Dissertation > Medicine, health > Oral Sciences > Oral Pathology

The Research of DPSCs and PDLSCs’Microenvirnment Construction by Cell Sheet Engineering Techniques

Author WangFang
Tutor ZhaoYiMin
School Fourth Military Medical University
Course Oral
Keywords Dental Pulp Stem Cells Periodontal ligament stem cells Scaffolds Conditioned medium Tissue Engineering Microenvironment Cell patch technology
CLC R780.2
Type Master's thesis
Year 2011
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Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) is an important seed cells for tissue engineering of tooth. Compared with other odontogenic seed cells (as SHEDs, SCAP, DFSCs), with access to simple, not patient age factors, tissue storage capacity and less invasive. The DPSCs and high value-added rate PDLSCs to repair dental periodontal tissue function, multi-differentiation potential and can be combined with the features making it tooth tissue engineering and future clinical potential huge teeth endogenous adult stem cells. The experimental cell patch technology tooth tissue engineering studies, to establish periodontal, endodontic conditions culture medium DPSCs PDLSCs co-culture system, detection of two cell microenvironment on cell morphology and function, further cell patch technology to build DPSCs PDLSCs-HA/TCP bracket complex in vivo histological studies to explore new approaches for the regeneration of dental tissue engineering. Part I: periodontal ligament stem cells (hPDLSCs) periodontal ligament cell sheets was prepared jointly by enzymatic digestion and tissue explant obtain primary cells using an inverted microscope cell morphology and growth conditions. Fourth generation of periodontal ligament cells were seeded into a Petri dish, after five weeks of continuous culture periodontal cell biological membrane can form machining performance, this cell patch wrapped HA / TCP scaffold implanted subcutaneously into nude mice, 4 weeks harvested after histological detection. This part of the experiment results show that: the periodontal ligament stem cell state, each layer of cells, both overlapping rules arranged between layers of cells closely linked. Histological examination showed that granular hydroxyapatite / tricalcium phosphate (HA / TCP) holder attached multilayer fibroblast-like cells outside the periodontal ligament-like tissue, and walk through visible capillaries, scaffolds teeth class cementum-like structure formed between the organization of film weeks. Part II: the study of the interaction of the microenvironment of dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLCS) were isolated and cultured dental pulp stem cells, periodontal ligament stem cells for use, preparation of dental pulp stem cell conditioned culture medium and periodontal ligament stem cells conditions culture solution. Each of the two types of cells in the two conditions in culture medium culture, divided into four groups. Inverted microscope, cells of four experimental groups morphological observation, MTT cell proliferation assay, ALP activity detection, and real-time quantitative PCR, which develop two sets of dental pulp stem cells detection DSP, Col ColIII gene; cultured periodontal ligament stem cells group detection ColI, OCN, scleraxis gene. The test results show that: the periodontal ligament cell conditioned culture fluid cultures pulp stem cells proliferation activity, alkaline phosphatase activity and DSP Col ColIII expression water were higher than those by the dental pulp cell conditioned culture fluid cultures pulp stem cells; culture fluid cultures periodontal ligament stem cells cultured dental pulp cells conditions the proliferation activity of periodontal ligament cells cultured liquid, alkaline phosphatase activity ColI, OCN, scleraxis, is lower than that by the periodontal ligament cells conditions. This part of the experiment was to investigate the effects of two types of stem cells and their microenvironment between experimental results show that the microenvironment of the periodontal ligament cell conditioned medium promoted the proliferation and differentiation of dental pulp cells and dental pulp cell conditioned medium The microenvironment Quedui periodontal ligament cells proliferation and differentiation inhibition. Part III: Building periodontal ligament cell patch -DPSCs-HA/TCP in vivo study of complex composite HA / TCP scaffold of dental pulp stem cells were cultured for 10 days in the bioreactor, the surface parcel periodontal ligament this complex cell patch transplanted into immunodeficient mice subcutaneous culture. Sacrificed six weeks after histological HA / TCP scaffold outside of the periodontal ligament-like tissue formation, stent outer surface and the surface of the internal voids pulp stem cells secrete mineralized matrix formation, but far more than the outer surface of the mineralized matrix internal pore surfaces of mineralized matrix; periodontal ligament-like tissue in contact with the pulp cells secrete large amounts of mineralized matrix, the nucleus is large and deeply stained; contact the dental pulp and periodontal ligament-like tissue adjacent cells hardly mineralization. The histological staining further validation periodontal ligament stem cells induce and promote the role of the dental pulp cells.

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