MICROSCOPY RESEARCH AND TECHNIQUE 78:792–800 (2015)

Ultrastructural Evaluation of Mesenchymal Stem Cells From Inflamed Periodontium in Different In Vitro Conditions RALUCA ZAGANESCU,1 LUCIAN BARBU TUDORAN,2† EMOKE PALL,3 ADRIAN FLOREA,4 ALEXANDRA ROMAN,5* ANDRADA SOANCA,5 AND CARMEN MIHAELA MIHU6† 1

Student, Faculty of Medicine, Department of Histology, Iuliu Hat¸ieganu University of Medicine and Pharmacy, Cluj-Napoca 400012, Romania 2 Department of Molecular Biology and Biotechnologies, Faculty of Biology and Geology, Babes¸-Bolyai University, Cluj-Napoca 400006, Romania 3 Department of Veterinary Reproduction, Obstetrics, and Gynecology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca 400372, Romania 4 Department of Cell and Molecular Biology, Faculty of Medicine, Iuliu Hat¸ieganu University of Medicine and Pharmacy, Cluj-Napoca 400349, Romania 5 Department of Periodontology, Faculty of Dental Medicine, Iuliu Hat¸ieganu University of Medicine and Pharmacy, Cluj-Napoca 400012, Romania 6 Department of Histology, Faculty of Medicine, Iuliu Hat¸ieganu University of Medicine and Pharmacy, Cluj-Napoca 400349, Romania

KEY WORDS

adult; granulation tissue; bone substitutes; brightfield microscopy; transmission electron microscopy; scanning electron microscopy

ABSTRACT This research aimed to observe the behavior of mesenchymal stem cells (MSCs) isolated from periodontal granulation tissue (gt) when manipulated ex vivo to induce three-dimensional (3D) spheroid (aggregates) formation as well as when seeded on two bone scaffolds of animal origin. Periodontal gt was chosen as a MSC source because of its availability, considering that it is eliminated as a waste material during conventional surgical therapies. 3D aggregates of cells were generated; they were grown for 3 and 7 days, respectively, and then prepared for transmission electron microscopic analysis. The two biomaterials were seeded for 72 h with gtMSCs and prepared for scanning electronic microscopic observation. The ultrastructural analysis of 3D spheroids remarked some differences between the inner and the outer cell layers, with a certain commitment observed at the inner cells. Both scaffolds showed a relatively smooth surface at low magnification. Macro- and micropores having a scarce distribution were observed on both bone substitutes. gtMSCs grew with relative difficulty on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds, demonstrating fibroblast-like or star-like shapes with elongated filiform extensions. Our results add other data on the possible usefulness of gtMSC and could question the current paradigm regarding the complete removal of chronically inflamed gts from the defects during periodontal surgeries. Until optimal protocols for ex vivo manipulation of MSCs are available for clinical settings, it is advisable to use biocompatible bone substitutes that allow the development of progenitor cells. Microsc. Res. Tech. 78:792–800, 2015. V 2015 Wiley Periodicals, Inc. C

INTRODUCTION Periodontitis is a chronic bacterial infection that leads to long-standing inflammation responsible for the irreversible destruction of the tooth supporting tissues (Taubman et al., 2007). Inflamed granulation tissue (gt) developed during the evolution of periodontitis is removed from the defects during conventional surgical interventions. Recent data have shown that this tissue contains a significant population of multipotent mesenchymal stem cell (MSC) that can be isolated and expanded under particular conditions (Hung et al., 2012; Park et al., 2011; Pall et al., under review; Ronay et al., 2014). It was shown that MSCs isolated from inflamed areas had similar tissue regeneration functions to MSCs derived from healthy sites (Hung et al., 2012; Park et al., 2011; Yu et al., 2014); however, some authors reported dysfunctional properties associated with MSCs from inflamed oral sites (Alongi et al., 2010; Liao et al., 2011; Pall et al., 2014 under review; C V

