Research Article, J Regen Med Vol: 6 Issue: 3

Effects of Polymer Encapsulated Glial Cell Line-Derived Neurotrophic Factor Secreting Cells on Odontoblast-like Cell Survival

Saito N¹, Washio A², Miyashita K², Wahlberg LU³, Emerich DF³, Morotomi T², Nishihara T⁴ and Kitamura C^2*

¹Department of Biological Endodontics, Graduate School of Biomedical & Health Sciences, Hiroshima University, Japan

²Division of Endodontics and Restorative Dentistry, Department of Oral Functions, Kyushu Dental University, Japan

³Gloriana Therapeutics Incorporation, Providence, USA

⁴Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Japan

*Corresponding Author : Dr. Chiaki Kitamura
Department of Oral Functions, Kyushu Dental University, Japan
Tel: 01-93-582-1131
E-mail: r06kitamura@fa.kyu-dent.ac.jp

Received: December 07, 2017 Accepted: December 19, 2017 Published: December 22, 2017

Citation: Saito N, Washio A, Miyashita K, Wahlberg LU, Emerich DF, et al. (2017) Effects of Polymer Encapsulated Glial Cell Line-Derived Neurotrophic Factor Secreting Cells on Odontoblast-like Cell Survival. J Regen Med 6:3. doi: 10.4172/2325-9620.1000140

Abstract

Keywords: GDNF; VEGF; Encapsulated cells; Odontoblast; Dental pulp; Starvation; Cell survival

Introduction

Dental pulp plays an essential role in tooth nutrition and detection of potential pathogens. Damage or loss of dental pulp frequently leads to increased fragility of the tooth and a reduction in quality of life. Accordingly, effective strategies are needed to enhance dental pulp wound healing and regeneration following damage or disease. Regenerative endodontics is a relatively new field of research with the goal of replacing damaged/diseased pulp tissues with regenerated pulp-like tissues. In general, the approaches under investigation include the use of stem cells to replace dental pulp cells [1] and the enhancement of regenerative signaling mechanisms often using material-based scaffolds to deliver therapeutic molecules capable of augmenting regrowth of damaged cells [2].

Our group is developing a delivery system based on implanting trophic factor-secreting cells that have been encapsulated in a polymer membrane. The pores of the membrane are sufficiently large to allow secreted molecules to cross the membrane and enter the surrounding host tissue, but small enough to protect the encapsulated cells from host immune identification. Encapsulated cell therapy provides a targeted, continuous, de novo synthesized source of molecules from a system that combines the potency of de novo in situ synthesis of cellderived molecules with the safety of an implantable, biocompatible, and retrievable medical device.

Glial cell line-derived neurotrophic factor (GDNF) is known as a member of the transforming growth factor-ß superfamily [3] and has well documented roles in cell survival and regeneration, especially within the central nervous system (CNS) [4]. In addition, GDNF has non-neuronal functions in tooth development [5] and in the survival of dental pulp cells [6], raising the possibility that GDNF could be used to improve wound healing and regeneration of dental pulp.

In the present study, we provide the first evidence that the sustained delivery of GDNF from encapsulated, genetically-modified cells can enhance and prolong the survival of odontoblast-like cells under in vitro long-term starvation conditions.

Material and Methods

Cell culture

ARPE-19 cells were cultured in T-175 flasks with growth medium; Dulbecco’s Modified Eagle Medium (DMEM) + glutamax (1x) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific Incorporaiton, MA, USA). Routine culture consisted of feeding the cells every 2-3 days and passaging them at 70-75% confluence. Cells were incubated at 37 ºC, 90% humidity and 5% CO₂. A rat clonal dental pulp cell line (KN-3 cells) with odontoblastic properties such as a high level of alkaline phosphatase (ALP) activity was previously established from dental pulp cells of rat incisors [7,8]. KN-3 cells were cultured with minimum essential medium Eagle alpha modification (αMEM) (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% heat-inactivated FBS (JRH Bioscience, Lenexa, KS, USA), 100 mg/mL streptomycin (Sigma-Aldrich), and 100 U/mL penicillin (Sigma-Aldrich) at 37ºC in an atmosphere of 5% CO₂ and air.

