The profiling and analysis of gene expression in human periodontal ligament tissue and fibroblasts

Abstract Objectives The periodontal ligament (PDL) is an important component of periodontium to support dental structure in the alveolar socket. Regeneration of PDL tissue is an effective treatment option for periodontal disease and the profiling of genes involved in this process will be informative. Therefore, our study aims to accurately delineate the profiling of gene expression for PDL tissue regeneration. Materials and Methods We isolated PDL tissues and PDL fibroblasts (PDLFs) from premolar teeth, which were extracted from healthy periodontal status patients undergoing orthodontic treatment. Messenger RNA (mRNA) expression in PDL tissue and PDLFs were analyzed using Cap analysis gene expression, which is a second‐generation sequencing technique to create profiling. We also determined the protein expression using Western blot. Results Collagens (type I, III, and VI), noncollagenous proteins (periostin and osteonectin), and proteoglycans (asporin, lumican, decorin, and osteomodulin) were highly expressed in PDL tissue. Integrin, β1 was also expressed in PDL tissue. On comparison of gene expression between PDL tissue and PDLFs, four PDL marker genes, osteopontin, asporin, periostin, and osteonectin, were decreased in PDLFs. The genes for gene regulation were also highly expressed. Conclusions Our study demonstrated the overall profiling of mRNA expression in PDL tissue and analyzed the important genes which may be useful for providing specific information for the reconstruction of PDL. We also identified the difference in gene expression between PDL tissue and PDLFs which might provide insights towards PDL regeneration.


| INTRODUCTION
Periodontium is a critical complex tissue that consists of gingiva, cementum, alveolar bone, and periodontal ligament (PDL). PDL connects the tooth into the alveolar socket by maintaining the attachment between the dental root and bone surface, and provides a cushion for occlusal loading in the masticatory system. Moreover, PDL is able to remodel itself, responding to mechanical stress and maintaining its structure and function under physiologic conditions (Chukkapalli & Lele, 2018;Smith et al. 2019). From a pathological perspective, mechanical overloading may result in the destruction of PDL structure. In current clinical practice, scaling and root planing are commonly performed. However, these treatments are not applicable in severed periodontitis status patients. Thus, PDL regeneration is required as a new treatment option.
Extracellular matrixes (ECMs) are considered as key factors for the tissue repair. In previous studies, collagens, proteoglycans, and noncollagenous proteins were identified as the key ECMs of PDL, (Berkovitz, 1990;Butler et al., 1975;Dublet et al., 1988;Embery, 1990) and most studies had focused on identifying genes from PDL fibroblasts (PDLFs) instead of tissue because PDLFs are the most abundant cells in PDL tissue. Collagen type I is the major collagen to form the main structure of PDL fiber while other types such as type III are codistributed with collagen type I. Proteoglycans (e.g., asporin, decorin, and versican (VCAN; Larjava et al., 1992;Yamada et al., 2006) and noncollagenous proteins (e.g., periostin; Xu et al., 2017) have also been reported in PDLFs. However, little is known about the overall profiling of gene expression in PDL tissue. According to a comparison of gene expression between PDLFs and PDL tissue, osteopontin was significantly high in PDL tissue (Lallier & Spencer, 2007). Other genes such as pleiotrophin, osteomodulin, alkaline phosphatase, bone sialoprotein 2, periostin, and fibromodulin (FMOD) were slightly high in PDL tissue as well.
Based on these results, there has been speculation that the cultured PDLFs may represent an immature cellular form of PDLFs (Lallier & Spencer 2007;Marchesan et al., 2011). Based on these results, there has been speculation that the cultured PDLFs in vitro may present a different nature compared from the PDLFs in PDL tissue under in vivo. These different cell conditions results the different gene expression. Thus, culturing PDLFs from PDL tissue may alter the ECMs properties due to the environmental changes from in vivo to in vitro. To regenerate PDL tissue using PDLFs, recovering the originated ECMs in vivo must also be considered. Furthermore, epithelial cell rest of Malassez, blood vessel, and lymphatic vessel are also present in the PDL tissue area. Including, osteoclasts, osteoblasts, and cementoblasts are located in the hard tissue surface. These components may provide molecules such as ECMs in PDL tissue to create a structure and support function. However, the gene profiling only from PDLFs in culture may be altered from the environmental change and cause insufficient data for the regeneration of PDL tissues.
Thus, gene expression in PDL tissue may include genes that are derived from these cells and it may be critical to identify the overall gene expression pattern in PDL tissue. The information of overall gene expression in PDL tissue may be necessary for PDL tissue regeneration.
Our study is the first study to profile and analyze gene expression of PDL tissue, using Cap analysis gene expression (CAGE) method. We analyzed (1) the profiling of whole messenger RNA (mRNA) expression of PDL tissue by second generation sequencing, (2) the comparison of mRNA expression between PDL tissue and PDLFs from the same patient samples. periodontitis, endodontic treated as well as an unerupted condition must be excluded. The female patients, age 12-26 years old, were included for this study and all of them are nonsmokers. Their identification information has been acquired for this study including, but not limited to, initials, hospital numbers, medical history, surgical history, and dental history. All the information was kept strictly confidential. The patients were deidentified into the code numbers specifically for this study. The patients' individual information was neither utilized for any purpose other than the purpose of the study nor published in this manuscript. The collected sample from the patients were applied to different methods (Table S1).