2015 WILEY PERIODICALS, INC.

Yazid et al., 2014) . MSCs are commonly cultured as two-dimensional (2D) monolayers using conventional tissue-culture techniques. In the meantime, MSCs exhibit the ability to aggregate into multicellular spherical clusters named spheroids when cultured in suspension or on nonadhesive surfaces (Yoon et al., 2012). These three-dimensional (3D) arrangements of the cells provide increased cell-to-cell interactions (Dissanayaka et al., 2015), mimic much better the in vivo environment of a real tissue in comparison with *Correspondence to: Alexandra Roman, Department of Periodontology, Faculty of Dental Medicine, Iuliu Hat¸ieganu University of Medicine and Pharmacy, 15 Victor Babes Street, Cluj-Napoca 400012, Romania. E-mail: [email protected] REVIEW EDITOR: Professor George Perry † L.B.T. and C.M.M. contributed equally to this work. Received 22 April 2015; accepted in revised form 17 June 2015 Contract grant sponsor: Iuliu Hatiganu University of Medicine and Pharmacy; Contract grant number: 1493/5/28.01.14. DOI 10.1002/jemt.22542 Published online 15 July 2015 in Wiley Online Library (wileyonlinelibrary.com).

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conventional monolayer cultures (Haycock, 2011) and exhibit an improved differentiation potential (Langenbach et al., 2013; Xiao and Tsutsui, 2013; Yoon et al., 2012). One particular challenge in obtaining periodontal regeneration has been the delivery of ex vivomanipulated MSCs grown on bone substitute scaffolds in periodontal defects (Chen et al., 2012; Ishikawa et al., 2009; Struillou et al., 2011; Yoshida et al., 2012) as an alternative to classical approaches associated with inconsistent outcomes (Tonetti et al., 2004; Trombelli et al., 2002). The biological properties of bone substitutes are essential for their osteoconductive function (Sollazzo et al., 2010) thus influencing the development of the cellular events that lead to periodontal regeneration. Actual difficulties prevent the widespread clinical implementation of MSC-delivery strategies for periodontal therapy (Yoshida et al., 2012) and more research is needed to advance in this field. One of the aims of this research was to evaluate, by ultrastructural analysis, the behavior of granulation tissue-derived MSCs (gtMSCs) when manipulated ex vivo to induce 3D spheroid formation, as an important property of these cells, which, to our knowledge, has not been reported so far. The previous research developed by our team on gtMSC’s properties investigated the specific antigen make-up, multipotent differentiation potential, functionality, and the ultrastructural characteristics (Pall et al., unpublished data). The data from this study would enlarge the area of comparison with MSCs isolated from healthy sites providing supplementary information regarding the characteristics of MSCs isolated from inflamed tissues. In addition, as 3D spheroids replicate more accurately MSC microenvironment they could be used in future investigations to ensure more accurately the in vivo developing conditions. The study also aimed to observe the behavior of gtMSCs cultured on two bone scaffolds of animal origin and to translate the knowledge into clinical practice as a potential approach to stimulate the intrinsic regenerative capacity of the periodontium. This study belongs to a large line of research aiming to have an insight into the characterization and behavior of oral MSCs isolated from some oral sources. MATERIALS AND METHODS Study Design MSCs were isolated from periodontal gts eliminated during a flap debridement surgery from a 39-year-old chronic periodontitis patient. The study was approved by the Ethical Board of the Iuliu Hat¸ieganu University (359/13.10.2014). The study protocol and the detailed procedure were explained and written informed consent was obtained from the patient. In obtaining informed consent and conducting the research, the study adhered to the principles outlined in the Declaration of Helsinki on experimentation involving human subjects.gtMSCs were grown on glass slides for histological observation and were used to generate 3day and 7-day 3D spheroids that were evaluated using transmission electron microscopy (TEM). Also, gtMSCs were seeded for 72 h onto two natural bone scaffolds and the assembles were observed using scanning electronic microscopic (SEM). Cells from the sixth Microscopy Research and Technique

