no contact to the oral cavity) and in vitro irradiation. Relative to controls, teeth subjected to 62 Gy in vivo showed higher UTV values for dentine and enamel. Sound teeth irradiated with 60 Gy in situ also showed higher UTV values for enamel, whereas dentine values were not significantly different from those of control. The mechanical properties of teeth irradiated in vitro were affected only after high experimental doses of up to 500 Gy. The difference between in vivo and in vitro mechanical properties may be due to radioxerostomia-induced damages as well as the status of dentine vitality. This supports the concept of direct radiation-induced damage in synergy with radioxerostomia-induced caries.

Histotomography of the odontoblast processes at the dentine–enamel junction of permanent healthy human teeth in the confocal laser scanning microscope

Abstract The translucency of teeth allows the non-destructive subsurface visualisation of their microstructure by confocal laser scanning microscopy (CLSM) at a level of about 150 μm below the surface. The dentine–enamel junction (DEJ) is accessible only directly adjacent to the cervix of the tooth. Therefore teeth have to be sectioned for studying marginal areas of the dental hard tissue. The potential of the technique for (pseudo) three-dimensional visualisation allows the study of an array of individual confocal images, the interpretation of which is similar to that of macroscopic tomographs (CT-scan, MRI). Additionally, the extended focus mode yields the overlay of individual confocal images in the form of a two-dimensional projection. This mode of operation proved to be particularly suited for the visualisation of odontoblast processes in their whole extension. The three-dimensional junction between enamel and dentine, the branches of the odontoblast processes and their interactions with the DEJ is demonstrable by CLSM without staining or other procedures of sample preparation. The direct microscopic comparison between samples, either fresh or kept in a humid chamber, and Technovit-embedded sample blocks gives evidence that the risk of artefacts by sample storage or by the embedding procedure is minimal. The tomographs limited to subsurface areas of the tissue also exclude mechanical surface artefacts due to grinding or cutting.

Histotomographische Untersuchungen zur Frage direkt radiogener Schmelzveränderungen

Das umfangreiche Schrifttum zur Strahlenkaries zeigt den kontroversen Forschungsstand. Eigene Untersuchungen haben direkt radiogene Veränderungen an der Schmelz-Dentin-Grenze histotomographisch nachweisen können (Atrophie der Odontoblastenfortsätze). Offen ist die Frage nach direkten Strahlenfolgen im Schmelz. Gesunde Zähne (Gr. 1, n = 10) wurden mit Proben nach hochdosierter In-vitro- (500–2500 Gy, Gr. 2, n = 20) und nach experimentell-enoraler In-situ-Bestrahlung (60 Gy, Gr. 3, n = 20) sowie mit Zähnen von Patienten nach präoperativer Strahlentherapie (36 Gy, Gr. 4, n = 20) und strahlenkariösen Zähnen nach tumortherapeutischer Strahlendosis (60 Gy, Gr. 5, n = 20) verglichen. Im direkten Parallelgruppenvergleich erfolgte eine standardisierte In-vitro-Demineralisierung (Milchsäuregel pH 5,0) an den vestibulären Schmelzoberflächen. Jede Probe wurde prospektiv histologisch durch konfokale Laser-Scanning-Mikroskopie (CLSM) nach 90, 180 und 270 min Entmineralisierung untersucht. Die Auswertung erfolgte mikromorphometrisch (Breite des Demineralisationssaums) und mikromorphologisch. Die Breite des Demineralisationssaums unterschied sich sowohl bei beiden in vivo bestrahlten Probengruppen (Gr. 4: p < 0,01; Gr. 5: p < 0,001) als auch bei den hochdosiert in vitro bestrahlten Zähnen (p < 0,01) signifikant von den Befunden der gesunden Kontrollen. Das histologische Bild der Demineralisation an den Zahnoberflächen von Radiotherapiepatienten zeigte dabei einen völligen Verlust der Prismenstruktur (homogen amorphe Substanz). Direkte Strahlenfolgen an der Schmelz-Dentin-Grenze wurden in früheren Untersuchungen dargestellt. Zusätzlich konnten jetzt signifikante mikromorphometrische Unterschiede im Demineralisierungsverhalten des bestrahlten Schmelzes gezeigt werden. Offensichtlich ist der Schmelz nach der Bestrahlung für einen Säureangriff weniger resistent. The volume of literature on radiation-induced caries is still inconsistent as regards the state of the art. Our histotomographic studies have shown direct radiogenic effects on the dentoenamel junction (atrophy of the odontoblast processes). Whether there are direct effects of radiation on dental enamel is not yet known. Sound teeth (group 1, n = 10) were compared either with enamel specimens after high dose in vitro radiation (500– 2500 Gy; group 2; n = 10) and after experimental enoral in situ irradiation (60 Gy; group 3; n = 20), or with teeth from patients after cancer radiotherapy (36 Gy; group 4; n = 20) and with teeth which had obviously decayed due to cancer radiotherapy (60 Gy; group 5; n = 20). Vestibular enamel surfaces of the teeth were exposed to identical in vitro demineralizations (lactic acid gel; pH = 5.0). Each of the samples was prospectively studied histologically by confocal laser scanning microscopy (CLSM) after 90, 180 and 270 min of acid interaction. Data were interpreted micromorphometrically (width of the demineralized area) and micromorphologically. The width of the demineralized area varies between the two in vivo irradiated groups of specimens (group 4: P < 0.01; group 5: P < 0.001) and the high dose in vitro irradiated teeth (P < 0.01) and is significantly different from the sound controls. The histological image of the demineralization in subsurface areas in the teeth of radiotherapy patients is characterized by a total loss of the prismatic structure (homogeneously, amorphous substance). Direct radiogenic effects at the dentoenamel junction have been described earlier. The additional information offered by this study is that there are significant micromorphometric differences in the demineralizing behaviour of irradiated enamel. Obviously, enamel is less resistant to acid attack after irradiation.

