Effects Of Lactoferrin On The Proliferation,Migration And Osteogenic Differentiation Of Human Periodontal Ligament Cells

Background and objectionPeriodontitis is one of the the most common oral diseases in adults and a major cause of human health hazards, which can cause damages of cementum, periodontal ligament, alveolar bone, gum tissue and result in teeth mobility and loss.Restoration of periodontal supporting tissues is the ultimate goal of the treatment of periodontal disease.Normal periodontal tissues have abilities of self-renewal and restoration, which could be achieved primarily through a variety of functions of human periodontal ligament cells (hPDLCs). hPDLCs is the most important cellular components of periodontal ligament with multiple differentiation potential and the function of secretion and degradation of collagen. It is the main source of the formation of periodontal ligament, cementum, periodontal alveolar bone and other periodontal attachment tissues and the foundation of periodontal supporting tissues' regeneration. The host's autoimmunity has been fully researched and confirmed in the development of periodontal disease. It has been known that Porphyromonas gingivalis Aeromonas (P.gingivalis) is the main pathogen of chronic periodontal disease, whose lipopolysaccharide (LPS) is the major virulence factor to cause an autoimmune response in hPDLCs. LPS plays a vital role in the periodontal disease. Therefore modulating inflammation and promoting periodontal supporting tissues' reconstruction are two critical aspects during the treatment of periodontal diseases which can not be ignored.Lactoferrin (LF) is one kind of glycoproteins presents in a variety of human tissues. It has antibacterial and antiviral activity, ability of inhibiting tumor cell proliferation and other characteristics. LF is part of the non-specific immune system of the host and also an important part of saliva defense system in the oral cavity. In recent years, studies suggest that LF also plays an important role in the growth and metabolism of bones. It has been confirmed that LF can induce bone marrow mesenchymal stem cells' expression of osteogenic markers, inhibit the formation of infection-related osteoclasts, promote the proliferation and differentiation of osteoblasts. In earlier studies of our research group it has been found that LF can cut the expression of Toll-like receptor 4 stimulated by lipopolysaccharide in hPDLCs, indicating that LF has a positive meaning in immunotherapy of periodontitis. An ideal drug for the treatment of periodontitis should be able to regulate inflammation and promote formation of periodontal supporting tissues. Therefore this study was designed to investigate the effects of LF on human periodontal ligament cells' proliferation, migration and differentiation, and to observe that whether LF could promote human periodontal ligament cells' osteogenic differentiation under inflammatory milieu stimulated by LPS.Chapter 1 The culture and identification of hPDLCs in vitroCulture hPDLCs in vitro, observe the features of hPDLCs' shape and identify its origin.Methods1. hPDLCs were obtained from periodontally healthy donors (age from 15-22), whose teeth were extracted for orthodontic reasons. The teeth should not have periodontal or pulp diseases and no pollution. Scraped the middle third of the root periodontal ligament tissue under sterile conditions, used modified tissue explant collagenase method to culture cells and subcultured them. Took primary and third generation of cells, used inverted phase contrast microscope to observe the growth and morphological characteristics.2. Immunohistochemistry-SP (streptavidin-perosidase) method:Selected the third generation of well-grew cells to make climbing film:Water bath 20 min in 37? 3% hydrogen peroxide deionized water, goat serum blocking for 30min, added the first antibody, placed in 37? wet box for 2 hours, washed, added the second antibody and incubated for 30 min at 37?. Used DAB Horseradish Peroxidase Color Development Kit for color development about 10 minutes, dehydrated and coverslipped with neutral resins. Observed with inverted microscope and photographed.3. Took the cells of third generation, made unicellular suspension with density of 5×104 mL-1. Added the mouse monoclonal antibodies against human CD29? CD44?CD90?CD 105 to cells respectively, then incubated the cells at room temperature for 1 hour; washed and added sheep against mice Ig-FITC, incubated at room temperature for 45 min away from light;washed with PBS at least 3 times, made unicellular suspension with PBS. Detected the cell surface molecule phenotype using Flow cytometry instrument.4. MTT assays to detect the cytoactive:cells of third generation were seeded in 96-well plates at 5×104 mL-1 and each well added 100?L cell suspension. Added 20?L