Effects Of Shuanghuangbu On Adhesion And Growth Of Human Periodontal Ligament Cells In Zein Scaffolds

Objective: Periodontal disease is an inflammatory destructive chronic disease, which can cause destruction and loss of attachment of periodontal tissue and eventually leading to tooth loss. The ultimate goal of periodontal therapy is to achieve regeneration of periodontal tissue, including periodontal ligament, alveolar bone and cementum. With the development of tissue engineering, application of scaffolds to repair periodontal tissue defects has become the focus of periodontal therapy. Zein is extracted from corn, it has good properties in film forming, bonding, waterproofing and moisture resistance, and also tolerance with acid and oils. Zein is widely used in food and medicine because of the characteristic, however it is less used in tissue engineering as scaffolds. In this research, Shuanghuangbu as a growth factor was combined with zein scaffolds, the aims were to study the effects of Shuanghuangbu on the adhesion, growth and proliferation of human periodontal ligament cells (HPDLCs) in zein scaffolds, and to evaluate the feasibility of shuanghuangbu-zein compound scaffold materials in periodontal tissue engineering.Methods:1 HPDLCs primary cultureHealthy premolars removed for orthodontic reasons from healthy adolescents were selected. The study was performed with the approval of the hospital's Human Investigations Committee. After washing with PBS or DMEM for 3 times, the periodontal membranes at 1/3 of roots were aseptically collected and used for primary cultures with the explant culture method.2 Source identification with immunocytochemistry and ABC staining method The second passage cells were cultured on slide for 72 h, and then it was washed with PBS 3 times. After immobilization of the cultured cells with 4% paraformaldehyde, the cell membranes were permeabilized for 10 min with 0.1% Triton X 100 in PBS and then blocked for 20 min with 3% H2O2, then it was washed with PBS three times. Mouse anti-Vimentin and rabbit anti-Cytokeratin (Zhongshan, Beijing, China) were diluted to their final concentration of 1:80 and 1:120 respectively. Both antibodies were incubated with the cells at room temperature for 1 h. Following a wash-out step with PBS, the second antibody was added and incubated with the cells at room temperature for 20 min, and then with DAB colouration and hematoxylin afterstain. Meanwhile, for negative controls, the specific primary antibody was replaced with PBS. That was observed under light microscope3 Shuanghuangbu preparationCoptidis rhizoma, scutellariae radix and rhizoma drynariae were weighed according to a certain proportion and mixed well, The mixture was soaked in distilled water for 30 min and then boiled in 8 volumes of water (v/w) for 2 h and 6 volumes of water (v/w) for 1.5 h, Shuanghuangbu decoction was prepared to concentration of 1 g/ml with water extraction and alcohol precipitation method. The decoction was discolored with 1% active carbon for three times, pH adjusted to 7.0 ~ 7.1, and then stored at 4°C.4 Preparation and pretreatment of zein scaffoldsSodium chloride according to a certain proportion was weighed and added to alcoholic solution of zein, the solution was then heated with stirring until evenly distributed, zein scaffolds were prepared by solvent casting/particle leaching, UV irradiated for 2 h and stored for use.5 MTT assay of periodontal ligament cell proliferationAt 1 cm~2/mL (v/v), the zein scaffold slices were soaked in DMEM containing 10% FBS at 37°C for 72 h. The zein scaffold extract were collected and then Shuanghuangbu was diluted to final concentrations of 50, 100, 150, 200, 500 and 1000μg/ml with the zein scaffold extract. The fifth passage cells were trypsinized, collected and made into cell suspensions at 1× 10~4/ml. Then the suspensions were seeded at 1×10~3 cells per well in 96-well plates. After culture at 37°C in 5% CO2 for 24 h. The wells were randomly divided into different concentrations of Shuanghuangbu group, zein scaffold extraction group and control group, MTT assay of periodontal ligament cell proliferati.6 Inverted phase contrast microscopy and scanning electron microscopy The zein scaffold slices were soaked in DMEM medium containing 10% FBS at 37°C for 24 h in a 24-well plate. The fifth passage cells were trypsinized,collected and made into cell suspensions at 1.5×105/ml. Then the suspensions were seeded at 3×104 cells per well in 24-well plates with zein scaffold slices, then 800μl of DMEM containing 10% FBS was added. Afterculture at 37°C in 5% CO2 for 24 h, the cells were divided into the experimental group and the control group in random. Cells were cultured for 3 weeks and then observed under inverted phase contrast microscope and scanning electron microscope.7 Statistical analysisAll data were presented as means±standard deviation (S.D.) and analyzed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Comparisons of group means were assessed with One-Way ANOVA. P < 0.05 was considered significant difference.Results:1 Cell culture and identificationPeriodontal ligament cells migrated from the tissue after 5 to 7 days in culture, oriented radially with growth; the cells were spindle or stellate in shape, with round cell body and even cytoplasm, round or oval nuclei. Identification result showed that the cells were vimentin-positive with brown staining in the cytoplasm and cytokeratin negative, confirming that the cells were fibroblasts of mesoderm origin and suitable for the experiments.2 The effect of Shuanghuangbu on HPDLCs proliferation in zein scaffoldsMTT assay result showed that compared with the control group and zein scaffold extract group, the different concentrations of Shuanghuangbu prepared with zein scaffold extract showed promotion of HPDLCs proliferation, and the most significant effect was achieved at 100μg/mL of Shuanghuangbu (P < 0.01), indicating that different concentrations of Shuanghuangbu significantly promoted the proliferation of HPDLCs in a dose-dependent manner. There was no significant difference between Zein scaffold extract group and control group (P > 0.05), which showed that zein scaffolds did not affect the proliferation of HPDLCs.3 Morphological observations of HPDLCs in zein scaffolds after Shuanghuangbu treatmentUnder inverted phase contrast microscope, Shuanghuangbu treatment increased the number of periodontal ligament cells in zein scaffolds, the cells were long spindle in shape, forming a net across the pores. Without Shuanghuangbu, a small number of cells were present in the zein scaffolds. Under scanning electron microscope, Shuanghuangbu treatment increased the number of periodontal ligament cells in zein scaffolds, the cells were mostly attached to the peripheral of the pores of the scaffold, spindle or star in shape; extending pseudopodia across the pores and forming a mesh; the cells were also on the scaffold surface and lacunae. Without Shuanghuangbu, a small number of cells were present in the zein scaffolds, mostly star in shape and mainly on lacunae. The cells in zein scaffolds under Shuanghuangbu treatment were mostly spindle shaped with stout cell body, uniform plasma and stereoscopic.Conclusion: Shuanghuangbu could promote the adhesion, growth and proliferation of human periodontal ligament cells in zein scaffolds, this indicates that Shuanghuangbu as a growth factor and shuanghuangbu-zein as composite scaffold materials used in periodontal tissue engineering were feasible.