Prognose und Prognosefaktoren enossaler Implantate im bestrahlten Kiefer

Zusammenfassung Einleitung: Die kaufunktionelle Wiederherstellung nach Kopf-Hals-Bestrahlung ist gegenüber anderen Tumorpatienten durch Strahlenkaries, Radioxerostomie und Osteoradionekroserisiko zusätzlich limitiert. Es wurde der Frage der Implantatprognose und potentieller Prognosefaktoren im bestrahlten Kiefer nachgegangen. Material und Methode: Die retrospektive Studie erfaßte von 1988–1997 197 Implantate (47 Patienten). Die Implantatprognose wurde durch Überlebenszeitanalyse (Kaplan-Meier) bestimmt, wobei implantatunabhängige Verluste zensiert wurden. Außerdem erfolgte eine Gruppenbildung nach potentiell die Prognose beeinflussenden Parametern. Grupppenunterschiede in den Überlebensraten wurden mit dem Log-Rank-Test auf Signifikanz geprüft. Ergebnisse: Von 197 Implantaten gingen 12 (6,1%) durch Periimplantitis und 8 (4,1%) durch mögliche biomechanische Überlastung verloren. Zensiert wurden 52 Verluste (26,4%) wegen Versterbens und 2 (1,0%) wegen Kieferresektion. 111 Implantate (56,3%) verblieben im Recall; die gemittelte Nachkontrollphase betrug 33 Monate. Die Überlebensrate (Kaplan-Meier-Analyse) betrug nach 1 und 2 Jahren 95%, nach 3 und 4 Jahren 92% und nach 5 und 6 Jahren 72%. Die univariate Analyse der Gruppenvergleiche zeigte eine lokal signifikant niedrigere Verlustrate nach periimplantärer Weichteilplastik (p = 0,036). Bestrahlungsdosis (p = 0,16), Chemotherapie (p = 0,9) und periimplantäre Osteoplastik (p = 0,84) beeinflußten die Prognose nicht. Obwohl kein vor der Bestrahlung gesetztes Implantat explantiert wurde, war der Unterschied bei sehr inhomogenen Gruppenstärken (prä radiationem: n = 29; post radiationem: n = 156) nicht signifikant (p = 0,13). Schlußfolgerungen: Im Literaturvergleich ist die Überlebensrate prae radiationem gesunder Zähne (1 Jahr: 75%; 5 Jahre: 45%) deutlich schlechter als die Prognose enossaler Implantate (1 Jahr: 95%; 5 Jahre: 72%). Die funktionell hochwertige Restitutio des Kauorgans durch implantatgetragenen Zahnersatz ist damit auch für bestrahlte Tumorpatienten bestätigt. Eine Kontraindikation enossaler Implantation konnte daneben für alle untersuchten zusätzlichen Prognosefaktoren ausgeschlossen werden. Summary In comparison to tumor patients not receiving radiotherapy, the rehabilitation of masticatory function after head and neck irradiation is limited due to radiation-induced caries, radioxerostomia, and the risk of osteoradionecrosis. This study focused on implants in the irradiated jaw and on the evaluation of the prognosis and the effect of potential factors on the prognosis. The retrospective study covered 197 implants (47 patients) from 1988 to 1997. The implant prognosis was determined by implant survival statistics (Kaplan-Meier). Losses not related to the implants were censored. In addition, groups were formed according to factors potentially affecting the prognosis. The significance of differences in the groups relative to survival were tested using the log-rank test. Twelve (6.1%) implants from a total of 197 were lost due to peri-implantitis, and eight (4.1%) due to possible biomechanical stress. A total of 52 losses (26.4%) due to death of patients and two (1.0%) due to resection of the jaw were censored; 111 (56.3%) implants remained at recall and