| PDLFs isolation from PDL tissue
PDL tissue was collected from teeth that were extracted for orthodontic treatment purposes. The extracted teeth were rinsed with HOSIRILUCK ET AL. | 659 10% penicillin-streptomycin-added phosphate-buffered saline. After rinsing, PDL tissue was removed from a tooth at the middle part of the dental root using a surgical scalpel blade (No. 11). For PDLFs, the PDL tissue was applied directly to a collagen-coated culture flask that contained 1 ml culture medium then the flask was flipped upsidedown for 1-2 h which caused PDL tissue to attach on the cultured surface of the flask. After the attachment of PDL tissue, the culture flask was flipped back and 3 ml culture medium was added.

| RNA isolation
RNA was isolated from PDL tissue using 1 ml of TRIzol reagent and homogenized with a homogenizer under instruction. For PDLFs, RNA was isolated using Qiagen, RNeasy Mini Kit. Both RNAs were solubilized with RNase-free water and stored at −80°C. The RNA concentration was measured with NanoDrop™ 1000 (Thermo Fisher Scientific). 3 | RESULTS

| Noncollagenous proteins
As shown in Figure 3b and were four major proteoglycans in PDL tissue, as shown in Figure 3c and

| Western blot analysis
Western blot analysis confirmed protein expression of the genes that were detected by CAGE analysis as mentioned above. In particular, as shown in Figure 7, collagen type VI (COL6A1) was highly expressed in PDL tissue. Integrin β1 was detected in both PDL tissue and PDLFs while integrin α5 was only detected in PDLFs. OPN, ASPN, POSTN, and SPARC were highly detected in PDL tissue but PDLFs.

| DISCUSSION
Our study demonstrated the profiling of the genes to play an important role for structure and function in PDL tissue. These genes can be categorized by their characteristics. We found over 17,000 expressed genes; of these, ECMs, in particular, collagen and noncollagenous ECMs, were the most highly expressed. ECMs play an essential role as biochemical and biomechanical initiators that are required for tissue morphogenesis, differentiation, and homeostasis.
ECM also support cell binding and regulate their function such as adhesion and migration. In PDL tissue, ECMs and other related genes also function to maintain the attachment between cementum and alveolar bone, and support daily occlusal loading.
4.1 | Type VI collagen was highly expressed, next to types I and III collagen, and may support the tight structure of PDL tissue In PDL tissue, collagen types I and III work as the main fibrilforming collagens to form and stabilize their structure. Unlike  other tissue, PDL highly expressed microfibril collagen which was collagen type VI, as shown in Figure 3a. Collagen type VI was distributed in ECMs (Cescon et al., 2015), as shown in Figure  type VI also binds with other ECMs such as BGN and WARP (Hansen et al., 2012) to make a connection to surrounding connective tissues and arrange their structure. Moreover, other studies have found an interaction between collagen type VI and other collagens such as collagen type II, IV (in basal laminar), and XIV (Bidanset et al., 1992;Bonaldo et al., 1990;Brown et al., 1994;Kuo et al., 1997). Taken together, these results suggest that PDL tissue may require collagen type VI to provide a tight structure and stabilize the PDL from mechanical stress, to prevent tissue destruction. Similar observations have been made in the temporomandibular Joint (data not shown).
4.2 | POSTN and SPARC were highly expressed in PDL tissue and might support the fibril forming collagen In Figure 3b, POSTN and SPARC were the major noncollagenous proteins in PDL tissue. POSTN is an ECM that is able to bind to multitargets such as collagen type I and V and integrin αVβ3 and αVβ5 (Zeltz et al., 2014). This type of binding may result in POSTN being able to establish a cross-linkage and distribution of ECM proteins (Du & Li, 2017). Likewise, SPARC is considered as a bifunctional protein and able to bind to both calcium and collagen type I. These effects may result in SPARC being able to play an essential role in PDL homeostasis and regulate the turnover rate of collagen in PDL tissue (Ribeiro et al., 2014;Trombetta & Bradshaw, 2010). Therefore, these two genes may work together to make a strong structure and support PDL tissue function.

