The Effect Of Dentin Derived Proteins On HPDLSCs In Vitro And In Vivo

During onset of root, the interaction of dental follicle cells (DFCs), Hertwig's epithelial root sheath (HERS) and dental papilla cells (DPCs) gives rise to formation of root and periodontium. Apart from above, it has been well recognized that DFCs which are the periodontal progenitor cells penetrate disintegrated HERS and contact with root dentin surface prior to any cementum formation. Our previous studies have proved that dentin non-collagenous proteins (DNCPs) can stimulate DFCs differentiate into cementoblast lineages. Furthermore, Sonoyama et al have successfully constructed bio-root periodontal complex in miniature pigs by developmental root apical papilla stem cells combining with periodontal stem cells.For true periodontium regeneration, direct contact between the conditioned or denuded root surface with periodontal cells seems to be a prerequisite. Clinically, disinfection and modification of the contaminated root including physical and chemical treatment to restore its biocompatibility and to favor the attachment of periodontal structures become necessity. After the smear layer removal, the new dentin collagenous and non-collagenous proteins are exposed and they are supposed to be a chemo-attractant for periodontal fibroblasts. DNCPs, which compromise glycoproteins/sialoproteins, phosphoroteins, proteoglycans and growth factors have been confirmed to act as an inductive agent in promoting many cells differentiation. Based on the fact of direct dentin to periodontal cell contact and the previous results, we proposed that dentin relative matrix may play an important role in periodontium formation and may benefit periodontal tissue regeneration. Therefore, it would be of great interest to investigate the effects of dentin matrix on periodontal stem cells.Taken into consideration that stem cells are capable of self-renewal and multi-lineage differentiation, which make them very promising in regenerating organs and tissues, these cells are of paramount importance in periodontal morphogenesis. The present study was designed to investigate the biological effects of DNCPs on human periodontal ligament stem cells (HPDLSCs) in vitro, and to determine the potential of reconstructing a periodontal complex with DNCPs treated HPDLSCs on chemical conditioned root dentin (CCRD) in vivo. It is the first time to applying active proteins derived from dentin with periodontal stem cells to construct periodontal structure. The strategy may be shed light on human periodontal tissue regeneration.1 Materials and methods:1.1 Isolation of HPDLSCs and HRAPSCsHPDLSCs and HRAPSCs were isolated from ten healthy donors, cultured, and expanded as previously described. Procedures were performed according to the approval of the institutional review board and the informed consent of the patients. Wisdom and premolar teeth intended for extraction due to orthodontic reasons were used as the cell source. Periodontal ligament tissue and apical tissues were obtained and cultured in MesenPRO RS medium containing 2% FCS, 100 units/mL penicillin, 100μg/mL streptomycin. Multiple colony-derived passage two were used in experiments.1.2 Adipogenetic/osteogenetic differentiation and immunohistochemistry of the stem cellsFor differentiation investigation, 1×104 cells/well were plated in a 12-well dish and cultured in DMEM supplemented with 2% FCS, 100 units/ml penicillin, 100μg/ml streptomycin, 50μg/ml anti ascorbic acid (medium A) for 1 day. Then, the cells were incubated with adipogenic or osteogenetic medium for another 21 days, and detected by Oil red O solution or Alizarin red staining respectively.16 Isolated putative stem cells were incubated with STRO-1 antibody, and subsequently incubated with FITC-conjugated anti-mouse secondary antibody. The surface marker STRO-1 positive cells were also analyzed by flow cytometry.1.3 Preparation of CCRDThe teeth for culturing cells were prepared for CCRD. Roots were sliced in longitudinal section. A series of physical and chemical procedures were performed according to reported protocols with minor modifications. In brief, dentinal materials were treated for 3 min with Chloroform-methanol (1:1), 0.6 M HCl for 5 min, 0.5M EDTA for 3 min, CaCl2 2M for 3 min, LiCl for 5 min, and absolute ethanol for 10 min at room temperature. Thereafter, the freeze-dried process was performed.1.4 Assessment of the effect of DNCPs on HPDLSCs1.4.1 Cell growth rate assay, flow cytometry and BrdU incorporationTo evaluate the proliferation potential and viability of the HPDLSCs treated or untreated with DNCPs, growth rate MTT assay, cell cycle analysis and BrdU incorporation into DNA were conducted. In the present study, DNCPs was dissolved in acetic acid solution. Two groups were set, to the test group, 1ug/ml DNCPs were added to medium A (medium B), and to the control group, only the medium A. The MTT assay was carried out for 5 days according to the cell proliferation kit protocol (Sigma). Flow cytometry analysis was respectively treated as previous report. The fractions of cells in the G1, S, and G2 phases of cell cycle were analyzed. DNA synthesis of the HPDLSCs in different medium was assessed by measuring bromodeoxyuridine incorporation (BrdU). Immunodetection of the incorporated BrdU into cells was performed as described previously. The BrdU-labelling index was determined as the percentage of BrdU-positive cells to the total cell number.1.4.2 Clonogenic assays and cells morphology observationTo assess colony-forming efficiency, single-cell suspensions within medium A or medium B (1×103 cells) were seeded into 6-well dishes. Day 7th cultures were stained with 0.1% toluidine blue. Cells morphology was examined under phase-contrast microscopy and scanning electron microscope.1.4. 