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Bio-Medical Materials and Engineering 24 (2014) 1803–1815 1803DOI 10.3233/BME-140991IOS Press

Infiltration of mesenchymal stem cellsinto PEGDA hydrogel

Gregory Yourek a,∗, Xuejun Xin b, Gwendolen C. Reilly c and Jeremy J. Mao d

a DL Biotech USA, Gaithersburg, MD, USAb Theracell, Northridge, CA, USAc Department of Materials Science and Engineering, INSIGNEO Institute for in silico Medicine,University of Sheffield, Sheffield, UKd College of Dental Medicine, Fu Foundation School of Engineering and Applied Sciences,Department of Biomedical Engineering, Columbia University, New York, NY, USA

Received 27 June 2012Accepted 11 March 2014

Abstract.BACKGROUND: Attachment of cells to fully hydrated hydrogel biomaterials, such as PEGDA, has proven challenging be-cause of the hydrophobic cellular membrane.OBJECTIVE: To demonstrate undifferentiated human mesenchymal stem cells (hMSCs) and hMSC-derived chondrocytesinfiltrated and attached to unmodified PEGDA hydrogel.METHODS: Human MSCs and MSC-Cys were cultured in and on PEGDA hydrogel. Attachment was verified by SEM andconfocal microscopy and was accompanied by vinculin expression, indicating the presence of focal contacts. Infiltration wasconfirmed by H&E and fluorescence staining.RESULTS: Cells cultured on top of PEGDA hydrogel infiltrated the material on the order of micrometers.CONCLUSIONS: These findings will aid in understanding the cell-scaffold interaction for regenerative medicine constructs.

Keywords: Mesenchymal stem cells, PEGDA, vinculin, differentiation, chondrogenesis, infiltration

1. Introduction

Articular cartilage is a metabolically active tissue that under normal conditions is maintained throughthe slow turnover of the extracellular matrix (ECM) by chondrocytes sparsely distributed throughout theECM [1]. Since cartilage tissue is avascular, once injured, it undergoes potentially irreversible necrosisrather than inflammation and repair as in vascularized tissues [2]. If a defect penetrates the underlyinglayer of subchondral bone, creating a full thickness defect or osteochondral lesion, limited repair occurs.However, this generally leads to the formation of less durable fibrocartilage rather than hyaline carti-lage [3]. When a cartilage defect is isolated, the ideal treatment strategy is to replicate normal tissueregeneration using autologous cells with the intention of creating a physiologically normal surface.

*Address for correspondence: Gregory Yourek, PhD, DL Biotech USA, Gaithersburg, MD, USA. Tel.: +1 630 728 3696;E-mail: gyourek@gmail.com.

0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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Mesenchymal stem cells (MSCs) are self-renewing multipotent cells with the capacity to differentiateinto several distinct mesenchymal cell types including cartilage [4] and bone [5] and can be isolated frombone marrow [6] and adipose tissue [7]. There is much interest in the potential of using MSC populationsin regenerative medicine of cartilage and for treatment of musculoskeletal trauma and disease. AdultMSCs are readily available from most individuals and can be used autologously [8] in concert withgrowth factors and biomaterials to create a tissue engineered construct.

Hydrogels are a hydrophilic polymer biomaterial that can absorb biological fluids and have been usedsuccessfully as scaffolds for MSCs in tissue engineered constructs. Many hydrogel compositions areFDA approved and have low inflammatory response, minimal initiation of thrombosis and cause onlyminor surrounding tissue damage [9,10]. Hydrogels have efficient gas and nutrient delivery and wasteexcretion. Since a hydrogel is a three-dimensional structure, it can mimic the extracellular matrix ofthe normal cellular environment [11]. In regenerative medicine, hydrogels have served three purposes:barriers following tissue injury [12], drug delivery systems [13] and cell encapsulation [14].

Many hydrogels are biocompatible and are capable of maintaining the viability of a variety of cells[4,15–21] encapsulated within them. A poly(ethylene glycol)-diacrylate (PEGDA) hydrogel has beenused to create a dual-tissue engineered mandibular condyle construct consisting of MSCs differentiatinginto bone and cartilage in two layers [14]. It has been shown that cells would not attach to this type ofgel because of limited protein adsorption [21], so ligands [21] or charges [22] have been attached to thehydrogels to promote cell attachment. In addition, some hydrogels include active growth factors such asepidermal growth factor [23] and transforming growth factor-β (TGF-β) [24] to guide cell behavior.