of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (thiazolyl blue) at a concentration of 5 mg/mL to each culture well at each indicated time points. After incubation at 37? for 4 hours, discarded the supernatant, and then added 150 ?L dimethyl sulfoxide to each well. The absorbances of cells were measured using the spectrophotometer with a test wavelength of 490 nm.5. Alizarin red staining method to detect the mineralization ability:choose the third generation hPDLCs and seeded at 6-well plates in the density of 5×104 mL-1 and cultured to 70%?80% confluence, then cultured with mineralization induction medium for 14 days. The induced cells were stained with 2% Alizarin Red, and were observed and taken images under the phase contrast microscope.Results1. hPDLCs cultured by modified tissue explant collagenase method had a higher grow and survival rate. The morphological observation showed that the cells were fibroblastic-like and spindle or start-shaped, had bigger volume and nuclear was in the center of the cell. The represented cells attached after 24 hours, gradually extended into irregular circle, polygon or spindle, and exhibited radial proliferation.2. SP method showed that after vimentin staining cytoplasm was deeply stained, which was positive; while after keratin staining cytoplasm was shallower stained, which meant the result was negative.3. The results of flow cytometry showed that:The positive cells rate signed with CD29?CD44?CD105?CD90 were 98.17%,99.13%,100% and 99.13% respectively, which illustrated that the cells were from mesenchymal.4. MTT assays showed that:The cells entered logarithmic phase after cultured for 72 hours, from which we could know that the cell activity was good.5. The mineralization nodules in the cells could be detected with the existence of mineralization medium after being cultured for 14 days.Chapter 2 The effects of lactoferrin on the proliferation and migration ofhuman periodontal ligament cellsMTT assay, wound healing assay, Transwell method are used to detect the effects of lactoferrin on proliferation and migration (horizontal migration and vertical migration) of hPDLCs, and to explore whether lactoferrin can promote the proliferation and migration of hPDLCs.Methods1. The cultivation of hPDLCs was the same as the chapter 1.2. The third generation of hPDLCs were seeded in 96-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS, DMEM containing 10% FBS+10 ?g/mL LF, DMEM containing 10% FBS+20 ?g/mL LF. And grouped cells according to different medium. Each group set 6 wells and took the MTT assay at the lth,3th,5th,7th day:added 20 ?L MTT solution to each well, incubated for 4 hours, discarded the medium and added 150 ?L dimethyl sulfoxide to each well. Slightly shaked 10 min, the spectrophotometer was used to measure the OD value at 490nm wavelength and drew the growth curve.3. Cells were inoculated in 24-well plates (5×104 mL-1), cultured in 1.5 mL DMEM containing 10% FBS. When the coverslips were 80%-90% covered with cells, abandoned the original culture medium, used 200 ?L micro pipette tip to draw a 1mm wide scratch at each bottom of the 6-well plates along a ruler and marked the observation points. PBS rinsed off the shedding cells, added culture medium and LF grouped as 0 ?g/mL LF,10 ?g/mL LF,20 ?g/mL LF. Each group had there wells, Photographed the observation points at 0,24,48 hours after the addition of stimulus.4. Took the third generation of hPDLCs and seeded in the upper transwell chamber at 5×104 mL-1, adjusted volume to 200?L using serum-free culture medium and added 500?L complete medium to the lower chamber containing 0?g/mL, 10?g/mL,20?g/mL LF. Discarded the culture medium both in the upper and lower chambers after 24 hours of incubation and wiped the cells that had not have migrated in the upper chamber with a cotton swab. Fixed in 4% paraformaldehyde for 10 min,1% crystal violet stained for 15 minutes, PBS washed. Observed at 200 X magnification and selected 5 vision randomly to read and calculate the mean number of cells.5. Statistical Analysis:All experiments were repeated three times. The data obtained were presented as the mean ± SD and set test level a=0.05. Used SPSS 19.0 software to evaluate the statistical significance.Results1. MTT assay showed that cells of the three groups all had a "S" shape growth curve, and went into the logarithmic growth phase on the third day, the plateau phase on the fifth day. The abilities of proliferation of 10 ?g/mL and 20 ?g/mL LF group were higher than 0 ?g/mL LF group at 3th,5th,7th day and the differences were significant (P<0.05).2. After 24 hours of incubation, the cells in each group all moved to the blank scratches, but 0 ?g/mL LF group's migration was slow,48 hours after the migration,10 ?g/mL,20 ?g/mL LF group almost covered the original scratches while scratches of 0 ?g/mL LF group could still be observed clearly.3.The number of migrating cells in 10 ?g/mL,20 ?g/mL LF group were higher than 0 ?g/mL LF group after 24 hours and the difference was significant (P<0.05).Chapter 3 The effects of lactoferrin on the osteogenic differentiation of human periodontal ligament cellsObserve the effects of lactoferrin on the formation of mineralized nodules and the expression of osteogenic related genes and proteins of hPDLCs by Alizarin Red staining, PNPP method, qRT-PCR and Western Blotting analysis to explore whether lactoferrin can promote hPDLCs' osteogenic differentiation.Methods1. The cultivation of hPDLCs was the same as chapter 1.2. