Bone, dentin and cementum differentially influence the differentiation of osteoclast-like cells

Bone, dentin and cementum differentially influence the differentiation of osteoclast-like cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bone, dentin and cementum differentially influence the differentiation of osteoclast-like cells Annika Both, Ghosn Ibrahim, Jana Marciniak, Birgit Rath-Deschner, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5492135/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Our aim was to investigate how different oral hard tissues determine the differentiation of osteoclast-like cells. Murine macrophage cells were stimulated for 12 d with RANKL and M-CSF on dentin slices. Morphological changes of cells and hard tissues were examined by electron microscopy and toluidine blue staining. Cells were stimulated with RANKL and M-CSF on pulverized bone, dentin, cementum or polystyrene – with and without stimulation. TRAP staining was performed. To elucidate total gene expression, RNA sequencing was carried out. Four target genes (CXCL2, IGF-1, GDF15, HSPA1b) were selected and their expression was analyzed by RT-PCR and ELISA. Statistics comprised One-way ANOVA and Tukey’s test (P < 0.05). Stimulation induced differentiation of mouse macrophages into TRAP-positive osteoclast-like cells forming resorption pits on dentin. Gene expression analysis revealed that 1930, 446 and 87 genes were differentially regulated by cultivation on cementum, bone or dentin respectively compared to polystyrene. Culture on bone or dentin caused CXCL2 upregulation. In all stimulated groups IGF-1 was downregulated while GDF15 expression was elevated in cultures on dentin. Cultivation of cells on cementum resulted in an upregulated HSPA1b expression. Our results indicate that extracellular matrix of different oral hard tissues plays an important role in differentiation processes of osteoclast-like cells. Biological sciences/Genetics/Gene expression Health sciences/Biomarkers Health sciences/Risk factors Health sciences/Medical research/Biomarkers Health sciences/Medical research/Genetics research Orthodontic root resorption osteoclast differentiation CXCL2 IGF-1 GDF15 HSPA1b Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Apical root resorption is among the most common and serious complications during orthodontic tooth movement. It can be assumed that there is a complex interplay of patient-specific and therapy related risk factors [1]. However, the biological etiology of this irreversible hard tissue loss of the tooth root by active osteoclast-like cells has not been adequately elucidated to date [2]. During orthodontic treatment, forces initiate a complex metabolic process in the periodontal ligament (PDL) [3]. Proinflammatory cytokines, prostaglandins, chemokines and growth differentiation factors (such as CXCL2 and GDF15) are released which represent a sterile inflammatory reaction and initiate osteoclastic bone resorption [3–5]. At the same time, protective molecules are expressed to preserve tissue homeostasis in the PDL during the force induced stress reaction. In this regard, IGF-1 and HSP70 maintain cell physiology by inhibiting apoptosis and promoting proliferation, chemotaxis, differentiation and cell survival [6, 7]. Studies show that orthodontically induced root resorption is often associated with localized over-compression and sterile coagulation necrosis in the PDL which subsequently turns into a cell-free hyaline zone [8]. As a result, multinucleated cells with osteoclast character, TRAP-negative fibroblast-like cells and macrophages migrate from the adjacent vital PDL and the blood to initiate the removal of the necrotic and hyalinized tissue. In addition, those multinucleated cells with osteoclast character participate in the reorganization of the periodontal ligament [9, 10]. However, it is mostly unavoidable that the degradation of the hyaline zones is accompanied by partial removal of the adjacent cementoid layer, an organic tissue covering the outer tooth root [10]. As a result, the mineralized root surface is denuded, allowing osteoclast-like cells to gain access and adhere to the dental hard tissues [11]. The recognition and attachment to mineral surfaces is mediated by integrins which interact with the extracellular matrix of the underlying substrate [12, 13]. Thereby, the direct contact with certain extracellular matrix proteins causes activation of osteoclast-like cells and thus initiates the resorption of the mineralized root tissues [12]. In the literature, these cells are called cementoclasts or odontoclasts/dentinoclasts which apparently originate from common hematopoietic precursor cells with osteoclasts [1]. Although they have been described to be smaller in size and to form smaller resorption pits on mineralized tissues, they share functional and morphological similarities with osteoclasts including the formation of ruffled borders and sealing zones, active secretion of acids and proteolytic enzymes as well as stimulation by the cytokines RANKL and M-CSF [14]. To date, the specific influencing factors that determine the individual differentiation pathways from hematopoietic precursor cells to specialized clastic cells are not sufficiently clarified. Despite the frequently observed damage to their roots if teeth are orthodontically moved through bone, the extent of resorption in the areas of cementum and dentin is typically significantly lower than in the area of bone. Thus, the process of attachment and activation on the different hard tissues seems to take place to different degrees. Thus, it was the aim of this study to elucidate if the hard tissue extracellular matrix in contact, namely that of bone, dentin or cementum, may have an influence on the differentiation pathway of the osteoclast-like cells. We hypothesize that the progenitor cells sense the respective hard tissue matrix and react with a corresponding differentiation which could explain the different susceptibility of the different hard tissues. Results Differentiation of osteoclast-like cells First, we wanted to evaluate the success of the chosen protocol to differentiate the murine macrophage cells into resorbing osteoclast-like cells. After 3 d of stimulation, attached cells were observed at the edge of dentin disks developing incipient cellular extensions. At the end of the incubation period (12 d), multinucleated cells forming podosomes were visualized by scanning electron microscopy (Fig. 1a). Furthermore, multiple resorption pits were detected on dentine surface by toluidine blue staining (Fig. 1b). Secondly, the murine macrophage cells were stimulated on pulverized bone, dentin and cementum tissue. After 12 d of stimulation, TRAP staining revealed numerous TRAP-positive cells in every hard tissue sample which corroborates the ability of differentiated murine macrophage cells to resorb the different hard tissue substrates they are cultured on (results not shown). Genome-wide gene expression analysis We aimed to investigate the effects of the three different hard tissue substrates on differentiating osteoclast-like cells at the transcriptional level. After RNA extraction and genome-wide gene expression analysis, differential gene expression analysis (DGE) was performed to analyze quantitative possible differentiated changes of gene expression between the different experimental groups (supplementary material S1-S10). In total, cells differentiated on cementum showed the highest number of significantly differentially expressed genes compared to the stimulation control group (1930 different transcripts) and to the negative control group (857 different trancripts), respectively (supplementary material S11-S12). Furthermore, 446 transcripts were significantly differentially regulated on bone and 87 on dentin in comparison to stimulation control (supplementary material S13-S14). Multiple comparisons between the different hard tissue groups revealed that there were 314 differentially expressed genes between the cementum and the dentin group, 252 between the bone and the cementum group and just one between the dentin and the bone group (supplementary material S15-S17). According to our sequencing results, we focused on four selected target genes for further investigation. The first gene was CXCL2, which was significantly upregulated due to our stimulation protocol. In the stimulation control, CXCL2 expression was upregulated by 5.2 fold compared to the negative control (supplementary material S18). We further analyze IGF-1 and GDF15 expression of the differentiated murine macrophage cells which were upregulated in every hard tissue sample in comparison to the negative control group (supplementary material S12, S19-S20). Furthermore, HSPA1b was found to be an interesting gene, as it showed the strongest upregulation in the cementum group in comparison to stimulation (227 fold) and negative control (43,8 fold) (supplementary material S11-S12). RT-PCR The CXCL2 expression was found to be significantly upregulated in cells stimulated on bone, dentin and cementum tissue compared to both control groups. CXCL2 expression was significantly higher in cells differentiated on bone tissue than in cells differentiated on cementum tissue. The comparison between the stimulation and negative control did not provide any significant differences in CXCL2 expression levels (Fig. 2a). RT-PCR revealed that IGF-1 expression was significantly downregulated in the stimulation control as well as in each hard tissue sample as compared to the negative control. The IGF-1 expression of cells cultured on dentin was significantly higher expressed in comparison to the stimulation control and the other two hard tissues (Fig. 2b). In comparison to unstimulated cells, stimulation of murine macrophage cells on polystyrene as well as on dentin tissue led to a significant upregulated GDF15 expression. Cells from the cementum group revealed a significantly decreased GDF15 expression in comparison to stimulation control (Fig. 2c). As indicated by the genome-wide gene expression analysis, the differentiation of murine macrophage cells on cementum tissue resulted in a significantly elevated HSPA1b expression in comparison to the bone, dentin as well as both control groups (Fig. 2d). ELISA Stimulation of the cells on polystyrene alone led to a significant upregulation of CXCL2 protein concentration in the supernatant in comparison to the unstimulated control. On dentin powder, protein formation of CXCL2 was strongly increased in comparison to both controls as well as to the cementum and bone group. Cells stimulated on