the average interval was 33 months. The rates of implant survival (Kaplan-Meier) after 1 and 2 years were 95%, after 3 and 4 years 92%, and after 5 and 6 years 72%. The univariate analysis of group comparisons showed a significantly lower rate of loss after perimplant flap reconstruction (p = 0.036). There was no effect due to the doses of irradiation (p = 0.16), chemotherapy (p = 0.90), or peri-implant osteoplasty (p = 0.84). Although none of the implants inserted before radiotherapy had to be explanted, the implant survival difference in the very heterogeneous groups was not significant (preirradiation, n = 29; postirradiation: n = 156; p = 0.13). According to the literature, the rate of survival of teeth which were sound before radiotherapy (1 year, 75%; 5 years, 45%) was distinctly lower than the survival of enossal implants (1 year, 95%; 5 years, 72%). The high-quality rehabilitation of masticatory function with implant-based protheses is the preferred method of treatment for irradiated tumor patients. In addition, contraindications for enossal implants were ruled out for all studied factors affecting prognosis.In comparison to tumor patients not receiving radiotherapy, the rehabilitation of masticatory function after head and neck irradiation is limited due to radiation-induced caries, radioxerostomia, and the risk of osteoradionecrosis. This study focused on implants in the irradiated jaw and on the evaluation of the prognosis and the effect of potential factors on the prognosis. The retrospective study covered 197 implants (47 patients) from 1988 to 1997. The implant prognosis was determined by implant survival statistics (Kaplan-Meier). Losses not related to the implants were censored. In addition, groups were formed according to factors potentially affecting the prognosis. The significance of differences in the groups relative to survival were tested using the log-rank test. Twelve (6.1%) implants from a total of 197 were lost due to peri-implantitis, and eight (4.1%) due to possible biomechanical stress. A total of 52 losses (26.4%) due to death of patients and two (1.0%) due to resection of the jaw were censored; 111 (56.3%) implants remained at recall and the average interval was 33 months. The rates of implant survival (Kaplan-Meier) after 1 and 2 years were 95%, after 3 and 4 years 92%, and after 5 and 6 years 72%. The univariate analysis of group comparisons showed a significantly lower rate of loss after perimplant flap reconstruction (p = 0.036). There was no effect due to the doses of irradiation (p = 0.16), chemotherapy (p = 0.90), or peri-implant osteoplasty (p = 0.84). Although none of the implants inserted before radiotherapy had to be explanted, the implant survival difference in the very heterogeneous groups was not significant (preirradiation, n = 29; postirradiation: n = 156; p = 0.13). According to the literature, the rate of survival of teeth which were sound before radiotherapy (1 year, 75%; 5 years, 45%) was distinctly lower than the survival of enossal implants (1 year, 95%; 5 years, 72%). The high-quality rehabilitation of masticatory function with implant-based protheses is the preferred method of treatment for irradiated tumor patients. In addition, contraindications for enossal implants were ruled out for all studied factors affecting prognosis.