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, changed the complete culture medium into osteogenic induction medium with different concentration of LF(0 ?g/mL,10 ?g/mL,20 ?g/mL). Cultured for 14 days and discarded the medium, PBS washed there times, fixed with 4% paraformaldehyde for 10 minutes,1% alizarin red stained for 30 minutes, photographed and calculated the area of mineralized nodules.3. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, changed the complete culture medium into osteogenic induction medium with different concentration of LF(0 ?g/mL,10 ?g/mL,20 ?g/mL). Cultured for 14 days and used cell lysis solution to lyse cells. Extracted the proteins and followed PNPP kit instruction to detect the alkaline phosphatase(ALP) activity. Alkaline phosphatase activity (U/gprot)=(absorbance measurement hole-absorbance of blank wells)/(absorbance of standard hole-absorbance of blank wells)×standard concentration of phenol (0.1 mg/mL)/test sample protein concentration (gprot/mL).4. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, changed the complete culture medium into osteogenic induction medium with different concentration of LF(0 ?g/mL,10 ?g/mL,20 ?g/mL). Cultured for 14 days. Took total RNA as a template for the reverse transcription to cDNA, formulated PCR reaction solution according to the instruction, the amplification was completed after initial denaturation, PCR reactions, melting curve analysis, then detected the expressions of ALP, OCN, OPN.5. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, changed the complete culture medium into osteogenic induction medium with different concentration of LF (0 ?g/mL,10 ?g/mL,20 ?g/mL). Cultured for 14 days. Proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction kit and the concentrations of fractionated proteins were measured with the BCA protein assay kit. Separated protein samples by SDS-PAGE and transferred them onto PVDF at 200 mA for 2 hours. Used mouse monoclonal antibodies against human OCN and mouse monoclonal antibodies against human OPN as the primary antibody and HRP-conjugated anti IgG as the secondary antibody. Signals of the results were developed on X-ray films. The densities of the developed bands were quantified with Quantity One software.6. Statistical Analysis:All experiments were repeated three times. The data obtained were presented as the mean SD and set test level ?=0.05. Used SPSS 19.0 software to evaluate the statistical significance.Results1. Alizarin Red staining showed that each group had formed mineralized nodules, but the area of mineralized nodules of 0 ?g/mL LF group was significantly smaller than the other two groups and the differences were significant (P<0.05). At the same time the area of mineralized nodules of 20 ?g/mL LF group was larger than the 10 ?g/mL LF group more and the difference was significant (P<0.05).2. Alkaline phosphatase activity detected by PNPP assay showed that the alkaline phosphatase activities of 10 ?g/mL LF group and 20 ?g/mL LF group were higher than 0 ?g/mL LF group, and the differences were significant (P<0.05).3. The expressions of osteogenesis-related genes:Real-time PCR results showed that under the stimulating of LF osteogenesis-related genes(ALP, OCN, OPN)were increased, and there were significant differences (P<0.05) between 10 ?g/mL,20 ?g/mL LF group and 0 ?g/mL LF group.4. Western Blot results showed that:10 ?g/mL LF group's and 20 ?g/mL LF group's gray value of protein bands were significantly higher than the 0 ?g/mL LF group, statistical analysis showed that the differences of gray value between the 10 ?g/mL LF group,20 ?g/mL LF group and 0 ?g/mL LF group were significant (P <0.05).Chapter 4 The effects of lactoferrin on the osteogenic differentiation of human periodontal ligament cells stimulated by LPSDetect the secretion of inflammatory cytokines in hPDLCs stimulated by LPS and the effects of LF on the osteogenic differentiation of hPDLCs under the inflammatory environment to explore that whether LF can promote the osteogenic differentiation of hPDLCs under inflammatory environment stimulated by LPS.Methods1. The cultivation of hPDLCs was the same as chapter 1.2. