3D透明质酸凝胶培养的成纤维细胞和间充质干细胞

Cell–cell interaction between vocal fold fibroblasts and bone marrow mesenchymal stromal cells in three-dimensional hyaluronan hydrogel Xia Chen and Susan L. Thibeault* Department of Surgery, University of Wisconsin at Madison, WI, USA Abstract Mesenchymal stromal cells (MSCs) are multipotential adult cells present in all tissues. Paracrine effects and differentiating ability make MSCs an ideal cell source for tissue regeneration. However, little is known about how interactions between implanted MSCs and native cells influence cellular growth, proliferation, and behaviour. By using an in vitro three-dimensional (3D) co-culture assay of normal or scarred human vocal fold fibroblasts (VFFs) and bone marrow-derived MSCs (BM-MSCs) in a uniquely suited hyaluronan hydrogel (HyStem–VF), we investigated cell morphology, survival rate, proliferation and protein and gene expression of VFFs and BM-MSCs. BM-MSCs inhibited cell proliferation of both normal and scarred VFFs without changes in VFF morphology or viability. BM-MSCs demonstrated decreased proliferation and survival rate after 7 days of co-culture with VFFs. Interactions between BM-MSCs and VFFs led to a significant increase in protein secretion of collagen I and hepatocyte growth factor (HGF) and a decrease of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6). In particular, BM-MSCs significantly upregulated matrix metalloproteinase 1 (MMP1) and HGF gene expression for scarred VFFs compared to normal VFFs, indicating the potential for increases in extracellular matrix remodelling and tissue regeneration. Application of BM-MSCs-hydrogels may play a significant role in tissue regeneration, providing a therapeutic approach for vocal fold scarring. Copyright © 2013 John Wiley & Sons, Ltd. Received 12 November 2012; Revised 7 March 2013; Accepted 25 March 2013 Keywords BM-MSCs; VFFs; three-dimensional co-culture; cell regulation; hydrogel 1. Introduction Wound healing is a complex, dynamic process of restoring cellular and tissue structure. It consists of inflammatory, proliferative and remodelling phases with well-organized interactions among various types of cells and cytokines, including platelets, macrophages, mesenchymal stromal cells (MSCs), resident cells (fibroblasts and epithelial cells), released platelet-derived growth factor (PDGF), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), transforming growth factor (TGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) (Hollinger et al., 2008). This intricate process is susceptible to interruption or failure, leading to the formation of non-healing chronic wounds and scar. Specific to vocal folds, scarring causes abnormal tissue structure and function resulting in vocal hoarseness and fatigue, significantly decreasing one’s quality of life (Ma and Yiu, 2001). To date, a number of regenerative medicine strategies have been investigated for the prophylaxis and treatment of vocal fold scarring. These include the application of synthetic extracellular matrix (ECM) hyaluronan hydrogel (Duflo et al., 2006b; Thibeault et al., 2010), cell implantation (Halum et al., 2007) and utilization of biomaterials and cells concurrently (Ohno et al., 2011). Further in vitro investigation is necessary to provide support for future in vivo regenerative medicine based therapies for vocal fold tissue fibrosis. *Correspondence to: S. L. Thibeault, Division of Otolaryngol- ogy–Head and Neck Surgery, Department of Surgery, University of Wisconsin Madison, 5107 WIMR, 1111 Highland Avenue, Madison, WI 53705-2275, USA. E-mail: thibeault@surgery. wisc.edu Copyright © 2013 John Wiley & Sons, Ltd. JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med (2013) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1757 Because wound healing and tissue regeneration involves interaction and regulation between cells, it is essential to understand how communication between