Confocal Laser Scanning Microscopy: A Nondestructive Subsurface Histotomography of Healthy Human Bone

Abstract. Microscopy of bony tissue usually requires special treatment for decalcification and processing of thin sections. Confocal laser scanning microscopy (CLSM) allows the nondestructive histotomography of organic hard tissue. The aim of this study was to visualize healthy human bone structures and to correlate identical areas in CLSM and conventional light microscopy. Each sample of healthy human lower jaw (n = 20) was divided into three parts: (1) fresh, untreated bony blocks studied by CLSM; (2) MMA-embedded thin sections (without decalcification), HE stained and studied by CLSM and conventional light microscopy (correlation of identical areas); (3) decalcificated, HE stained, histological sections studied by conventional light microscopy. In untreated bony blocks, microstructures such as osteocytes and lamellae were identified by CLSM. These structures could be correlated with conventional light microscopy. In CLSM, subcellular structures cannot yet be interpreted, whereas cytoplastic processes of osteocytes were seen with high contrast. With CLSM, nondestructive histology of cortical bone can be obtained. The risk of artifacts due to pretreatment is minimized, and subsurface visualization does not affect the interpretation.

Mikromorphologische Kieferveränderungen nach Bestrahlung

Für die infizierte Osteoradionekrose (IORN) des Kiefers werden unterschiedliche pathogenetische Mechanismen initialer Strahlenfolgen im Knochen diskutiert: Wichtige Parameter sind dabei die direkte Zellschädigung der Osteozyten [28] und die Gefäßfibrosierung [21]. Bislang liegen wenige Kenntnisse zum Ausmaß der initialen, subklinischen Veränderungen vor. 4 Probengruppen wurden anhand verschiedener Patientenkollektive definiert: Gruppe 1: gesunde Unterkieferproben meist von orthodontischen Operationen (n = 10 Patienten); Gruppe 2: Unterkieferproben mit manifester IORN (n = 12 Patienten); Grruppe 3: Unterkieferproben nach 36 Gy präoperativer Radio(chemo)therapie (n = 9 Patienten); Gruppe 4: Unterkieferproben nach 60–70 Gy Bestrahlung ohne Hinweise auf IORN (n = 9 Patienten). Alle Proben wurden histotomographisch mit konfokaler Laser-Scanning-Mikroskopie (CLSM), durchlichtmikroskopisch (DL) und dunkelfeldmikroskopisch (DFM) nach Fluoreszenzmarkierung des intakten Zellkerns (Bisbenzimid H 33258) untersucht. Korrelierende Histologien identischer Probenareale erfolgten an Trenndünnschliffen [8]. Als Ergebnisse wurden gefunden: 1. Alle unterschiedlich bestrahlte Knochenproben zeigten Areale mit weitgehendem bis vollständigem Verlust vitaler Osteozyten. Diese Strahlenfolge trat auch schon nach 36 Gy und kurzem Intervall bis zur Operation (Gruppe 3) auf. 2. Im CLSM zeigten sich zusätzlich mikromorphologische Veränderungen der lamellären Struktur, die zwischen den Probengruppen progredient waren. Hieraus können folgende Schlußfolgerungen gezogen werden: 1. Der vorbeschriebene Vitalitätsverlust der Osteozyten [10, 23, 28] konnte als sehr frühe, initiale, radiogene Läsion bestätigt werden. 2. Zusätzlich zeigten sich Veränderungen der lamellären Mikroarchitektur des Knochens als frühe und im Gruppenvergleich progrediente Strahlenläsion. Die mikromechanisch funktionelle Bedeutung dieser Veränderung muß in weiterführenden Untersuchungen eruiert werden. Early radiation effects on human bone may lead to osteoradionecrosis (ORN). Direct bone cellular lesions [28] as well as fibrous degeneration of blood vessels [21] are considered to be pathologically relevant. Only few data on initial subclinical radiation effects are available. Patients were grouped according to the dose of radiation and clinical findings. Group 1: sound human bone of lower jaw, mostly collected during orthodontic surgery (n = 10 patients); group 2: specimens of lower jaw from patients with ORN (n = 12 patients); group 3: specimens of lower jaw from patients with head and neck cancer who were preoperatively treated with 36 Gy radiation; group 4: specimens of lower jaw from patients with head and neck cancer (n = 9) who were treated with 60–70 Gy radiation. Specimens were studied by confocal laser scanning microscopy (CLSM), by conventional light microscopy (DL) and by flourescence darkfield microscopy (DFM) after bisbenzimide staining (H 33258) of the viable cellular nuclei. For the correlating study of identical areas in CLSM and DL the specimens were prepared according to the sawing and grinding technique [8]. All the radiated bony specimens, regardless of the dose of radiation, showed areas of extensive or total loss of vitality of the osteocytes. This finding was also evident after 36 Gy and a short interval between radiation and sample collection (group 3). Additionally, in CLSM micromorphologic lesions of the lamellate structure were seen. With these results we can confirm the loss of vitality of the osteocytes as an initial radiation effect as described earlier [10, 23, 28]. In addition to these findings, alteration of the lamellate microstructure was found in the early phase after radiation. The functional and mechanical significance of these findings should be the subject of further studies.