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, divided the cells into blank control group, 0.01?g/mL LPS group, 0.1 ?g/mL LPS group, 1?g/mL LPS group. Total RNA was extracted after 24 hours of each group, took reverse transcription of cDNA and used real-time fluorescent quantitative PCR to detect the expressions of TLR4, IL-6, and IL-8.3. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, changed the complete culture medium into osteogenic induction medium, and divided the cells into osteogenic induction medium(OM) group, LPS+osteogenic induction medium group, LPS+LF+osteogenic induction medium group. After being cultured for 7 days, stained with Alizarin Red, observed the formation of mineralized nodules under an inverted microscope and photographed, calculated the area of mineralized nodules to get the average value.4. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, changed the complete culture medium into osteogenic induction medium, and the way of grouping the cells was the same as previous experiment. Cultured for 7 days and used cell lysis solution to lyse cells. Extracted the proteins and followed PNPP kit instruction to detect the ALP activity.5. The third generation hPDLCs were seeded in 6-well culture plates (5×104 mL-1) and cultured in DMEM containing 10% FBS. After the well has a monolayer of adherent cells, changed the complete culture medium into osteogenic induction medium, and the way of grouping the cells was the same as previous experiment. After 7 days removed the medium and collected cells, extracted the total protein and used Western Blotting analysis to detected the expressions of OCN and OPN in each group.6. Statistical Analysis:All experiments were repeated three times. The data obtained were presented as the mean ± SD and set test level a=0.05. Used SPSS 19.0 software to evaluate the statistical significance.Results1. TLR4 and inflammatory factors were expressed in each group, in 0.01?g/mL LPS group the expression of TLR4 had no difference with the control group (P>0.05), while IL-6 and IL-8's expressions had significant differences with the control group (P<0.05). TLR4, IL-6, IL-8's expressions of 0.01?g/mL LPS group and 1?g/mL LPS group had significant differences with the control group (P<0.05). TLR4 and IL-8's expressions of 0.1?g/mL LPS group had relatively high volume compared to 1?g/mL LPS group and the differences were significant(P<0.05).2. Mineralized nodules can be observed in all groups. LPS+LF+OM group had a larger number of mineralized nodules compared with LPS group and the difference was significant (P<0.05). LPS group's mineralized nodules area was less than the OM group, and the difference between them was significant (P<0.05).But the difference between LPS+LF+OM group and OM group was not significant(P<0.05).3. LPS+OM group had the minimum expression of ALP activity while LPS+ LF+OM group had the highest expression, and the difference between them was significant (P<0.05). Difference between OM group and LPS+OM group was not significant (P>0.05).4. Western Blot results showed that LPS+OM group's OCN, OPN protein expressions declined slightly compared with OM group, but the difference was not statistically significant (P>0.05). LPS+LF+OM group's OCN, OPN protein expressions were higher than OM group and LPS+OM group, and the differences were significant (P<0.05).Conclusion1. Cells isolated and cultured with modified tissue explant collagenase method has advantages in swimming out, survival rate, infection rate, etc. And the 6th or 7th generation of cells still has proliferation and mineralization capacity. Flow cytometry phenotypic analysis of cell surface molecules and immunohistochemistry can prove that the cultured cells are hPDLCs which have reliable sources and the abilities of proliferation and mineralization differention.2. The proliferation of hPDLCs can be enhanced when adding LF, as well as the capacities of horizontal and vertical migration. But there is no obvious trend shows that the proliferation and migration will increase with the increasing of the concentration of LF.3. In the process of mineralization in normal state hPDLCs, LF can promote the formation of mineralized nodules, improve the expressions of osteogenesis-related genes and proteins, suggesting that LF can promote the osteogenic differentiation of hPDLCs. Wherein the expressions of osteogenesis-related proteins associated with the concentration of LF which proves that there is relationship between the concentration of LF and the osteogenic differentiation of hPDLCs to some extent.4. It is more consistent with the hPDLCs in the development of periodontitis that hPDLCs has raised secretion of inflammatory cytokines stimulated by LPS. And the osteogenic differentiation capacity of hPDLCs in inflammatory environment stimulated by LPS can be promoted by LF, Which provides a theoretical basis for the clinical treatment of periodontitis.