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Wehrhan et al.- 1

Bisphosphonate- associated osteonecrosis of the jaw is linked to suppressed TGFβ1-signaling and increased Galectin-3 expression: A histological study on biopsies

Falk Wehrhan MD, DMD1; Peter Hyckel MD,DMD, PhD2; Arndt Guentsch DMD3, Emeka Nkenke MD, DMD, PhD1, Phillip Stockmann MD, DMD1; Karl A. Schlegel MD, DMD, PhD1, Friedrich W. Neukam, MD, DMD, PhD1, Kerstin Amann MD,PhD4

1 Department of Oral and Maxillofacial Surgery

University of Erlangen-Nuremberg

2 Department of Plastic Surgery/ St. Georg-Hospital Eisenach

University of Jena

3 Department of Conservative Dentistry

University of Jena

4 Institute of Pathology

University of Erlangen-Nuremberg

Address Correspondence and request for reprints to:

Falk Wehrhan MD, DMD

Department of Oral and Maxillofacial Surgery,

Friedrich-Alexander-University of Erlangen

Glueckstrasse 11, 91054 Erlangen, Germany

Phone: +49-9131-853-3616

Fax: +49-9131-853-4219

Email:

Short running title: Altered TGF(1 and Galectin-3 in BRONJ and oral soft tissue

Number of words in abstract: 227

Total number of figures: 4

Keywords: BRONJ, oral soft tissue, transforming growth factor beta 1, galectin-3, oral surgery

Abstract[mlr1] :

Background/ aim of the study:

Bisphosphonate associated osteonecrosis of the jaw (BRONJ) implies an impaired impairment in oral hard- and soft tissue repair. An understandingnalysis of related the signal transduction -alterations involved can informenables therapeutical strategies. Transforming Growth growth Factor factor β1 (TGFβ1) is a critically regulates regulator of tissue repair;. Galectingalectin -3 mediates tissue differentiation and specifically modulates periodontopathic bacterial infection. The aim of thise study was to compare the expression of TGFβ1-related signaling molecules and the expression of Galectin-3 in BRONJ- affected mucosal tissue and healthy mucosal tissues. To discriminate between BRONJ- specific impairment ofs in TGFβ1 signaling from and secondary inflammatory changes, the results were compared with to the expression ofg TGFβ1 and Galectin-3 in mucosal tissues with osteoradionecrosis. affected mucosal tissue (ORN).

Material and Methods:

Oral mucosal tissue samples Twenty with histologically- provenconfirmed BRONJ (n=20), 20 osteoradionecrosis (n=20) related, and 20 healthyno lesions (normal, n=20) oral mucosal tissue samples were processed for immunohistochemistry. For targeting of TGFβ1, Smad-2/3, Smad-7 and Galectin-3, an aAutomated staining with-based an APAAP (alkaline phosphatase-anti- alkaline phosphatase) kit -staining kit was used to detect TGFβ1, Smad-2/3, Smad-7, and Galectin-3. Semiquantitative We semiquantitatively assessment measureded the ratio of stained cells/ total amount number of cells (labeling index, Bonferroni-adjustment).

Re[mlr2]sults:

In the BRONJ-related samples TGFβ1 and Smad-2/3 presented were a significantly decreased (p(0.032 and p(0.028, respectively) in the BRONJ samples and in the ORN group a significantly increased (p(0.04 and p(0.043, respectively) in the osteoradionecrosis samples labeling index compared with to normal tissue. Smad-2/3 labeling index was found to be significantly decreased (p(0.028) in the BRONJ samples and significantly increased (p(0.043) in the ORN tissue compared to the normal tissue. Smad-7 showed awas significantly increased (p(0.031) staining in the BRONJ group and significantly decreased (p(0.026) staining in the osteoradionecrosisORN group. Galectin-3 staining was significantly (p(0.025) increased in both the BRONJ- and the osteoradionecrosisORN (p(0.038) groups compared to the normal mucoperiosteal tissue group. However, Galectin-3 expression was significantly higher in the BRONJ-group samples compared to than in the osteoradionecrosisORN-tissue samples (p( 0.044).

