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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: J Tissue Eng Regen Med. 2020 Mar 4;14(4):622–632. doi: 10.1002/term.3026

A Proof of Concept Gene-Activated Titanium Surface for Oral Implantology Applications

Noah Zachary Laird 1, Walla I Malkawi 1, Jaidev Leela Chakka 1, Timothy Acri 1, Satheesh Elangovan 1,2,*, Aliasger K Salem 1,3,*
PMCID: PMC7180124  NIHMSID: NIHMS1573389  PMID: 32078257

Abstract

Dental implants are very successful medical devices, yet implant failures do occur due to biological and mechanical complications. Peri-implantitis is one such biological complication that is primarily caused by bacteria and their products at the implant-soft tissue interface. Bacterial infiltration can be prevented by the formation of a reliable soft tissue seal encircling dental implants. Platelet derived growth factor-BB (PDGF-BB) has significant chemotactic and proliferative effects on various mesenchymal cell types, including fibroblasts, and therefore can be an effective molecule to enhance the peri-implant soft tissue seal. To overcome the limitations of the recombinant protein form of PDGF-BB, such as cost and the need for supraphysiological doses, we have developed and characterized a titanium surface that is rendered bioactive by coating it with polyethylenimine (PEI)-plasmid DNA (pDNA) nanoplexes in the presence of sucrose. Human embryonic kidney 293T (HEK293T) cells and human primary gingival fibroblasts (GFs) were successfully transfected in culture with enhanced green fluorescent protein (EGFP)-encoding pDNA or platelet derived growth factor subunit B (PDGFB)-encoding pDNA loaded into nanoplexes and coated onto titanium discs in a dose-dependent manner. GFs were shown to secrete PDGF-BB for at least 7 days after transfection, and displayed both minimal viability loss and increased integrin-α2 expression 4 days post-transfection.

Keywords: dental, oral, coating, implant, titanium, gene-therapy, transfection, polyethylenimine

1. Introduction:

Titanium dental implants are now one of the most successful medical devices, with a success rate upwards of 90% (van Velzen et al., 2015). Despite their high survival rates, implant failures do occur and implants are not immune to mechanical and biological complications. Peri-implant mucositis (the implant equivalent of gingivitis) and peri-implantitis (the implant equivalent of periodontitis) represent the bulk of biological complications, and are more prevalent than previously estimated (Derks et al., 2015). Similar to periodontitis, the primary etiological factor for these peri-implant pathologies is bacterial plaque but the tissue destruction endpoint is host-mediated. For effective management of peri-implantitis, surgical intervention is warranted in many clinical situations. Currently there is a lack of evidence demonstrating that current reconstructive approaches are effective in predictably resolving the disease process and completely restoring the lost bone support (Khoshkam et al., 2013). Therefore, it is recommended that the best way to manage peri-implantitis is to prevent it from occurring (Jepsen et al., 2015).

The soft tissue seal encircling dental implants constitutes an important barrier in preventing the down growth of bacterial biofilms that can initiate gingival recession and peri-implant pathologies around dental implants (Atsuta et al., 2016). In the microbe-rich oral environment, rapid formation of a high quality circumferential soft tissue barrier at the supra-crestal component of the dental implant soon after its placement is key to the establishment and long-term maintenance of peri-implant health (Tomasi et al., 2016). Previous studies have clearly identified the inferiority of peri-implant soft tissue seals and the change in collagen fiber orientation from radial about the tooth to circular around the implant when compared to natural gingival seals (Arvidson et al., 1996; Cochran et al., 1997; Ikeda et al., 2002; Schierano et al., 2002). The inferiority of adhesion between peri-implant mucosa and implant surface relative to natural teeth in combination with the reduced vascularity in the former makes the peri-implant soft tissue seal vulnerable to rapid tissue breakdown (Ikeda et al., 2002; Moon et al., 1999; Tetè et al., 2009). Therefore, strategies to enhance the rapidity of formation and quality of the peri-implant soft tissue seal are vital to the maintenance of peri-implant health.