A recent focus in tissue engineering osteochondral tissues is to use hydrogels as scaffolds for tissue-engineered constructs. However, while there are numerous reports as to how cells respond biochemicallyafter being encapsulated in the hydrogels, little is known about the interactions the surrounding cells havewith their non-physiological environment. The hypothesis of this study was that human MSCs (hMSCs)can interact with and infiltrate PEGDA hydrogel. This material is used for regenerative medicine appli-cations such as cartilage tissue replacement. Previously we described in vivo cell infiltration into PEGDAhydrogel which had not been reported prior to that work [18]. We attempted to recreate this migration inthe laboratory under in vitro conditions. Using multiple microscopic techniques, we acquired a better un-derstanding of the interactions and infiltration of PEGDA hydrogels by hMSCs and their chondrogeniccounterparts (hMSC-Cys).

2. Methods

2.1. Cell isolation and culture

Human MSCs were isolated from commercially available whole bone marrow (AllCells, Berkeley,CA) using “RosetteSep®” (StemCell Technologies; Vancouver, BC) according to the manufacturer’s in-structions. Briefly, bone marrow was mixed with phosphate buffered saline (PBS; Cambrex; East Ruther-ford, NJ), containing 2% fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA), 1 mM EDTAsolution. This solution was gently layered on “Ficoll-Paque®” (StemCell Technologies; Vancouver, BC).The solution was centrifuged and mononuclear enriched cells were removed and centrifuged to separatethe cells.

The cells were counted and plated in basal culture media, which was Dulbecco’s Modified Eagle’sMedium-Low Glucose (DMEM-LG; Sigma, St. Louis, MO), 1% antibiotic (containing 10 units/l Peni-cillin G sodium, 10 mg/ml Streptomycin sulfate and 0.25 mg/ml amphotericine B, Gibco, Invitrogen

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Corporation, Carlsbad, CA) and 10% FBS. Media was changed every two days to remove non-adherentcells. At passaging, cells were plated in tissue culture flasks and incubated in 5% CO2 at 37◦C, withfresh media changes every 3–4 days.

Chondrogenic supplemented media was DMEM-high glucose, 1% 1× ITS+ (BD Biosciences,Franklin Lakes, NJ; containing human recombinant insulin and human transferrin (12.5 mg each), sele-nous acid (12.5 µg), Bovine Serum Albumin (2.5 g), and linoleic acid (10.7 mg)), 1% antibiotic (contain-ing 10 units/l Penicillin G sodium, 10 mg/ml Streptomycin sulfate and 0.25 mg/ml amphotericine B),100 µg/ml sodium pyruvate, 50 µg/ml ascorbic acid, 40 µg/ml L-proline, 0.1 µM dexamethasone and10 ng/ml TGF-β3.

2.2. PEGDA

Commercially available PEGDA (Nektar Therapeutics, Huntsville, AL), MW 3400 Da, was sterilizedin powder form under UV light overnight. The chemical structure of PEGDA is shown below:

A total of 1 g PEGDA was dissolved in 9.8 ml sterile PBS and 100 µl Pen/strep. A total of 100 µl of 5%photoinitiator (2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone; Irgacure 2959) solutionin 100% ethanol was added to PEGDA solution and stored in the dark at 4◦C until use.

Cells were mixed with PEGDA at 1 × 106 cells/ml. A total of 100 µl of cell/PEGDA solution (100 ×103 cells) was loaded in liquid phase in a sterile microcentrifuge tube cap (6 × 3 mm: dia.×height)and polymerized for 10 min using 365 nm long wavelength UV light. Cell-free PEGDA hydrogel wasprepared as above, except using 50 µl liquid PEGDA hydrogel instead of 100 µl of the hydrogel. Cellswere plated on top of the polymerized PEGDA and Thermanox© cell culture treated coverslips at (1–2)×103 cells/cm2 for undifferentiated hMSCs and (5–10)×103 cells/cm2 for hMSC-Cys. Since hMSC-Cys proliferate more slowly in serum-free media, they were plated at higher density than hMSCs sosimilar confluency for all cell types could be obtained at the time of experimentation. Cells were allowedto attach for at least 4 h at which time they were flooded with 1 ml of respective media for cells on discsand PEGDA constructs. Constructs of encapsulated cells were cultured in 2 ml of respective media usingthe previously described conditions and media formulas. Empty PEGDA scaffolds in each type of mediaserved as cell-free controls. Cells were cultured in unsupplemented and supplemented media for 2 weeksbefore analysis.