different cell types can affect regenerative outcomes. Vocal fold fibroblasts (VFFs), the main cellular component of vocal fold lamina propria, plays a vital role in the maintenance, development and repair of the ECM of vocal fold lamina propria (Gray et al., 2000). MSCs are multipo- tential adult stromal cells present in all tissues, which can be activated upon entering wounds or other damaged tissue, producing a broad range of bioactive molecules that have roles in immunomodulation, anti-apoptosis, angiogenesis, support of the growth and differentiation of local stem and progenitor cells, anti-scarring and chemoattraction (Caplan, 2010). Since 1995, bone marrow-derived MSCs (BM-MSCs) have been used in Phase I/II clinical trials in other parts of the body to treat scarring and tissue deficits (Langston, 2005; Mazo et al., 2012). Moreover, attention has been directed at the potential of BM-MSCs therapy for the prevention and treatment of vocal fold scar (Hong et al., 2011; Svensson et al., 2011). To date there is a paucity of data defining interactions between BM-MSCs and native tissue cells, specifically in a biomaterial environment. Investigations regarding in vitro stromal cell communication and thera- peutics for vocal fold scar require complex multicellular structures – multiple cell types and a three-dimensional (3D) ECM. For this investigation, we developed an in vitro 3D co-culture assay using VFFs, BM-MSCs and hyaluronan hydrogel HyStem–VF. HyStem–VF has been shown previously to be biocompatible with human VFFs (Chen and Thibeault, 2010a) to regulate human VFFs function, enhance ECM remodelling (Chen and Thibeault, 2010b) and improve tissue regeneration and vocal fold scarring (Duflo et al., 2006b). The purpose of this investi- gation was to elucidate in vitro cooperative aspects of VFFs and BM-MSCs in HyStem–VF and to characterize cellular behaviour parameters, including cell morphol- ogy, proliferation, viability and profiling of various bioac- tive proteins and genes. Our hypothesis was that in 3D, BM-MSCs and VFFs regulate each other’s proliferation rates without a significant effect on cell morphology and viability. We further hypothesize that through paracrine effects BM-MSCs regulate VFFs ECM production to promote tissue regeneration, providing in vitro support for our long-term goal – employing BM-MSCs in combination with hydrogels as an injectable therapeutic for vocal fold scarring. 2. Materials and methods 2.1. Human vocal fold fibroblasts and BM-MSCs BM-MSCs were derived from bone marrow of healthy donors, based on protocols approved by the University of Wisconsin Health Science Institutional Review Board (IRB) after obtaining informed consent from the donors (Hanson et al., 2010). VFFs were isolated from normal and scarred vocal folds of human donors (Chen and Thibeault, 2008; Jette et al., 2013), based on protocols approved by the University of Wisconsin Health Science IRB. BM-MSCs and VFFs were expanded using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Passages 4–8 of these primary cells were used for all experiments. 2.2. Hyaluronan–gelatin hydrogel HyStem–VF is an injectable chemicallymodified hyaluronan- gelatin hydrogel (Biotime Inc., Alameda, CA, USA), which was obtained by mixing 1ml 1.4% w/v thiol-modified semi-synthetic glycosaminolycan analogous (Glycosil) with 75ml 1.0% w/v thio-modified gelatin (Gelin-S) and crosslinking this mixture with 8.2% w/v Extralink (PEGDA), as previous described (Shu et al., 2003). The final concentra- tion of HyStem–VF was 1.2% Glycosil, 0.06% Gelin-S and 0.8% PEGDA. All components were dissolved in sterile water in a cell culture hood to ensure sterility. At room tempera- ture, HyStem–VF casts in about 5min. 2.3. 