Previous studies have reported coating titanium with recombinant biological molecules such as platelet-derived growth factor-BB (PDGF-BB) and bone morphogenetic protein 2 (BMP-2) to enhance peri-implant tissue healing (Bates et al., 2013; Chong et al., 2006; Froum et al., 2015; Yoo et al., 2015a; Yoo et al., 2015b). The major barriers to using recombinant proteins are cost and the supraphysiological amounts required to compensate for their reduced bioavailability (Lee et al., 2011). Viral gene therapy has the potential to overcome these barriers, but concerns about immune reactions and insertional mutagenesis will hinder its translational potential (Evans, 2012). Non-viral gene therapy that utilizes non-viral vectors to deliver DNA of interest is an attractive alternative to viral gene delivery. Compared to other strategies, non-viral gene delivery systems are inexpensive and are customizable, however their major limitation is lower transfection efficiency (Al-Dosari et al., 2009). Despite this, using pDNA complexed with a cationic polymer such as PEI to form nanoplexes remains a popular and viable transfection method that yields high transfection efficiency and sustained expression of transfected genes (Mellott et al., 2013).

In this proof of concept study, our objective was to develop and characterize coated titanium surfaces that contain PEI-pDNA nanoplexes stabilized by a lyoprotectant and are similar in structure to those of titanium dental implants. Rapid delivery of nanoplexes coding for therapeutic proteins to mucosal tissue could induce sustained expression and release of those proteins soon after implantation. The use of sucrose as a lyoprotectant has already been shown to enable the stabilization of particle formulations and PEI-pDNA nanoplexes in other settings utilizing lyophilization, however, it has not been investigated in the context of surface coatings designed for gene therapy (Huang et al., 2003; Kasper et al., 2011; Mukalel et al., 2018). We demonstrate that this lyoprotectant-stabilized surface coating can successfully transfect the robust HEK293T cell line with nanoplexes containing EGFP pDNA. We also demonstrate that GFs can be successfully transfected with nanoplexes containing PDGFB pDNA and that the secreted PDGF-BB can be detected for at least 7 days after transfection. We additionally report that GFs transfected to express PDGF-BB have enhanced integrin-α2 mRNA expression and minimal viability loss after 4 days, as compared to control groups.

2. Materials and Methods:

2.1. Preparation of Titanium Discs:

Titanium discs made of commercially pure titanium were prepared as described previously (Atluri et al., 2017). Briefly, discs were sanded with a variable speed grinder-polisher (Ecomet 3, Buehler, Lake Bluff, IL, USA) using grinding papers (CarbiMet, Buehler) ascending to grit number 600. The titanium discs were then sandblasted (EWL Type 5423, KaVo, Germany) using 50 μm white aluminum oxide blasting compound (Ivoclar Vivadent, Liechtenstein). The sandblasted discs were then sonicated (Branson 5200, Branson Ultrasonics, Danbury, CT, USA) twice in ultrapure water for 5 minutes each to remove any remnants of blasting compound. The discs were degreased with acetone for 15 minutes, then acid etched with 30% nitric acid for 30 minutes. The discs were then rinsed with ultrapure water and stored in 70% ethanol. Sandblasting followed by acid etching is a common method used in the fabrication of dental implants with roughened titanium surface and has been used extensively in implantology research to create titanium surfaces that mimic commercial dental implant surface for in vitro evaluations (Bowers et al., 1992; Ko et al., 2010; Schneider et al., 2004).

2.2. Purification of Plasmid DNA:

A plasmid encoding EGFP (Plasmid #13031, Addgene, United Kingdom), a plasmid encoding mouse PDGFB (RefSeq BC064056, Origene, Rockville, MD, USA), and an empty vector plasmid (Plasmid #52535, Addgene) were purified from DH5α Escherichia coli that had been previously transformed. Purification was performed using a GenElute HP Endotoxin-Free Plasmid Maxiprep Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol.

2.3. Fabrication of PEI-pDNA Nanoplexes With and Without Sucrose:

Nanoplexes were prepared as described previously (Elangovan et al., 2014). Briefly, two 500 μL solutions containing either 130 μg of 25 kDa branched PEI (Sigma-Aldrich) or 100 μg of pDNA were prepared in DNAse/RNAse-free water (ThermoFisher Scientific, Waltham, MA, USA). The PEI solution was added to the pDNA solution, vortexed for 30s, and incubated for 30 minutes to allow for complexation between the pDNA and PEI. The resulting 1 mL nanoplex solution had a nitrogen (N) to phosphate (P) ratio (N/P ratio) of 10, which was shown to yield maximum transfection efficiency with minimal cytotoxicity (D’Mello et al., 2016; Elangovan et al., 2014). Varying volumes of 40% sucrose solution in DNAse/RNAse-free water (ThermoFisher Scientific) were added to the nanoplex solution to yield the desired sucrose concentrations (10%, 7.5%, 5%, 2.5%, 2%, 1%).