Conclusion:

Disrupted Our results showed that disrupted TGFβ1 signaling characterizes was associated with delayed periodontal repair in BRONJ sitessamples. The findings also indicated that a different impairments of in TGFβ1- signaling were different in BRONJ compared to osteoradionecrosis. BRONJ seems appeared to be associated with increased terminal osseous differentiation and decreased soft tissue proliferation. Increased The increase in Galectin-3 demonstrates reflected the increase in osseous differentiation of mucoperiosteal progenitors, and this might explain the inflammatory anergy of observed in BRoral ONJ-affected soft tissues. The results substantiated the clinical success of treating BRONJ-treatment by with seqestrotomysequestrectomy, followed by strict mucosa closure. BRONJ can be further elucidated by Investigation investigatingof the the specific intraoral osteoimmunologic situationstatus will. elucidate BRONJ further.
Introduction

Regarding the etiology ofNumerous attempts have been made to explain the development of BRONJ (Bbisphosphonate-related associated osteonecrosis of the jaw (BRONJ), numerous attempts have been made to explain the development of BRONJ, but until now there is nothe formal pathology remains unknown [1]. Previous studies have described the concordance of lLocal ONJ -accompanyingand an inflammatory reaction that was induced by an superinfection of the tissue lesions by intraoral, gram-negative bacteriabacteria superinfection of the tissue has been concordantly described [1, 2]. Although Alternatively, there is increasing evidence that BRONJ is caused by bisphosphonate (BP)-related impairment of the cellular interplay of among osteoblasts, osteoclasts, fibroblasts, and keratinocytes during tissue remodelingremodeling. However,, it remains unclear if whether BRONJ originates inarises from the a laceration of in the oral mucosa or in from the underlying jaw bone tissue [1]. Recently, BRONJ was related to an impairment of in Msx-1-related osteoblast proliferation has been described [3]. There are cHowever, results are contradictionoary data describing regarding the biologic impact of BP on periodontal epithelial and connective tissue cells. BP gel formulations, topically applicated in periodontal lesions, did have not caused any adverse effects [4]. Despite these findingsIn contrast, when necrosis was casually described holding alendronate tablets were held under a denture in contact with the oral mucosa, necrosis occurred [5]. BP was shown to Osteogenetic stimulation of e bone progenitor cells by toward osteogenesis BP was shown in vitro [6]. AlsoIn addition, the administration of zoledronic acid to oral gingival fibroblasts in vitro reduced expression of ECM (Eextracellular matrix (ECM) ) proteins such as, including collagens I, II, and III has been described in-vitro following administration of zoledronic acid to oral gingival fibroblasts [7].

TGFβ1 (Transforming Growth growth factor β1 (TGFβ1) is a pleiotropic cytokine that, mediating mediates fibroblast differentiation and proliferation of fibroblasts and regulating regulates EMT (the epithelial-to-mesenchymal -transition (EMT) during wound repair {Heldin, 2009 #3986;Huminiecki, 2009 #3987}. TGFβ1It exerts its intracellular actions by through Smad proteinsprotein signaling. Whereas Smad 2/3 is described to bewas identified as the TGFβ1 downstream TGFβ1 effector, and Smad, Smad 7 inhibits inhibited intracellular TGFβ1-related signaling [8]. Increased TGFβ1- and Smad-2/3 expression have beenwas shown to be related to fibrocontractive wound healing disorders [9]. Loss of TGFβ1 has been implicated in delayed wound healing and impaired extracellular matrix ECM deposition [10]. TGFβ1 was shown to differentially affect epithelial and fibrous connective tissues; by it inhibiting inhibited the migration of epithelial cells during wound healing, but stimulating stimulated proliferation of fibroblasts [11]{Schultze-Mosgau, 2002 #2666;Schultze-Mosgau, 2003 #2672}. Since TGFβ1 and Smad signaling is were shown to be involved in both osseous and connective tissue remodelling; thus,, knowledge of possible BP-related alterations of thein TGFβ1 signaling could help tomight explain the BP- associated changes of in the oral mucosa tissues in of BRONJ affected jaws {Kanaan, 2006 #3989;Wu, 2009 #3990}. The oral diseaseFurthermore, osteoradionecrosis has been described to be associated with increased TGFβ1 expression [12].