bone powder showed a significantly upregulated CXCL2 synthesis in comparison to the negative control and a significant lower one in comparison to the stimulation control (Fig. 3a). The protein formation of IGF-1 was significantly downregulated due to stimulation with RANKL and M-CSF on polystyrene in comparison to the unstimulated cells. In addition, IGF-1 formation was significantly downregulated in every hard tissue group in comparison to both controls. The cells which were stimulated on dentin tissue showed a significant higher IGF-1 synthesis in comparison to the cementum and bone groups (Fig. 3b). Cells from the stimulation control group showed a downregulation in GDF15 protein formation compared to the negative control. The protein synthesis of GDF15 was significantly increased in the dentin group in comparison to the unstimulated and stimulated control. Cells which were cultured on bone tissue as well revealed a significant downregulated GDF15 synthesis compared to both controls. In the cementum culture, no detectable GDF15 protein concentration was measurable (Fig. 3c). Finally, a significant increased protein level of HSPA1b was found in the supernatant of the cementum cultures in comparison to all other experimental groups (Fig. 3d). Discussion This study looked for a possible explanation for the different susceptibility of the 3 different oral hard tissues in the context of orthodontically induced periodontal remodeling processes. We provide evidence that the differentiation of osteoclast-like cells is affected by the hard tissue substrate to which they adhere. Our results suggest a particular importance of HSPA1b for resorptive cells cultivated on cementum as compared to the other hard tissues. The resorption of dental hard tissues is caused by active osteoclast-like cells which are supposed to originate from a common hematopoietic progenitor cells with osteoclasts [15]. To date, it is not clear which influencing factors determine the development into specialized clastic cells. Furthermore, there is disagreement in the literature whether the resorption of cementum and dentin is caused either by one common or two specialized types of osteoclast-like cells [1, 14, 16, 17]. The differentiation of osteoclasts is subject to a strict regulation by numerous cytokines and molecular factors. Especially cytokines M-CSF and RANKL are responsible for maturation and cell fusion into differentiated TRAP-positive multinuclear cells which adhere to mineralized tissues via podosomes and induce resorption [18, 19]. Since we used a macrophage cell line in our study, we first wanted to investigate whether these murine macrophage cells can transform into mature osteoclast-like cells through cultivation with RANKL and M-CSF. After 12 d of stimulation, we were able to confirm the differentiation of murine macrophage cells by detecting multinucleation, cell expansion and podosome formation as well as the appearance of resorption lacunae on the dentin surface representing the typical characteristics of active mature osteoclasts. Thus, the stimulation of murine macrophage cells by RANKL and M-CSF proved to be a suitable model for our further experiments on osteoclast differentiation. Next, we wanted to investigate whether the different hard tissues to be resorbed have an influence on the differentiation pathway of maturing osteoclast-like cells. To complement and expand the existing knowledge on osteoclast differentiation mainly on bone and dentin [20–22], we additionally used cementum tissue as an interesting growth matrix for differentiating osteoclast-like cells since the resorption of the cementum layer is described as a decisive factor of external root resorption [1, 23]. Moreover, we were the first to pulverize the hard tissue samples in order to increase the contact area for the adherent cells. Studies provide evidence that chemical and physical properties of the extracellular matrix influence the formation and activity of adherent osteoclasts [20, 22, 24]. Interestingly, Rumpler et al. demonstrated that the osteoclast formation rate was higher on dentin than on bone slices and hypothesized that the amount of non-collagenous proteins such as osteopontin as well as unknown osteocyte-derived proteins could influence osteoclast activity [21]. After cultivation and stimulation of murine macrophage cells on bone, dentin and cementum, we were able to detect TRAP-positive cells on all three tested hard tissue substrates. Thereby, our stimulation protocol proved to be suitable for cell stimulation on bone, dentin and cementum. Differential gene expression analysis revealed significant quantitative changes in gene expression levels between the experimental groups confirming that osteoclastic differentiation was clearly affected by the different hard tissue substrates. Furthermore, the striking variability of gene expression between bone, dentin and cementum resorbing cells indicated that these cells had undergone an individual differentiation process and should therefore be considered as three distinct types of clastic cells after the differentiation process is completed. Therefore, we expand existing knowledge about the physiology of clastic cells affirming studies that claimed dentin and cementum are resorbed by a different type of clastic cells [1, 17]. Based on our sequencing results, we selected four conspicuous target genes for further gene and protein expression analysis: Firstly, we selected the chemokine CXCL2 which is known as a stimulating factor on osteoclast differentiation by promoting proliferation, adhesion and migration of osteoclastic precursor cells [25]. During the early stage of orthodontic tooth movement, chemokines, like CXCL2, are released in the periodontal ligament as part of the acute inflammatory response. These chemokines activate and recruit cells from the murine macrophage line especially to the pressure side of periodontal ligament [26] which mature and fuse into osteoclasts and exert their resorptive activity on alveolar bone as well as on the tooth root surface [4, 27]. Interestingly it was shown that CXCL2 expression is differentially regulated by RANKL through JNK and NF-κB signaling pathways in mouse macrophagic precursor cells to promote osteoclast differentiation [25]. In accordance with the literature, our sequencing results revealed a significant upregulation of CXCL2 in osteoclast-like cells after stimulation by RANKL and M-CSF on polystyrene which was confirmed at the protein level. Furthermore, RT-PCR and ELISA test revealed that osteoclast-like cells which had been cultured on bone and dentin tissue, significantly upregulated CXCL2 gene expression and protein formation in comparison to the unstimulated control. This suggests that CXCL2 is involved in bone as well as in tooth root resorption. Moreover, our sequencing results showed that CXCL2 gene expression was significantly downregulated in osteoclast-like cells which were cultured on cementum tissue in comparison to the stimulation control. This finding was supported at the protein level. Therefore, we hypothesize that cementum may provide anti-inflammatory and thus anti-resorptive effects on differentiating osteoclast-like cells. On the other hand, the low CXCL2 protein concentration in the supernatant of the cementum cultures could also be influenced by the smaller number of differentiated cells grown on cementum. Although we performed a normalization of protein concentration for our analyses, the CXCL2 protein concentration in total was outside the optimal assay range. Further investigations are required to understand how CXCL2 protein expression is involved in clastic differentiation due to contact of the cells with different surfaces. IGF-1 is known as an important growth factor in bone matrix and thus is responsible for the skeletal development [28]. Moreover, the IGF family regulates distinct functions in oral biology including tooth development and growth [29]. During experimental tooth movement, increased IGF-1 expression levels have been detected in PDL-cells providing antiapoptotic and homeostatic effects on the periodontal ligament in response to mechanical strain [30–32]. Furthermore, previous studies reported on the stimulating effect of IGF-1 on osteoclast formation. [33–35]. On the one hand, IGF-1 is released by osteoblasts and binds to the IGF-1 receptor which is expressed on osteoclast precursor cells to stimulate osteoclastogenesis [35]. On the other hand, studies have shown that IGF-1 is secreted by osteoclasts themselves and thus acts in an autocrine manner on osteoclast differentiation [35–37]. Moreover, Götz et al. immunohistochemically detected IGF family members in cementum- and dentin-resorbing odontoclasts as well as in resorption lacunaes during external root resorption [30]. Our genome-wide sequencing points into the direction of an upregulation of IGF-1 expression due to cultivation in the presence of RANKL and M-CSF. In contrast, validation by RT-PCR and ELISA test revealed that cell stimulation by RANKL and M-CSF demonstrated a decreased IGF-1 expression in the osteoclast-like cells in all groups as compared to the negative control. According to the latest literature, Ma et al. demonstrated that IGF-1 expression in RAW264.7 osteoclast-like cells was significantly downregulated at both RNA and protein levels due to stimulation with RANKL [38]. In addition, the reciprocal interaction between IGF-1 and RANKL expression was described in vivo by Xu et al. regarding to the OPG/RANKL/RANK/IGF-1 pathway [39]. Although RNA sequencing has become the gold standard for transcriptome studies, it is a very complex and expensive technique that usually prevents several repetitions of the experiment. Thus, our results emphasize the importance of a repeated validation of the Seq based expression profiles by RT-PCR and the protein assays with respect to IGF-1. Growth differentiator factor 15 (GDF15), also called Macrophage Inhibitory cytokine-I, is a member of the transforming growth factor (TGF-)β and bone morphogenic protein (BMP) superfamily. Initially detected in activated macrophages and described as an autocrine regulator of macrophage activation [40], GDF15 is mainly expressed under pathological states such as tissue injury and inflammation [41]. With respect to orthodontic tooth movement, GDF15 was shown to be secreted by hPDL fibroblasts under mechanical stress and in the following stimulated the GDF15 expression of osteogenic marker genes to increase osteoblast differentiation [42]. Besides, studies proved that GDF15 acts as a proinflammatory promoter for osteoclast