2.3. Characterization of Nanoplexes Pre- and Post-Lyophilization:

Nanoplexes were prepared as described above, then frozen in a −80°C freezer and lyophilized in microfuge tubes (FreeZone -105 4.5, Labconco, Kansas City, MO, USA). Their zeta potential and hydrodynamic size (pre- and post-lyophilization) were then measured via dynamic light scattering (DLS) and electrophoretic light scattering with a Zetasizer Nano-ZS (Malvern Instruments, United Kingdom) according to the manufacturer’s protocol.

2.4. Three Dimensional (3D) Printing and Preparation of Disc Suspension Devices:

“Suspension snaps” and “suspension bases” were designed with software (Fusion 360, AutoDesk, San Rafael, CA, USA) (Figure S1) and printed from 1.75mm poly-lactic acid (PLA) thermoplastic filament (Prusa Research, Czech Republic) using a fused deposition modeling (FDM) 3D printer (Original Prusa i3 MK3, Prusa Research). Printed suspension bases were glued to the lid of a 24-well tissue culture plate (Dot Scientific, Burton, MI, USA) with super glue such that all bases were oriented in the same direction, creating the “suspension lid” (Figure S2).

2.5. Coating of Titanium Discs with Nanoplex-Sucrose Solution:

Before use in transfection experiments, discs were sonicated twice for 15 minutes in ultrapure water, then rinsed with 70% ethanol, transferred to a biosafety cabinet, then rinsed thrice with sterile DNAse/RNAse-free water (ThermoFisher Scientific). The washed discs were fitted into the printed snaps, which were then slid into the bases on the suspension lid (Figures 1, S2). The discs, snaps, and suspension lid were all sterilized within the biosafety cabinet with UV light at 300μW/cm2 for 20 minutes (Figure 1). Nanoplex solution was pipetted onto the surface of the sterilized discs and spread across the entire surface of the disc using a pipette tip (Figure 1). The suspension lid and discs covered with nanoplex solution were covered with an inverted 24-well tissue culture microplate (DOT Scientific), and the nanoplex solution was frozen on the discs in a −80°C freezer. The discs were then lyophilized (FreeZone 4.5 -105, Labconco).

Figure 1:

Figure 1:

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Schematic of disc coating and transfection procedure. In brief, titanium discs were placed in 3D printed snaps affixed to the lid of a plate, UV sterilized, and then coated with nanoplex solution containing sucrose (blue) and lyophilized to coat the surface with nanoplexes solidified in sucrose (white). Discs were then suspended immersed in the medium containing cells for 4 hours to transfect cells that had been seeded 24 hours prior.

2.6. Release of Nanoplexes from Coated Discs:

Nanoplexes were prepared as described above, and 25.6 μLs of a 40% sucrose solution was added to 1mL of prepared nanoplexes to yield a final sucrose concentration of 1%. Discs were placed in a 24-well tissue culture microplate (DOT Scientific) and coated with nanoplex-sucrose solution (56.4 μLs) as described above, with the final coating of nanoplexes containing 5.5 μg of pDNA. After lyophilization, 0.8 mLs of DNAse/RNAse free water (ThermoFisher Scientific) was added to each disc and the microplate was shaken at 100 rpm. Every 0.5 hours a 100 μL aliquot was removed from each well and replaced with 100 μL of fresh DNAse/RNAse free water (ThermoFisher Scientific). The removed aliquot was combined with 100 μLs of a 3.57 mg/mL aqueous solution of heparin sulfate (Sigma-Aldrich) and stored at 4°C until all aliquots had been removed. A freshly made nanoplex-sucrose solution was prepared as described above and serially diluted to create a standard curve comprised of solutions containing 2.0, 0.67, 0.22, 0.074, and 0.025 μg of pDNA per 1 mL. One-hundred μL aliquots from the standard curve solutions were also removed, mixed with 100 μLs of heparin sulfate solution, and stored at 4°C until all aliquots had been removed. DNA content of each heparin-aliquot solution was quantified with the Quant-iT™ PicoGreen™ dsDNA Assay Kit according to the manufacturer’s protocol (ThermoFisher Scientific). Release of DNA from the discs was calculated by determining the concentration of DNA present in each sample well at the time of sampling according to the standard curve and controlling for dilution from replacement of removed medium.

