The Combination Of Aligned Scaffolds And Gene Therapy In Periodontal Tissue Engineering

Periodontal disease is one of the major causes of tooth loss due to the destruction of the tooth-supporting periodontal tissue. Hence, periodontal tissue regeneration plays an important role for the treatment of periodontitis. However, current clinical therapies, such as guided tissue regeneration (GTR), cannot achieve the complete regeneration of periodontal tissue. Cell-based periodontal tissue engineering has been proposed as an alternative approach. The principle of tissue engineering is to seed a biodegradable scaffold with multipotent cells containing growth factors into bone defects to regenerate the lost tissue. Currently there are some limitations for tissue engineering, such as difficult control of stem cell differentiation, poor retention of growth factor in defect site and so on. These problems may be circumvented by gene therapy to some extent, so the combination of gene therapy and tissue engineering technique may be a promising approach to the regeneration of periodontal tissue.It is well known that the microstructure of periodontal tissue have a specific aligned arrangement. However, the fibres of current scaffolds applied in periodontal regeneration were disorder and random arrangement, which could not mimic the natural periodontal microstructure and would limit the aligned tissue regeneration. Aligned scaffolds of tissue engineering not only can induce the cells’growth direction, but also can promote the cell proliferation and migration. Therefore, two type of aligned scaffolds were designed in this study, including parallel scaffolds that mimic the microstructure of periodontal ligament (parallel arrangement) and cross scaffolds that mimic the trabecular structure of alveolar bone (cross arrangement). It is expected that those kinds of aligned scaffolds could guide the cells growth direction, enhance the cell proliferation and migration, and then promote the orderly periodontal regeneration.Scaffolds typically used in tissue engineering could function as carriers, facilitating the introduction of gene transfected cells into the body. It has been verified that scaffold made by different biomaterials generated different effect of gene therapy. However, the influence of scaffolds’fibre alignment on gene therapy is still unknown.Hence, the combination of aligned scaffolds and gene therapy was designed in this study in order to investigate the i) potential application of gene therapy-aligned scaffolds composite in periodontal regeneration and ii) whether aligned scaffolds may exert influences on gene therapeutic effect.Due to the three important factors (scaffolds, cells and growth factors) of tissue engineering and target gene and cells of gene therapy, this study designed aligned scaffolds and cultured the PDLSCs over-expressed BMP2 (BMP2/PDLSCs). Meanwhile, we also investigated osteogenic potentials of BMP2/PDLSCs/aligned scaffolds composite and the influence of gene therapy by aligned scaffolds.Experiment oneDesign and characterization of aligned electrospun scaffoldsObjective:To design and detect the aligned electrospun scaffolds to mimic the microstructure of periodontal tissue.Materials and methods:In order to mimic the microstructure of natural periodontal tissue, two kinds of aligned scaffolds (parallel and cross random) and two unaligned scaffolds (random and film scaffolds) were designed in this study. Except the different fibre arrangement, the other surface characterization of those four kinds of scaffolds, such as fibre diameter, pore size and distribution, should have no significant difference. Cooperative institutions (Department of Biomaterials, Radboud University Nijmegen Medical Centre) prepared four kinds of scaffolds by modified electrospinning. The surface morphology of scaffolds was observed by optical microscopy, scanning electron microscopy (SEM) and atomic force microscope (AFM). Fast Fourier Transform (FFT) was used to characterize fibre alignment. Image J was used to analyze the fibre diameter, pore size and distribution on surface of scaffolds.Results:1. Under the optical microscopy, SEM and AFM, large bundles of fibres in the parallel scaffold were oriented in a single direction. The normalized intensity values (or FFT alignment value) of parallel scaffolds with a single peak was the highest.2. The fibres in cross scaffold were aligned in two directions under the optical picture and SEM, while the fibres were oriented in one direction under AFM. There were two peaks on FFT alignment value for cross scaffolds.3. In the case of the random scaffold, even if they appeared smooth in optical pictures, randomly oriented fibres could be found using SEM and AFM. Random scaffolds did not show overt evidence of alignment by FFT analyze.4. As for the solvent-cast smooth film, it was translucent and presented with very smooth surface properties under the optical microscope and with wave-like features in the SEM and AFM. The FFT alignment value of film scaffold did not show overt evidence of alignment.5. The average fibre diameter, pore sizes and distribution on the parallel cross, and random scaffolds were not significantly different (p> 0.05).Conclusions:Aligned scaffolds (parallel and cross scaffold) were successfully got in this part, which is benefit for the following research.Experiment twoIsolation and identification of human periodontal ligament stem cells Objective:To isolate and identify of human periodontal ligament stem cells.Materials and methods:PDLSCs were digested, isolated and cultured by enzyme-digested culture method. Cells were identified by following three parts. Stem cell markers (STRO-1, CD44 and CD 146) of cultured cells were analyzed by flow cytometry. Alizarin red and oil red staining were used to detect