Study On Constructed Tissue Engineered Dentin By Gene Transfected Fibroblasts

The development of tissue engineering enhances the possibility and feasibility for damaged oral tissue reparation and regeneration. Currently, the research on tissue engineering is one of the most fascinating topics in the field of oral and maxillofacial plastic surgery. Many scientists pay more attentions on regenerating tooth by using tissue engineering technology worldwide, since teeth play the important role in the development of jaws, the maintenance of maxillofacial profile, the function of language and chewing. As the main ingredient of the tooth, dentin constructs the outlook of tooth and its regeneration is the key point in the research of tissue engineered tooth. Therefore, in this study the construction of seed cells, biological scaffold and tissue engineered dentin were investigated.Objectives:Oral mucosa fibroblasts are easier to be obtained and cultured. To establish the dentin matrix protein1-porcine oral mucosa fibroblasts (DMP1-POMF) as seed cells for tissue engineered dentin, the oral mucosa fibroblasts were transfected by dentin matrix protein 1( DMP1). To evaluate the feasibility of using DMP1-POMF as seed cells for tissue engineered dentin, the biological function of DMP1-POMF and the expression of odontoblasts specific protein was detected.DMP1-POMF cells which were established as seed cells were seeded onto acellular dermal matrix (ADM) scaffold. In order to evaluate the feasibility of using ADM as scaffold for tissue engineered dentin, the biocompatibility between the seed cells (DMP1-POMF) and scaffold was detected by HE staining, scanning electron microscopy and flow-cytometric analysis. To investigate the construction of tissue engineered dentin, the ectopic dentinogenesis of seed cells (DMP1-POMF) compound ADM was evaluated by the observation of growing of the compound in vivo, the change of histologic structure and the expression of dentin specific protein.DMP1-POMF cells which were established as seed cells were cultured as three-dimensional pellet. To evaluate the accumulation of extracellular matrix protein in three-dimensional pellet, the cells morphologic characteristics was observed and the flow-cytometric analysis was carried out. The histological and immunohistochemistric detection was performed after the pulp capping with DMP1-POMF cells pellet to investigate the function of DMP1-POMF pellet on dentin regeneration in vivo.Methods:1 Construction of pEGFP-DMP1 PlasmidThe full length of dentin matrix protein 1( DMP1) cDNA was linked into an eukaryotic expression vector pEGFP-Cl . The recombinant plasmid pEGFP -DMP1 was then amplified and tested by an enzyme cutting technique in vitro.2 Cell Culture, Dentin Matrix Protein 1 Transfection2.1 Porcine Oral Mucosa Fibroblasts (POMF) Primary CulturePrimary fibroblast cell cultures were established from porcine oral mucosa biopsies. The samples of lamina propria connective tissue of oral mucosa were subsequently rinse three times with l ml ice-cold PBS supplemented with penicillin (100IU/ml)and streptomycin(100ug/ml)(Sigma, USA). Then connective tissue fragments were transferred to 60-cm2 cell culture bottles containing 2ml Dulbecco,s modified Eagle,s medium(DMEM) (Gibco BRL),10% fetal calf serum (FCS) (Gibco BRL), penicillin (100IU/ml)and streptomycin (100ug/ml; Sigma). Cells were cultured at 37°C and 5% CO2 until reaching 60% confluence. Growing cells were trypsinized and transferred to new cell culture bottles without the original connective tissue fragments.2.2 Mesenchymal Stem Cells (MSC) CultureMSC were isolated from bone marrow and purified by centrifuge in vitro.The proliferation and growth characteristics were observed in primary and passage culture.2.3 Dentin Matrix Protein 1 TransfectionGroup A: Porcine oral mucosa fibroblasts (POMF) were transfected by dentin matrix protein1(DMP1) and were named dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) in this research.Group B: Porcine oral mucosa fibroblasts (POMF) were transfected by empty vector pEGFP-C1 and were named pEGFP-C1-porcine oral mucosa fibroblasts(C1-POMF) in this research.Group C: Mesenchymal stem cells (MSC) were transfected by dentin matrix protein1(DMP1) and were named dentin matrix protein1- mesenchymal stem cells(DMP1-MSC) in this research.POMF,and MSC named group A and C between the 4th and 6th passages, were plated at 1×105 into 6-well culture dishes and transfected with 2μg pEGFP-DMP1 and Lipofectamine 5μl/ml (Invitrogen, USA). Solutions consisted of 150μl DMEM with no FCS were added to the plasmid vector in a concentration of 2μg/ml, mixed 150μl DMEM containing Lipofectamine for a concentration of 5μl/ml. After 20 min at 22°C, the DNA/lipofectamine complexes in a final volume of 300μl were added to the cell culture wells (3.5cm in diameter) containing 1×105 fibroblasts and cultured at 37°C and 5%CO2. After 