2.7. Culture of HEK293T and GF Cells:

HEK293T and GF cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) containing 1% sodium pyruvate, 1% HEPES buffer, 1% Glutamax (all from ThermoFisher Scientific), 0.05mg/mL gentamycin sulfate (IBI Scientific, Dubuque, IA, USA), and 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, USA) in a humidified incubator at standard culture conditions (37°C and 5% CO2, Sanyo Scientific, Japan). Cells were passaged with 0.25% trypsin-EDTA (ThermoFisher Scientific).

2.8. Transfection of Cells with Coated Titanium Discs:

The transfection protocol (Figure 1) was adapted from methods described previously (D’Mello et al., 2016; Khorsand et al., 2017). Briefly, cells were seeded in 24-well tissue culture plates (DOT Scientific) 24 hours prior to transfection at a seeding density of 50,000 and 80,000 cells per well for HEK293T and GF cells, respectively. For each well, existing medium was replaced with 1.2 mL of complete DMEM lacking FBS. Coated discs attached to the suspension lid were removed from the lyophilizer and quickly placed on the plate containing the cells, resulting in the coated surface of the titanium discs being 4 mm from the bottom of the well. The plate with suspension lid and discs suspended above cells was gently shaken horizontally in two directions to ensure the medium contacted the entire surface of the discs. The plate was incubated under standard culture conditions for 4 hours, after which the suspension lid and discs were removed and the medium in each well was replaced with 1 mL complete DMEM containing 10% FBS. The cells were cultured under standard culture conditions until they were analyzed.

2.9. Measurement of Transfection Efficiency of Coated Discs:

Flow cytometry was used to quantitatively determine transfection efficiency of cells after transfection with nanoplexes containing EGFP pDNA delivered from titanium discs, as described above. Forty eight hours post-transfection, cells were trypsinized with 200 μL of 0.25% trypsin-EDTA (ThermoFisher Scientific), and 1 mL complete DMEM medium was added to each well to neutralize the trypsin. The detached cells were then suspended via pipetting. The resulting cell suspensions were transferred to 1 mL tubes and analyzed with a FACScan (Beckton Dickinson, Franklin Lakes, NJ, USA) flow cytometer equipped with a 15 mW 488 nm excitation laser. Forward scatter (FSC), side scatter (SSC), and green fluorescence (FL1, 560nm filter) parameters were measured. Cell debris was excluded through analysis with FlowJo software. A fluorescence threshold based on the negative control was created, and the percentage of cells fluorescing above the threshold was determined for each sample. Fluorescence microscopy (EVOS FL, ThermoFisher Scientific) was used to qualitatively assess transfection efficiency after transfection with nanoplexes containing EGFP pDNA delivered from titanium discs.

2.10. Determination of Cytotoxicity of Coated Discs:

MTS cell proliferation assay reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) was used to determine viability of GF cells after treatment with nanoplexes containing PDGFB pDNA or empty vector pDNA delivered from titanium discs. Four days after transfection cells were detached with 0.25% trypsin-EDTA (ThermoFisher Scientific) and the trypsin was neutralized with 1 mL complete DMEM, then the detached cells were suspended via pipetting. Two 100 μL aliquots of each resulting cell suspension were placed into 96-well plates, after which 20 μL of MTS assay reagent was added to each well. Cell viability was determined according to the manufacturer’s protocol using a microplate reader (SpectraMax Plus 384, Molecular Devices, San Jose, CA, USA).

2.11. Determination of PDGF-BB Secretion:

The enzyme-linked immunosorbent assay (ELISA) was used to quantify the secretion of rodent PDGF-BB by cells transfected with nanoplexes containing PDGFB pDNA delivered from titanium discs. On days 1, 2, and 7 after transfection, 600 μL of medium was collected from each well and replaced with fresh DMEM medium. The collected medium was centrifuged at 230G for 5 minutes to remove detached cells, and 500 μL of the resulting supernatant was transferred to a separate vial. The collected supernatants were flash frozen with liquid nitrogen and stored at −80°C until all media collections had been performed. All samples were then thawed on ice and the amount of PDGF-BB in the medium was quantified with an ELISA kit (Quantikine ELISA, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.