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passage were used for all experiments. The experiments were performed in triplicate. Cell Obtaining and Characterization The periodontal gt samples were waste materials. Tissue samples were introduced in transport medium (DMEM 13—Dubelcco’s modified Eagle medium [Sigma-Aldrich, St. Louis, MO] plus 10% fetal bovine serum [Sigma-Aldrich, St. Louis, MO] plus 1% antibiotic/antimycotic [Gibco Life Technologies, Paisley, United Kingdom]), were transported to the tissue culture laboratory immediately after harvesting, and isolated after 2 h. The isolation and characterization of MSCs are detailed elsewhere (Pall et al., unpublished data; Roman et al., 2012, 2013). Briefly, cells were isolated using the explant method. Collected gt samples were minced and added on T25 flasks. The propagation medium was represented by DMEM/F12 (Nutrient F12 Ham) (Sigma-Aldrich, St. Louis, MO) supplemented with 10% of fetal calf serum (EuroClone, Milano, Italy), 2 mM of glutamine, 1% of nonessential amino acids (Sigma-Aldrich, St. Louis, MO). After 5 days of culture, the medium was replaced and tissue pieces were removed. The cells were grown until confluence (70–80%) and then subcultured (1:2). Immunophenotype analysis and trilineage differentiation assays used cells at the sixth passage of culture. Flow cytometry evaluated the following antigens: CD34/45, CD49, CD73, CD90, CD44, CD105, and HLA-DR (BD Biosciences, San Jose, CA) by following the manufacturer’s recommendation. Trilineage differentiation assay was performed by culturing the cells in osteogenic, adipogenic, and chrondrogenic induction media. Cytochemical staining was further realized to confirm the successful differentiation of the cells. Histological Analysis Sterile silanized (superFrost) glass slides (Dako Cytomation, Santa Clara, CA) were placed in 10-mm Petri dishes and covered with a suspension of cells at sixth passage at a density of 1 3 105 cells in normal propagation medium. The plates were maintained at 378C in fully humidified atmosphere at 5% CO2 in air for 72 h. The slides were removed and fixed with buffered neutral 4% of paraformaldehyde (Sigma-Aldrich) solution for 20 min. Then, the slides were washed with sterile saline solution, processed using progressing concentrations of ethanol (80, 95, and 100%) and xylene (Leica TP 1020, Leica Microsystems 157NusslochGMbH, Nussloch, Germany), and then embedded in paraffin and sectioned. The slides were treated with xylene, rehydrated using descending concentration of ethanol (100, 95, and 80%) washed, and stained with hematoxylin and contrastained with eosin and then coverslipped with NeoMountV (Merk KGaA, Darmstadt, Germany). R

MSC Spheroid Generation The hanging drops aggregation technique was used for the generation of spheroids because it is associated with more uniform spheroid dimensions and the number of the cells is well established after the initial quantification compared with the suspension method (Kurosawa, 2007). Dissociated MSC monolayers

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Fig. 1. gtMSCs grown on glass slides. a: Spindle-shaped cells alternating with star-shaped cells (Ob, 403); b: Interconnected cell ramifications and undifferentiated characteristics (Ob, 1003). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

(passage 6) were resuspended in culture medium to obtain a single-cell suspension. In brief, 20–30 lL of drops containing 4 3 103 cells were placed on the lid of a 100-mm Petri dish in regular arrays. The lid was inverted and placed over the bottom of the Petri dish. The dish with hanging drops was incubated for 2 days. The spheroids were harvested and the suspension containing the spheroids was subsequently transferred into two bacterial-grade dishes and cultivated for 3 days (Kurosawa, 2007). Transmission Electron Microscopy The cell aggregates were processed for TEM according to the usual protocols (Hayat, 2000; Watt, 2003). The cell aggregates were prefixed with 2.7% of glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M of phosphate-buffered saline (PBS) for 2 h, washed four times with 0.1 M of PBS, postfixed with 1% of osmium tetroxide (Fluka, Buchs, Switzerland) in 0.15 M of PBS for 1.5 h, and washed with 0.15 M of PBS. Next, they were dehydrated in an acetone series and embedded in Epon 812 (Fluka). Ultrathin sections obtained using glass knives on a Bromma 8800 ULTRATOME III (LKB, Stockholm, Sweden) were contrasted with alcoholic uranyl acetate (Merck, Darmstadt, Germany) and lead citrate (Fluka). The sections were examined on a JEOL JEM 1010 transmission electron microscope (JEOL, Tokyo, Japan), and the images were captured using a Mega VIEW III camera (Olympus, Soft Imaging System, M€ unster, Germany). Bovine Bone Substitutes Two bone substitutes were used. Bio-OssV (BO) (Geistlich Pharma AG, Wolhusen, Switzerland) consists of granules size of 0.25–1 mm. BO is a natural hydroxyapatite bone substitute (Roberts et al., 2011). The highly purified osteoconductive mineral structure is produced from natural bone in a multistage purification process, adhering to the strictest safety regulations; during this process, all organic components are removed. The inorganic bone matrix of Bio-OssV has a