| Asporin was the most highly expressed proteoglycan in PDL tissue and may play a major role as a negative regulator for mineralization
Compared to proteoglycans, ASPN, LUM, DCN, and OMD were highly expressed in PDL tissue, as shown in Figure 3c. ASPN suppressed BMP-2 activity and acted as a negative regulator for PDL mineralization to prevent nonphysiological mineralization such as an ankyloses (Yamada et al., 2007). This character resulted in PDL being an unmineralized tissue despite being surrounded by mineralized structures, and worked as the cushion between cementum and alveolar bone to absorb the mechanical force.

| LUM and DCN might be involved in the ECM arrangement while OMD might regulate collagen fibril growth of PDL tissue
DCN and OMD were involved with the structural organizing of ECMs in PDL tissue while LUM bound to collagen type I via a leucine-rich repeat to organize the collagen fibril (Kalamajski & Oldberg, 2009;Svensson et al., 2000). Similar to LUM, DCN is bound mainly to collagen type I through the leucine-rich repeat and to collagen type VI at the N terminal region to regulate the normal growth of PDLFs. They organized the collagen fibrils in PDL tissue using these connections (Alimohamad et al., 2005;Hakkinen et al., 2000;Kalamajski et al., 2007). OMD can also bind to collagen type I via a leucine-rich repeat to control the optimal collagen fibril growth in PDL tissue (Tashima et al., 2018). Taken together, LUM, DCN, and OMD are mainly bound to collagen to regulate and arrange the structure of ECMs.

| Integrin β1 was the most highly expressed integrin subunit and might be a major contributor for integrin subunit combination in both PDL tissue and PDLFs
In vertebrates, the integrin family has two different subunits, α and β, and they make a heterodimer to bind to ECMs through an RGD motif (Barczyk et al., 2013(Barczyk et al., , 2010. We identified α and β subunits in both PDL tissue (16 α subunits and 8 β subunits) and PDLFs (13 α subunits and 7 β subunits). As shown in Figure 6a,b, integrin β1 was the most expressed subunit in both PDL tissue and PDLFs and may be considered to be the major component of the heterodimer. Barczyk et al. (2010) showed that integrin β1 was the main β subunit for the possible integrin combination. In our data for the β subunit, β1 was 57.52% and β5 was 17.86% in PDL tissue. Similarly, β1 was 91.29% The ratio of integrin α and β in PDL tissue and PDLFs. Integrin α and β were equally expressed in PDL tissue while integrin β was relatively highly expressed in PDLFs. PDL, periodontal ligament; PDLF, PDL fibroblast 4.6 | Genes for gene regulation were highly expressed and may be important to promote a high turnover rate in PDL tissue Besides ECMs, genes that regulate transcription and translation such as MALAT1 and SCARNA2 were highly expressed in PDL tissue as well.
MALAT1 is a trans-acting factor which regulates alternative splicing of pre-mRNA by interacting with serine/arginine proteins and influencing their distribution (Gutschner et al., 2013;Tripathi et al., 2010). A previous study found that MALAT1 upregulated fibroblast growth factor 2 which promoted human PDL stem cells proliferation (Chen et al., 2019).
SCARNA2, a product of independent transcription, might alter small nucleolar ribonucleoprotein activity and affect the level of modifications within ribosomal RNA (Gerard et al., 2010). Taken together, these data suggest that these genes might promote a high turnover rate in PDL tissue by regulating transcription and translation, including cell proliferation. The turnover rate of collagen in PDL tissue was rapid with a turnover time of 13.5 days, (Orlowski, 1978) comparing to hemin degradation of red blood cells (4 months; Shemin & Rittenberg 1946). Thus, PDL tissue might have a high turnover rate by these factors.

