Cell Adhesion & Migration ISSN: 1933-6918 (Print) 1933-6926 (Online) Journal homepage: https://www.tandfonline.com/loi/kcam20 Hyaluronan-CD44 interactions mediate contractility and migration in periodontal ligament cells Zeinab Al-Rekabi, Adriane M. Fura, Ilsa Juhlin, Alaa Yassin, Tracy E. Popowics & Nathan J. Sniadecki To cite this article: Zeinab Al-Rekabi, Adriane M. Fura, Ilsa Juhlin, Alaa Yassin, Tracy E. Popowics & Nathan J. Sniadecki (2019) Hyaluronan-CD44 interactions mediate contractility and migration in periodontal ligament cells, Cell Adhesion & Migration, 13:1, 139-151, DOI: 10.1080/19336918.2019.1568140 To link to this article: https://doi.org/10.1080/19336918.2019.1568140 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. View supplementary material Accepted author version posted online: 24 Jan 2019. Published online: 08 Feb 2019. Submit your article to this journal Article views: 534 View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kcam20 CELL ADHESION & MIGRATION 2019, VOL. 13, NO. 1, 139–151 https://doi.org/10.1080/19336918.2019.1568140 RESEARCH ARTICLE Hyaluronan-CD44 interactions mediate contractility and migration in periodontal ligament cells Zeinab Al-Rekabia, Adriane M. Furab, Ilsa Juhlina, Alaa Yassinc, Tracy E. Popowicsd, and Nathan J. Sniadecki a,b,e aDepartment of Mechanical Engineering, University of Washington, Seattle, WA, USA; bDepartment of Bioengineering, University of Washington, Seattle, WA, USA; cDepartment of Periodontics, University of Washington, Seattle, WA, USA; dDepartment of Oral Health Sciences, University of Washington, Seattle, WA, USA; eInstitute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA ABSTRACT ARTICLE HISTORY The role of hyaluronan (HA) in periodontal healing has been speculated via its interaction with the Received 8 August 2018 CD44 receptor. While HA-CD44 interactions have previously been implicated in numerous cell Revised 12 November 2018 types; effect and mechanism of exogenous HA on periodontal ligament (PDL) cells is less clear. Accepted 2 January 2019 Herein, we examine the effect of exogenous HA on contractility and migration in human and KEYWORDS murine PDL cells using arrays of microposts and time-lapse microscopy. Our findings observed Traction forces; cell HA-treated human PDL cells as more contractile and less migratory than untreated cells. migration; hyaluronan; Moreover, the effect of HA on contractility and focal adhesion area was abrogated when PDL CD44; periodontal ligament cells were treated with Y27632, an inhibitor of rho-dependent kinase, but not when these cells cells were treated with ML-7, an inhibitor of myosin light chain kinase. Our results provide insight into the mechanobiology of PDL cells, which may contribute towards the development of therapeutic strategies for periodontal healing and tissue regeneration. Introduction facilitates healing [8]. In spite of its abundance in many types of malignant tumors and its promotion of tumor The periodontal ligament (PDL) connects the tooth to progression [9], HA is safe for periodontal treatments. the alveolar bone and transmits occlusal forces to the Its application has shown early promise in clinical periodontium, both of which are critical for mastica- studies, but with moderate results [10]. HA is thought tion. Damage to the PDL, either through disease or to be involved in stimulating proliferation, differentia- injury may impair mastication and/or lead to tooth tion, contraction, and/or migration in many cell types loss. Clinical therapies for periodontal regeneration, [11–19]. However, there has been discrepancy in results such as open flap debridement, guided tissue regenera- with HA, which has been attributed to its cell-specific tion (GTR), or tissue-engineering approaches, share the response and the molecular weight of HA [20]. Thus, common goal of harnessing the native process of defining the responses of PDL cells to HA is important wound healing in order to restore the PDL to its origi- to improve the strategies for periodontal regeneration. nal form and function [1–3]. After the inflammation PDL cells express CD44 [21,22], which is the princi- phase, new PDL cells, namely stem cells and fibroblasts, pal receptor to HA. CD44 is a single-chain molecule migrate into the wound site and rebuild the tissue [4]. composed of an N-terminal extracellular domain con- PDL cells are critical to wound healing, for they synthe- taining the ligand-binding sites, a membrane-proximal size new collagen fibers and align the fibers by their region, a transmembrane segment, and a cytoplasmic contractile activity [5,6]. As a result, both cellular con- tail [23]. The molecular size of CD44 ranges from 80 to tractility and migration are key components in PDL 150 kDa depending on variable splicing of at least 11 of regeneration, so mechanisms that regulate their