Ultrasound transmission velocity of the irradiated jaw bone in vivo.

Rose et al. [1] recently showed that bone elasticity of the jaw as measured by UTV is dependent on the age of the patient. This has earlier been shown for phalanx of the hand [2]. However, due to the anisotrophic architecture of bone the elastic behaviour of jaw bone can not be predicted by just transferring earlier results of the measurement of limbs to the lower jaw. Moreover, as the authors correctly state, the ultrasound signal does not stringently pass the specimen through the shortest but the fastest way. Therefore the signal is described as apparent UTV. Rose et al. found increasing UTV values during early bone maturing. This was seen at the phalanx as well as at the premolar region of the mandible. Up to the age of 30 years increasing UTV values are found although the increase was less striking at the mandible. Beyond the age of 30 years only a small number of patients is found in their study. This group seems to show decreasing UTV values. The applicability of UTV to jaw bone can be confirmed by our findings, in which we measured UTV in the median of the mandible (chin region) of patients before and after therapeutic irradiation. Fifty-six patients (27–77 years; 41 men) were enrolled into this study. Fourteen patients did not receive irradiation (control); 15 underwent pre-therapeutic irradiation with 36 Gy; 14 received ≥60 Gy. Sixteen patients were followed intra-individually before and after preoperative irradiation with 36 Gy. UTV was measured using a 2-MHz device (Krautkrämer USD 10, Hürth, Germany). The transducers which are mounted on a calliper were applied at the chin (mental spine and pogonion). All measurements were repeated five times and the median value was calculated. The second phalanx of the non-dominant hand served as control. The results are given in Fig. 1 as a box plot. The Kruskal-Wallis test showed no significant difference between the control, 36-Gy and ≥60-Gy group. In the group of 16 patients who could be followed intraindividually no statistically significant difference in UTV values after irradiation with 36 Gy was found. In the control group no significant difference between the UTV values of men (n=40) and women (n=15) was found. A linear correlation between the age of patient in the control group and the UTV was found at the chin (r=–0.6; P=0.006) and less striking at the second phalanx (r=–0.49; P=0.076).