| The different gene expression patterns between PDL tissue and PDLFs might provide important information about PDL tissue regeneration
From CAGE data, the highly expressed genes were different between PDL tissue and PDLFs because of the environmental changes ( known as tenascin-W ( Barczyk et al., 2013), are glycoproteins that exist in chordates. TNN not only supported cell adhesion and migration but also promoted bone development and angiogenesis, as an adhesion modulatory protein. At the end stage of PDL differentiation, TNN was highly expressed (Nishida et al., 2007). However, TNN disappeared in most adult tissues. On the other hand, a recent study found that TNN was highly expressed in tumor cells and TNN may possibly function as a tumor marker for anticancer therapies (Tucker & Degen, 2019). Thus, TNN may be one of the more important ECMs that promotes cellular attachment in PDL tissue. As mentioned above, we also found other decreased noncollagenous ECMs in PDLFs (ASPN, POSTN, and OMD) although these genes were also highly expressed in PDL tissue. ASPN functioned as a negative regulator for mineralization (Yamada et al., 2007), POSTN supported a strong structure in PDL tissue (Du & Li, 2017), and OMD played an important role for the ECM organization and controlled the optimal growth rate of collagen fibrils (Tashima et al., 2018). These differences may be essential for PDL regeneration.
4.8 | Two possibilities in the difference of gene profiling between PDL tissue and PDLFs PDL tissue is considered to be a complex structure that contains not only PDLFs but also other components such as epithelial cell rest of Malassez, blood, and lymph vessels, including those surrounding the hard tissues, cementum, and bone. Each cell expresses different genes which result in different functions in PDL tissue. The combination of gene expression from these cells helps to keep PDL function. When we compared the gene expression patterns between PDL tissue and PDLFs, we noticed that most genes maintained the same expression level but some genes such as TNN, OPN, and OCN were decreased in PDLFs. This result implied two possibilities. One possibility is that the decreased genes may be expressed by the other cells instead of PDLFs. In fact, OPN was found in porcine epithelial cell rests of Malassez and blood vessels. TNN was found in the primary culture of osteoblasts which promotes cell migration and mineralization (Tucker & Degen, 2019). OCN was mainly expressed in osteoblasts (Patti et al., 2013).
The lymphatic vessel was also found in PDL tissue and may be necessary to maintain PDL tissue function (Berggreen & Wiig, 2013;Levy & Bernick 1968). In prior study, TNN expression indicated angiogenesis function by commonly found adjacent to the blood vessel in tumor samples. TNN is also used as a specific marker of gliomaassociated blood vessels and stimulates angiogenesis (Martina et al., 2010) However, we did not find highly expressed lymphaticrelated genes in our study.
On the other hand, TNN, OPN, and OCN were also functionally expressed in PDLFs (Alves et al., 2015;Barczyk et al., 2013)  These results indicated that OPN might respond to mechanical stress and promote remodeling in PDL. According to the molecular structure, OCN contained Gla motif which was able to bind to hydroxyapatite (Razny et al., 2017). This stimulation might partially contribute bone remodeling that surrounds PDL tissue. The replenishment of these genes may recover PDL function during F I G U R E 7 The western blot analysis for collagen type6A1 (a), integrin β1 (b), integrin α5 (c), POSTN (d), OPN (e), SPARC (f), ASPN (g), and beta-actin (h). Protein expression showed that collagen type6A1 and integrin β1 was highly expressed in both PDL tissue and PDLFs. OPN, POTN, ASPN, and SPARC were highly expressed in PDL tissue while integrin α5 was highly expressed in PDLFs. As positive controls, the extracts from mouse lung, mouse bladder, mouse liver, and Hela cells were utilized in each blotting separately. PDL, periodontal ligament; PDLF, PDL fibroblast HOSIRILUCK ET AL.

| CONCLUSION
Our study is the first study to profile and analyze gene expression of PDL tissue, using second generation sequencing, CAGE. We also compared gene expression between PDL tissue and PDLFs from the same patient for the first time, which is more critical to know the gene profiling between two different environmental conditions.
These findings may provide more critical and significant information for PDL tissue reconstruction.

ACKNOWLEDGMENTS
This study was supported in part by Grant-in-aid for the 2018-2019 Research Project of the Research Institute of Health Sciences, Health Sciences University of Hokkaido. Our analytic contribution from Dr. Rie Takai.

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS
All authors have made substantial contributions to the conception and design of the study. Nattakarn Hosiriluck, Ayuko Takada, and Haruna Kashiohave been involved in data collection and data analysis. Nattakarn Hosiriluck, Ayuko Takada, Itaru Mizuguchi, and Toshiya Arakawa have been involved in data interpretation, drafting the manuscript, and revising it critically. Nattakarn Hosiriluck and Toshiya Arakawa have given final approval of the version to be published.

DATA AVAILABILITY STATEMENT
The data that support the finding of this study are available from the first author (Nattakarn Hosiriluck) or the corresponding author (Toshiya Arakawa), upon your request.