5h, the DNA/lipofectamine complexes were removed, and a fresh medium with 10% FCS was added to stop transfection. The expression of recombinant plasmid pEGFP -DMP1 and transfer efficiency were evaluated by fluorescent microscope. Transfected cells achieved subconfluence after 7 days. At the 3rd passage, the cells were diluted 1:10, cultured with G418 sulfate at 0.8μg/ml of medium. Individual colonies were isolated and expanded by plating cells at low density. POMF named group B were mock transfected with empty vector pEGFP-C1 and selected identically. Cells were harvested for further analysis.3 The biological influence on fibroblasts of gene transfection3.1 RT-PCRTotal RNA was isolated from cultured cells during transfection on 24h, 48h and G418-resistant with the use of Trizol reagent (Invitrogen, USA). First-strand cDNA syntheses were performed by reverse transcription with the SuperScript pre-amplification system.The expression of DMP1, ameloblastin (Ambn) and dentin sialoprotein (DSP)was detected.3.2 Flow-cytometric Analysis of Cell-cycle and DNA PloidCell-cycle phases and DNA ploid were determied by flow-cytometric analysis based on expression of the ki-67 antigen (G1, G2/M, S) and the S-phase-specific proliferating cell nuclear antigen (PCNA) (Landberg et al., 1990). DMP1-POMF and C1-POMF were digested with trypsin/ethylene -dianmine tetraacetic acid (EDTA) into single cell suspensions (2×105 cells). Then the cells were fixed with cold ethanol (70%) on ice and incubated with the monoclonal antibodies specific to the cell cyucle-associated antigens ki-67 conjugated to FITC and PCNA conjugated to PE(1/10) (Dako corp) respectively. After being washed twice in PBS, the cells were analyzed with the use of a FACSCalibur flow cytometer.3.3 In vitro Assay for lnducement of Mineralized Nodule Formation and von Kossa StainingThe mineralization microenvironment was created by treating the DMP1-POMF , C1-POMF and DMP1-MSC (80-90% confluent) with 10mMβ-glycerophosphate and 100μg/ml ascorbic acid along with 10nM dexamethasone (Sigma, USA). The cells undergoing mineralization (40 days in culture) were fixed with 10% formalin in neutral buffer (Sigma) for 15min. The slides were washed with distilled water and then treated with 1% AgNO3 for 1h, washed again with distilled water, and treated with 2.5% sodium thiosulfate for 5 min. The specimens were counterstained and then examined under a light microscope.3.4 Transmission Electron Microscope (TEM)The DMP1-POMF , C1-POMF and DMP1-MSC cultured 21d were digested into single cell suspensions. Then the cells were washed twice, fixed in cold glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in an ethanol series, embedded in epoxy resin, and then observed under transmission electron microscope.4 An experimental study on fibroblasts transfected by DMP1 loading on acellular dermal matrix4.1 Acellular dermal matrix (ADM) scaffold CultureFor loading of cells into transplant vehices, multi-fracture ADM cut by ophthalmologic scissors were prewetted in complete medium.Group A: The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) were seeded directly onto ADM scaffold.Group B: Porcine oral mucosa fibroblasts (POMF) were seeded directly onto ADM scaffold.Approximately 2×105 cells were loaded per 0.2×0.2cm2 ADM by capillary action and then incubated at 37°C for 7 days.4.2 Histological ObservationThe transplants were fixed in 4% paraformal dehyde overnight and processed for paraffin-embedding. The sections of 4.5μm thickness were stained in hematoxylin and eosin.4.3Scanning Electron Microscopy (SEM)To evaluate the characteristics of the cellular proliferation and cell-material attachment electron microscopy imagine was conducted on carbon-coated, polished block faces of poly-methyl-methacrylate (PMMA) embedded ADM scaffold with DMP1-POMF, the POMF as control. The samples were imaged in a Zeiss DSM 962 digital scanning electron microscope operated at 20 or 30 KV in the back scattered electron mode with the use of a KE (Toft, cambs, UK) solid-state back-scatter electron detector. 4.4 Flow-cytometric Analysis of Cell-cycle and Protein Quantification of DMP1, DSP and Collagen Type ICell-cycle phases were determied by flow-cytometric analysis based on expression of the ki-67 antigen (G1, G2/M, S) and the S-phase-specific proliferating cell nuclear antigen (PCNA) (Landberg et al., 1990). The adherent monolayer DMP1-POMF and the DMP1-POMF transplanted in ADM scaffold were digested with trypsin/ethylene-dianmine tetraacetic acid (EDTA) into single cell suspensions (2×105 cells). Then the cells were fixed with cold ethanol (70%) on ice and incubated with the monoclonal antibodies specific to the cell cyucle-associated antigens ki-67 conjugated to FITC and PCNA conjugated to PE(1/10) (Dako corp) respectively. DMP1, DSP and collagen Type I polyclonal antibodies was added directly to the above-mentioned 2×105 cells for