differentiation [5, 43, 44]. After RANKL induced stimulation of RAW264.7 cells in the presence of recombinant GDF15, Li et al. detected an increased number of TRAP-positive cells as well as a higher expression of osteoclast differentiation marker genes in comparison to control cells. Thereby, they were able to demonstrate that GDF15 contributes to the force-induced activation of NF-κB and ERK signaling pathways to promote osteoclast differentiation [45]. Regarding to its stress-induced expression and its different roles in regulating cell functions, development and survival, Symmank et al. proposed that GDF15 could be an interesting therapeutic approach for the treatment of bone and dental root resorptions [42]. Our sequencing data revealed a significant upregulation of GDF15 expression in every hard tissue group in comparison to the negative and to the stimulation control group. These results were completed and expanded by the results of the RT-PCR and by ELISA: GDF15 expression was upregulated by cultivation on dentin tissues on the gene and protein level in comparison to the stimulation and to the negative control. As already mentioned, cultivation on cementum resulted in fewer cells and therefore we suspect that the GDF15 protein expression was below the assay range. The HSP70 family is highly conserved during evolution and has been extensively studied in the literature [46, 47]. In the human genome, about 17 genes of the HSP70 family have been identified, including HSPA1b [48]. While some HSP70 genes are constitutively expressed and serve as housekeeping genes, others, like HSPA1b, are inductively expressed in response to environmental stresses such as thermal heat, hypoxic conditions or mechanical strain [49, 50]. Previous studies demonstrated the cytoprotective role of HSP70 in the periodontal ligament [51]. During orthodontic tooth movement, increased HSP70 expression levels were detected in the pressure zone [52] and revealed anti-inflammatory effects to the mechanically loaded periodontal ligament. Thus, HSP70 is supposed to dampen the host's inflammatory tissue response and to prevent excessive tissue loss in the hPDL during orthodontic tooth movement [7, 53, 54]. Furthermore, studies indicated a dampening effect on osteoclast formation which can be related to the suppression of the NF-κB and MAPK signaling pathways [54]. Inhibition of HSP70 clearly increased the number of osteoclasts in mechanically stimulated periodontal ligament cells [7, 51]. Moreover, heat pre-treatment, which induces cytoplasmic upregulation of heat shock proteins, resulted in reduced osteoclast formation [55]. Our sequencing results revealed that culturing mouse macrophage cells on cementum tissue resulted in a strong upregulation of HSPA1b (227 fold) compared to the stimulation control which was confirmed by RT-PCR. The same results were obtained at the protein level. In this context, the strong upregulation of HSPA1b which was induced by direct contact of the cells with the cementum matrix could indicate an autologous inhibition of cementum-resorbing osteoclast-like cells. As the outer root surface usually remains almost undamaged during physiological as well as pathological resorption of the alveolar bone, e.g. periapical periodontitis, the cementum layer is described in literature as a natural protective shield against external root resorption [56–58]. Firstly, pre-cementum and cementoblasts form a non-mineralized organic layer. Clastic cells, however, only seem to resorb anorganic substrates [59]. Secondly, cementocytes upregulate Tnfrsf11b expression which increases the OPG level in the cementum matrix and thus inhibits surrounding osteoclast differentiation [60]. Furthermore, local damage to the cementum is subject to an immediate repair mechanism by cementoblasts to prevent further invasion of clastic cells into deeper layers of the tooth root [1]. Our study suggests that the cementum matrix slows down the resorption process by upregulating HSPA1b expression in maturing osteoclast-like cells and thus acts as natural defense mechanism against the progression of external root resorption. Future studies must show whether this finding can be implemented as a clinical avoidance strategy in the sense of a translation process with local pharmacological induction of HSPA1b. Conclusion In summary, the present results indicate an influence of the different oral hard tissue substrates bone, dentin and cementum on the activity and differentiation of osteoclast-like cells. We identified IGF-1, GDF15 and CXCL2 as significantly regulated target genes in bone-, dentin- and cementum-resorbing osteoclastic cells which represents a useful basis for further investigations elucidating the molecular mechanisms of external root resorption. In addition, we were the first to analyze the expression of HSPA1b in the context of dental root resorption. The clearly increased expression of HSPA1b, which was activated by the cells in contact with the cementum matrix, indicates an autoinhibitory effect in osteoclast-like cells which could attenuate the progressive degradation of the tooth root. This highlights HSPA1b as a possible target gene for therapeutic approaches. Materials and methods Cells and differentiation protocol A murine macrophage cell line has been a widely accepted model for osteoclast maturation and function for 20 years and was used in our study [61]. Firstly, we wanted to evaluate the success of the chosen protocol to differentiate the macrophages into resorbing osteoclast-like cells. The murine macrophage cell line (American Type Culture Collection, #TIB-71, Manassas, VA, USA) was cultured on dentine discs (Immunodiagnostic Systems, #AE-8050, Boldon Colliery, UK) in 24-well plates (30.000 cells/well). The cells were incubated in Dulbecco’s Modified Eagle Medium (Thermo fisher scientific, #11965092, Waltham, MA, USA) supplemented with 10% FBS, 1% Penicillin/ Streptomycin, Plasmocin and Vitamin C at 37°C in an atmosphere of 5% CO 2 . Osteoclast differentiation was induced by the addition of RANKL (Enzo, #ALX-522-131-C010, Farmingdale, NY, USA) and M-CSF (R&D Systems, # 416-ML, Minneapolis, MN, USA). The medium was replaced every 72 h. After 3 and 12 d of stimulation, morphological changes were assessed under light reflection microscope. In addition to morphological changes, resorption pits were analyzed under scanning electron microscope after 12 d of stimulation. Furthermore, toluidine blue staining was used (Sigma Aldrich, #T3260, St. Louis, MO, USA) to identify the resorption pits initiated by the differentiated osteoclast-like cells. Secondly, we wanted to analyze the osteoclast-like character of cells differentiated on three different hard tissue powders. The murine macrophage cells were seeded into 6-well plates (n=6) in a density of 30.000 cells per well and stimulated with 30 ng/mL RANKL (Enzo, #ALX-522-131-C010) and 20 ng/mL M-CSF (R&D Systems, # 416-ML) per well on the three different hard tissues bone, cementum and dentin under the same experimental conditions as described before. After the approval of the Ethics Committee of the University of Bonn and written informed consent by the patients (# 458/22), dentin and cementum were obtained from teeth that had to be extracted for medical reasons. Residual iliac crest bone was obtained from augmentation and dysgnathia surgeries, which would normally have been discarded. To increase the contact area between the hard tissue and the differentiating murine macrophage cells, bone, dentin, and cementum were pulverized using a surgical Lindemann bur (Komet Dental, Lemgo, Germany). Scanning electron microscopy was performed to visualize the different hard tissue powder particles (Fig. 4a-f). The plates containing the hard tissue powder (0.01 g/well) were sterilized under UV light for one hour before cell seeding. As stimulation control group, murine macrophage cells were stimulated with RANKL (Enzo, #ALX-522-131-C010) and 20ng/mL M-CSF (R&D Systems, # 416-ML) on polystyrene without any hard tissue substrate. Cells cultivated on polystyrene without further stimulation served as negative control. After 12 d, tartrate-resistant acid phosphatase (TRAP) staining was performed on the differentiated cells using the Acid Phosphatase Leukocyte Kit (Sigma Aldrich, #387A, St. Louis, MO, USA) regarding to manufacturer’s instructions. The stained cells were analyzed using reflected light microscopy. RNA extraction and genome-wide gene expression analysis The murine macrophage cells were cultured under the same experimental conditions as described before. After 12 d, cells were lysed in RLT-buffer (QIAGEN, #79216, Hilden, Germany), RNA was extracted using the QIAshredder (QIAGEN, #79656, Hilden, Germany) and RNeasy Mini-Kit (QIAGEN, #74106, Hilden, Germany). RNA concentration and purity were measured with Nanodrop (PeqLab, Erlangen, Germany). Afterwards, the extracted RNA was converted into a cDNA library using QuantSeq 3’mRNA-Seq Library Prep Kid FWD (Lexogen, #015.96, Vienna, Austria) for genome-wide gene expression analysis by sequencing according to manufacturer’s instructions. Cluster generation and the sequencing protocol was created by the Next Generation Sequencing (NGS) Core Facility of the Medical Faculty of the University of Bonn. Further information on this step can be found at www.illumina.com . Determination of gene and protein expression The previous experiment was repeated three times for each hard tissue, and the RNA was extracted. Afterwards, the RNA was converted into cDNA with iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, #4106228, Munich, Germany) and PTC-200 Peltier Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). Based on the sequencing results, the expression of conspicuous genes from the genome-wide analysis namely CXCL2, IGF-1, GDF15 and HSPA1b was analyzed by quantitative Realtime-PCR (RT-PCR). For this purpose, 1 µL of cDNA was used in a 25-µL reaction mixture containing 2.5 µL of QuantiTect Primer Assay (CXCL2 #QT00113253, IGF1 #QT02423379, GDf15 #QT00124481, HSPA1b #QT00254436, QIAGEN, Hilden, Germany), 12.5 µL of iQ SYBR Green Supermix (Bio-Rad Laboratories, #1708880, Hercules, CA, USA) and 9 µL of nuclease free water (QIAGEN #129117, Hilden, Germany). For data normalization, a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#QT01658692, QIAGEN, Hilden, Germany) was included in the plate setup and employed for comparative △△-CT analysis with the Software CFX-Manager (Bio-Rad Laboratories, Hercules, CA, USA) and iCycler (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instruction. The applied protocol consisted