2.12. Determination of Integrin α-2 Gene Expression:

Quantitative polymerase chain reaction with reverse transcriptase (qRT-PCR) was used to determine the gene expression of integrin-α2 in GFs transfected with nanoplexes containing PDGFB pDNA delivered from titanium discs. Four days after transfection, each well containing cells was treated with 400 μL of TRIzol™ reagent (ThermoFisher Scientific) and homogenized, then collected and stored at −80°C. Samples were then thawed, and RNA was isolated according to the manufacturer’s protocol. RNA was reverse transcribed using a Genius thermocycler (Techne, United Kingdom) and reagents from a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Integrin-α2 gene transcripts were quantified against an 18sRNA control with a QuantStudio 3 PCR system (ThermoFisher Scientific) using the following PrimeTime qPCR Probes (Integrated DNA Technologies, Coralville, IA, USA):

Primer 1: 5’-AACCTCCAGTTCCCATGTTC-3’

Primer 2: 5’-AATGTCCTGTTGACCTATCCAC-3’

Probe: 5’-/56-FAM/TGGTGAGGA/ZEN/TCAAGCCGAGGC/3IABkFQ/-3’

2.13. Culture and Imaging of GFs Seeded on Coated and Uncoated Titanium Surfaces:

Bare titanium discs were prepared as described above and placed in 24-well tissue culture microplates (DOT Scientific). Coated titanium discs were prepared in 24-well tissue culture microplates (DOT Scientific) by coating bare titanium discs with nanoplexes containing 5.5 μg of PDGFB pDNA according to the coating protocol described above. GFs were seeded onto coated and uncoated titanium discs and cultured in complete DMEM medium under standard culture conditions for 4 days. Each well was then washed twice with PBS, then fixed in 4% paraformaldehyde in PBS at room temperature for 30 minutes. Samples were dehydrated by a series of 15 minute incubations in solutions with increasing ethanol concentrations (25, 50, 75, 95, and 100% ethanol). Samples were rinsed once in hexamethyldisilazane (Sigma-Aldrich), then incubated in hexamethyldisilazane for 15 minutes. All liquid present in each sample well was aspirated and samples were dried overnight. Samples were sputter coated with a gold-palladium alloy and imaged with field emission scanning electron microscopy (SEM) (Figure S3) (Hitachi S-4800, Hitachi, Japan).

2.14. Statistical Analysis:

Statistics were performed with Prism 8 software (GraphPad, San Diego, CA, USA) using two-tailed student’s t-tests (Figure 2), 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons tests (Figures 4A, 4B, 5A, 5B, 6B, 6C), and 2-way ANOVA with Tukey’s multiple comparisons tests (Figure 6A) as necessary. Significance was set at the α<0.05 level (p<0.05).

Figure 2:

Figure 2:

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Average particle diameter and polydispersity index (A) and zeta potential (B) of nanoplexes before and after lyophilization with varying sucrose concentrations. Values are expressed as mean + SD or mean ± SD (n=4). Significant differences between samples particle diameter and zeta potential were assessed by student’s t-tests (ns: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 4:

Figure 4:

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(A) Fluorescence microscopy images of HEK293T cells transfected with nanoplex solutions containing 2.5 μg of EGFP pDNA and containing sucrose in a range of concentrations (1-10%) that were lyophilized in microfuge tubes and reconstituted. Scale bar represents 1000 μm. Transfection efficiencies of HEK293T cells (B) and GFs (C) transfected with either plain nanoplex solution (sucroseneg) or nanoplex solution containing 1% sucrose (sucrosepos) that had each been lyophilized on a titanium disc. All transfected cells were treated with nanoplexes containing 5.5 μg of pDNA. Values are expressed as mean + SD (n=3). Significant differences between samples were assessed with 1-way ANOVA Tukey’s multiple comparisons test (ns: no significance, ****p<0.0001).

Figure 5:

Figure 5:

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Transfection efficiencies resulting from transfection of HEK293T cells (A) and GFs (B) with titanium discs coated with 1% sucrose nanoplexes containing indicated amounts of EGFP pDNA. Values are expressed as mean + SD (n=3). Significance differences were assessed with 1-way ANOVA Tukey’s multiple comparisons tests (# and @ represent a significant difference from the 2.5 μg dose at the p<0.01 level and p<0.0001 level, respectively; *p<0.05, **p<0.01).