GDNF cell line establishment

Human GDNF cDNA optimized for human cell line expression was produced by Invitrogen (Thermo Fisher Scientific Incorporation, MA, USA) and subsequently cloned to replace NGF in the expression vector pT2.CAn.hNGF, resulting in the plasmid pT2.CAn.hoG. ARPE- 19 cells were transfected with this vector using the Sleeping Beauty (SB) transposase system [9,10]. Briefly, cells were co-transfected with plasmids pT2.CAn.hoG and the SB vector pCMV-SB-100x. As the SB vector does not contain a eukaryotic selection marker cassette, it is only transiently expressed. The transient expression window allows for the active, transposase-mediated integration of the SB transposon, i.e. the inverted repeat SB substrate sequences and the sequences contained within these repeats, including the GDNF expression and neomycin antibiotic resistance cassettes. Clones were selected using G418 (Sigma-Aldrich) and single colonies were expanded and isolated based on their GDNF release levels.

Device fabrication

Cells were encapsulated into porous hollow fiber membranes (the typical pore diameter is <1 μm) as previously described [11]. Devices were manufactured from polysulfone membrane internally fitted with filaments of polyethylene terephthalate yarn scaffolding for cell adhesion. Prior to filling, cultured cells were suspended in human endothelial serum free medium (heSFM) (Gibco) at a density of 10,000 cells/μL and injected into each device using a custom manufactured automated cell-loading system. Devices were loaded with either GDNF-producing cells (GDNF-device) or with non-modified ARPE- 19 cells (ARPE-control device) and were separately maintained in a well of 24-well plate with 1 mL of heSFM at 37ºC in an atmosphere of 5% CO₂ and air. Maintenance medium of the device was changed in every 5 days.

Scanning electron microscopy analysis

Scanning electron microscopy (SEM) was used to evaluate the ultrastructure of the device membrane wall, as well as the inner and the external surfaces of the device membrane. For visualization of the wall, a freeze-fracture technique was employed prior to coating with metal. Samples were immersed in liquid nitrogen for approximately 2 min and fractured using two pairs of jeweler’s forceps. Samples were mounted on carbon adhesive backing on SEM stubs and sputter-coated with a Pd-Au target for 3.5 min. The Hitachi 2700 Scanning Electron Microscopy (Hitachi High Technologies America Incorporation, Clarksburg, MA, USA) was used with an accelerating voltage of 8 kV.

Enzyme-linked immunosorbent assay

Concentrations of GDNF and VEGF secreted from devices were analyzed by enzyme-linked immunosorbent assay (ELISA). GDNFand ARPE-control devices were individually maintained in 1 mL of heSFM or αMEM supplemented with 0.1% FBS for 7 days. The media was collected and the concentrations of GDNF and VEGF in the medium were determined using human GDNF Emax Immuno Assay System (Promega, Madison, WI, USA) and Quantikine ELISA Human VEGF Immunoassay (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s protocol, respectively. Absorbance was determined using a microplate reader (Bio-Rad iMark, Bio- Rad Laboratories, Hercules, CA, USA). GDNF and VEGF in the maintenance medium treated with either soluble Flt-1 or soluble GFRα-1 were also analyzed by ELISA.

Morphological analysis of the cells co-cultured with devices

Twenty four hours before assay, GDNF-device and ARPE-control devices were individually placed into wells of 24-well plate with 1 mL of αMEM supplemented with 0.1% FBS. KN-3 cells (3×10³ cells/well) in 24-well plates were cultured in αMEM with 10% FBS for 24 hours, followed by a change to αMEM with 0.1% FBS, and then each device was gently immersed into an individual well. During the co-culture, the medium was not exchanged. Seven days after the co-culture, the cells proximal and distal to each device were observed using phasecontrast microscopy.

Cell viability assay

Cell viability was analyzed by direct counting of viable cells that excluded trypan blue. A diagram of this experiment is shown in Figure 1. The cells (3×10³ cells/well for 7 days and 1×103 cells/well for 14 days) were cultured in 24-well plates containing αMEM with 10% FBS for 24 hours, then the medium was changed to one of the following; 1% FBS-αMEM, 0.1% FBS-αMEM, 0.1% FBS heSFM control; 0.1% FBS mixture of αMEM and heSFM in a ratio of 50:50, 0.1% FBS ARPE-19 control; 0.1% FBS mixture of αMEM and the maintenance medium of ARPE-19-control devices in a ratio of 50:50, and 0.1% FBS ARPE-19 GDNF; 0.1% FBS mixture of αMEM; and the maintenance medium of GDNF-devices in a ratio of 50:50.