3 Cell adhesion assayA cell adhesion assay was performed using the methods of Rodrigues. Briefly, 24-well culture plates were incubated with DNCPs, Nonspecific binding sites were blocked with bovine serum albumin. Cells suspension (5×104/ml) was plated into plates. Attached cells were harvested with trypsin, and counted in a haemocytometer. Data are expressed as the percentage of attached cells compared with negative control.1.4. 4 Analysis of alkaline phosphatase (ALP) activity, quantitative real time PCR and MineralizationTo investigate the potential of HPDLSC treated with DNCPs to differentiate into mineralizing cementoblast-like lineages in vitro, the ALP activity, real time RT-PCR and mineralization behavior were analyzed. The quantitative ALP activity was measured at 1, 7 and 14 days. The mRNA expressions of several osteoblastic markers, including COL ?, ALP and OCN were evaluated by real-time quantitative RT-PCR analysis following stimulated by DNCPs. cells were maintained in medium A or medium B and supplemented with osteogenetic media for 21 days. Mineralized formation was identified by Alizarin red staining. Amount of Alizarin red bound to the mineral in each dish was quantified according to Jo.1.4.5 In vitro Cells attachment to CCRDPrepared and sterilized CCRD were soaked in medium A or medium B for 24h. HPDLSCs were seeded on each CCRD surface. The morphologies of the CCRD and CCRD-cells composite were examined under SEM.1.4. 6 Statistical analysisData were analysed using SPSS version 10.0 (Chicago, IL, USA). Statistical analysis of the data was performed by student's t-test. For all tests, significance level was set at P < 0.05 for all tests.1. 5 In vivo studyHigh density of HPDLSCs and ECM were harvested by TrypLETM Express and were seeded on each CCRD which having infiltrated with medium A or medium B. All the CCRD-cells composite were implanted into dorsal subcutaneous area of athymic mice. CCRD were also implanted into dorsal subcutaneous area of athymic mice as negative control. The specimens were harvested at 4 weeks post-transplantation, and stained with H&E.1.6 Preparation of Collagen/HA/GM controlled release TGF-β1 scaffoldsPreparation of gelatin microsphere TGF-β1 composite were fabricated according to the method described by Sha Huang and Faming Chen in our lab. The HA powders were prepared by a simple aqueous precipitation method as described in our previous reports. An 5%(w/w) equal volume of both nano-CDHA and gelatin microsphere- TGF-β1 was put into solution and stirred for 1 hour, Every 1 ml of composite solution was poured into one well of 24-well plate, and then kept at -200C overnight. These complexes were then frozen by immersion into -800C for 2 h and transferred into a freezedrying vessel (OHRIST BETA 1-15, Germany) for 24 h until dry. HPDLSCs were seeded onto scaffolds composite and cultured for 3 days. Then the scaffolds and cells compounds were observed under SEM and also traplanted into athymic mice for 2 weeks.2 Results2.1 Characteristics of HPDLSCs and HRAPSCsWe isolated postnatal stem cells from periodontium and apical tissues, and the isolated cells formed single-cell-derived colonies and most of the cells retained their fibroblastic spindle shape. Ex-vivo expanded HPDLSCs and HRAPSCs expressed the cell surface molecules STRO-1 by immunohisto- chemical staining. After 3 weeks of culture with an adipogenic inductive cocktail, stem cells developed into oil red O-positive lipid fat cells. Small round alizarin red-positive nodules formed in the cultures after 3 weeks of osteoblastic induction, indicating calcium accumulation in vitro.2.2 SEM observation of CCRDWhen the specimens were examined under SEM, CCRD demonstrated clean surface which was eliminated smear layer, opened and widened dentin tubules. Cells showing well growth were seen on all specimens. In dense cell area, cell sheets were formed and covered dentin surface. In sparse cell area which CCRD and cells treated with DNCPs, cells showed polygonal or cubical shape in stead of spindly shape. 2.3 Effect of DNCPs on HPDLSCsMTT assays showed that cells cultured with DNCPs demonstrated a statistically significant increase in proliferation at 3d and 5d as compared to control group (P < 0.05). In addition, the former showed a significantly higher rate of BrdU uptake than the later. The higher proliferation activity of induced HPDLSCs was further confirmed by flow cytometry.Both group of HPDLSCs showed the ability to form adherent clonogenic cell clusters. These results showed no statistically significant difference between the two groups. Observed under phase-contrast microscope, untreated cells were fibroblastic and bipolar in shape. While treated cells became flatter and most of them were cuboidal or polygonal after the induction of DNCPs for 7 days. The SEM results were coincident with above light microscope findings, there were