of a heating phase at 95°C for 5 min for enzyme activation, 40 cycles including a denaturation step at 95°C for 10 s and a combined annealing/ extension step at 60°C for 30 s per cycle with subsequent melting pot analysis after each run. Finally, protein expression of CXCL2, IGF-1, GDF15 and HSPA1b was quantitatively detected from the supernatant of the cell cultures using commercially available enzyme-linked Immunosorbent Assay (ELISA) kits (CXCL2 #MM200, IGF1 #MG100, GDF15 #MGD15, R&D systems, Minneapolis, USA and HSPA1b #E2120Mo, BT LAB, Shanghai, China), regarding to the manufacturer’s instructions. The photometric measurement was performed by the microplate reader PowerWave X (BioTek Instruments, Winooski, VT, USA) at absorbance of 450 nm. The DNA concentration of differentiated cells was used to normalize the measured protein concentrations. Statistical analysis The transcriptional data from RNA-Seq were compared by differential gene expression analysis (DGE) using the statistical software R ( www.r-project.org ). The PCR and ELISA experiments were repeated at least three times. Descriptive analyses of data were presented as means ± standard errors of the mean (SEM). For statistical analysis, we used GraphPad Prism statistics software (Version 7.00 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com ). Normal distribution was examined using the Kolmogorov–Smirnov test. Multiple comparisons were conducted by ANOVA and Tukey’s multiple comparisons test. Differences with P < 0.5 were considered significant. Declarations Acknowledgement: We would like to thank the Next Generation Sequencing Core Facility of the Medical Faculty/West German Genome Center Bonn at the University of Bonn for providing support and instrumentation for the 3'mRNASeq analysis. Author contributions: Conceptualization: S.BM., E.CK., C.K., L.G., A.J.; Methodology: A.B., J.M., G.I., L.G., S.BM.; Formal Analysis and Investigation: A.B., G.I., J.M., L.G., S.BM.; Writing – original draft preparation: A.B., S.BM.; Writing – review and editing: G.I., J.M., B.RD., E.CK., C.K., L.G., A.J; Funding acquisition: C.K., A.J.; Resources: C.K., A.J.; Supervision: S.BM., E.CK., C.K., B.RD., L.G., A.J. All authors read and approved the final manuscript. Competing Interests: The authors declare no competing interests. Funding: This study was supported by the Medical Faculty of the University of Bonn. Data availability: The datasets presented in this study can be found in online repositories. The datasets generated and/or analysed during the current study are available in the Zenodo repository https://doi.org/10.5281/zenodo.14512793. 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Epimedii Folium and Ligustri Lucidi Fructus Promote Osteoblastogenesis and Inhibit Osteoclastogenesis against Osteoporosis via Acting on Osteoblast-Osteoclast Communication. Oxid Med Cell Longev . 2023 , 7212642 (2023). Xu, X. et al. Oral Exposure to ZnO Nanoparticles Disrupt the Structure of Bone in Young Rats via the OPG/RANK/RANKL/IGF-1 Pathway. Int J Nanomedicine . 15 , 9657–9668 (2020). Bootcov, M.R. et al. MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci U S A . 94 , 11514–11519 (1997). Unsicker, K., Spittau, B., Krieglstein, K. The multiple facets of the TGF-β family cytokine growth/differentiation factor-15/macrophage inhibitory cytokine-1. Cytokine Growth Factor Rev . 24 , 373–384 (2013). Symmank, J. et al. Mechanically-induced GDF15 Secretion by Periodontal Ligament Fibroblasts Regulates Osteogenic Transcription. Sci Rep . 9 , 11516 (2019). Hinoi, E. et al. Positive regulation of osteoclastic differentiation by growth differentiation factor 15 upregulated in osteocytic cells under hypoxia. J Bone Miner Res . 27 , 938–949 (2012). Steinmetz, J., Stemmler, A., Hennig, C.L., Symmank, J., Jacobs, C. GDF15 Contributes to the Regulation of the Mechanosensitive Responses of PdL Fibroblasts through the Modulation of IL-37. Dent J (Basel) . 12 , 39 (2024). Li, S., Li, Q., Zhu, Y., Hu, W. GDF15 induced by compressive force contributes to osteoclast differentiation in human periodontal ligament cells. Exp Cell Res . 387 , 111745 (2020). Fernández-Fernández, M.R., Valpuesta, J.M. Hsp70 chaperone: a master player in protein homeostasis. F1000Res . 7 , F1000 Faculty Rev-1497 (2018). Hu, C. et al. Heat shock proteins: Biological functions, pathological roles, and therapeutic opportunities. MedComm . 3 , e161 (2022). 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Yamaguchi, M., Mishima, H. The Role of RANKL and Involvement of Cementum in Orthodontic Root Resorption. Appl. Sci . 11 , 7244 (2011). Aidos, H., Diogo, P., Santos, J.M. Root Resorption Classifications: A Narrative Review and a Clinical Aid Proposal for Routine Assessment. Eur Endod J . 3 , 134–145 (2018). Zhao, N. et al. Isolation and Functional Analysis of an Immortalized Murine Cementocyte Cell Line, IDG-CM6. J Bone Miner Res . 31 , 430–442 (2016). Kong, L., Smith, W., Hao, D. Overview of RAW264.7 for osteoclastogensis study: Phenotype and stimuli. J Cell Mol Med . 23 , 3077–3087 (2019). Additional Declarations No competing interests reported. Supplementary Files S1Neg.ctrlStim.ctrl.xls S1: Transcripts induced in murine macrophage cells stimulated on polystyrene (n=6), fold of stimulation control S2Neg.ctrlBone.xls S2: Transcripts induced in murine macrophage cells stimulated on bone (n=6), fold of negative control S3Neg.ctrlDentin.xls S3: Transcripts induced in murine macrophage cells stimulated on dentin (n=6), fold of negative control S4Neg.ctrlCementum.xls S4: Transcripts induced in murine macrophage cells stimulated on cementum (n=6), fold of negative control S5Stim.ctrlBone.xls S5: Transcripts induced in murine macrophage cells stimulated on bone (n=6), fold of stimulation control S6Stim.ctrlDentin.xls S6: Transcripts induced in murine macrophage cells stimulated on dentin (n=6), fold of stimulation control S7Stim.ctrlCementum.xls S7: Transcripts induced in murine macrophage cells stimulated on cementum (n=6), fold of stimulation control S8BoneDentin.xls S8: Transcripts induced in murine macrophage cells stimulated on dentin (n=6), fold of bone S9BoneCementum.xls S9: Transcripts induced in murine macrophage cells stimulated on cementum (n=6), fold of bone S10CementumDentin.xls S10: Transcripts induced in murine macrophage cells stimulated on dentin (n=6), fold of cementum S11Stim.ctrlCementum.xls S11: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on cementum (n=6), fold of stimulation control S12Neg.ctrlCementum.xls S12: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on cementum (n=6), fold of negative control S13Stim.ctrl.Bone.xls S13: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on bone (n=6), fold of stimulation control S14Stim.ctrl.Dentin1.xls S14: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on dentin (n=6), fold of stimulation control S15CementumDentin1.xls S15: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on dentin (n=6), fold of cementum S16BoneCementum.xls S16: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on cementum (n=6), fold of bone S17BoneDentin.xls S17: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on dentine (n=6), fold of bone S18Neg.ctrlStim.ctrl.xls S18: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on polystyrene (n=6), fold of negative control S19Neg.ctrl.Dentin.xls S19: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on dentin (n=6), fold of negative control S20NegctrlBone.xls S20: Significant transcripts (P<0.05) induced in murine macrophage cells stimulated on bone (n=6), fold of negative control Cite Share Download PDF Status: Published Journal Publication published 05 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 Feb, 2025 Reviews received at journal 14 Jan, 2025 Reviews received at journal 13 Jan, 2025 Reviewers agreed at journal 02 Jan, 2025 Reviewers agreed at journal 01 Jan, 2025 Reviewers invited by journal 31 Dec, 2024 Editor assigned by journal 31 Dec, 2024 Editor invited by journal 19 Dec, 2024 Submission checks completed at journal 18 Dec, 2024 First submitted to journal 20 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5492135","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":392524959,"identity":"65aaa3dd-6b7f-422b-b0a6-9fede7416d30","order_by":0,"name":"Annika Both","email":"","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":false,"prefix":"","firstName":"Annika","middleName":"","lastName":"Both","suffix":""},{"id":392524960,"identity":"305b6475-f6ff-45af-951c-c5ddb145c274","order_by":1,"name":"Ghosn Ibrahim","email":"","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":false,"prefix":"","firstName":"Ghosn","middleName":"","lastName":"Ibrahim","suffix":""},{"id":392524961,"identity":"01ce8208-417f-4a35-8542-662623fbe9cf","order_by":2,"name":"Jana Marciniak","email":"","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":false,"prefix":"","firstName":"Jana","middleName":"","lastName":"Marciniak","suffix":""},{"id":392524962,"identity":"83465d35-5869-4a5c-aacf-a8be96956ac3","order_by":3,"name":"Birgit Rath-Deschner","email":"","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":false,"prefix":"","firstName":"Birgit","middleName":"","lastName":"Rath-Deschner","suffix":""},{"id":392524963,"identity":"870305a6-91f9-4fc2-b4a3-afcce43288d8","order_by":4,"name":"Erika Calvano Küchler","email":"","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"Calvano","lastName":"Küchler","suffix":""},{"id":392524964,"identity":"990dd9bb-8c33-4d74-bc6a-4482ad281a4c","order_by":5,"name":"Christian Kirschneck","email":"","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Kirschneck","suffix":""},{"id":392524965,"identity":"54d483b5-bd3e-4b1c-b59d-162557b9f580","order_by":6,"name":"Lina Gölz","email":"","orcid":"","institution":"Department of Orthodontics and Orofacial Orthopedics, Friedrich-Alexander-University Erlangen-Nürnberg, Gluecksstrasse 11, 91054, Erlangen","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Gölz","suffix":""},{"id":392524967,"identity":"a3fb9b70-8ec4-48a6-8183-65eca104fd99","order_by":7,"name":"Andreas Jäger","email":"","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Jäger","suffix":""},{"id":392524969,"identity":"e28896d9-acff-4bc3-b861-e02f54ff8e7f","order_by":8,"name":"Svenja Beisel-Memmert","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYLACCQTThkECwmMmVktCGpFaECDhMGEt8jOSjz2wqGGI5hc7nfi48sf5xJmzm489LmCwlsOlxeBGWrqBxDGG3Jmzczcbnkm4nThb5li68QyGdGOcWnjOmElIsDHkbridu02yAahlnkSOmTQPw+HEBlwO6zn/TULiH0Pu/tu52382JJyDa6nHpYXheA+bhGQb0Bbp3G2MDQkHEmdDtSTgdNjxNjMJyT6J3Bm3czdLNqQlG8+ccyxNmscg3RCnw5qZn0lLfLPJ7Z+du/Fjg42d7IzbzcekeSqs5XHZAgLMEsjxD7UdnwYGBsYP+OVHwSgYBaNgpAMAIkVS7Hyv164AAAAASUVORK5CYII=","orcid":"","institution":"Department of Orthodontics, Medical Faculty, University Hospital Bonn, Welschnonnenstr. 