The cells were cultured in each medium for 7 or 14 days with the medium change every 2 days. Attached cells were washed with phosphate buffered saline and detached after a brief exposure to trypsin/EDTA. An aliquot (10 mL) of cell suspension was mixed with an equal volume of trypan blue and placed into a TC10 automated cell counter (Bio-Rad Laboratories). Viable cells were counted using trypan blue exclusion. The proliferation of KN-3 cells following the end of long-term starvation was also examined. Each medium was changed to αMEM with 1% FBS at the end of long-term starvation, and cultured for 2 days, followed by counting of viable cells using trypan blue exclusion.

ALP staining and activity

KN-3 cells (3×10³ cells/well) were cultured in 24-well plates in each conditioned medium for 7 days, then the medium was changed to αMEM with 1% FBS and cultured for additional 1 and 3 days. At each time point, the adherent cells were fixed and stained for ALP using a NBT/BCIP Stock Solution (Sigma-Aldrich) according to the manufacturer’s instructions. Also, the cells (4.5×10³ cells/well) in 6-well plates were cultured in each conditioned medium for 7 days, followed by additional 1 and 3 days in αMEM with 1% FBS, at which time ALP activity was measured using a p-nitrophenylphosphate assay (LabAssay ALP Kit; Wako Pure Chemical Industries, Tokyo, Japan). After 15 min of incubation at 37°C, absorbance of p-nitrophenylphosphate at 405 nm was determined using a microplate reader (Bio-Rad iMark, Bio-Rad Laboratories), and the specific activity of ALP (U/μL) was calculated. One unit of the enzymatic activity is defined as release of 1 nmol p-nitrophenyl per min at pH 9.8 and 37°C.

Neutralization of VEGF by soluble Flt-1 and GDNF by soluble GFRα-1

Before the cell viability assay, 2 mg/mL of soluble Flt-1 (recombinant human VEGF R1/Flt-1 Fc Chimera, 321-FL, R&D systems) was added to each maintenance medium and incubated for 3 hours to neutralize VEGF [12]. The cells were then cultured in each conditioned medium pretreated with soluble Flt-1 for 7 days. Each medium was changed to fresh soluble Flt-1-neutralized medium every 2 days. Seven days after culture, cell viability was analyzed by trypan blue exclusion. Also, the maintenance medium of GDNF-device was pretreated by 3 mg/mL of soluble GFRα-1 (recombinant rat GFRα-1/ GDNF Rα-1 Fc Chimera, 560-GR, R&D systems) for 3 hours before the cell viability assay. The cells were cultured in soluble GFRα-1- neutralized medium for 7 days, with the change of the medium to fresh soluble GFRα-1-neutralized medium every 2 days. The cell viability was analyzed by trypan blue exclusion 7 days after the culture.

Statistical analysis

All experiments were performed at least three times to test the reproducibility of results, and the representative results are shown. Results of ELISA, the cell viability assay, and ALP activity are presented as mean ± standard deviation (SD) of three replicates. Statistical significant differences were determined using one-way ANOVA computation combined with Tukey’s post hoc tests (IBM SPSS Statistics, Version 19; IBM SPSS, Chicago, IL, USA). Values of p<0.05 and <0.01 were regarded as significant.

Results

SEM analysis of a device

Figure 2 shows the general appearance (Figure 2A) and SEM cross sections of an encapsulation device. The membrane of the device possessed a typical isoreticulated morphology with a relatively dense, thin outer skin and an open, much thicker macroporous substructure (Figures 2B, C, D). Measures of membrane cross sections revealed an inner diameter of approximately 480 μm, an outer diameter of 660 μm, and a corresponding wall thickness of approximately 90 μm. In Figure 2E, the function of a device is conceptualized. Geneticallymodified GDNF-secreting ARPE-19 cells or non-modified ARPE-19 cells are individually loaded into a device. The cells are retained within the device, and GDNF and/or VEGF produced by encapsulated cells are secreted to the surrounding medium through the pores of the device. Nutrition for encapsulated cells is taken through pores.

