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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 6  |  Page : 888-892
Histochemical characterization of bone and cementum using modified gallego's iron fuschin stain and van gieson stain and its application in fibro-osseous lesions


Department of Oral Pathology, Government Dental College and Hospital, Aurangabad, Maharashtra, India

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Date of Submission05-Dec-2018
Date of Decision21-Mar-2020
Date of Acceptance31-Aug-2020
Date of Web Publication22-Mar-2021
 

   Abstract 


Objectives: Fibro-osseous lesions (FOLs) of the jaws exhibit an overlapping histomorphologic spectrum with respect to nature of calcifications. Sometimes these calcifications may be difficult to characterize as bone and cementum on routine Hematoxylin and Eosin (H&E) staining. This causes difficulty in assessing the origin and diagnosis of these lesions. Thus the study aimed to characterize bone, cementum, and hard tissue components in FOLs using special stains. Method: The study included a histochemical evaluation of 20 samples of bone and cementum and 12 cases each of fibrous dysplasia (FD) and ossifying fibroma (OF). Three consecutive sections of each tissue were stained with H and E, modified Gallego's iron fuschin stain and Van Gieson stain. H and E and modified Gallego's iron fuschin stained sections were analyzed under light microscope whereas Van Gieson stained section was analyzed under polarizing microscope. Results: It was found that cementum stained red and bone stained greenish-yellow in color. The calcifications seen in fibrous dysplasia stained greenish-yellow in color. Three cases of OF showed greenish-yellow calcifications and nine cases showed reddish calcifications. Polarization study of bone showed lamellar pattern and tooth cementum showed quilt pattern. Four cases of FD showed lamellar pattern and eight cases showed haphazard pattern. In OF, three cases showed lamellar pattern and nine cases quilt pattern. Conclusion: Calcifications having lamellar pattern and greenish-yellow color suggest their osteogenic origin thus having aggressive nature and requiring aggressive treatment. Calcifications having quilt pattern and reddish color suggest periodontal ligament origin thus having less aggressive behavior and less extensive treatment.

Keywords: Bone, calcification, cementum, stain

How to cite this article:
Agrawal RS, Bhavthankar JD, Mandale MS, Thakur A, Bhagde PA, Salve S. Histochemical characterization of bone and cementum using modified gallego's iron fuschin stain and van gieson stain and its application in fibro-osseous lesions. Indian J Dent Res 2020;31:888-92

How to cite this URL:
Agrawal RS, Bhavthankar JD, Mandale MS, Thakur A, Bhagde PA, Salve S. Histochemical characterization of bone and cementum using modified gallego's iron fuschin stain and van gieson stain and its application in fibro-osseous lesions. Indian J Dent Res [serial online] 2020 [cited 2021 Apr 15];31:888-92. Available from: https://www.ijdr.in/text.asp?2020/31/6/888/311667



   Introduction Top


Fibro-osseous lesions of the jaws form an intriguing group of pathologies that are a diagnostic challenge owing to nature of mineralized tissue (ranging from bone: woven/lamellar bone, cementum, or dystrophic calcifications). They have a wide overlapping histo-morphologic spectrum leading to difficulty in assessing the origin, pathogenesis, and rendering a confirmed diagnosis. An in-depth analysis of histopathologic characteristics of the fibrous and osseous components will help in a better understanding of fibro-osseous lesions.[1] The nature of the fibrous tissue and the organic matrix in the ossifications differ considerably. Fibrous dysplasia (FD) is a genetic disorder that arises from basal bone and exhibits predominantly metaplastic woven bone. Ossifying fibroma (OF), on the other hand, is considered to be a highly cellular neoplasm exhibiting cementicles, woven and lamellar bone components.[2] However, it is often challenging to differentiate between bone and cementum in routine histological H and E staining technique. This leads to risk of misinterpretation of histological data. Histological stains help in the microscopic study of the tissues. They contain dyes that bind to the tissue. Hematoxylin & Eosin is the most commonly used histological stain. Along with the routine staining techniques, there are alternative staining procedures that aid in highlighting the particular features of the tissues that are not seen with routine H and E.[3],[4] Therefore these special staining methods can be used as an adjunct for predicting the histopathological diagnosis.[5] Several stains have been modified and used with various other stains to improve their effectiveness.[4] One such stain is modified Gallego's stain that has been derived from the modification of Lille's stain which uses basic reagents like Hematoxylin, Carbol fuschin and Aniline blue.[6] Another stain that is used is Van Gieson stain.[7] These stains can be used as the differential stains for the hard tissues like bone and cementum.[3] Thus these two alternative staining techniques are used for identification of bone and cementum and also for the various pathological lesions that are characterized microscopically by calcifications.[3],[8] The present study was done to differentiate between bone and cementum and identify the nature of hard tissue seen in pathological lesions like fibrous dysplasia and OF using special stains. These stains were modified Gallego's stain seen under light microscopy and Van Gieson Stain seen under polarizing microscopy. An attempt was made to correlate these findings with the origin and aggressiveness of these lesions. Thus, the study aimed at characterizing normal bone, cementum and hard tissues components in FOLs using special stains.