3D co-culture of VFFs and BM-MSCs In co-culture, primary normal or scarred VFFs were seeded at 1�105 cells/well in a six-well plate [tissue culture polystyrene (TCP)] in DMEM–10% FBS medium. After incubation at 37 �C for 4h, a transwell insert with 500ml mixture of BM-MSCs and HyStem–VF (2�106 cells/ml) was plated into the well with VFFs (Figure 1). All cells and hydrogel were covered by medium and co-cultures were maintained for 7days. 3D co-culture allows cells tomaintain contact distance without physical contact. Cell concentra- tion was calculated to maintain constant cell–cell distance; a cell concentration of 1�106/ml in 3D is equivalent to a plating density of 1�104 cells/cm2 (Semino et al., 2003). As controls, monocultures of VFFs on TCP or BM-MSCs in 3D HyStem–VF were established using the same methods as noted above. All experiments were performed in triplicate. 2.4. Immunostaining and confocal microscopy After 1week of co-culture, VFFs and BM-MSCs were sepa- rately fixed in 4% paraformaldehyde for 30min. After washing, the cells were permeabilized three times with 1� PBS with 0.1% Triton X-100 for 5min. Following blocking (in 5% normal goat serum), the cells were incu- bated with 1:200 mouse anti-human-prolyl 4-hydroxylase antibody for 90min (hPH; Millipore, Billerica, MA, USA) and detected with 1:100 Alexa488-conjugated goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR, USA) for 60min at room temperature. Nuclei were labelled with DAPI (mounting medium with DAPI, Vector Laboratories, Burlingame, CA, USA). Cell morphological features were examined and the images X. Chen and S. L. Thibeault Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013) DOI: 10.1002/term were captured using an inverted confocal microscope (Nikon A1R, Melville, NY, USA). 2.5. Cell viability assay Cell viability of BM-MSCs and VFFs was separately semi- quantified using a Live/Dead Viability/Cytotoxicity Assay (Invitrogen, Carlsbad, CA, USA) according to the manufac- turer’s instructions. This assay is based on the simultaneous determination of live and dead cells with two-colour fluorescence probes (calcein acetoxymethyl ester–calcein AM and ethidium homodimer 1-EthD-1) that measures recognized parameters of cell viability–intracellular esterase activity and plasma membrane integrity. This assay has been used to quantify apoptotic cell death and cell- mediated cytotoxicity (Lichtenfels et al., 1994). After 7 days of co-culture, VFFs and BM-MSCs incubated separately with staining solution (2mM calcein AM and 4mM EthD-1 in PBS) at room temperature for 30min. Following incubation, cells were imaged using a Nikon E600 fluorescence microscopy-equipped (Nikon Instruments, Melville, NY, USA) Olympus DP71 CCD (Olympus America, San Jose, CA, USA) at� 10 magnification, using green and red filters. The percentage of live and dead cells was determined using MetaMorph software for each condition in quadruplicate. A minimum of 100 cells were counted for each image. 2.6. Cell proliferation assay VFFs were seeded at 1�104 cells/well in 24-well plates and grown for 4h prior to adding the insert with BM-MSCs and HyStem–VF (1�105 cells/well in 200ml HyStem–VF). After 1, 4 and 7days of co-culture, cell numbers of VFFs and BM-MSCs were separately monitored in quadruplicate, using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Briefly, 200 ml medium was gently removed from each well and 200 ml CellTiter- Glo reagent was added into each well with VFFs or BM-MSCs. After 10min of incubation at room tempera- ture, the luminescent output was read on a Flex Station III plate-reader (Molecular Devices, Sunnyvale, CA, USA). The luminescent signal reflects the ATP level and is proportional to the number of viable cells (Chen and Thibeault, 2010a; Crouch et al., 1993). 