engleski

Histological methods for osteoid and mineralized bone tissue labeling

Objectives: Bone deposition is the main sign of chronic periapical lesion healing. It can be assessed through radiological and histological analysis. In histological analysis various dyes are used, routinely or exceptionally. The aim of our research was to assess experimental value of standard Toluidine Blue (TB) staining, and bone-specific vital stains, Procion Red and doxycycline, in labeling osteoid and newly deposited mineralized bone tissue. Methods: Pulp chambers of two experimental animals (9 roots each) were exposed to the oral environment. After 35 days, endodontic therapy was performed using ProFile&reg ; Ni-Ti rotary instruments and Thermafill&reg ; obturators, and access cavities sealed with amalgam. At this point animal 1 was given an intraperitoneal injection of Procion Red (10 mg/kg), while animal 2 received a 12-day peroral administration of doxycycline (100 mg/kg). Animals were sacrificed 35 days following therapy, and analysis performed on histological sections. Qualitative analysis included TB stained sections using light microscope and unstained sections using fluorescent microscope. Histomorphometric indices were measured on TB stained sections using computer program ISSA (Vams, Zagreb, Croatia). Results: TB staining enables qualitative analysis of osteoid deposits as well as histological features of periapical lesion. Fluorescent deposits of Procion Red and doxycycline as vital stains mark the border of newly-formed mineralized bone. Quantitative histomorphometric analysis of TB stained sections showed greater osteoid surface (animal 1: 4, 26 % &plusmn ; 4, 03 ; animal 2: 30, 85 % &plusmn ; 15, 07 ; p<0, 01)and osteoid thickness (animal 1: 12, 55 &#956; m &plusmn ; 4, 94 ; animal 2: 13, 99 &#956; m &plusmn ; 5, 31 ; p<0, 01)and lower osteoclast index in the animal that received doxycycline (animal 1: 72, 78 mm-2 &plusmn ; 45, 40 ; animal 2: 38, 92 mm-2 &plusmn ; 34, 70 ; p<0, 01. Statistical analysis was performed using Mann-Whitney U test. Conclusion: TB staining enables quantification of osteoid tissue but for mineralized bone analysis vital stains are indispensable. Measured indices show statistically significant difference in periapical lesion healing between two animals. It proves that doxycycline cannot be considered an independent marker of newly deposited bone because it interferes with bone metabolism through inhibition of bone resorption.

histological labeling; osteoid; bone; dog; fluorescent dyes

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