Figure 6:

Figure 6:

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GFs were treated with 5.5 μg PDGFB pDNA via coated titanium discs (nanoplexes), 5.5 μg of empty vector pDNA via coated titanium discs (empty vector), an equivalent amount of free PEI via coated titanium discs (PEI alone), or untreated (control). The sucrose concentration for all coatings was 1%. (A) Relative viability of transfected GFs 4 days after transfection. Values are expressed as mean + SD (n=3). (B) Relative absorbances from ELISA targeting PDGF-BB protein in the culture media of GFs 1, 2, and 7 days after transfection. The * symbol indicates significant difference from “Control” and “PEI Alone” groups at the p<0.05 level. Values are expressed as mean + SD (n=3). (C) Fold change in GF expression of integrin-α2 4 days after transfection. Values are expressed as mean ± SD, where SD was determined from Ct values and converted to fold-change. Significant differences between samples were assessed with 1-way and 2-way ANOVA with Tukey’s multiple comparisons (ns=no significance, *p<0.05, **p<0.01).

3. Results:

3.1. Characterization of Nanoplexes:

Nanoplexes were prepared using sucrose concentrations between 0 and 10% and the zeta potential and hydrodynamic size were measured before and after lyophilization. Samples containing no sucrose showed high aggregation, as did samples containing only 1% sucrose (Figure 2A). The zeta potential post-lyophilization decreased only for the 0% sucrose group, despite the 1% sucrose group displaying aggregation after the lyophilization process (Figure 2B).

3.2. Release of nanoplexes from coated discs:

Titanium discs were coated with 1% sucrose nanoplex solution containing 5.5 μg of pDNA and the release of those nanoplexes from the coated discs was characterized by assaying DNA concentration in removed aliquots after treatment with heparin. The release profile displays an immediate release of nanoplexes followed by a steady decline as determined via measurement of DNA concentration in removed aliquots (Figure 3).

Figure 3:

Figure 3:

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Release profile of DNA from titanium discs coated with a 1% sucrose nanoplex solution containing 5.5 μg of pDNA. Values represent mean ± SD (n=4).

3.2. Transfection using discs coated with nanoplex solution with and without sucrose:

HEK293T cells were transfected with nanoplexes containing 2.5 μg of EGFP pDNA with varying concentrations of sucrose that were lyophilized and reconstituted in microfuge tubes. Transfection occurred at all sucrose concentrations tested, with no visually discernible difference in transfection between concentrations as shown in fluorescence microscopic images (Figure 4A).

HEK293T cells and GFs were transfected with nanoplexes containing 5.5 μg of EGFP pDNA via treatment with either plain nanoplex solution (sucroseneg) or nanoplex solution containing 1% sucrose (sucrosepos) that had been lyophilized on titanium discs (Figure 4B). The 5.5 μg dose was chosen in an attempt to yield detectable transfection from the samples lacking sucrose. Sucroseneg samples yielded negligible transfection (Figure 4B). Sucrosepos samples yielded an average transfection efficiency significantly greater than the control for both HEK293T cells and GFs (p<0.0001).

3.3. Dose dependence of transfection efficiency in multiple cell types:

HEK293T cells and GFs were treated with titanium discs coated with 1% sucrose nanoplex solution containing 2.5 to 6.5 μg of EGFP pDNA (Figure 5). Transfection efficiencies of HEK293T cells showed incrementally increasing transfection efficiencies until the 5.5 μg dose, after which the transfection efficiency decreased (Figure 5A). There was approximately an order of magnitude difference in transfection efficiency when comparing HEK293T cells and GFs for all doses. The GFs displayed more variable transfection efficiency, with no significant differences at the α=0.05 level being found between any doses. The difference between the 2.5 μg dose and the 5.5 μg dose in GFs approached significance (p=0.063) and for both HEK293T cells and GFs the 5.5 μg dose yielded the highest mean transfection efficiency.

3.4. Viability of transfected GFs 4 days after transfection with nanople4xes containing PDGFB pDNA:

GFs were transfected with nanoplexes containing 5.5 μg of PDGFB pDNA via titanium discs coated with nanoplex solution containing 1% sucrose and cell viability was measured 4 days afterward. GFs treated with discs coated with nanoplexes containing PDGFB pDNA had a reduced average viability compared to the control (84%), but the difference was not statistically significant (p>0.2) (Figure 6A). GFs treated with an equivalent amount of nanoplexes containing empty vector pDNA had an average viability of 38%, and were significantly different from both the control and the “nanoplexes” experimental group (p<0.01) (Figure 6A).