Retrospektive Analyse zur prognostischen Bedeutung der Art der Unterkieferresektion und einer adjuvanten Strahlentherapie bei unterkiefernahen Karzinomen

Durch die retrospektive Auswertung von 100 Patienten mit unterkiefernahen Karzinomen (1983–1994) sollte eruiert werden, inwieweit die Art der Unterkieferresektion (Kasten- vs. Kontinuitätsresektion) den Erkrankungsverlauf prognostisch beeinflusst. Als potenziell prognosebeeinflussende Parameter wurden das Tumorstadium, der Unterkieferresektionsmodus, der Umfang der Lymphknotendissektion, die Strahlentherapiedosis und die Chemotherapie einbezogen. Zur Prognosebewertung dienten die Endpunkte „Versterben“, „Metastase“ und „Rezidiv“. 32 pT2-Karzinome, die jeweils zur Hälfte kasten- bzw. kontinuitätsreseziert wurden, zeigten bezüglich der Endpunkte „Versterben“, „Metastase“ und „Rezidiv“ gleiche Ereignisquoten. Überlebenszeiten und Metastasenfreiheitszeiten (Kaplan-Meier) verwiesen univariat auf einen tendenziellen Vorteil der Kontinuitätsresektion. Die relativen Risiken ergaben dagegen keinen Hinweis auf einen relevanten Effekt der Resektionsform. Da sich in ¶der multivariaten Analyse neben dem pTNM-Stadium die Bestrahlungsdosis als unabhängiger Prognosefaktor präsentierte, wurden hierzu zusätzlich ¶3 Gruppen gebildet. Der Vergleich von 62 nicht strahlentherapierten Patienten gegenüber 19 niedrig dosiert ¶(36u2002Gy) und 19 hoch dosiert (63u2002Gy) Bestrahlten zeigte univariat in den Überlebenszeiten einen Vorteil nach Ausbestrahlung. Dieser Vorteil wurde multivariat durch ein signifikant geringeres relatives Risiko für „Versterben“ und „Metastase“ nach Ausbestrahlung bestätigt. Die diskordante Datenlage der Studie zur Unterkieferresektion ist keine verlässliche Basis, eine Therapieempfehlung zu stützen, die vom etablierten Konzept abweicht. Bei aller Zurückhaltung aufgrund der retrospektiven und monozentrischen Ergebniseruierung kann aber ein Hinweis auf einen möglichen relevanten Prognosevorteil durch eine hochdosierte, adjuvante Bestrahlung abgeleitet werden, der multizentrisch retrospektiv und ggf. in prospektiven, randomisierten Studien geprüft werden sollte. The prognostic effect of bone resection (continuous vs non-continuous) was analysed in a retrospective study of 100 patients who were treated for squamous cell carcinoma located close to the lower jaw, between 1983 and 1994. Tumour stage, type of bone resection, extent of lymphatic node resection, dose of radiotherapy and chemotherapy were documented. Prognosis was characterised by the statistical end points “death”, “metastasis” and “relapse”. Thirty-two stage pT2 carcinomas, half of which were treated by continuous and half by non-continuous resection of the lower jaw, showed the same occurrence of the statistical end points death, metastasis or relapse. There was a trend towards a significantly longer survival time and metastasis-free interval in the group of continuous resection. The hazard ratios, however, showed no effect depending on the type of resection. However, both tumour stage (pTNM) and dose of radiation independently influenced prognosis in multivariate analysis. Consequently, three groups were defined. Univariate analysis of 62 patients without radiotherapy vs 19 with low-dose radiotherapy (36u2002Gy) and 19 with high-dose (62u2002Gy) showed a positive effect on the rate and time of survival in the group treated with high-dose radiotherapy. This was confirmed by multivariate analysis showing significantly lower hazard ratios for death and metastasis in the high-dose radiotherapy group after adjusting to cofactors (e.g. tumour stage). The data of this study challenge the current concept of resection of the lower jaw. However, due to the low number of patients and the retrospective character of the study, it is not possible to give suggestions regarding established therapy concepts. The advances of a high-dose radiotherapy in this study should be the subject of further multicentre retrospective and prospective randomised trials.