Concentrations of GDNF and VEGF secreted from devices

GDNF and VEGF secretion from devices in human endothelial serum free medium (heSFM) was quantified by ELISA. GDNF levels were 5.9 mg/day for GDNF-devices and 0 mg/day for ARPE-control devices. Because ARPE-19 cells normally secrete low levels of VEGF, VEGF was also measured by ELISA and found that VEGF levels were ranged from 0.59-0.67 ng/day, for GDNF- and ARPE-control devices, respectively. When devices were maintained in αMEM with 0.1% FBS, the secretion of both growth factors decreased. In αMEM with 0.1% FBS, concentrations of GDNF and VEGF secreted from GDNFdevices were 0.15 mg/day and 0.4 ng/day, and VEGF secreted from ARPE-control devices was 0.37 ng/day, respectively.

Cell proliferation in co-culture of KN-3 cells with devices

The survival of KN-3 cells was examined after the co-culture with GDNF- or ARPE-control device in αMEM with 0.1% FBS for 7 days (Figure 3). When KN-3 cells were co-cultured with GDNFdevice, proliferation of the cells with notable processes was observed in surrounding area of the device (Figure 3B-a), whereas only a few living cells were observed in the far area of the devices (Figure 3Bb). A similar concentration gradient-dependent effect was seen when KN-3 cells were co-cultured with ARPE-control devices. Proliferation of the cells with processes was observed in surrounding area of the device (Figure 3C-a), but not in the far area (Figure 3C-b).

Cell survival after long-term starvation

From the result of ELISA, the secretion of GDNF and VEGF from devices decreased in αMEM with 0.1% FBS than in heSFM, so the cell viability assay was carried out using the medium which the maintenance medium of the device and αMEM were mixed in an equal amount (Figure 1).

Figure 4 shows the survival of KN-3 cells under normal (1% FBS-αMEM) and starvation conditions. After 7 days of culture, the number of living cells in 0.1% FBS heSFM control group was about one-third that of the 1% FBS-αMEM group, and the numbers of living cells in both 0.1% FBS ARPE-19 control and 0.1% FBS ARPE- 19 GDNF groups were significantly greater than those in both 0.1% FBS-αMEM and 0.1% FBS heSFM control groups (Figure 4A). After 14 days of culture, the number of living cells in 0.1% FBS ARPE-19 control group appeared to be greater than that of 0.1% FBS heSFM control group, and the number of living cells in 0.1% FBS ARPE-19 GDNF group was significantly greater than that of 0.1% FBS heSFM control group (Figure 4B).

Cell proliferation after long-term starvation

To analyze the proliferative ability of surviving KN-3 cells post long-term starvation, the cells after the end of long-term starvation were cultured in αMEM with 1% FBS for 2 days and the numbers of living cells were determined (Figure 5). After 7 day starvation, the numbers of living cells from the 0.1% FBS heSFM control, 0.1% FBS ARPE-19 control, and 0.1% FBS ARPE-19 GDNF groups were significantly greater than that from 0.1% FBS-αMEM group (Figure 5A). After 14 days of starvation, the number of living cells of 0.1% FBS ARPE-19 GDNF group was significantly greater than that from 0.1% FBS heSFM control group, and the number of living cells of 0.1% FBS ARPE-19 control group was comparable to that of 0.1% FBS-αMEM control (Figure 5B).

Surviving KN-3 cells post 7 day starvation were cultured in αMEM with 1% FBS for an additional 1 and 3 days, and then processed for ALP staining and quantification of ALP activity. ALP staining was observed in the cells of 0.1% FBS-αMEM, 0.1% FBS ARPE-19 control, and 0.1% FBS ARPE-19 GDNF groups, but not in that of 0.1% FBS heSFM control group (Figure 6). ALP activity of 0.1% FBS-αMEM, 0.1% FBS ARPE-19 control, and 0.1% FBS ARPE-19 GDNF groups increased in a time dependent manner (Figure 7).

Effects of neutralization of VEGF and GDNF on the cell survival

Figure 8 shows the effects of either soluble Flt-1 or soluble GFRα- 1 on cell survival after long-term starvation. In the 0.1% FBS ARPE-19 control group, neutralization of VEGF with soluble Flt-1 significantly reduced the number of living cells. In contrast, there was no difference in the number of living cells in the 0.1% FBS ARPE-19 GDNF group with or without the neutralization of VEGF by soluble Flt-1. When the maintenance medium of GDNF-device was pretreated by soluble GFRα-1 to neutralize GDNF, the number of living cells in the 0.1% FBS ARPE-19 GDNF group was intensely reduced.