The possible role of BP-related changes of in Smad-2/3 expression is of interest sincemay also affect TGFβ1-independent Smad activation has been demonstrated by the glycoprotein, Galectin-3, in a TGFβ1-independent pathway [13]. Galectin-3 is, involved in the regulation of epithelial and bone differentiation and of plays a pivotal role in inflammatory responses and fibrotic tissue remodelling; it has been demonstrated shown to inhibit the cytokine activation of cytokines by periodontopathic gram negative bacteria [14-16]. Galectin-3 expression has been demonstrated to bewas increased in radiation- impaired epithelial tissues. The degree of Galctin-3 expression in squamous epithelial tissues was was shown to correspondpositively associated with differentiation positively and negatively associated with proliferation negatively {Kasper, 2000 #3883;Szabo, 2009 #4519}.Therefore, the roles of the TGFβ1- and Galectin-3-related signaling network ofin cellular differentiation, tissue regeneration, and inflammation is may of relevance in order to elucidatebe relevant to the mechanisms underlying BRONJ-underlying cellular mechanisms.

The American Society for Bone and Mineral Research The has formed a BRONJ-task force that demanded requires clinical and basic research inaddressing jaw- specific biology [17].[mlr3] This study was aimed to compare the cellular expression levels of TGFβ1, Smad 2/3, Smad 7, and Galectin 3 in BRONJ-related periodontal tissues compared to healthy oral mucosa. We This semiquantitative immunohistochemical analysis was carried out to assessed the impact of intravenous BP-therapy on the spatial distribution and protein expression of the TGFβ1 signaling cascade molecules and Galectin-3 in BRONJ sites with semiquantitative immunohistochemical analysis. To di[mlr4]scriminate between BRONJ-specific impairments and secondary inflammatory changes that could affect TGFβ1 signaling, the results were compared to the expression of TGFβ1 and Galectin-3 in mucosal tissues with osteoradionecrosis.

Materials and Methods:

Patients and material tissue harvesting

Oral mucosa specimens from 60 patients have beenwere included in this study. Twenty specimens have been were obtained from 20 consecutive patients with clinically and histologically evident BRONJ of that 20 patients undergoingwent radical sequestrecotomy. The ethical aspects of the study have beenwere approved by the local ethical committee of the University of Erlangen-Nuremberg (Ref.-Nr. 4272). The specimens were harvested in 20 consecutively treated patients. The specimens used in this study were from part of the tissue samples provided collected for routine histopathological diagnostics. Each specimen included in this investigation was confirmed to be related to theexhibit histopathologic aspects of BRONJ. Besides In addition to the histopathological characteristics of BRONJ in specimens related bone tissue, the inclusion criteria for including the specimens in this investigation were: patients received intravenous application of either pamidronate or zoledronate for at least 12 months in for treating carcinoma treatment, and patients showed clinical evidence of an exposed jaw bone for at least 8 weeks. Specimens from patients with Any former radiotherapy was were excluded. The clinical data and the exact description of treatment procedures of for the patients included in this study has beenwere documented previously [18]. Since aAll of the specimens were obtained during clinically routine clinical procedures, where and the material tissue was harvested from the same tissue neededcollected for standard diagnostics. Thus,, no surgical procedure specific to this study was performed, and no additional material was harvested collected from patients.

The controls comprised 20 alveolar mucosal specimens that were collected, harvested during intraoral surgery procedures in patients negative for anywith no BP-history and presenting no clinical signs of intraoral inflammatory processesion or periodontitis. Of these 20 control samples, 13 specimens were harvested from the alveolar crest after a tooth extraction of teeth when that required the removal of sharp bone ridges were removed and the adaptation of soft tissues were adapted;. 4 specimens were harvested from mucoperiosteal tissue extracted during orthognatic surgery in the lower jaw.;. In 3 cases the control tissue samplesspecimens were from mucoperiosteal tissue that covered wisdom teeth harvested duringthat required removal of wisdom teeth infrom the lower jaw, when mucoperiosteal tissue covering the wisdom tooth, had to be removed. The gender and the age of the patients were matched in the BRONJ – and the control groups have been matched, with exeptexception of the the 4 samples resulting from the orthognatic surgery procedure. The average age of the patients in the BRONJ group was higher than that in the 4 normal patients that underwent orthognatic surgery.