2.3. Imaging

After two weeks in culture, cells were fixed in formaldehyde, permeabilized with Triton X-100 andblocked with 1% BSA. Hydrogels were embedded in paraffin, sectioned and stained with H&E byHistoserv, Inc. (Germantown, MD). Cells were stained with fluorescent “Alexa Fluor” 488 Concav-alin A and 568 Phalloidin conjugates (Molecular Probes) and FITC-conjugated vinculin monoclonalantibody (Sigma) per the manufacture’s recommendations. Additionally, cells with PEGDA hydrogelswere stained with “Cell Tracker Orange Fluorescent Probe” (Cambrex; East Rutherford, NJ). Histologyimages were obtained using a Nikon Eclipse E800 microscope. Phase contrast images were obtainedusing a Leica DMIRB microscope. Fluorescence images were obtained using a Nikon Eclipse E800and Leica DMIRB microscope. Multiphoton confocal images were obtained using a Mai-Tai multipho-

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ton laser coupled with visible laser (Bio-Rad, UK) into an inverted laser scanning confocal microscope(Nikon, Japan). Images presented are representative images of three independent experiments.

For SEM imaging, fixed constructs were rinsed twice with PBS, and dehydrated in ethanol solutionsof 70%, 80%, 90% and three times 100%, for 10 min each. Constructs were immersed for 3 min in 100%hexamethyldisilazane and transferred to a desiccator to avoid water contamination until SEM analysis.After drying, the samples were mounted on stubs and sputter coated with platinum. Samples were ex-amined with a Hitachi S-3000N SEM. Images presented are representative images of three independentexperiments.

For infiltration measurements, images were imported into Google SketchUp and distance was mea-sured using the tape measure tool.

3. Results

3.1. Superior light imaging

When hMSCs were cultured on tissue culture-treated coverslips, the cells demonstrated a fibroblast-like and spindle shaped appearance with lengths from 50–100 µm. This was visible both under phasecontrast (Fig. 1(A)) and fluorescence (Fig. 1(D)) microscopy. However, when the cells were mixed with

Fig. 1. Phase contrast and fluorescence images of hMSCs on and in PEGDA hydrogel. Human MSCs were seeded onThermanox® disc (A, D) and encapsulated within (B, E) and on top of (C, F) PEGDA hydrogel in standard culture condi-tions. After 2 weeks, images were obtained for phase contrast (A–C) and fluorescence (D–F) (stain for concavalin A whichstains membrane sugars, images were converted to grayscale where the membrane is white) microscopy. Cells exhibited typicalfibroblast-like morphology on Thermanox® disc (A, D); however, became spheroids when encapsulated within PEGDA hydro-gel (B, E). When seeded on top of PEGDA hydrogel (C, F), cells displayed round morphology similar to those encapsulated inPEGDA. Cells in different focal planes in C and F are infiltrating the PEGDA hydrogel.

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PEGDA solution and were encapsulated within the material by photopolymerization of the hydrogel, theshape of the cells became spherical (Fig. 1(B) and (E)). The diameter of the cells was ∼10 µm in threedimensions. Cell volume was estimated based on the surface area being on the order of hundreds of µm2

and an assumed height of 1–2 µm as found in previous AFM experiments [25], indicating that typicalcell volume of the cell in 2-D and 3-D is of the same order of magnitude. When cells were cultured ontop of photopolymerized cell-free PEGDA hydrogel, the cells acquired a shape that was neither long andflat as in 2-D nor short and round as in 3-D, but somewhere between those two morphologies (Fig. 1(C)and (F)). These images also identified cell infiltration into the PEGDA hydrogel, as cells are in differentfocal planes of the 2-D image, most apparent in the fluorescence image (Fig. 1(F)).

3.2. Lateral light imaging

Human MSCs and hMSC-Cys were encapsulated in and seeded on top of PEGDA hydrogel and cul-tured for 2 weeks in respective media. After that time, the hydrogels were fixed, embedded, sectionedand stained for H&E, which stains for cell nucleus and associated cellular proteins. Staining of thecells encapsulated within the PEGDA hydrogels indicated homogeneous positioning of each type of cellwithin the material (Fig. 2(A) and (B)) as we have previously noted in our laboratory [14]. When thecells were seeded on top of the cell-free polymerized PEGDA hydrogel, both cell types infiltrated thePEGDA hydrogel from the original topmost position to the interior of the hydrogel (Fig. 2(C) and (D)).This was evident by staining present near the top, but under the surface, of the PEGDA hydrogel, withan absence of staining in the remainder of the material. The cells migrated an average of 10–40 µm intothe PEGDA hydrogel (Fig. 2(E)).

3.3. SEM imaging

Using SEM, we observed that hMSCs originally seeded on top of PEGDA hydrogel infiltrated thehydrogel (Fig. 3). Interestingly, the cells displayed a round morphology similar to that seen of cells en-capsulated within the hydrogel, possibly due to the weak attachment of the cells to the PEGDA hydrogel.