3.5. PDGF-BB secreted from GFs transfected with nanoplexes containing PDGFB pDNA:

GFs were transfected with nanoplexes containing 5.5 μg of PDGFB pDNA via titanium discs coated with nanoplex solution containing 1% sucrose. Media samples were collected on days 1, 2, and 7, and the presence of PDGF-BB measured with ELISA. Relative absorbances show that the cells transfected with nanoplexes containing PDGFB pDNA had elevated levels of PDGF-BB in their media, suggesting expression and secretion of the protein in response to transfection (Figure 6B). The elevated levels are significantly different from the controls on days 2 and 7 (* = p<0.05) (Figure 6B).

3.6. Increased expression of integrin-α2 in GFs after transfection with nanoplexes containing PDGFB pDNA:

GFs were transfected with nanoplexes containing 5.5 μg of PDGFB pDNA via titanium discs coated with nanoplex solution containing 1% sucrose and cells were collected 4 days afterward. RT-qPCR analysis showed that integrin-α2 expression was upregulated 2.4 fold compared to the negative control (p<0.05) (Figure 6C).

4. Discussion:

Titanium discs coated with nanoplex solution that did not have any sucrose did not show any meaningful transfection (Figure 4B). This demonstrates that sucrose is a necessary additive to conserve the transfection activity of nanoplexes during freezing and lyophilization on a titanium surface, a novel finding that is consistent with what is previously reported in different settings (Huang et al., 2003; Kasper et al., 2011). Interestingly, despite high aggregation being observed after lyophilization of nanoplexes in a 1% sucrose solution (Figure 2A), the 1% sucrose nanoplex coating on titanium discs still retained strong transfection efficiency (Figure 4A, 3B). Since this coating is intended for use in the oral environment, limiting the total amount of sucrose available to saccharolytic oral bacteria is an important consideration. While sucrose alone has not been found to induce inflammation in gingival tissue, growth of saccharolytic bacteria on titanium surfaces has been shown to be enhanced by repeated exposure to high concentration sucrose solutions and the resulting biofilms can have inflammatory effects on local tissue (Della Corte et al., 2018; Hert et al., 2014; Khan et al., 2017; Murakami et al., 2018, p.; Souza et al., 2019). The 1% sucrose solution was therefore selected for use in later experiments to improve the translatability of this coating by reducing total sucrose available to oral bacteria. The release profile of coated nanoplexes was found to resemble that of immediate release, with the maximal DNA concentration being reached very rapidly (within just 0.5 hours) after which the DNA concentration steadily declines. This downward trend is likely due to the well documented instability of nanoplexes in solution (Anchordoquy et al., 2000).

Different doses of nanoplexes in 1% sucrose solutions were coated onto titanium discs and tested to determine the optimal dose for transfection. Based on the HEK293T experiment, the 5.5 μg of pDNA in nanoplexes dose yielded the highest transfection efficiency (Figure 5A), and the GFs appear to match this optimal dose (Figure 5B). There is a notable decrease in transfection efficiency with the 6.5μg dose, possibly a result of increasing cell death due to exposure to an increased amount of PEI, which is known to be cytotoxic to some cells (Putnam et al., 2001). These results demonstrate that the nanoplex coating is capable of transfecting both the robust HEK293T cell line (Figure 5A) and application-relevant human primary oral cells in vitro (Figure 5B). The transfection efficiencies reported here are within expected limits given previous transfection studies performed on HEK293T and other primary cells. These studies demonstrate high transfection efficiencies in HEK293T cells (ranging from 70-90%) and single digit transfection efficiency percentages in various primary cells (Acri et al., 2019; Guerra-Crespo et al., 2003; Mellott et al., 2013; Thomas et al., 2005). Despite the typically low transfection efficiencies obtained with primary cells in vitro, studies have shown that nanoplexes can still elicit clinically relevant effects in vivo (Khorsand et al., 2017). However, the in vitro transfection efficiencies presented here may differ from in vivo transfection efficiencies because these transfections were performed using serum-free conditions. The presence of serum proteins during transfection has been shown to reduce overall transfection efficiency in vitro, and so the proteins present in saliva may also reduce transfection efficiency in the oral environment (Zhu et al., 2018).

The transfection experiments with GFs indicate that PDGF-BB is secreted by transfected cells and that the secreted PDGF-BB likely stimulates the proliferation of local cells and enhances their expression of integrin-α2, an integrin involved in cell adhesion (Boisvert et al., 2010). The ELISA results indicate that GFs were successfully transfected with nanoplexes containing PDGFB pDNA and secreted PDGF-BB protein (Figure 6B). This establishes that the nanoplex coating of titanium surfaces is a potential alternative to directly using recombinant proteins in augmenting the peri-implant soft-tissue seal. Transfection of local gingival fibroblasts can induce sustained secretion of PDGF-BB, maintaining therapeutic action at the titanium surface-mucosal interface without the need for additional dosing. This sustained secretion resulted in cellular responses indicative of potential therapeutic effects for in vivo applications. These include enhanced cell proliferation, reduced loss of cell viability and increased integrin-α2 expression.