l hr on ice. The cells were then incubated with goat anti-mouse IgM conjugated to FITC (1/50 dilution, DAKO Corp.) for 45min on ice. After being washed twice in PBS, the cells were analyzed with the use of a FACSCalibur flow cytometer. Positive expression was defined as the level of fluorerscence greater than 99% of the corresponding isotype-matched control antibodies.4.5 Subcutaneous Transplantation ProcedureThe DMP1-POMF with ADM scaffold were transplanted into 10-week-old immuncompromised beige mice. Nine immuncompromised beige mice were used. Muscle of thigh incisions were made on each mouse and sarcous pockets were made by blunt dissection. A single transplant was placed in each pocket with up to 2 transplants per animal. The transplants were recovered at 7, 14 and 21 days post-transplantation, fixed with 4% formalin, decalcified with buffered 10% EDTA (PH8.0), and then embedded in paraffin. The sections of 4.5-μm thickness were stained in hematoxylin and eosin .The avidin-biotin peroxidase complex method was used for immunohistochemistry. The POMF with ADM scaffold and The DMP1-POMF single cell suspensions were as control.5 The dentin formation by dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) pellete5.1 Cell Pellet CultureGroup A: The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) pellet culture.Group B: The adherent monolayer DMP1-POMF culture.Group C: Porcine oral mucosa fibroblasts (POMF) pellet culture.The pellet culture of the cells was performed. Briefly l-ml aliquots containing 2×105 cells were centrifuged in a 15ml conical polypropylene tube at 1000 rpm for 5 min. Pellets were maintained in DMEM supplemented with 10% heat-inactivated bovine calf serum and 50μg/ml L-ascorbic acid phosphate and penicillin-streptomycin. The medium was changed two times per wk.5.2 Histological ObservationPellets on days 7,14 and 21 were fixed in 4% paraformal dehyde overnight and processed for paraffin-embedding. The sections of 4.5μm thickness were stained in hematoxylin and eosin.5.3 Cells CountedThe cells were dispersed by trypsin and counted at each time point during pellet culture and monolayer culture.5.4 Flow-cytometric Analysis of Cell-cycle and Protein Quantification of DMP1, DSP and Collagen Type ICell-cycle phases were determied by flow-cytometric analysis based on expression of the ki-67 antigen and the S-phase-specific proliferating cell nuclear antigen. The adherent monolayer DMP1-POMF and the DMP1-POMF pellet were digested with trypsin/ethylene-dianmine tetraacetic acid (EDTA) into single cell suspensions (2×105 cells). The adherent monolayer POMF and the POMF pellet were as control. Then the cells were fixed with cold ethanol (70%) on ice and incubated with the monoclonal antibodies specific to the cell cyucle-associated antigens ki-67 conjugated to FITC and PCNA conjugated to PE(1/10) (Dako corp) respectively. DMP1, DSP and collagen Type I polyclonal antibodies was added directly to the above-mentioned 2×105 cells for l hr on ice. The cells were then incubated with goat anti-mouse IgM conjugated to FITC (1/50 dilution, DAKO Corp.) for 45min on ice. After being washed twice in PBS, the cells were analyzed with the use of a FACSCalibur flow cytometer. Positive expression was defined as the level of fluorerscence greater than 99% of the corresponding isotype-matched control antibodies.5.5 The dentin formation by DMP1-POMF pellete in vivo Group A: The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) pellet reparationGroup B: Porcine oral mucosa fibroblasts (POMF) pellet reparationGroup C: Ca(OH)2 reparationA total of 48 permanent teeth were used from 4 adult miniature pigs with a mean age of 1 year. Animals were randomly divided into two groups.Observation time was 1 month group (n=2),3 months group (n=2).The animals were anesthetized with an injection of 3% pentobarbital sodium (lml/kg).The viable pulp tissue was exposed through a central cavity prepared with a sterie,round steel bur,2mm in diameter. Bleeding after pulpotomy was controlled with sterile cotton pellets.After the bleeding had stopped, the test materials, transfected POMF pellets (group A), non-transfected POMF pellets (group B) or calcium hy droxide.