Discussion

Cell encapsulation is a maturing platform technology that has emerged as a viable therapeutic option capable of providing targeted, long-term, continuous, de novo synthesized delivery of very high levels of therapeutic molecules in regenerative therapy [13]. The size of encapsulation devices can be tailored for implantation directly into the desired region of virtually any organ or tissue where the structure of the device retains the enclosed cells, allows the molecules of interest to be secreted, and also prevents immunological reactions of host; all while maintaining the viability of the encapsulated cells [14-16]. In the present study, the in vitro effects of GDNF and VEGF delivered from encapsulation devices were examined, and several basic findings were noted; (a) GDNF and VEGF from the device were capable of imparting significant survival benefits to odontoblast-like cells under long-term starvation conditions, (b) these benefits were concentration gradient-dependent and most pronounced in the region proximal to the device and diminished with increasing distance from the device, and (c) the surviving cells retained their function as demonstrated by normal proliferative and process elongation capabilities.

Surprisingly, odontoblast-like cells were significantly protected from the consequences of long-term starvation when the maintenance medium of both GDNF- and ARPE-control devices were used for the mixed medium. The maintenance medium of GDNF-device included both GDNF and low levels of VEGF, whereas the maintenance medium of ARPE-control device included only low levels of VEGF. The initial results of the cell viability assay suggested that VEGF contributed to the survival of odontoblast-like cells but the contribution of was unclear since the engineered ARPE-10 cells secreted both GDNF and VEGF. To clarify effects of GDNF secreted from GDNF-device on the cell survival, each maintenance medium was pre-treated with soluble Flt-1 as an inhibitor of VEGF, or soluble GFRα-1 as an inhibitor of GDNF, to neutralize each growth factor in the maintenance medium. As a result, the survival of odontoblast-like cells was suppressed by the addition of soluble Flt-1 to the maintenance medium of ARPEcontrol device, whereas the cell survival by the maintenance medium of GDNF-device was not inhibited by soluble Flt-1. On the other hand, the cell survival was completely inhibited by the addition of soluble GFRα-1 to the maintenance medium of GDNF-device. These results indicate that GDNF itself secreted from GDNF-device prolongs the survival of odontoblast-like cells with or without VEGF. In the neutralization assay, soluble Flt-1 did not completely inhibit the survival of odontoblast-like cells. Also after 7 days of starvation, some living cells were observed when cultured in the mixed medium of αMEM and human endothelial serum free medium, suggesting some benefit of the media itself. This effect dissipated after 14 days, further suggesting that the prolongation of the cell survival was induced by GDNF and VEGF secreted from devices, not by human endothelial serum free medium.

Multiple roles of GDNF and VEGF have been demonstrated in a variety of studies. Several research have indicated that the delivery or the induction of GDNF can prolong and protect the survival of photoreceptors of eye [17], spinal cord [18], and bone marrow mesenchymal stem cells [19]. During tooth development, GDNF and its receptors are expressed in dental pulp supporting a role for GDNF in epithelial mesenchymal interactions [20]. In GDNF knockout mice odontoblast differentiation is disrupted and GDNF inhibits dental pulp cell death induced by serum deprivation, suggesting that GDNF may play a cytoprotective role in dental pulp homeostasis during stress conditions and pulpal necrosis [5]. VEGF is known to affect the survival and the proliferation of endothelial cells [21]. In addition to its potent angiogenic abilities, VEGF produced by dental pulp cells can act in an autocrine manner to promote the chemotaxis, proliferation, and/or differentiation of dental pulp cells [22]. Further ongoing experiments are now attempting to clarify the mechanism (s) underlying the effects of GDNF and VEGF secreted from encapsulation devices on the survival of odontoblast-like cells, as well as to clarify effects of them on other cell types such as cementoblasts and periodontal ligament cells.

Conclusions

The in vitro results of the present study indicate that both GDNF and VEGF delivered from encapsulated cells exerted positive survival and proliferative influences on odontoblast-like cells. When GDNFand/ or VEGF-secreting devices are used for dental pulp in the clinic, it should be size-adapted and structurally optimized, however, the present results also suggest the possibility of using this encapsulated cell-based delivery system for multimodal polypharmacy in dentistry.

Acknowledgements

This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to C.K.: 26293406, N.S.: 16K20460). The authors declare that they have no conflicts of interest related to this study.

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