2.7. Gene expression analysis Total RNAwas separately extracted fromVFFs and BM-MSCs after 24 h of co-culture, using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and reverse-transcribed using a QuantiTect Reverse Transcription Kit (Qiagen). mRNA from the cDNA sample was applied with specific primer pairs (Table 1) for matrix metalloproteinase 1 (MMP1), tissue inhibitor of metalloproteinase 3 (TIMP3), collagen I a-2 (Col1), collagen III a-1 (Col3), IL-6, VEGF, HGF and the housekeeping gene b-actin (internal control). Reactions were performed using SYBR-Green PCR Master Mix (Roche, Basel, Switzerland) in the Light Cycler System (Roche) with a standard curve method, as described previously (Chen and Thibeault, 2010a). Results are calculated by each target gene mRNA (ng/ml) normalized to the housekeeping gene b-actinmRNA (ng/ml). 2.8. Secreted cytokines and proteins After 48 h of co-culture, conditioned media were collected for all conditions. Secreted cytokines and proteins, which included MCP-1, IL-6, VEGF, HGF, TGFb1, HGF, collagen I and collagen III, were analysed by enzyme-linked immuno- sorbent assay (ELISA; Invitrogen; except for collagen I, MD Bioproducts, St. Paul, MN, USA; and collagen III, My Biosource, San Diego, CA, USA), according to manufac- turers’ instructions. 2.9. Statistical analyses Values of cellular ATP, cell survival rate, protein and gene expression were expressed as mean� standard deviation (SD). Analysis of variance (ANOVA) with Fisher’s protected least significant difference tests was performed to examine: Figure 1. Schematic of 3D co-culture Table 1. Primer sequences and products of RT–PCR Gene GeneBank no. Forward primer Reverse primer Size (bp) Collagen I-a2 NM_000089 50-AACAAATAAGCCATCACGCCTGCC-30 50-TGAAACAGACTGGGCCAATGTCCA-30 101 Collagen III-a1 NM_000090 50-CCATTGCTGGGATTGGAGGTGAAA-30 50-TTCAGGTCTCTGCAGTTTCTAGCGG-30 187 MMP1 NM_002421 50-TGCAACTCTGACGTTGATCCCAGA-30 50-ACTGCACATGTGTTCTTGAGCTGC-30 122 TIMP3 NM_000362 50-TGATGCAGCACACACAATTCCC-30 50-AAGCTCTGTTATTCTGGCCTGGGT-30 102 VEGF NM_003376 50-ACACATTGTTGGAAGAAGCAGCCC-30 50-AGGAAGGTCAACCACTCACACACA-30 179 HGF NM_000601 50-GGCCCACTTGTTTGTGAGCAACAT-30 50-TGGTGGGGTGCTTCAGACACACTTA-30 84 b-Actin NM_001101 50-ACGTTGCTATCCAGGCTGTGCTAT-30 50-CTCGGTGAGGATCTTCATGAGGTAGT-30 188 Fibroblasts and MSCs in 3D HA culture Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013) DOI: 10.1002/term (a) effect of co-culture on cell proliferation at different time points: (b) effect of co-culture on cell survival rate; (c) effect of co-culture on protein expression of cytokines and collagens; and (d) effect of co-culture on gene expression of cytokines, collagens and growth factors. Prior to all analyses, data were rank-transformed. p≤0.05 was considered significant. All analyses were performed using SAS statistical software (SAS Institute, Cary, NC, USA). 3. Results 3.1. Morphological features of co-cultured VFFs and BM-MSCs Representative photographs for each type of cell under different culture conditions are presented in Figure 2. After 1week of culture with and without BM-MSCs, normal and scarred VFFs maintained their typical spindle shape (Figure 2A–D). BM-MSCs in 3D HyStem–VF demonstrated roundedmorphological features (Figure 2E); after co-culture with VFFs (normal and scarred) BM-MSCs sustained this rounded morphology (Figure 2 F, G). After a 2week culture period, VFFs and BM-MSCs (including controls and co-cultured cells) maintained similar morpho- logical features as described for 1week (data not shown). 3.2. Effect of co-culture on cell proliferation In order to investigate the effect of co-culture on cell proliferation, total ATP values, which are proportional to the number of viable cells, were investigated separately for VFFs and hydrogel-encapsulated BM-MSCs (Figure 3). After 1week of culture, scarred VFFs growth was slower than normal VFFs (p< 0.001), proliferation rates for both normal and scarred VFFs were significantly suppressed by BM-MSCs compared to their monoculture controls (p< 0.001; Figure 3A). In contrast, proliferation of BM-MSCs on days 1 and 4 was not significantly affected by either VFFs (Figure 3B). On day 7, proliferation of BM-MSCs was significantly inhibited by both normal and scarred VFFs compared to control (p< 0.001 and p< 0.05, respectively). 