The osteoradionecrosis group specimens (ORN) included patients (n = 20) were from patients who that had been treated with radiotherapy prior to surgery of for oral squamous epithelial carcinoma. with These patients received a mean total reference dose of 68 Gy in the lower jaw region. The specimens used in this study were collected after a mean interval of 36 months between radiotherapy and secondary surgery steps, when the samples used in this study were harvested. Tissue samples were obtained from the soft tissue that surrounding surrounded the exposed bone that was exposed during a sequestrecotomy of osteoradionecrosis -affected mandibular bone. The osteoradionecrosisORN group consisted of 12 males and 8 female patientss at with a median age at of 57 years[mlr5]. The 60 specimens to be used in this study were measured on (average size: 5 ×x 3 x × 3 mm) and were then immediately fixed in 4% formalin.

Immunohistochemical staining

4%-The formalin- fixed, paraffin-embedded tissue samples were prepared for immunohistochemical staining assliced in consecutive sections using with a microtome (Leica, Nussloch, Germany) and then dewaxed in graded alcohol in preparation for immunohistochemical staining. Immunohistochemical staining was performed using with the alkaline phosphatase-anti-alkaline phosphataseAPAAP method and an automated staining device (Autostainer plus, DakoCytomation, Hamburg, Germany). We used according to the standard protocol, recommended for the staining kit used (Dako Real, Cat. K5005, DakoCytomation). Proteins were detected by incubating tissues in the autostainer (20 °C, 1 h) with specific antibodies. TGFβ1 was detected using with a polyclonal rabbit-IgG anti-human TGFβ1 antibody (anti-TGFβ1; sc-146, Santa-Cruz, Santa Cruz, USA; dilution 1:100) in the autostainer (20°C, 1h). Smad-2/3 was detected by with a polyclonal goat-IgG (anti-human Smad-2/3, sc-6033, SantaCruz, Santa Cruz, USA; dilution: 1:100). Smad-7 was targeted usingdetected with a polyclonal goat-anti-human antibody (sc-9183, Santa Cruz, dilution 1:100). Galectin-3 was stained usingdetected with a polyclonal rabbit-anti-human antibody (sc-20157, Santa Cruz, dilution 1:100). The sSecondary antibodiesy was were used according toincluded in the staining kit; (biotinylated polyclonal, goat-anti-rabbit was used for (TGFβ1 and, Galectin-3) and; rabbit anti-goat IgG was used for (Smad-2/3 and, Smad-7)) (E 0466, DAKO, dilutions 1:100). Visualisation of staining was performed usingStains were visualized with the Fast Red Solution, localized by biotin-associated activation of the staining kit secondary antibodies (ChemMate-Kit, Dako). This was, followed by incubation in hematoxylin for nuclear counterstaining the nucleus. Two consecutive tissue samples were processed per immunohistochemical staining;, 1 one served as a negative control in each case (identical treatment, but replacement of the incubation step with a primary antibody by incubation with an IgG-istotype-IgG of the primary antibody). A positive control sample that was known to stain positive staining for a given antibody sample was also included in each series as a positive control.

Semiquantitative immunohistochemical analysis

The BRONJ-related and healthy oral mucosa sections were examined qualitatively under a bright-field microscope (Axioskop, Zeiss, Jena, Germany) at 100-400× x magnification for changes differences in the numbers and localization of stained mucosa cells, which comprising comprised fibroblasts, fibrocytes, and periosteal progenitor cells in samples of BRONJ-related and healthy oral mucosa. In the healthy jawsamples, subepithelial tissues were examined, was observed, including connective, submucous, and epiperiosteal structures. Bone tissue was excluded from any the analysis. In BRONJ samples, soft tissues attached next to the necrotic zone were examined was located for observation. For each sampleWithin these areas, three visual fields per section for each sample were digitized at 200× x magnification using with a CCD camera (Axiocam 5, Zeiss, Jena, Germany) and the program Axiovision program (Axiovision, Zeiss, Jena, Germany). The dimensions of the digitized images were 800 ×x 500 µm at the original 200× magnification x 200). For this purpose, Rrandomiszed, systematic subsampling was performed based on the method by of Weibel and as practiced in own former research [19] [20-22]. Semiquantitative A semiquantitative analysis was performed of to determine the cytoplasmic expression levels of TGFβ1, Smad-2/3, Smad-7, and Galectin-3. Twas performed determing the labeling index was defined as the percentage of expressing cells (ratio of positively stained cells to the total number of cells per visual field, multiplied by 100). Cells of fibroblast lineage, including perisoteal progenitor cells, were recognized by their spindle shape and by excluding cells of epithelial and endothelial origin.[mlr6] Endothelial cells and epithelial cells were excluded from counting. Cell counting was performed by 3 independendent observers that were, not engaged in the project, but; all were familiar with tissue morphology analyseis and immunohistochemistry immunohistochemical methods.