macro- and microporous structure (total volume, 70– 90%) similar to human spongious bone. Owing to the large interconnecting pore volume and the natural com position, the formation and ingrowth of new bone at the implantation site is encouraged. BO has a compressive strength of 35 MPa (Bio-OssV-Technical details.pdf/Manufacturer information). Natural Bovine BoneV (NB) (aap Biomaterials GmbH, Dieburg, Germany) is an interconnecting macroporous and microporous hydroxyapatite system, produced from bovine cancelous bone (spongiosa) in a high-temperature process of several hours. The bone substitute consists of granules size of 0.5–1 mm. The porosity lies within a range of 65–80% vol % and the pore size lies within a range of approximately 100– 1,500 lm (Natural Bovine BoneV-Technical detailsManufacturer information) R

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Cell Seeding on Scaffold Biomaterials The clusters of granules from both natural bovine bone substitutes (0.01 g) were placed in sterile conditions in multicompartmented dishes (four-well) with culture medium. After enzymatic treatment and centrifugation, a concentration of 2 3 105 cells/culture well (sixth passage) was seeded over the biomaterials. The negative control was represented by a piece of biomaterial in culture medium without cells and the positive control was represented by a well containing only cells. The cultures were maintained at 378C in fully humidified atmosphere at 5% of CO2 in air for 72 h. The cultures were observed on a daily basis during the culture period with an inversed phase microscope (Nikon TS100, Nikon Instruments, Europe).

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Scanning Electron Microscopy For SEM analyses, negative controls and bovine bone particles seeded with MSCs were washed twice with PBS and fixed for 2 h in 2.5% of glutaraldehyde in PBS (0.1 M), rinsed three times with PBS, and dehydrated in a graded ethanol series. The samples were critical-point dried and gold-sputtered (AGAR Automatic Sputter-Coater, Agar Scientific, Stansted, Microscopy Research and Technique

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Fig. 2. Ultrastructural aspects of the inner region of MSC spheroids after 3 days of culture (a–c) and 7 days (d). a: Cells with a lower deggree of differentiation showed large euchromatic nuclei (n) with prominent nucleoli (nu). b: Detailed view of cytoplasm containing a

relative high amount of rough endomplasmic reticulum (rer) and a few mitochondria (m). c: Plasma membranes of adjacent cells are in close contact and attached by tight junctions (arrowheads). d: A high amount of lipid droplets (l) were observed after 7 days of culture.

United Kingdom), and then examined with a JEOL JSM-5510LV Scanning Electron Microscope (Jeol, Tokyo, Japan).

designed nuclear membrane (Figs. 1a and 1b). The extensions showed an interconnected aspect (Fig. 1a).

RESULTS Isolation of gtMSCs and Histological Observations gtMSCs at sixth passage were used for the experiments. The ability of isolated cells to form adherent clonogenic cell clusters of fibroblast-like morphology, similar to those recorded for different MSC populations, was shown by the formation of about 170 single colonies, generated from 104 single cells cultured at low density. The histological examination of gtMSCs grown on glass slides revealed that the cells were confluent and adherent to the substrate. The shape of the cells was variable spindle-shaped cells alternating with starshaped cells. The aspect of gtMSCs on the slides sustained their undifferentiated state: increased number of cell ramifications, a pale-stained cytoplasm, a voluminous nucleus in comparison with the cell’s size. The nuclei were euchromatic, nucleolated, with a wellMicroscopy Research and Technique