the potential of osteogenic and adipogenic differentiation of PDLSCs, respectively.Results:During culturing, single-cell-derived colonies formed. PDLSCs showed the positive expression of STRO-1 (6.5±0.5%), CD44 (99±0.5%), CD146 (45±1%). Alizarin red-positive mineral deposits gradually formed. Oil red O positive lipid clusters could be observed and grown.Conclusions:PDLSCs were successfully isolated and identified, which also expressed STRO-1/CD44/CD146 and had the potential of multipotent differentiation.Experiment threeComparison of different kinds of vectors for gene delivery to human periodontal ligament stem cellsObjective:To compare the transfection efficiency and toxicity of gene transfer methods on PDLSCs and search a best approach and optimal protocol for transfering genes into PDLSCs.Materials and method:PDLSCs were transfected by (1) Lipofectamine 2000, (2) polyethylenimine, (3) GBfectene-Elite transfection reagent, (4) X-tremeGENE HP DNA Transfection Reagent, (5) MATra Magnet Assisted Transfection (MATra), compared to (6) lentiviral vectors harboring a GFP gene. In order to better evaluate the optimal transfection dose of each reagent for PDLSCs, three group were designed for every transfection method according to the transfection reagent dose:1) double of recommended doses on their respective instruction (2X),2) recommended doses (1×), 3) a half of recommended doses (0.5×). Transfection efficiency was measured by fluorescence microscope and flow cytometry. Meanwhile, cell morphology and growth status were observed by optical microscope to estimate the cytotoxicity.Results:Among these methods, the transfection efficiency of the former four methods was not very satisfactory(less than 6%) compared to that of lentiviral vectors (positive control,95%). However, MATra was the most effective non-viral method (11%). Moreover, the cellular toxicity was lower than that of the former four methods. Moreover, recommended doses (1×) showed the best transfection efficiency among the three different transfection reagent doses.Conclusions:The transfection efficiency of PDLSCs with MATra was higher than the other non-viral transfection regents in this study, but it was far less than viral vectors.Experiment fourLentiviral vectors production and PDLSCs transfection andidentificationObjective:To search a best transfection regent for lentiviral vectors package. Meanwhile, PDLSCs were transfected by BMP2 lentiviral vectors and identified.Materials and methods:293T producer cells were transfected by TurboFect Transfection Reagent, EntransterTM-D4000, NeofectTM DNA transfection reagent, Lipofectamine 2000 and X-tremeGENE HP DNA Transfection Reagent. Transfection efficiency was measured by fluorescence microscope. Then, PDLSCs were transfected by BMP2 lentiviral. BMP2 gene (3days) and protein (7days) of BMP2/PDLSCs were detected by Q-PCR, real-time PCR and western blot, respectively. Meanwhile, non-transfected PDLSCs and PDLSCs transfected by lentiviral containing only GFP were used as control.Results:The transfection efficiency of TurboFect Transfection Reagent was higher than the other transfection reagents in this study. The results of Q-PCR and real-time PCR showed that the BMP2 gene expression of the BMP2/PDLSCs was significantly higher than in the control groups (p<0.001). Similarly, after 7 days of transfection, the western blot showed that the BMP2 protein expression of the BMP2/PDLSCs was higher than control groups.Conclusions:TurboFect Transfection Reagent was commended for lentiviral vectors package. PDLSCs were successfully transfected by BMP2 lentiviral and over expressed the BMP2 gene and protein for the following research.Experiment fiveThe influence of BMP2/PDLSCs by aligned electrospun scaffoldsObjective:To explore whether aligned scaffolds could influence the effect of gene therapy through observing the osteogenesis potential of BMP2/PDLSCs seeded on different fibrous scaffolds. The orientation, proliferation, and osteogenesis of cells under the influence of the aligned electrospun scaffold were investigated.Materials and methods:BMP2/PDLSCs were seeded onto two types of aligned scaffolds (parallel and cross scaffolds) and two types of unaligned scaffolds (random scaffold and smooth film) in this study. Cell morphology was observed by confocal microscope and SEM. Cell proliferation also tested by PicoGreen assay. Osteogenesis-related gene and protein expression (BMP2, ALP, BSP, Col I OCN and Runx2), ALP activity, collagen Ⅰ and calcium content were investigated. SEM also been used to observe the mineralization on the surface of scaffolds.Results:BMP2/PDLSCs on the aligned scaffold showed elongation along the axes of the scaffold. The DNA content, osteogenesis-related gene and protein expression, ALP activity, collagen Ⅰ and calcium content of the BMP2/PDLSCs seeded on the aligned scaffold were significantly higher than those on the unaligned scaffold(p0.05). Under SEM, more calcified nodules had formed on the surface of the parallel and cross scaffolds than that on unaligned scaffolds.Conclusions:Aligned scaffolds could induce the orientation, enhance the proliferation, and promote the osteogenic differentiation of BMP2/PDLSCs, which indicated aligned scaffolds could enhance the effect of ex vivo gene therapy. There was no significantly different between parallel and cross scaffolds in the aspect of inducing cell orientation, enhancing cell proliferation, and promoting cell osteogenic differentiation.All in all, this study firstly demonstrated that aligned scaffolds, not just vectors for ex vivo gene therapy, could promote the impact of ex vivo gene therapy. The finding provides a new method for improving the function of ex vivo gene therapy. This composite of BMP2/PDLSCs/aligned scaffold will be a bright future in periodontal tissue engineering.