The difference in cytotoxicity of empty vector nanoplexes versus nanoplexes containing PDGFB pDNA could be explained by the proliferative effect of PDGF-BB (Battegay et al., 1994; Gallego-Muñoz et al., 2017). In those samples that were transfected with nanoplexes containing PDGFB pDNA, the presence of PDGF-BB may have been able to stimulate proliferation such that the viability loss upon exposure to the PEI from the nanoplexes was mostly recovered. Meanwhile, the samples exposed to nanoplexes, but not transfected with PDGFB pDNA, displayed a reduced viability compared to the control (Figure 6A).

Integrin-α2 is a cell adhesion protein whose expression has been previously reported to be enhanced by exposure to PDGF-BB (Åhlén et al., 1994). Previous studies have identified different subunits of integrin (α2, α4, α5, β1 and β3) to be expressed by the cells of the periodontium (Hormia et al., 1990; Steffensen et al., 1992). These integrins play a key role in cellular adhesion, extracellular matrix (ECM) homeostasis and therefore, in tissue healing. The enhanced expression of integrins such as integrin-α2 could result in improved adhesion of ECM cells onto the titanium surface, thus improving the peri-implant soft tissue seal required to prevent bacterial downgrowth. A previous investigation had demonstrated the presence of multiple integrin subunits (including α-2) expressed by GFs when they are grown in contact with titanium implant surfaces, indicating the role of these adhesion proteins in establishing this complex bio-device seal (Oates et al., 2005). Our proof of concept study points to a novel way to increase the expression of integrin-α2, and therefore has promising potential. Future in vitro work will explore the use of other lyoprotectants while in vivo investigations testing implants with our bioactive coating will provide key information on the feasibility and clinical translatability of this approach. In some implant systems, the smooth surface of the abutment is what will facilitate this mucosal seal and therefore, there is a greater need to develop bioactive smooth titanium surfaces (in addition to bioactive roughened titanium surface) to enhance the clinical applicability of this approach. The approach evaluated in this study is mainly focused on developing a bioactive surface to enhance soft tissue attachment but this methodology can be adapted to deliver osteogenic factors to enhance bone to implant contact and eventually osseointegration, which is a current focus of our research group.

5. Conclusion:

Coating titanium surfaces by simply lyophilizing a nanoplex solution containing 1% sucrose is a simple yet effective way of activating the surface to have therapeutic properties. Based on the ability of a 4-hour transfection to generate sustained PDGF-BB secretion up to 7 days after transfection, this coating technique has promise for application in implant dentistry to augment peri-implant soft tissue seal by stimulating proliferation of local cells and increasing their adhesion potential. The presence of regenerative factors such as PDGF-BB produced and secreted by local cells circumvents the need for the delivery of recombinant proteins. Further studies should investigate the possibility of using non-nutritive lyoprotectants to reduce the potential for stimulation of bacterial growth and determine whether they can enhance the low transfection efficiency seen in the GF transfection results presented here. Additionally, future studies should investigate whether transfection from coated titanium implants in vivo can mimic the results reported here and whether increased adhesion is induced by induced expression of PDGF-BB. Specifically, future studies should assay effects of coated titanium dental implants on properties of the supra-crestal soft tissue seal including rate of formation, collagen fiber orientation, and overall quality.

Supplementary Material

1

Acknowledgements:

This project was supported by a grant from the ITI Foundation, Switzerland. Data presented herein were obtained at the Flow Cytometry Facility, which is a Carver College of Medicine/Holden Comprehensive Cancer Center core research facility at the University of Iowa. The facility is funded through user fees and the generous financial support of the Carver College of Medicine, Holden Comprehensive Cancer Center, and Iowa City Veteran’s Administration Medical Center. Noah Z. Laird acknowledges the support of a training fellowship from the University of Iowa Center for Biocatalysis and Bioprocessing and of the NIH-sponsored Predoctoral Training Program in Biotechnology (T32 GM008365). The authors acknowledge Sean Geary for his help with reviewing and editing during preparation.

Footnotes

Disclosure:

The authors declare no conflict of interest

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