Ultrastructural Aspects of gtMSC Spheroids After 3 days of culture, the spheroids (100–150 lm) were harvested and processed for TEM. On TEM images, distinct ultrastructural features were observed in different regions of the 3D MSC spheroids. After 3 days, cells exhibiting a low degree of differentiation were observed in the inner parts of the spheroids having large, euchromatic nuclei and prominent nucleoli (Fig. 2a). Their cytoplasm was rich in rough endoplasmic reticulum and had a moderate amount of lipid droplets (Figs.2a–2c); rare autophagosomes and a few mitochondria (Figs. 2a–2c) were present. The cells were tightly packed, with narrow extracellular spaces, and were attached in several points by tight junctions (Fig. 2c). No cell prolongations were present in the inner regions, and thus witnessing a certain commitment (Fig. 2c). After 7 days of development of MSC spheroids, the cells of the inner regions preserved the same ultrastructural architecture, but significantly increased amounts of lipid droplets were present (Fig. 2d).

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Fig. 3. Ultrastructural aspects of the outer region of MSC spheroids after 3 days of culture (a–e) and 7 days (f). a: Undifferentiated, less packed cells, with large nuclei (n) and prominent nucleoli (nu), a few autophagosomes (ap) and rare lipid droplets (l). b: Adherent cells were observed toward the inner region of MSC spheroids attached in some points with tight junctions (arrowheads); c: Peripheral cells

showed extensions of different sizes containing glycogen granules (g). d: Detailed view of an autophagosome; e: Low amounts of lipids (l) present in peripheral cells. f: Very high amounts of lipid droplets were observed after 7 days (rer, rough endoplasmic reticulum; ly, lysosomes).

In the sections corresponding to the more external parts of MSC spheroids, the same aspect of undifferentiated cells could be observed (Fig. 3a, right-hand side). As shown in Figures 3a and 3d, cells with some

lipid droplets and autophagosomes were present. The cells were not tightly packed and several cell extensions could be noticed (Fig. 3b). In the mean time, for cells placed several layers beneath, it was observed a Microscopy Research and Technique

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Fig. 4. SEM images of bovine bone substitutes. a: A relative smooth surface of BO at low magnification. b: The rough aspect and continuous grains on the surface of BO. c: A relative smooth surface of NB at low magnification. d: The continuous grains on the surface of NB.

tendency of cell aggregation with a close relationship between cell membranes (Fig. 3b). In the peripheral regions of the spheroids, cell extensions of different sizes were present, containing glycogen granules, which were also observed in the cytoplasm (Fig. 3c). After 3 days of development, the outer cells of MSC spheroids displayed several lipid droplets (Fig. 3e), but after 7 days lipid droplets were more abundant (Fig. 3f), suggesting a differentiation tendency for the outer cells, too. Scanning Electron Microscopy SEM analysis of BO and NB samples was performed to achieve a morphologic characterization of the scaffolds after 72 h of incubation in culture medium without cells. Both scaffolds showed a 3D structure and a relatively smooth surface at low magnification (Figs. 4a and 4c). Macropores of about 300 lm and micropores in the range of 1–20 lm were observed on BO samples having a scarce distribution (Figs. 4a and 5a). When viewed closer, BO hydroxyapatite consisted of continuous grains and occasional clusters of spikes. A rough aspect characterized the surface of BO particles (Fig. 4b). Microscopy Research and Technique

SEM observation of NB particles revealed the presence of scarce macropores of about 100 lm and of micropores in the range of 1–10 lm (Figs. 4c and 5c). A rough surface and the presence of continuous grains characterized the surface of NB particles (Fig. 4d). SEM investigation of the scaffolds seeded with gtMSCs showed that cells exhibited a certain degree of proliferation on the biomaterials. After 72 h of proliferation, gtMSCs scarcely covered the surface of bovine bone scaffolds. SEM demonstrated a fibroblast-like shape of the cells grown on BO, with an approximate dimension of the cell body of about 30 lm (Figs. 5a and 5b). SEM analysis observed that cells grown on NB showed a star-like shape, with elongated filiform extensions. The dimension of the cell bodies was approximately 40 lm (Figs. 5c and 5d). A lack of concordance between micropores and cell dimensions could be observed for both biomaterials (Figs. 5a and 5c). The same mode of adhesion of the cells on both biomaterials was observed. However, the SEM analysis revealed that after 72 h gtMSCs grew with relative difficulty on the biomaterials.