Statistical analysis

In order to analyzse thecytoplasmic immunohistochemical cytoplasmic staining and the spatial pattern of expression, the labeling index was determined as the number of of positively stained cells per total cells in the visual field was taken. Multiple measurements per group of investigation were aggregated pooled for each sample group prior to analysis. The aggregation of data was performedpooled in each group for each addressed protein and group as follows:: 20 analysed analyzed specimens ×x 3 analyzsed visual fields = 60 counts of positively stained cells and 60 counts of the total number of cell counts; this resulting resulted in 60 labeling indices per group. Descriptive analysis of lTheabeling index data results were performedare expressed as using the median (ME), the interquartile range (IQR), standard deviation (SD), and range. Graphical Box plot description was performed on diagrams representing represent the median, the interquartile range, minimum (Min), and maximum (Max). Confirmatory comparisons were made performed between treatment and control groups using with generalizsed estimating equations (GEE) with that included the “treatment modality” and the “subject id” as independent factors for appropriate analysis of repeated measurements per individual. Multiple p values were adjusted according to Bonferroni by multiplying each p value obtained by the number of confirmatory tests performed (n=10). Two-sided, adjusted p-values ≤of p <= 0.05 were considered to be significant. All calculations were made usingAnalyses were performed with SPSS 17.0 for Windows (SPSS Inc, Chicago, USA).

Results

Analysis of TGFβ1- expression

Histochemical assessment revealedThe tissue sections comprised connective tissue of variable width between thickened bone formation and the a layer of epithelial layerum (Fig. 1). Observation We consistently showed observed partially confluent necrotic lesions of partial confluency in BRONJ-related bone tissue. The connective tissue and the ECM presented a vVariable densitiesy of inflammatory infiltrates were contained within the connective tissue layers and the ECM. Multinucleated cells were present in all BRONJ samples of BRONJ. Capillaries were seen observed in both BRONJ-related mucoperiosteal specimens and healthy jaw connective tissue[KA7]. In normal jaw mucoperiosteal tissue, TGFβ1 expression was localized toin the cytoplasm of fibroblasts and progenitors within the connective tissue layer of normal jaw mucoperiosteal tissue (Fig. 2a). The spatial pattern of TGFβ1 staining showedwas homogenously distribution distributed within the connective tissue. In the BRONJ-related tissue, a reduced cellular density of TGFβ1 expressing fibroblasts and progenitor cells was noted (Fig. 2b). In the BRONJ group,The staining of connective tissue-related cells was rarewere rarely stained, and the cellular density of TGFβ1 expressing fibroblasts in the fibrous and inflammatory tissue surrounding the bone matrix were less dense was decreased comparedthan those observed in to normal and osteoradionecrosis-related tissue (Fig. 2b, c). Next, we counted tThe number of TGFβ1 expressing cells in the fibrous soft tissue structures, which compriseding periosteal progenitors, fibroblasts, and fibrocytes, and compared to the total number of connective tissue-related cells were counted next. The labeling index of TGFβ1 expressing cells, giving the labeling index relative cellular expression (ratio of TGFβ1 expressing cells/total number of fibrous tissue-related cells) was significantly diminished (p ( 0.032) in the BRONJ group and significantly increased (p( 0.04) in the osteoradionecrosis group compared with to the control mucoperiosteal tissue (Table 1; Fig 2d).