   Methods Top


This retrospective study was conducted in the Department of Oral Pathology and Microbiology following institutional ethical committee clearance. Control group included 20 samples of teeth and 20 samples of bone of which decalcified sections were prepared. Formalin fixation was done. Decalcification procedure was done using 2% nitric acid for four days. Twenty four samples of pathologic tissue comprising 12 cases each of Fibrous Dysplasia and OF were retrieved from the departmental archives. Three consecutive sections each of 5 μm thickness were stained with the following:

  1. One section was stained with Hematoxylin and Eosin to recognize the histological differences between all the tissues
  2. Second section was stained with Van Gieson stain and assessment of fibrillar component was done under polarized light. The orientation of collagen fibers was categorized as parallel, random or quilt arrangement. This was seen in normal hard tissue (bone and cementum) and within calcifications in the pathologic tissues
  3. Modified Gallego's iron fuschin stain was used to assess difference between bone and cementum. Sections were stained by using the following technique: The sections were initially deparaffinised, stained in Hematoxylin for 8-12 minutes and rinsed in distilled water. Then, they were sensitised in mordant for two minutes and rinsed in distilled water (Mordant was prepared by mixing 200 ml of distilled water in 1.5 ml of concentrated Nitric acid with 1 ml of 40 percent formaldehyde and 1.5 ml of Iron chloride). Later they were stained with 3 ml of carbolfuschin diluted in 50 ml of 0.2 percent acetic acid, rinsed in distilled water and washed in mordant for 1-2 minutes. Finally, these sections were stained with 0.01 per cent aniline blue in saturated picric acid solution for 30 seconds, dehydrated, cleared with xylene and mounted in DPX mounting media. The stained sections were viewed using routine light microscopy and were interpreted under 4X, 10X and 40X magnification. Slides were viewed under light microscope. The various colours taken by different hard tissues were observed and interpreted. Bone, dentin appeared greenish yellow and cementum appeared reddish pink in color.



   Results Top


A decalcified section of a tooth and bone stained with modified Gallego's iron fuschin stain, shows the deeply stained layer of cementum contrasting markedly with the bone [Table 1]. The cementum is deep red [Figure 1]a in contrast to the green stain of the bone seen in [Figure 1]b. The mineralizations of FD showed greenish yellow coloration [Figure 2]a. The mineralizations of OF showed greenish yellow as well as reddish coloration [Figure 2]b,[Figure 2]c,[Figure 2]d. In the present study, fibrous dysplasia was mainly comprised of mature trabeculae of bone along with woven bone. Cases of OF showed both mature lamellar bone, cementicles and both the cementicles with bone at the periphery. [Table 1] shows the details of the cases of FD and OF that showed greenish yellow and reddish calcifications.
Figure 1: Modified Gallego's Iron Fuschin stained section of cementum that stained red in color (a). Modified Gallego's Iron Fuschin stained section of bone that stained green in color represented by star mark (b)

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Figure 2: Fibrous dysplasia showing greenish yellow calcifications (a). Ossifying fibroma showing Greenish yellow calcifications (b). Ossifying fibroma showing reddish pink calcifications (c). Ossifying Fibroma showing calcifications that are yellow with reddish outline at periphery (d)

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Table 1: Modified Gallego's iron fuschin stain, Normal Tissue and Pathologic Tissue