We detected attachments made by the cells in the PEGDA hydrogel using scanning electron mi-croscopy (SEM). This was evident in both hMSCs and hMSC-Cys (Fig. 4). Attachments of both celltypes emanated from various locations of the cells to the hydrogel and were on the orders of hundredsof nanometers in width.

3.4. Confocal imaging

Human MSC-Cys that were seeded on top of a PEGDA hydrogel were stained with “Cell TrackerOrange” and a series of images was obtained using confocal microscopy (Fig. 5). Arranging these imagesinto a 3-D reconstruction corroborates our conventional light microscopy observances of cell infiltrationinto the hydrogel. The cell noted by an arrow is under the surface of the hydrogel, which can be detectedby the faint artifactual staining of the hydrogel. An estimate of the distance of infiltration from thesetypes of images is difficult because of the lack of a landmark denoting the top of the hydrogel as notedin the previous images. However, it seems to be on the same order (micrometers) as the above notedmigration by H and E staining.

Actin cytoskeleton (Fig. 6(A) and (E)), vinculin (Fig. 6(B) and (F)) and nucleus (Fig. 6(C) and (G))were localized in both cell types. Typical long, distinct actin fibers of hMSCs and hMSC-Cys as pre-viously found by us [25,26] were not observable. Instead, the actin cytoskeleton was evenly distributed

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Fig. 2. H&E staining of hMSCs infiltrating PEGDA hydrogel. Human MSCs (A and C) and hMSC-Cys (B and D) encapsulatedwithin PEGDA (A and B) and seeded on top of PEGDA (top noted by dashed lines) (C and D) hydrogel after 2 weeks standardculture conditions. Cells were fixed, sectioned and stained with H&E for cell locations. Cells encapsulated within PEGDAhydrogel were homogeneous throughout the hydrogel. Cells seeded on top of PEGDA hydrogel infiltrated the material as therewere cells 100–200 µm below the surface of the PEGDA hydrogel (noted by arrows). The PEGDA surface is denoted by thedotted line. Distance infiltrated measured in Google SketchUp shown in E.

throughout the cells. The DAPI stain for the nucleus was not present in all cells as the nucleus of allthe cells in the image may not have been in the same focal plane. There was homogeneous staining ofvinculin throughout each cell type (Fig. 6(B) and (F)) with occasional locations of intense staining in

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Fig. 3. SEM of hMSCs seeded on top of and attaching to PEGDA hydrogel. Human MSCs were cultured on top of cell-freePEGDA hydrogel and cultured in standard culture conditions for 2 weeks as described above. The cells and material were fixed,dehydrated and observed using SEM. Images B and C are magnified view of boxes in A and B, respectively. Cells displayedattachment noted by arrows (C).

the vinculin and merge images of each cell type (Fig. 6(D) and (H)). There was also alignment (yel-low) noted in the merge images (Fig. 6(D) and (H)) of the actin (red) and vinculin (green), denoting thetermination of actin fibers into focal contacts.

4. Discussion

Recently, PEGDA hydrogels have become of interest in regenerative medicine for use as a biomaterial,along with cells and growth factors, to replace or repair failing tissue [14]. However, there is a lack ofbasic literature describing how cells interact with this type of material. In our laboratory, we observedabundant host cell infiltration into cell-free PEGDA hydrogel loaded with growth factor and implantedsubcutaneously in a murine model [18]. Hydrogels do not generally promote cell attachment and thusthere has been a motivation to develop a suitable modification method to encourage better interactions ofcells with this material [20,22–24]. By seeding cells on top of empty hydrogel, as well as encapsulating

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Fig. 4. SEM of cells encapsulated within and attaching to PEGDA hydrogel. Human MSCs (A–B) and hMSC-Cys (C–D) wereencapsulated within PEGDA hydrogel and cultured in standard culture conditions for 2 weeks as described above. The cellsand material were fixed, dehydrated and observed using SEM. Images B and D are magnified views of boxes in A and C,respectively. Attachments by cells are noted by arrows (B, D).

cells within the hydrogel, we recreated in vivo cell infiltration into PEGDA hydrogel in vitro. Usingmultiple forms of microscopy, we observed cell infiltration into, and attachment to, a PEGDA hydrogelby undifferentiated and chondrogenically differentiating hMSCs. This infiltration occurred after only2 weeks in culture, was on the order of 10–100 µm and was facilitated by cell attachment to the PEGDAvia focal contacts.