17, 53111 Bonn","correspondingAuthor":true,"prefix":"","firstName":"Svenja","middleName":"","lastName":"Beisel-Memmert","suffix":""}],"badges":[],"createdAt":"2024-11-20 15:38:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5492135/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5492135/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-04874-9","type":"published","date":"2025-06-05T15:57:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71998840,"identity":"772767e2-09c1-4e4d-907e-0a3fe05d369a","added_by":"auto","created_at":"2024-12-20 12:57:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2205528,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentiation of osteoclast-like cells. (a) Scanning electron microscopy of differentiated osteoclast-like cell on dentin disc with developed podosomes (green arrow) at 1600x magnification. (b) Toluidine blue staining of differentiated osteoclast-like cells on dentin disc visualized by light reflection microscopy at 30x magnification. Resorption pits (RP), multinucleated cells (MC); modified after Ibrahim et al. (2020).\u003c/p\u003e","description":"","filename":"fig1forscirep.png","url":"https://assets-eu.researchsquare.com/files/rs-5492135/v1/97421f7b349180b51fd00c48.png"},{"id":71998866,"identity":"cc9fa5ac-011c-41b3-8d40-01742bbcf994","added_by":"auto","created_at":"2024-12-20 12:57:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4884442,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative gene expression of CXCL2 (a), IGF-1 (b), GDF15 (c) and HSPA1b (d). Statistically significant differences between experimental groups according to Tukey’s multiple comparisons test Data are represented as mean ± SEM; \u0026nbsp;\u003cem\u003en\u003c/em\u003e = 18. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; **** p \u0026lt;0.0001\u003c/p\u003e","description":"","filename":"fig2forscirep.png","url":"https://assets-eu.researchsquare.com/files/rs-5492135/v1/3b1a25b5ca804b28cff16de9.png"},{"id":71998843,"identity":"5a22eb4a-4687-4b0b-ac57-624850e9c3a0","added_by":"auto","created_at":"2024-12-20 12:57:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2380262,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative protein formation of CXCL2 (a), IGF-1 (b), GDF15 (c) and HSPA1b (d). 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control\u003c/p\u003e","description":"","filename":"S20NegctrlBone.xls","url":"https://assets-eu.researchsquare.com/files/rs-5492135/v1/ba0a3b04670105728b43eba5.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bone, dentin and cementum differentially influence the differentiation of osteoclast-like cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eApical root resorption is among the most common and serious complications during orthodontic tooth movement. It can be assumed that there is a complex interplay of patient-specific and therapy related risk factors [1]. However, the biological etiology of this irreversible hard tissue loss of the tooth root by active osteoclast-like cells has not been adequately elucidated to date [2].\u003c/p\u003e \u003cp\u003eDuring orthodontic treatment, forces initiate a complex metabolic process in the periodontal ligament (PDL) [3]. Proinflammatory cytokines, prostaglandins, chemokines and growth differentiation factors (such as CXCL2 and GDF15) are released which represent a sterile inflammatory reaction and initiate osteoclastic bone resorption [3\u0026ndash;5]. At the same time, protective molecules are expressed to preserve tissue homeostasis in the PDL during the force induced stress reaction. In this regard, IGF-1 and HSP70 maintain cell physiology by inhibiting apoptosis and promoting proliferation, chemotaxis, differentiation and cell survival [6, 7].\u003c/p\u003e \u003cp\u003eStudies show that orthodontically induced root resorption is often associated with localized over-compression and sterile coagulation necrosis in the PDL which subsequently turns into a cell-free hyaline zone [8]. As a result, multinucleated cells with osteoclast character, TRAP-negative fibroblast-like cells and macrophages migrate from the adjacent vital PDL and the blood to initiate the removal of the necrotic and hyalinized tissue. In addition, those multinucleated cells with osteoclast character participate in the reorganization of the periodontal ligament [9, 10]. However, it is mostly unavoidable that the degradation of the hyaline zones is accompanied by partial removal of the adjacent cementoid layer, an organic tissue covering the outer tooth root [10]. As a result, the mineralized root surface is denuded, allowing osteoclast-like cells to gain access and adhere to the dental hard tissues [11]. The recognition and attachment to mineral surfaces is mediated by integrins which interact with the extracellular matrix of the underlying substrate [12, 13]. Thereby, the direct contact with certain extracellular matrix proteins causes activation of osteoclast-like cells and thus initiates the resorption of the mineralized root tissues [12].\u003c/p\u003e \u003cp\u003eIn the literature, these cells are called cementoclasts or odontoclasts/dentinoclasts which apparently originate from common hematopoietic precursor cells with osteoclasts [1]. Although they have been described to be smaller in size and to form smaller resorption pits on mineralized tissues, they share functional and morphological similarities with osteoclasts including the formation of ruffled borders and sealing zones, active secretion of acids and proteolytic enzymes as well as stimulation by the cytokines RANKL and M-CSF [14].\u003c/p\u003e \u003cp\u003eTo date, the specific influencing factors that determine the individual differentiation pathways from hematopoietic precursor cells to specialized clastic cells are not sufficiently clarified. Despite the frequently observed damage to their roots if teeth are orthodontically moved through bone, the extent of resorption in the areas of cementum and dentin is typically significantly lower than in the area of bone. Thus, the process of attachment and activation on the different hard tissues seems to take place to different degrees. Thus, it was the aim of this study to elucidate if the hard tissue extracellular matrix in contact, namely that of bone, dentin or cementum, may have an influence on the differentiation pathway of the osteoclast-like cells. We hypothesize that the progenitor cells sense the respective hard tissue matrix and react with a corresponding differentiation which could explain the different susceptibility of the different hard tissues.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDifferentiation of osteoclast-like cells\u003c/h2\u003e \u003cp\u003e First, we wanted to evaluate the success of the chosen protocol to differentiate the murine macrophage cells into resorbing osteoclast-like cells. After 3 d of stimulation, attached cells were observed at the edge of dentin disks developing incipient cellular extensions. At the end of the incubation period (12 d), multinucleated cells forming podosomes were visualized by scanning electron microscopy (Fig.\u0026nbsp;1a). Furthermore, multiple resorption pits were detected on dentine surface by toluidine blue staining (Fig.\u0026nbsp;1b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecondly, the murine macrophage cells were stimulated on pulverized bone, dentin and cementum tissue. After 12 d of stimulation, TRAP staining revealed numerous TRAP-positive cells in every hard tissue sample which corroborates the ability of differentiated murine macrophage cells to resorb the different hard tissue substrates they are cultured on (results not shown).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenome-wide gene expression analysis\u003c/h3\u003e\n\u003cp\u003eWe aimed to investigate the effects of the three different hard tissue substrates on differentiating osteoclast-like cells at the transcriptional level. After RNA extraction and genome-wide gene expression analysis, differential gene expression analysis (DGE) was performed to analyze quantitative possible differentiated changes of gene expression between the different experimental groups (supplementary material S1-S10). In total, cells differentiated on cementum showed the highest number of significantly differentially expressed genes compared to the stimulation control group (1930 different transcripts) and to the negative control group (857 different trancripts), respectively (supplementary material S11-S12). Furthermore, 446 transcripts were significantly differentially regulated on bone and 87 on dentin in comparison to stimulation control (supplementary material S13-S14). Multiple comparisons between the different hard tissue groups revealed that there were 314 differentially expressed genes between the cementum and the dentin group, 252 between the bone and the cementum group and just one between the dentin and the bone group (supplementary material S15-S17).\u003c/p\u003e \u003cp\u003eAccording to our sequencing results, we focused on four selected target genes for further investigation. The first gene was CXCL2, which was significantly upregulated due to our stimulation protocol. In the stimulation control, CXCL2 expression was upregulated by 5.2 fold compared to the negative control (supplementary material S18). We further analyze IGF-1 and GDF15 expression of the differentiated murine macrophage cells which were upregulated in every hard tissue sample in comparison to the negative control group (supplementary material S12, S19-S20). Furthermore, HSPA1b was found to be an interesting gene, as it showed the strongest upregulation in the cementum group in comparison to stimulation (227 fold) and negative control (43,8 fold) (supplementary material S11-S12).\u003c/p\u003e\n\u003ch3\u003eRT-PCR\u003c/h3\u003e\n\u003cp\u003eThe CXCL2 expression was found to be significantly upregulated in cells stimulated on bone, dentin and cementum tissue compared to both control groups. CXCL2 expression was significantly higher in cells differentiated on bone tissue than in cells differentiated on cementum tissue. The comparison between the stimulation and negative control did not provide any significant differences in CXCL2 expression levels (Fig.\u0026nbsp;2a).