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The Van Gieson stained slides of all the 40 normal samples were subjected to evaluation under polarized light microscope which showed varying orientation of collagen fibers. Cementum presented quilt pattern [Figure 3]a. Bone showed lamellar pattern [Figure 3]b. The collagen fibres seen in mineralizations of FD showed randomly arranged parallel lamellations [Figure 3]c. The collagen fibres seen in mineralizations of OF showed quilt arrangement [Figure 3]d, [Table 2]. In the present study, fibrous dysplasia was mainly comprised of mature trabeculae of bone along with woven bone. Cases of OF showed mature lamellar bone, presence of cementicles and cementicles with mature bone at the periphery.
Figure 3: Van Gieson stained section of cementum that showed quilt pattern of collagen fibres (a). Van Gieson stained section of bone that showed lamellar pattern of collagen fibres (b). Fibrous Dysplasia showing Lamellar arrangement of collagen fibres (c). Ossifying Fibroma showing quilt pattern of collagen fibres (d)

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Table 2: Van Gieson stain, normal tissue and pathologic tissue

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Statistical analysis was done and it was found that the sensitivity for the bone and cementum using modified Gallego's iron fuschin stain and Van Gieson stain was 100% [Table 3].
Table 3: Statistical analysis

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   Discussion Top


Fibro-osseous lesions of jaws includes FD and OF. OF in turn is classified as Ossifying/Cementifying Fibroma, NOS and Juvenile Ossifying Fibroma (JOF). Juvenile JOF is categorized into Trabecular Juvenile Ossifying Fibroma and Psammomatoid Juvenile Ossifying Fibroma (PsJOF). The aggressiveness of these lesions may depend on the tissue of origin.[9]

Oral hard tissues like bone and cementum vary in their composition. The major differences lies in the non-collagenous extracellular matrix proteins like concentration of phosphoserine, phosphothreonine and gamma carboxyglutamic acid. Amino acid composition of core proteins associated with bone and cementum proteoglycans are also dissimilar. This difference helps in differential staining of bone and cementum with modified Gallego's iron fuschin stain.[3],[10],[11] While on the other hand, the principle of Van Gieson stained polarization is on the basis of strong reaction of acidic dyes, for example, Sirius red with collagen molecules that are rich in basic amino acids. The elongated dye molecules get aligned parallel to the long axis of each collagen molecule thus promoting enhancement of its normal birefringence when viewed under polarizing light.[12],[13]

Fibro-osseous lesions of the craniofacial bones are a behaviouraly diversified array of diseases that share common microscopic features. All lesions are composed of a hypercellular fibrous element and an osseous element both of which exhibit a wide spectrum of variations.[14],[15] The more significant lesions from management point of view are fibrous dysplasia and ossifying fibroma as they are two entirely separate pathogenetic diseases with behavioral differences that dictate specific management treatment strategies.[16] Fibrous dysplasia is a benign dysplastic process of altered osteogenesis that may occur within a single bone (monostotic) or in multiple bones (polyostotic).[17] Ossifying fibroma has been considered as a neoplasm in the true sense, exhibiting progressive proliferative capabilities and bony expansion.[18] They exhibits an overlapping histomorphologic spectrum with respect to nature of calcification. These calcifications may be difficult to characterize as bone and cementum on routine staining. This causes difficulty in assessing the histogenesis and diagnosis of these lesions.[19],[20] Various special stains are used, for example, Von kossa, Alizarin red, Picro-thionin, Toluidine blue and Alcian blue are available but the use of single histo-chemical stain that differentiates between the hard tissues of tooth are rare.[21] Modified Gallego's's stain is also similar type of stain that not only stains the decalcified sections but also differentially stains the hard tissues of tooth and the calcified structures present in the pathological lesions thus helps in obtaining a clear histological picture.[3],[5],[22]

In the present study, the FOL's encompassing hard tissue components were taken. This lesions develops the proliferation of both epithelial and mesenchymal components which results in the formation of hard tissues like enamel, dentin and cementum in an abnormal pattern.[23] When stained with modified gallego stain, these components were stained with different colours, dentin stained green and cementum red.[3]