Vinculin is a component of focal contacts [27] and can bind to F-actin [28]. Induced changes invinculin gene expression play a role in adhesion and motility by regulating the number of focal contacts[29]. We have demonstrated vinculin expression in both hMSCs and hMSC-Cys encapsulated withinPEGDA hydrogel, as well as actin cytoskeleton reminiscent of cells in a 3-D matrix. This expression ofvinculin, a constituent of focal contacts which allow the cell to attach to its environment, may providesome insight into the migration of the cells through the PEGDA hydrogel. The cells may migrate throughthe material by attaching to the polymer chains of the hydrogel and taking advantage of the degradationof the hydrogel [30], or possibly by production of proteinases [31].

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Fig. 5. Confocal stack montage of hMSC-Cys infiltrating PEGDA hydrogel. Human MSCs were cultured on top of PEGDAhydrogel and cultured in chondrogenic supplemented media for 2 weeks as described above. The cells and material were fixedand stained with “Cell-Tracker Orange” and images were converted to grayscale where cells are white. Group of images is 3-Dreconstruction of slices beginning from the top of the PEGDA hydrogel looking down (A) with sequential clockwise rotationsaround the vertical axis of 7.5◦ (B–K) to a complete 90◦ rotation displaying a cross section of the PEGDA hydrogel (L). Cellnoted by arrow in G–L migrated from its original position on top of the material to becoming embedded within the hydrogel.

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Fig. 6. Actin/vinculin/nucleus stain of hMSCs and hMSC-Cys encapsulated within PEGDA hydrogel. Human MSCs wereencapsulated at in PEGDA hydrogel and cultured in unsupplemented (hMSCs, A–D) and chondrogenic supplemented (hMSC–Cys, E–H) media for 2 weeks as described above. The cells and material were fixed and stained with “Alexa Fluor” phalloidin(actin, red, A and E), FITC-conjugated vinculin (green, B and F) and DAPI (nucleus, blue, C and G). Representative imagesof each stain as well as an overlay of all colors for each cell type (D and H) are shown. Both cell types showed focal contactpresence, exemplified by yellow color as a result of green and red (vinculin and actin stains, respectively) colors overlaid inimages (noted by arrows). (The colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-140991.)

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There is a possibility that infiltration would have progressed longer with a longer time in culture.However, the purpose of this study was to determine if we could reproduce in vitro cell migration intoPEGDA hydrogel noted in vivo. It may be noted that if the mesh size of the polymer chains formingthe hydrogel was on the order of the size of the cells used (10 µm diameter when encapsulated in a 3-Denvironment), the cells may have simply moved through the pores immediately upon seeding. While afinite pore size was not determined in this group of experiments, the relatively smooth appearance ofthe PEGDA hydrogel specifically in the SEM images (see Figs 3, 5 and 6) indicate an absence of poresequal to or greater than the size of the encapsulated cells. Likewise, the mesh size of similar PEGDAhydrogels has been found to be on the order of angstroms [32]. This is much smaller than the size of anycell, so we can conclude with confidence the cells actively migrated through the hydrogel as opposed topassively moving through it. The hydrogel shrunk to about one third of its original size after dehydrationwith HMDS during preparation for the SEM study. Since the cells in the hydrogel still displayed intactconnections to the hydrogel and typical cell morphology for cells encapsulated in this type of material,the loss of size of the hydrogel did not impact the results.

There should be some stimulus present for cells to migrate into the hydrogel. In the in vivo study,the PEGDA hydrogel loaded with growth factor may have acted as a chemotactic factor for the hostcell infiltration [33]. However, in the current in vitro study, growth factor was not added to the PEGDAhydrogels. Growth factors present in the experiments were in the serum in the basal (hMSC) media andthe TGF-β3 in the chondrogenic (hMSC-Cy) media. Growth factor trapped within the PEGDA hydrogelmay have acted as a chemotactic factor for the cells seeded on top of the hydrogel. The cells would movefrom lower concentrations of growth factor to higher concentrations if growth factor became trapped inthe hydrogel [34].

5. Conclusions

In conclusion, we found in vitro attachment and infiltration of hMSCs and their chondrogenic counter-parts in PEGDA hydrogel. This interaction of these cells with unmodified PEGDA hydrogel has not, toour knowledge, been reported in the literature. These findings aid in the understanding of how cells inter-act with the scaffold in which they are placed to create a tissue engineered construct and has implicationsin the development of successful biomaterials.

Acknowledgements

This work was funded by NIH grant EB006261 to JJM. We are indebted to Drs. Shan Sun, IgorTitushkin and Michael Cho for assistance with staining and confocal microscopy.

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