\u003c/p\u003e \u003cp\u003eRT-PCR revealed that IGF-1 expression was significantly downregulated in the stimulation control as well as in each hard tissue sample as compared to the negative control. The IGF-1 expression of cells cultured on dentin was significantly higher expressed in comparison to the stimulation control and the other two hard tissues (Fig.\u0026nbsp;2b).\u003c/p\u003e \u003cp\u003eIn comparison to unstimulated cells, stimulation of murine macrophage cells on polystyrene as well as on dentin tissue led to a significant upregulated GDF15 expression. Cells from the cementum group revealed a significantly decreased GDF15 expression in comparison to stimulation control (Fig.\u0026nbsp;2c).\u003c/p\u003e \u003cp\u003e As indicated by the genome-wide gene expression analysis, the differentiation of murine macrophage cells on cementum tissue resulted in a significantly elevated HSPA1b expression in comparison to the bone, dentin as well as both control groups (Fig.\u0026nbsp;2d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eStimulation of the cells on polystyrene alone led to a significant upregulation of CXCL2 protein concentration in the supernatant in comparison to the unstimulated control. On dentin powder, protein formation of CXCL2 was strongly increased in comparison to both controls as well as to the cementum and bone group. Cells stimulated on bone powder showed a significantly upregulated CXCL2 synthesis in comparison to the negative control and a significant lower one in comparison to the stimulation control (Fig.\u0026nbsp;3a).\u003c/p\u003e \u003cp\u003e The protein formation of IGF-1 was significantly downregulated due to stimulation with RANKL and M-CSF on polystyrene in comparison to the unstimulated cells. In addition, IGF-1 formation was significantly downregulated in every hard tissue group in comparison to both controls. The cells which were stimulated on dentin tissue showed a significant higher IGF-1 synthesis in comparison to the cementum and bone groups (Fig.\u0026nbsp;3b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCells from the stimulation control group showed a downregulation in GDF15 protein formation compared to the negative control. The protein synthesis of GDF15 was significantly increased in the dentin group in comparison to the unstimulated and stimulated control. Cells which were cultured on bone tissue as well revealed a significant downregulated GDF15 synthesis compared to both controls. In the cementum culture, no detectable GDF15 protein concentration was measurable (Fig.\u0026nbsp;3c).\u003c/p\u003e \u003cp\u003eFinally, a significant increased protein level of HSPA1b was found in the supernatant of the cementum cultures in comparison to all other experimental groups (Fig.\u0026nbsp;3d).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study looked for a possible explanation for the different susceptibility of the 3 different oral hard tissues in the context of orthodontically induced periodontal remodeling processes. We provide evidence that the differentiation of osteoclast-like cells is affected by the hard tissue substrate to which they adhere. Our results suggest a particular importance of HSPA1b for resorptive cells cultivated on cementum as compared to the other hard tissues.\u003c/p\u003e \u003cp\u003eThe resorption of dental hard tissues is caused by active osteoclast-like cells which are supposed to originate from a common hematopoietic progenitor cells with osteoclasts [15]. To date, it is not clear which influencing factors determine the development into specialized clastic cells. Furthermore, there is disagreement in the literature whether the resorption of cementum and dentin is caused either by one common or two specialized types of osteoclast-like cells [1, 14, 16, 17].\u003c/p\u003e \u003cp\u003eThe differentiation of osteoclasts is subject to a strict regulation by numerous cytokines and molecular factors. Especially cytokines M-CSF and RANKL are responsible for maturation and cell fusion into differentiated TRAP-positive multinuclear cells which adhere to mineralized tissues via podosomes and induce resorption [18, 19]. Since we used a macrophage cell line in our study, we first wanted to investigate whether these murine macrophage cells can transform into mature osteoclast-like cells through cultivation with RANKL and M-CSF. After 12 d of stimulation, we were able to confirm the differentiation of murine macrophage cells by detecting multinucleation, cell expansion and podosome formation as well as the appearance of resorption lacunae on the dentin surface representing the typical characteristics of active mature osteoclasts. Thus, the stimulation of murine macrophage cells by RANKL and M-CSF proved to be a suitable model for our further experiments on osteoclast differentiation.\u003c/p\u003e \u003cp\u003eNext, we wanted to investigate whether the different hard tissues to be resorbed have an influence on the differentiation pathway of maturing osteoclast-like cells.\u003c/p\u003e \u003cp\u003eTo complement and expand the existing knowledge on osteoclast differentiation mainly on bone and dentin [20\u0026ndash;22], we additionally used cementum tissue as an interesting growth matrix for differentiating osteoclast-like cells since the resorption of the cementum layer is described as a decisive factor of external root resorption [1, 23]. Moreover, we were the first to pulverize the hard tissue samples in order to increase the contact area for the adherent cells.\u003c/p\u003e \u003cp\u003eStudies provide evidence that chemical and physical properties of the extracellular matrix influence the formation and activity of adherent osteoclasts [20, 22, 24]. Interestingly, Rumpler et al. demonstrated that the osteoclast formation rate was higher on dentin than on bone slices and hypothesized that the amount of non-collagenous proteins such as osteopontin as well as unknown osteocyte-derived proteins could influence osteoclast activity [21].\u003c/p\u003e \u003cp\u003eAfter cultivation and stimulation of murine macrophage cells on bone, dentin and cementum, we were able to detect TRAP-positive cells on all three tested hard tissue substrates. Thereby, our stimulation protocol proved to be suitable for cell stimulation on bone, dentin and cementum.\u003c/p\u003e \u003cp\u003eDifferential gene expression analysis revealed significant quantitative changes in gene expression levels between the experimental groups confirming that osteoclastic differentiation was clearly affected by the different hard tissue substrates. Furthermore, the striking variability of gene expression between bone, dentin and cementum resorbing cells indicated that these cells had undergone an individual differentiation process and should therefore be considered as three distinct types of clastic cells after the differentiation process is completed. Therefore, we expand existing knowledge about the physiology of clastic cells affirming studies that claimed dentin and cementum are resorbed by a different type of clastic cells [1, 17].\u003c/p\u003e \u003cp\u003eBased on our sequencing results, we selected four conspicuous target genes for further gene and protein expression analysis:\u003c/p\u003e \u003cp\u003eFirstly, we selected the chemokine CXCL2 which is known as a stimulating factor on osteoclast differentiation by promoting proliferation, adhesion and migration of osteoclastic precursor cells [25]. During the early stage of orthodontic tooth movement, chemokines, like CXCL2, are released in the periodontal ligament as part of the acute inflammatory response. These chemokines activate and recruit cells from the murine macrophage line especially to the pressure side of periodontal ligament [26] which mature and fuse into osteoclasts and exert their resorptive activity on alveolar bone as well as on the tooth root surface [4, 27]. Interestingly it was shown that CXCL2 expression is differentially regulated by RANKL through JNK and NF-κB signaling pathways in mouse macrophagic precursor cells to promote osteoclast differentiation [25]. In accordance with the literature, our sequencing results revealed a significant upregulation of CXCL2 in osteoclast-like cells after stimulation by RANKL and M-CSF on polystyrene which was confirmed at the protein level. Furthermore, RT-PCR and ELISA test revealed that osteoclast-like cells which had been cultured on bone and dentin tissue, significantly upregulated CXCL2 gene expression and protein formation in comparison to the unstimulated control. This suggests that CXCL2 is involved in bone as well as in tooth root resorption. Moreover, our sequencing results showed that CXCL2 gene expression was significantly downregulated in osteoclast-like cells which were cultured on cementum tissue in comparison to the stimulation control. This finding was supported at the protein level. Therefore, we hypothesize that cementum may provide anti-inflammatory and thus anti-resorptive effects on differentiating osteoclast-like cells. On the other hand, the low CXCL2 protein concentration in the supernatant of the cementum cultures could also be influenced by the smaller number of differentiated cells grown on cementum. Although we performed a normalization of protein concentration for our analyses, the CXCL2 protein concentration in total was outside the optimal assay range. Further investigations are required to understand how CXCL2 protein expression is involved in clastic differentiation due to contact of the cells with different surfaces.\u003c/p\u003e \u003cp\u003eIGF-1 is known as an important growth factor in bone matrix and thus is responsible for the skeletal development [28]. Moreover, the IGF family regulates distinct functions in oral biology including tooth development and growth [29]. During experimental tooth movement, increased IGF-1 expression levels have been detected in PDL-cells providing antiapoptotic and homeostatic effects on the periodontal ligament in response to mechanical strain [30\u0026ndash;32]. Furthermore, previous studies reported on the stimulating effect of IGF-1 on osteoclast formation. [33\u0026ndash;35]. On the one hand, IGF-1 is released by osteoblasts and binds to the IGF-1 receptor which is expressed on osteoclast precursor cells to stimulate osteoclastogenesis [35]. On the other hand, studies have shown that IGF-1 is secreted by osteoclasts themselves and thus acts in an autocrine manner on osteoclast differentiation [35\u0026ndash;37]. Moreover, G\u0026ouml;tz et al. immunohistochemically detected IGF family members in cementum- and dentin-resorbing odontoclasts as well as in resorption lacunaes during external root resorption [30]. Our genome-wide sequencing points into the direction of an upregulation of IGF-1 expression due to cultivation in the presence of RANKL and M-CSF. In contrast, validation by RT-PCR and ELISA test revealed that cell stimulation by RANKL and M-CSF demonstrated a decreased IGF-1 expression in the osteoclast-like cells in all groups as compared to the negative control. According to the latest literature, Ma et al. demonstrated that IGF-1 expression in RAW264.7 osteoclast-like cells was significantly downregulated at both RNA and protein levels due to stimulation with RANKL [38]. In addition, the reciprocal interaction between IGF-1 and RANKL expression was described \u003cem\u003ein vivo\u003c/em\u003e by Xu et al. regarding to the OPG/RANKL/RANK/IGF-1 pathway [39]. Although RNA sequencing has become the gold standard for transcriptome studies, it is a very complex and expensive technique that usually prevents several repetitions of the experiment. Thus, our results emphasize the importance of a repeated validation of the Seq based expression profiles by RT-PCR and the protein assays with respect to IGF-1.