Modified Gallego's, stain is a variant of Lille's stain that uses basic reagents hematoxylin, carbol fuschin and aniline blue. The advantage of this stain is that it differentially stains the hard tissues seen in tooth, dental and other pathological lesions and gives a clearer understanding of the histological picture of the hard tissue deposits in oral lesions. With modified Gallego's stain, bone and dentine appears green. Whereas, cementum like deposition appears red in colour.[3] Levy RJ 1956[22] also found that with modified Gallego's stain, cementum stained red and bone stained green in color. Pathala VM et al. 2017[5] found the similar findings where cementum stained red in color and bone stained green in color. Bone showed lamellar arrangement of collagen fibers. On the other hand, cementum showed quilt pattern of collagen fibers. Kulkarni RR et al. 2014[1] found that collagen fibres in cementum showed quilt pattern and in bone showed lamellar pattern. In our study, mature trabecular bone with lamellar pattern was seen in four cases of FD with eight cases showing woven bone along with haphazard arrangement of collagen fibers. The three cases showed lamellar pattern and nine cases of OF showed droplet-shaped acellular cementum-like material seen in H and E stained sections that appeared delicate lines ('quilt' pattern) under polarized light. This 'quilt' pattern of cementum under polarized microscopy can be used to distinguish it from bone. In our study the 'quilt' was seen along with woven bone in some of the cases of ossifying fibroma and was completely absent in cases of FD.[1],[8] Kulkarni RR et al. 2014[1] found lamellar arrangement of collagen fibres in all 6 cases of fibrous dysplasia and quilt arrangement in all 11 cases of ossifying fibroma.

Fibrous dysplasia forms from the basal alveolar bone and not from PDL tissue origin. This can be concluded from the fact that mutation (GNAS1) of the germ cells osteoblast occurs at an early stage of embryogenesis when the alveolar bone proper has still not formed. This mutation leads to formation of abnormal bone seen in fibrous dysplasia. On the other hand, OF originates from alveolar bone proper in juxtaposition to the periodontal tissue.[24] Apart from this, the increased frequency of cementicle formation directs their PDL origin. Mesenchymal blast cells that are existing within the periodontal ligament are capable of being stimulated to produce tumors composed of cementum, lamellar bone, fibrous tissue, or any combination of these tissues. Distinction between tumors of periodontal membrane origin and tumors of medullary bone origin is significant because the latter tumors usually behave in a more aggressive fashion.[1],[5],[23]

Among the ossifying fibroma, JOF have aggressive behavior. The aggressive behavior of trabecular variant is attributed to its osteogenic origin.[9] In PsJOF, psammoma bodies are concentric or laminated ossicles that contain osteocytes. Immunohistochemical studies have shown positivity for osteonectin thus suggesting its osteogenic origin.[24],[25],[26],[27],[28],[29],[30] The reason for the aggressiveness of the lesion is unknown. But the tissue of origin may be the reason for the aggressive behavior of the lesion. This may change the treatment plan of the lesion. Thus the tumors of osteogenic origin are more aggressive in nature thus requiring extensive treatment whereas tumors of PDL origin are less aggressive thus requiring less extensive treatment. In the present study, presence of bony calcification in Ossifying fibroma was suggestive of either trabecular variant or psammomatoid variant directing its osteoprogenitor cell origin. The presence of cemental calcifications suggested the conventional variant of ossifying fibroma thus redirecting its PDL origin. Presence of bone with periphery of cementum was seen in psamommatoid variant suggestive of both origins and supports cemento-ossifying fibroma terminology. Presence of bone and cementum indicated osteoprogenitor and cemental origin which means origin from more primitive cells i.e., less differentiated and more aggressive tumors. thus, psammomatoid variant is more aggressive than trabecular variant.


   Conclusion Top


Special stains like modified Gallego's Iron Fuschin stain and Van Gieson stain offers insight into the nature of calcifications in FOL's. This may help to understand the histogenesis of these lesions, thus will help to determine treatment plan. They can also be used to differentiate hard tissue components in odontogenic tumors. Further research in various pathologies of head and neck region such as FOL's, Cementoblastoma and osteoblastoma should be evaluated with larger sample size.

Limitations

It is a technique sensitive and objective procedure. The stain fades away with increased time duration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Ruchika Sanket Agrawal
Flat No. 801, Tulsi Apt., C Wing, Maitri Vatika, Parsik Nagar, Kalwa, Thane, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_889_18

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