3.3. Effect of co-culture on cell survival rate After 1week of co-culture, cellular live/dead indicator dyes (calcein AM and EthD-1) were directly added to each kind of cell from different conditions and after 30min images were captured and analysed (Figure 4). For day 7, normal and scarred VFFs with or without co-cultured BM-MSCs demonstrated few dead (red) cells among many live (green) cells (Figure 4A–D, H). There was no significant differences in VFFs survival rate between monoculture and co-culture (p> 0.05). For BM-MSCs monoculture there was a significant increase in the number of dead cells compared to monocultured VFFs (Figure 4E; p< 0.0001). Co-culture with both normal and scarred VFFs (Figure 4F, G) significantly reduced BM-MSCs survival percentage from 80.5% to 64.0–65.4%, respectively (Figure 4H; p< 0.01). 3.4. Effect of co-culture on the gene expression of VFFs and BM-MSCs In order to examine the effect of co-culture on gene expression of VFFs and BM-MSCs, mRNA transcript expression was measured independently for VFFs and BM-MSCs, using real-time PCR. Investigated genes included wound healing-related VEGF and HGF ECM Figure 2. Cell morphology after 1week of culture: (A) monoculture normal VFFs; (B) normal VFFs after co-culture with BM-MSCs; (C) monoculture scarred VFFs; (D) scarred VFFs after co-culture with BM-MSCs; (E) monoculture BM-MSCs in 3D HyStem–VF; (F) BM-MSCs in 3D HyStem–VF co-culture with normal VFFs; (G) BM-MSCs in 3D HyStem–VF co-culture with scarred VFFs. Cells were stained with p4h (green) and nuclei were counterstained with DAPI (blue). Scale bar=10mm X. Chen and S. L. Thibeault Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013) DOI: 10.1002/term regulation-related collagen type I a-2 (Col1), collagen type III a-1 (Col3), MMP1 and TIMP3, and an inflamma- tory cytokine, IL-6. After 24 h, monocultured scarred VFFs demonstrated significantly higher expression of Col1, Col3, MMP1 and TIMP3 compared to monocultured normal VFFs (p< 0.05, p< 0.01, p< 0.01 and p< 0.05, re- spectively), and co-culture with BM-MSCs caused further increases in expression of Col1,MMP1 and TIMP3 compared to scarred VFFs monoculture (p< 0.05, p< 0.01 and p< 0.05, respectively; Figure 5A and Table 2). In particular, MMP1 expression from scarred VFFs increased 4.19 times in the presence of BM-MSCs. We also observed that signifi- cantly lower levels of VEGF and HGF genes were expressed in monocultured scarred VFFs compared to monocultured normal VFFs (both p< 0.05), and BM-MSCs significantly upregulated their expression levels for both VFFs compared Figure 4. Effect of 3D co-culture on cell viability. After 1week of monoculture and co-culture, live/dead microscopy images of VFFs and BM-MSCs in HyStem–VF were taken: (A) monocultured normal VFFs; (B) co-cultured normal VFFs; (C) monocultured scarred VFFs; (D) co-cultured scarred VFFs; (E) monocultured BM-MSCs in HyStem–VF; (F) normal VFFs co-cultured BM-MSCs in HyStem– VF; (G) scarred VFFs co-cultured BM-MSCs in HyStem–VF. Green, viable cells; red, dead cells. Scale bar=500mm. (H) Live cells (%) were analysed by Metamorph software. *p<0.05; **p<0.01 Figure 3. Effect of co-culture on cell proliferation. VFFs were seeded at 10 000 cells/well and grown for 4h prior to co-culture with BM-MSCs (100 000 cells/well in 3D HA). On days 1, 4 and 7, viable cell numbers of VFFs and BM-MSCs were separately evaluated by ATP amount (RLU) in quadruplicate. Data shown represent mean�SD of a single representative experiment. (A) ATP levels of VFFs (normal and scarred VFFs) in monoculture and BM-MSCs co-culture conditions; (B) ATP levels of BM-MSCs in monoculture and co- culture conditions. *p<0.05; **p<0.01 Fibroblasts and MSCs in 3D HA culture Copyright © 2013 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2013) DOI: 10.1002/term to their monoculture (p< 0.05 and p< 0.01, respectively). IL-6 gene expressio