\u003c/p\u003e \u003cp\u003eGrowth differentiator factor 15 (GDF15), also called Macrophage Inhibitory cytokine-I, is a member of the transforming growth factor (TGF-)β and bone morphogenic protein (BMP) superfamily. Initially detected in activated macrophages and described as an autocrine regulator of macrophage activation [40], GDF15 is mainly expressed under pathological states such as tissue injury and inflammation [41]. With respect to orthodontic tooth movement, GDF15 was shown to be secreted by hPDL fibroblasts under mechanical stress and in the following stimulated the GDF15 expression of osteogenic marker genes to increase osteoblast differentiation [42]. Besides, studies proved that GDF15 acts as a proinflammatory promoter for osteoclast differentiation [5, 43, 44]. After RANKL induced stimulation of RAW264.7 cells in the presence of recombinant GDF15, Li et al. detected an increased number of TRAP-positive cells as well as a higher expression of osteoclast differentiation marker genes in comparison to control cells. Thereby, they were able to demonstrate that GDF15 contributes to the force-induced activation of NF-κB and ERK signaling pathways to promote osteoclast differentiation [45]. Regarding to its stress-induced expression and its different roles in regulating cell functions, development and survival, Symmank et al. proposed that GDF15 could be an interesting therapeutic approach for the treatment of bone and dental root resorptions [42].\u003c/p\u003e \u003cp\u003eOur sequencing data revealed a significant upregulation of GDF15 expression in every hard tissue group in comparison to the negative and to the stimulation control group. These results were completed and expanded by the results of the RT-PCR and by ELISA: GDF15 expression was upregulated by cultivation on dentin tissues on the gene and protein level in comparison to the stimulation and to the negative control. As already mentioned, cultivation on cementum resulted in fewer cells and therefore we suspect that the GDF15 protein expression was below the assay range.\u003c/p\u003e \u003cp\u003eThe HSP70 family is highly conserved during evolution and has been extensively studied in the literature [46, 47]. In the human genome, about 17 genes of the HSP70 family have been identified, including HSPA1b [48]. While some HSP70 genes are constitutively expressed and serve as housekeeping genes, others, like HSPA1b, are inductively expressed in response to environmental stresses such as thermal heat, hypoxic conditions or mechanical strain [49, 50]. Previous studies demonstrated the cytoprotective role of HSP70 in the periodontal ligament [51]. During orthodontic tooth movement, increased HSP70 expression levels were detected in the pressure zone [52] and revealed anti-inflammatory effects to the mechanically loaded periodontal ligament. Thus, HSP70 is supposed to dampen the host's inflammatory tissue response and to prevent excessive tissue loss in the hPDL during orthodontic tooth movement [7, 53, 54]. Furthermore, studies indicated a dampening effect on osteoclast formation which can be related to the suppression of the NF-κB and MAPK signaling pathways [54]. Inhibition of HSP70 clearly increased the number of osteoclasts in mechanically stimulated periodontal ligament cells [7, 51]. Moreover, heat pre-treatment, which induces cytoplasmic upregulation of heat shock proteins, resulted in reduced osteoclast formation [55]. Our sequencing results revealed that culturing mouse macrophage cells on cementum tissue resulted in a strong upregulation of HSPA1b (227 fold) compared to the stimulation control which was confirmed by RT-PCR. The same results were obtained at the protein level.\u003c/p\u003e \u003cp\u003eIn this context, the strong upregulation of HSPA1b which was induced by direct contact of the cells with the cementum matrix could indicate an autologous inhibition of cementum-resorbing osteoclast-like cells. As the outer root surface usually remains almost undamaged during physiological as well as pathological resorption of the alveolar bone, e.g. periapical periodontitis, the cementum layer is described in literature as a natural protective shield against external root resorption [56\u0026ndash;58]. Firstly, pre-cementum and cementoblasts form a non-mineralized organic layer. Clastic cells, however, only seem to resorb anorganic substrates [59]. Secondly, cementocytes upregulate Tnfrsf11b expression which increases the OPG level in the cementum matrix and thus inhibits surrounding osteoclast differentiation [60]. Furthermore, local damage to the cementum is subject to an immediate repair mechanism by cementoblasts to prevent further invasion of clastic cells into deeper layers of the tooth root [1]. Our study suggests that the cementum matrix slows down the resorption process by upregulating HSPA1b expression in maturing osteoclast-like cells and thus acts as natural defense mechanism against the progression of external root resorption. Future studies must show whether this finding can be implemented as a clinical avoidance strategy in the sense of a translation process with local pharmacological induction of HSPA1b.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the present results indicate an influence of the different oral hard tissue substrates bone, dentin and cementum on the activity and differentiation of osteoclast-like cells. We identified IGF-1, GDF15 and CXCL2 as significantly regulated target genes in bone-, dentin- and cementum-resorbing osteoclastic cells which represents a useful basis for further investigations elucidating the molecular mechanisms of external root resorption. In addition, we were the first to analyze the expression of HSPA1b in the context of dental root resorption. The clearly increased expression of HSPA1b, which was activated by the cells in contact with the cementum matrix, indicates an autoinhibitory effect in osteoclast-like cells which could attenuate the progressive degradation of the tooth root. This highlights HSPA1b as a possible target gene for therapeutic approaches.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eCells and differentiation protocol\u003c/h2\u003e\n\u003cp\u003eA murine macrophage cell line has been a widely accepted model for osteoclast maturation and function for 20 years and was used in our study [61].\u003c/p\u003e\n\u003cp\u003eFirstly, we wanted to evaluate the success of the chosen protocol to differentiate the macrophages into resorbing osteoclast-like cells. The murine macrophage cell line (American Type Culture Collection, #TIB-71, Manassas, VA, USA) was cultured on dentine discs (Immunodiagnostic Systems, #AE-8050, Boldon Colliery, UK) in 24-well plates (30.000 cells/well). The cells were incubated in Dulbecco\u0026rsquo;s Modified Eagle Medium (Thermo fisher scientific, #11965092, Waltham, MA, USA) supplemented with 10% FBS, 1% Penicillin/ Streptomycin, Plasmocin and Vitamin C at 37\u0026deg;C in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. Osteoclast differentiation was induced by the addition of RANKL (Enzo, #ALX-522-131-C010, Farmingdale, NY, USA) and M-CSF (R\u0026amp;D Systems, # 416-ML, Minneapolis, MN, USA). The medium was replaced every 72 h. After 3 and 12 d of stimulation, morphological changes were assessed under light reflection microscope. In addition to morphological changes, resorption pits were analyzed under scanning electron microscope after 12 d of stimulation. Furthermore, toluidine blue staining was used (Sigma Aldrich, #T3260, St. Louis, MO, USA) to identify the resorption pits initiated by the differentiated osteoclast-like cells.\u003c/p\u003e\n\u003cp\u003eSecondly, we wanted to analyze the osteoclast-like character of cells differentiated on three different hard tissue powders. The murine macrophage cells were seeded into 6-well plates (n=6) in a density of 30.000 cells per well and stimulated with 30 ng/mL RANKL (Enzo, #ALX-522-131-C010) and 20 ng/mL M-CSF (R\u0026amp;D Systems, # 416-ML) per well on the three different hard tissues bone, cementum and dentin under the same experimental conditions as described before. After the approval of the Ethics Committee of the University of Bonn and written informed consent by the patients (# 458/22), dentin and cementum were obtained from teeth that had to be extracted for medical reasons. Residual iliac crest bone was obtained from augmentation and dysgnathia surgeries, which would normally have been discarded. To increase the contact area between the hard tissue and the differentiating murine macrophage cells, bone, dentin, and cementum were pulverized using a surgical Lindemann bur (Komet Dental, Lemgo, Germany). Scanning electron microscopy was performed to visualize the different hard tissue powder particles (Fig. 4a-f). The plates containing the hard tissue powder (0.01 g/well) were sterilized under UV light for one hour before cell seeding.\u003c/p\u003e\n\u003cp\u003eAs stimulation control group, murine macrophage cells were stimulated with RANKL (Enzo, #ALX-522-131-C010) and 20ng/mL M-CSF (R\u0026amp;D Systems, # 416-ML) on polystyrene without any hard tissue substrate. Cells cultivated on polystyrene without further stimulation served as negative control.