mineralized secretary matrix granules can be found on the surface of the treated cellsCell adhesion assay showed both groups have proper adhesive ability. But increased cells attachment presented statistically in the group whose plates and cells were incubated with DNCPs (Fig. 4H; P<0.05).ALP activity of hPDLSCs in response to different DNCPs concentrations was increased. ALP activity of HPDLSCs was obviously higher under the induction of DNCPs with respect to non-induced group at 7 and 14 days (P<0.05), After incubation in mineralized culture for 21 days, HPDLSCs cultured in presence of DNCPs produced extensive sheets of calcified deposits whereas deposits of the HPDLSCs in absence of DNCPs were sparsely scattered. Quantification of the amount of Alizarin red also showed significantly difference.The mRNA expressions of COL I, ALP and OCN in presence of DNCPs caused a 1.7, 2.2, 1.2 folds increase compared with those in absence of DNCPs respectively for 2 day incubation.2.4 Historical observations of tissue samples in vivoIn 2 out of 6 DNCPs treated specimens for 4-week transplantation, new cementum-like tissue with cell-rich fibrous tissue adjacent or inserted into it formed along the CCRD surface. In 3 out of 6 DNCPs treated specimens, there are obvious separations between neo-formed tissues with dentin surface, only showing monostratified or stratified cubical cementoblast-like cells regularly aligned on dentin surface. While to the all DNCPs untreated group and one DNCPs treated specimen, no obvious cementum-like structure or periodontal fibrous tissue formed along the CCRD surface. To the negative control of naked CCRD transplantation, there was no host cells attachment or tissue regeneration on all CCRD surface.2.5 characteristics of Collagen/HA/GM controlled release TGF-β1 scaffoldsScaffolds exhibited macroporous microstructure. The pores were interconnected with pore size about 100-200 mm. scaffolds demonstrated superior biocompatibility.The amount of TGF-β1 was detected for 2 weeks in vitro and in vivo.3 Discussion:Natural dentin contains numerous collagenous protein and non-collagenous signaling molecular protein, sequestered in mineralized matrix, which includes BMPs, TGF-β, DSP, DPP, DMP-1 and others. Accordingly, we hypothesized that dentin molecules bound to matrix components or to hydroxyapatite crystals may be exposed or released as a consequence of injury to the periodontal ligament. 12,13,26,27,28,29 We hypothesized that dentin matrix produced by odontoblasts could migrate through and diffuse to periodontal tissues, which is similar to the well-known phenomenon of diffusion of enamel matrix proteins through the pre-mineralizing mantle dentin into the odontoblast layer.Based on previous studies and theory of root onset, we managed to determine whether the DNCPs derived from dentin provided additional advantages for the cellular events associated with periodontal regeneration. The present study indicated that DNCPs can promote proliferation potential and viability of HPDLSCs. In addition, induced HPDLSCs presented several features of cementoblast differentiation, as indicated by morphologic changes, enhanced alkaline phosphatase (ALP) activity, increased matrix mineralization, and up-regulated the expressions of mineralization associated genes such as Collagen I, OCN and ALP. Furthermore, our adhesion assay showed that DNCPs can slightly promote HPDLSCs attachment ability, compared with control treatment. It is suggested that, although the underlying mechanisms are still not well understood, cemento/osteoblastic differentiation of HPDLSCs may be affected by the dose of DNCPs according to our results.Clinically, organic or inorganic acid characterized as partly demineralization and deproteinization which is frequently used as root conditionding. They may partly demineralize the planed root surfaces, eliminate the smear layer, and expose some components of the extracelular matrix of dentin or cementum. Our SEM observations of CCRD demonstrated elimination of smear layer, opening and widening the tubules. Cell spreading was already evident and the penetration of cytoplasmic process into dentinal tubules was frequently observed.Ex-vivo-expanded HPDLSCs aggregate treated or untreated by DNCPs combining with CCRD were transplanted into immunocompromised mice. 4 weeks post-transplantation, histological findings demonstrated that new cementum-like tissue formed on CCRD surfaces. In light of these in vivo evidence, we testified again that DNCPs have provided additional advantages for the cellular events associated with periodontal regeneration.We developed a novel three-dimensional special scaffold consists of collagen, nano-calcium deficient hydroxyapatite and gelatin microsphere combining with controlled release growth factors, which provided structural support and stimulated repair, and within certain parameters, the ability to use it to promote periodontal tissue regeneration.In conclusion, our study has demonstrated that DNCPs is an effective stimulator of HPDLSCs proliferation, morphological changes, attachment as well as cenmento/osteoblastic differentiation. DNCPs facilitate HPDLSCs forming cementum-like adjacent fibrous tissue on CCRD. These findings therefore provide convincing evidence and useful data for DNCPs as a potent tool to facilitate periodontal regeneration.