(group C),were applied directly onto the exposed pulp tissue respectively. After a 1-minute waiting period, the cavities were sealed with a glass-ionomer cement.At 1 or 3 months after surgery, animals were killed. Experimental teeth were removed in toto and fixed in cold 4% neutral buffered formaldehyde for 24 hours. The teeth were then demineralized in 12.5% ethylenediaminetetraacetic acid (EDTA) and subsequently embedded in paraffin. Following longitudinal serial sectioning, the section was stained with hematoxylin and eosin. Unstained sections were submitted to immunohistochemistry.Results:1 Plasmid Identification The constructed pEGFP-DMP1 could produced 1.5kb and 4.7kb fragments by an enzyme cutting technique at Xho I and EcoR I sites and their size were same as those of DMP1 and pEGFP–Cl. This indicated that DMP1 cDNA was successfully subcloned into pEGFP–Cl.2 DMP1 TransfectionThe fluorescence microscopy showed that fluorescence light was spread over a broader cellular distribution both in the nucleus and cytoplasm in all of POMF transfected with pEGFP–Cl, but fluorescence light was focused on the cellular membrane and cytoplasm of POMF and MSC transfected with pEGFP -DMP1. The transfection showed an efficiency of 36.5% at day 2 in porcine fibroblasts transfected DMP1. Cells transfected with pEGFP -DMP1 exhibited profound changes in their morphology. These cells were columnar and polarized tended to align themselves in straight parallel lines and had long dendrite-like processes. In contrast, cells transfecting mock pEGFP–Cl did not exhibit any characteristic morphological change with a silm spindle pattern.3 The variation of fibroblasts by gene transfectionRT-PCR analysis demonstrated that transfection of DMP1 triggered the differentiation of DMP1-POMF into odontoblast-like cells, with expression of Ambn corresponding to early stage markers and DSP corresponding to the terminally differentiated state. It was the most unexpected finding in this study that the expression of odontoblast-specific markers Ambn and DSP during the cellular differentiation process. As expected the mock fibroblasts (expressing the vector alone) did not express any of the odontoblast-specific genes. 4 In vitro Assay for lnducement of Mineralized Nodule Formation and von Kossa StainingAn in vitro nodule formation assay was carried out to determine whether the up-regulation of odontoblast-specific gene transcription resulted in a mineralized matrix formation by the von Kossa staining. Nodule formation due to secretion of extracellular matrix proteins in the presence of phosphate ions and ascorbic acid has been considered to be an important feature for mineralization and precedes mineralization. It was observed that transfection of DMP1 enhanced the onset of mineralization in fibroblasts, the size of the nodule formed was significantly large and the overall kinetics of nodule formation was favored by at least 4- to 5- fold in transgenic cell lines when compared with the mock cells.5 The Result of Transmission Electron Microscope (TEM) Discovery The ultrastructural features of pEGFP-DMP1 transfected fibroblast cells showed that there was massive expansive rough endoplasmic reticulum and incremental myelin sheath-like figures and matrix vesicles in the cytoplasm. The cisternae were dilated to varying degrees and were full of abundant proteinoid substances. The collagenous fibrillae was distributed widely intercellular.6 The effect of DMP1 on generation of ectopic osteodentin in transplantation Transfected cells adhered on the fiber of ADM for 24 hours began to proliferate and grow in the framework of three-dimension. The results of this transplantation demonstrated that the fibroblasts transplants had not yet generated mineralized tissues at 7 days. At 14 days ADM post-transplantation with transfected POMF generated osteodentin on the surface of ADM in all transplants. At 21 days ADM post-transplantation, abundant and large osteodentin formation was formed. Immunohistochemical staining showed that DMP1, DSP and collagen type I was positive in the connective tissue compartment prior to the generation of osteodentin formation in fibroblasts transplants. These proteins were not expressed in the mock fibroblasts transplants.5 The dentin formation induced by transfected fibroblasts pellet The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) in the pellet were oval or polygonal, and the nuclei contained a few round or ovoid nucleoli. The pellet progressively became spherical on days 14 and the extracellular matrix accumulated. The histopathological results revealed that the restorative dentin bridge was observed a month later and the bridge was mainly tubular dentin in 3 month. There were well developed odontoblasts under the bridge . The cases of dentine bridge formation of non-transfected POMF pellet and Ca(OH)2 group were less than that of DMP1 transfected pellct .Conclusion:1 DMP1,Ambn and DSP gene could be expressed stably in Porcine oral mucosa fibroblasts (POME) transfected by dentin matrix protein1 (DMP1) (DMP1-POMF cells).2 The DMP1-POMF cells have biological characteristics of mineralization.3 DMP1-POMF cells could be used as seed cells for dentin formation.4 DMP1-POMF could be attached on the surface of acellular dermal matrix(ADM) and proliferated actively.5 DMP1-POMF cells attached on the surface of acellular dermal matrix could be successfully constructed to the dentin formation by implanted subcutaneously into nude mice.6 ADM could be used as scaffold material for dentin formation.7 DMP1-POMF pellet could induce the formation of reparative dentin in vivo.