\u003c/p\u003e\n\u003cp\u003eAfter 12 d, tartrate-resistant acid phosphatase (TRAP) staining was performed on the differentiated cells using the Acid Phosphatase Leukocyte Kit (Sigma Aldrich, #387A, St. Louis, MO, USA) regarding to manufacturer\u0026rsquo;s instructions. The stained cells were analyzed using reflected light microscopy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA extraction and genome-wide gene expression analysis\u003c/h2\u003e\n\u003cp\u003eThe murine macrophage cells were cultured under the same experimental conditions as described before. After 12 d, cells were lysed in RLT-buffer (QIAGEN, #79216, Hilden, Germany), RNA was extracted using the QIAshredder (QIAGEN, #79656, Hilden, Germany) and RNeasy Mini-Kit (QIAGEN, #74106, Hilden, Germany). RNA concentration and purity were measured with Nanodrop (PeqLab, Erlangen, Germany).\u003c/p\u003e\n\u003cp\u003eAfterwards, the extracted RNA was converted into a cDNA library using QuantSeq 3\u0026rsquo;mRNA-Seq Library Prep Kid FWD (Lexogen, #015.96, Vienna, Austria) for genome-wide gene expression analysis by sequencing according to manufacturer\u0026rsquo;s instructions. Cluster generation and the sequencing protocol was created by the Next Generation Sequencing (NGS) Core Facility of the Medical Faculty of the University of Bonn. Further information on this step can be found at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.illumina.com\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eDetermination of gene and protein expression\u003c/h2\u003e\n\u003cp\u003eThe previous experiment was repeated three times for each hard tissue, and the RNA was extracted. Afterwards, the RNA was converted into cDNA with iScript\u0026trade; Select cDNA Synthesis Kit (Bio-Rad Laboratories, #4106228, Munich, Germany) and PTC-200 Peltier Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA).\u003c/p\u003e\n\u003cp\u003eBased on the sequencing results, the expression of conspicuous genes from the genome-wide analysis namely CXCL2, IGF-1, GDF15 and HSPA1b was analyzed by quantitative Realtime-PCR (RT-PCR). For this purpose, 1 \u0026micro;L of cDNA was used in a 25-\u0026micro;L reaction mixture containing 2.5 \u0026micro;L of QuantiTect Primer Assay (CXCL2 #QT00113253, IGF1 #QT02423379, GDf15 #QT00124481, HSPA1b #QT00254436, QIAGEN, Hilden, Germany), 12.5 \u0026micro;L of iQ SYBR Green Supermix (Bio-Rad Laboratories, #1708880, Hercules, CA, USA) and 9 \u0026micro;L of nuclease free water (QIAGEN #129117, Hilden, Germany).\u003c/p\u003e\n\u003cp\u003eFor data normalization, a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#QT01658692, QIAGEN, Hilden, Germany) was included in the plate setup and employed for comparative △△-CT analysis with the Software CFX-Manager (Bio-Rad Laboratories, Hercules, CA, USA) and iCycler (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer\u0026rsquo;s instruction. The applied protocol consisted of a heating phase at 95\u0026deg;C for 5 min for enzyme activation, 40 cycles including a denaturation step at 95\u0026deg;C for 10 s and a combined annealing/ extension step at 60\u0026deg;C for 30 s per cycle with subsequent melting pot analysis after each run.\u003c/p\u003e\n\u003cp\u003eFinally, protein expression of CXCL2, IGF-1, GDF15 and HSPA1b was quantitatively detected from the supernatant of the cell cultures using commercially available enzyme-linked Immunosorbent Assay (ELISA) kits (CXCL2 #MM200, IGF1 #MG100, GDF15 #MGD15, R\u0026amp;D systems, Minneapolis, USA and HSPA1b #E2120Mo, BT LAB, Shanghai, China), regarding to the manufacturer\u0026rsquo;s instructions. The photometric measurement was performed by the microplate reader PowerWave X (BioTek Instruments, Winooski, VT, USA) at absorbance of 450 nm. The DNA concentration of differentiated cells was used to normalize the measured protein concentrations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eThe transcriptional data from RNA-Seq were compared by differential gene expression analysis (DGE) using the statistical software R (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.r-project.org\u003c/span\u003e\u003c/span\u003e). The PCR and ELISA experiments were repeated at least three times. Descriptive analyses of data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the mean (SEM). For statistical analysis, we used GraphPad Prism statistics software (Version 7.00 for Windows, GraphPad Software, San Diego, California, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.graphpad.com\u003c/span\u003e\u003c/span\u003e). Normal distribution was examined using the Kolmogorov\u0026ndash;Smirnov test. Multiple comparisons were conducted by ANOVA and Tukey\u0026rsquo;s multiple comparisons test. Differences with P\u0026thinsp;\u0026lt;\u0026thinsp;0.5 were considered significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e We would like to thank the Next Generation Sequencing Core Facility of the Medical Faculty/West German Genome Center Bonn at the University of Bonn for providing support and instrumentation for the 3\u0026apos;mRNASeq analysis. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eConceptualization: S.BM., E.CK., C.K., L.G., A.J.; Methodology: A.B., J.M., G.I., L.G., S.BM.; Formal Analysis and Investigation: A.B., G.I., J.M., L.G., S.BM.; Writing \u0026ndash; original draft preparation: A.B., S.BM.; Writing \u0026ndash; review and editing: G.I., J.M., B.RD., E.CK., C.K., L.G., A.J; Funding acquisition: C.K., A.J.; Resources: C.K., A.J.; Supervision: S.BM., E.CK., C.K., B.RD., L.G., A.J. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by the Medical Faculty of the University of Bonn.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e The datasets presented in this study can be found in online repositories. The datasets generated and/or analysed during the current study are available in the Zenodo repository https://doi.org/10.5281/zenodo.14512793.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement:\u003c/strong\u003e All methods were carried out in accordance with the relevant guidelines and regulations from the Ethics Committee of the University of Bonn. All experimental protocols were approved by the Ethics Committee of the University of Bonn. Written informed consent was obtained from all subjects and/or their legal guardian(s).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Feller, L., Khammissa, R.A., Thomadakis, G., Fourie, J., Lemmer, J. Apical External Root Resorption and Repair in Orthodontic Tooth Movement: Biological Events. \u003cem\u003eBiomed Res Int\u003c/em\u003e. \u003cb\u003e2016\u003c/b\u003e, 4864195 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sameshima, G.T., Iglesias-Linares, A. Orthodontic root resorption. \u003cem\u003eJ World Fed Orthod\u003c/em\u003e.\u003cb\u003e10\u003c/b\u003e, 135\u0026ndash;143 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li, Y., Jacox, L.A., Little, S.H., Ko, C.C. 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Overview of RAW264.7 for osteoclastogensis study: Phenotype and stimuli. \u003cem\u003eJ Cell Mol Med\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e, 3077\u0026ndash;3087 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Orthodontic root resorption, osteoclast differentiation, CXCL2, IGF-1, GDF15, HSPA1b","lastPublishedDoi":"10.21203/rs.3.rs-5492135/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5492135/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOur aim was to investigate how different oral hard tissues determine the differentiation of osteoclast-like cells. Murine macrophage cells were stimulated for 12 d with RANKL and M-CSF on dentin slices. Morphological changes of cells and hard tissues were examined by electron microscopy and toluidine blue staining. Cells were stimulated with RANKL and M-CSF on pulverized bone, dentin, cementum or polystyrene \u0026ndash; with and without stimulation. TRAP staining was performed. To elucidate total gene expression, RNA sequencing was carried out. Four target genes (CXCL2, IGF-1, GDF15, HSPA1b) were selected and their expression was analyzed by RT-PCR and ELISA. Statistics comprised One-way ANOVA and Tukey\u0026rsquo;s test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Stimulation induced differentiation of mouse macrophages into TRAP-positive osteoclast-like cells forming resorption pits on dentin. Gene expression analysis revealed that 1930, 446 and 87 genes were differentially regulated by cultivation on cementum, bone or dentin respectively compared to polystyrene. Culture on bone or dentin caused CXCL2 upregulation. In all stimulated groups IGF-1 was downregulated while GDF15 expression was elevated in cultures on dentin. Cultivation of cells on cementum resulted in an upregulated HSPA1b expression. Our results indicate that extracellular matrix of different oral hard tissues plays an important role in differentiation processes of osteoclast-like cells.\u003c/p\u003e","manuscriptTitle":"Bone, dentin and cementum differentially influence the differentiation of osteoclast-like cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-20 12:57:10","doi":"10.21203/rs.3.rs-5492135/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-14T08:52:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-14T08:47:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-13T14:42:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139177640156036906530225270443887161412","date":"2025-01-02T08:41:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9429097159227337634616711686790653905","date":"2025-01-01T23:07:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-01T00:06:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-01T00:05:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-12-19T18:46:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-18T11:02:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-11-20T15:23:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b718a5d-57a3-4a10-a97c-63f585897458","owner":[],"postedDate":"December 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41846022,"name":"Biological sciences/Genetics/Gene expression"},{"id":41846023,"name":"Health sciences/Biomarkers"},{"id":41846024,"name":"Health sciences/Risk factors"},{"id":41846025,"name":"Health sciences/Medical research/Biomarkers"},{"id":41846026,"name":"Health sciences/Medical research/Genetics research"}],"tags":[],"updatedAt":"2025-06-09T16:08:12+00:00","versionOfRecord":{"articleIdentity":"rs-5492135","link":"https://doi.org/10.1038/s41598-025-04874-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-05 15:57:53","publishedOnDateReadable":"June 5th, 2025"},"versionCreatedAt":"2024-12-20 12:57:10","video":"","vorDoi":"10.1038/s41598-025-04874-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-04874-9","workflowStages":[]},"version":"v1","identity":"rs-5492135","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5492135","identity":"rs-5492135","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}