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Fig. 5. SEM images of gtMSCs grown on bovine bone substitutes. a: Fibroblast-like aspect of a cell grown on BO. b: Cell grown on BO—closer view. c: Star-like shaped cells grown on NB. d: Ramifications of a cell on NB.

DISCUSSIONS This research was undertaken to determine the capacity of gtMSCs to generate 3D spheroids to observe their ultrastructure and also to investigate the behavior of gtMSCs when seeded on two bovine bone scaffolds. Periodontal gt was chosen as a MSC source because gtMSCs present in periodontal defects could increase the local intrinsic regenerative potential if properly stimulated by biomaterial scaffolds used in surgical approaches. In addition, periodontal gt is easily available as a waste material during conventional surgical therapies opening thus promising perspectives to be used in regenerative medicine. The cells used in this study were previously isolated and characterized by our team (Pall et al., unpublished data); gtMSCs fulfilled minimal standard criteria suggested by the International Society for Cellular Therapy (Dominici et al., 2006) to be considered as MSCs. This research addressed 3D spheroid formation as actually it is considered an in vitro highly sophisticated cell culture system, replicating the complex in vivo microenvironment of MSCs, offering a study tool to explore stem cell homeostasis and differentiation

capabilities. In addition, 3D environment of spheroids positively influences the fate of scaffold-based tissue constructs (Laschke et al., 2006) as MSCs exhibit vessel-forming capacities and thus directly contribute to the formation of an intrinsic microvasculature within the matrices (Lin et al., 2012). Seeding 3D MSC spheroids onto scaffolds markedly improved the vascularization of tissue constructs when compared to the conventional seeding with single-cell suspensions (Laschke et al., 2013). TEM analysis of 3D spheroids enabled us to remark some differences between the inner and the outer cell layers. Although all the cells exhibited a low degree of differentiation, the more peripheral cells presented many large extensions and a few cell organelles that are indicatives for an undifferentiated state. No aggregation tendency was remarked in this region. On the other hand, the lack of cell prolongations, the establishment of tight junctions between the cells, and the abundance of lipid droplets were indicative for a certain commitment of the inner cells. The same pattern for inner and outer regions of the spheroids was previously reported by our group when ultrastructural analysis investigated 3D aggregates generated from Microscopy Research and Technique

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palatal-derived MSCs (Roman et al., 2013). Other observations reported the presence of tight junctions between the outer layers of the 3D colonies formed by embryonic stem cells (Mumaw et al., 2010). Two biomaterials from the same class (bovine bone substitutes) were chosen because different manufacturing protocols could induce particular properties to the biomaterials, and thus influencing differently the cellular response. BO is the most documented oral bone substitute; it is widely supported by scientific literature after being tested extensively in vitro and in vivo, from preclinical animal studies to human clinical investigations (Baldini et al., 2011; Camelo et al., 1998; Jafarian et al., 2008; Mellonig, 2000); in addition, there are extensive reports on its use in tissue engineering approaches using ex vivo-manipulated cells (Ac¸il et al., 2000; Asti et al., 2008; Wiedmann-AlAhmad et al., 2005). On the other hand, little research has been done on the use of NB. In this study, SEM analyses depicted the presence of macro- and micropores associated with both bovine bone substitutes. The presence of the microarchitecture is important as it can influence chemoattraction, adhesion, and migration of the cells which in turn will affect matrix deposition and mineralization (Keogh et al., 2010; Ravindran et al., 2010; Tierney et al., 2009; Zeltinger et al., 2001), but also the formation of new vascular structures. An increased microporosity augments the surface area for cell adherence (O’Brien et al., 2005) and cytokine adsorption (Weibrich et al., 2000), but in the meantime limits choice in the cell movement and creates a greater resistance to scaffold penetration (Harley et al., 2008). Growth of bone tissue into the pores is possible only if pore diameter is at least 100 lm; pores of