Konfokale Laser-Scanning Mikroskopie (CLSM) Validierung der zerstörungsfreien Histotomographie gesunden Knochengewebes

Durch Fixierung (Formalin), Entkalkung (Schnitte) oder mechanische Bearbeitung (Schliffe) besteht bei der Hartgewebshistologie das Risiko physikalisch-chemischer oder mechanischer Artefakte. Da Studien zur Ätiopathogenese pathologischer Veränderungen meist auf subklinische Läsionen zielen, besteht die Gefahr, daß Artefakte frühe Veränderungen vortäuschen oder vorhandene überdecken. Es wird der Frage einer artefaktminimierten Histologie nachgegangen. Bei der konfokalen Laser-Scanning-Mikroskopie (CLSM) rastert ein monochromatischer Laserstrahl über die Probenoberfläche und dringt in das Gewebe ein. Die Intensität der remittierten Strahlung wird in einem Detektor gemessen. Durch eine konfokale Blende (pinhole) erreicht nur Laserlicht aus einer extrem dünnen In-Fokus-Ebene den Detektor, so daß schrittweises Bewegen des Objekttischs eine tomographische Serie von Einzelbildern visualisiert. Gesunde kortikale Knochenproben des Unterkiefers (n = 20) wurden durchlichtmikroskopisch und im CLSM untersucht: Dabei wurden identische Probenareale an Dünnschliffen nach HE-Färbung visualisiert. Außerdem wurden eingebettete und frische Gewebeblöcke derselben Knochenproben im CLSM histotomographiert. Als Ergebnisse wurden gefunden: 1. Lichtmikroskopische Mikromorphologie kortikalen Knochens ist im CLSM valide visualisierbar. 2. Viele lichtmikroskopisch erst nach Spezialfärbungen darstellbare Strukturen (z.B. Osteozytenfortsätze) können im CLSM an Probenblöcken ohne Vorbereitung mikroskopiert werden. Hieraus können folgende Schlußfolgerungen gezogen werden: 1. Die zerstörungsfreie Histotomographie des CLSM unter der Probenoberfläche vermeidet mechanische Artefakte völlig. 2. Physikalisch-chemische Artefakte werden kontrollierbar, da auch frische Proben mikroskopiert werden können. 3. Die pseudo-dreidimensionale Darstellung erlaubt eine histologische Gewebeinterpretation, die makroskopischen Schichtbildverfahren (CT, MRT) vergleichbar ist. Fixation (formalin), decalcification (sections) or mechanical treatment (grinding) all bear the risk of artifacts occurring during hard-tissue histology. Because studies on the etiology of pathological changes mostly focus on subclinical lesions, artifacts can simulate early changes or even be superimposed on existing changes. The objective of this study was to determine how artifacts can be reduced. In confocal laser scanning microscopy (CLSM) a focused laser beam scans the surface of the specimens and penetrates into the tissue. The intensity of the remitted light is recorded. The confocal effect is due to an extremely small aperture (pinhole), excluding light from out-of-focus planes of the sample. By stepwise movement of the object table, a tomographic series of tomographic images is obtained. Sound cortical bone samples of the lower jaw (n = 20) were studied by light microscopy and by CLSM, visualizing identical areas of a ground sectioned sample after H & E staining. Additionally, embedded and fresh blocks of tissue of the same bone sample were studied histotomographically in the CLSM. (1) Light microscopic micromorphology of cortical bone can be visualized adequately in the CLSM; (2) many structures that can be visualized by light microscopy only after special staining (e.g., osteozyte processes) can be visualized by the CLSM using sample blocks without pretreatment. Conclusion: (1) Nondestructive subsurface histotomography by CLSM totally excludes mechanical artifacts; (2) physicochemical artifacts can be handled more easily because fresh samples can be studied; (3) pseudo-three-dimensional imaging allows histological interpretation of the tissue that is equivalent to macroscopic tomographic techniques (CT, MRT).