activity the 21 exons coding for CD44 and post-translational may provide new clinical strategies. modifications [24]. In particular, PDL cells express iso- Hyaluronan (HA) is a nonsulfated, linear glycosami- forms CD44s and CD44H [21,22]. CD44 expression noglycan found abundantly in the PDL [7]. HA has upregulates proliferation and mineralization in PDL been linked to periodontal regeneration by playing an cells [24], but its effects on PDL contractility and anti-inflammatory role that prevents tissue damage and migration are less clear. CONTACT Nathan J. Sniadecki [email protected] Department of Mechanical Engineering, University of Washington, Seattle, WA, USA Supplemental data can be accessed here. © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 140 Z. AL-REKABI ET AL. The binding of HA to CD44 triggers signals pro- are treated with a ROCK inhibitor, Y27632, but not posed to affect cell migration and contractility through when treated with ML-7, an inhibitor of MLCK. RhoA and Rac1 [9,25,26]. In particular, RhoA activates downstream effector rho-dependent kinase (ROCK) Results [27]. ROCK phosphorylates the regulatory light chain of nonmuscle myosin and prevents myosin phosphatase Exogenous HA increases contractility and reduces activity, both of which lead to a greater amount of migration in human PDL cells nonmuscle myosin bipolar filaments. In another path- The overall expression of the CD44 receptor in human way, myosin light chain kinase (MLCK) regulates the PDL cells was characterized using flow cytometry formation of nonmuscle myosin bipolar filaments by (Figure 1(a)) and the data showed that 97.8% of the phosphorylating the regulatory light chain of myosin cells expressed this receptor. Furthermore, we found through a Ca2+/calmodulin-dependent pathway [28]. that 1.60% of the cells in the population were positive Bipolar filaments interact with F-actin, leading to acto- for CD31 (Figure 1(b)), an endothelial cell marker, and myosin-mediated contractility and cell migration 43.9% were positive for CD146 (Figure 1(c)), a stem cell [27,29]. Due to the fact that ROCK and MLCK have marker. In addition, human PDL cells cultured in vitro distinct roles in contractility and migration [30], it is showed a spindle-shaped, fibroblast-like phenotype. plausible that HA-CD44 interactions in conjunction These findings indicate that PDL cells were comprised with the ROCK pathway mediate PDL cell contractility largely of fibroblasts and some expressed stem cell and migration; however, this relationship has yet to be markers. Moreover, the CD44 receptor is present in established. almost the entire population. The objective of this study is to define the effects and To examine changes in contractility and migration mechanism of action of exogenous HA on PDL cells in response to exogenous, low molecular weight HA, such that an understanding of HA-CD44 interactions we seeded human PDL cells onto arrays of PDMS may be applied to the development of clinical techni- microposts or onto glass-bottom dishes coated with ques for periodontal regeneration. Here, we hypothe- PDMS. The surface of the PDMS of the microposts size that HA-CD44 interactions mediate contractility and glass-bottom dishes were coated with plasma- and migration in PDL cells. As a first step, we examine derived fibronectin to promote cell attachment. PDL the effect of exogenous HA on contractility and migra- cells appeared to grow normally on the microposts, tion of human PDL cells. Our results demonstrate that displaying similar morphological features to cells cells supplemented with exogenous HA appear more grown on culture dishes. In order to limit any exogen- contractile and less migratory. To further establish the ous HA, hyaluronidase (HYAL) was applied to human role that HA plays on PDL cells, CD44 knockout (KO) PDL cells for 1 hour prior to treating with HA. In mice are used as an in vitro model. Herein, we find that comparison to the controls (Figure 2(a)), we observed CD44-KO PDL cells appear more migratory and less an increase in stress fibers in these cells in response to contractile, even following exogenous stimulation with either exogenous HA (Figure 2(b)) or a sequential com- HA when compared to wild-type (WT) cells. Finally, bination of exogenous HYAL and HA (Figure 2(c)). HA-CD44 interactions are abrogated when PDL cells Next, we examined whether exogenous HA affected contractility, and measured the traction forces of PDL Figure 1. Characterization of human PDL cells using flow cytometry. The data shows that (a) 97.8% of human PDL cells expressed the CD44 receptor, (b) 1.60% of the cells expressed the CD31 receptor (endothelial cell line marker) and (c) 43.9% of the population expressed the CD146 receptor (stem cell marker). Red is the untagged control cell population and blue is the cell population tagged for CD44, CD31 or CD146.