Tissue Engineer Tooth Root Structure

Tooth depends on the anchorage effect of root to be hold in the aveolar bone. Tooth root is composed of dentin-pulp complex, cementum and periodontal ligament. The dentin-pulp complex occupies the volumic main part of tooth root, while the cementum acts as as integral part of periodontal tissue to link the periodontal ligament with the root dentin. Tissue engineering of tooth root is of significance. Tissue engineered tooth root with certain morphology combining with traditional post crown could be used to restore tooth loss, on the other hand tissue engineered root structure could be utilized to improve the biological interface between the implant post and alveolar bone.The anatomical and physical features of dentin-pulp complex determine the principles that should be applied in dentin tissue engineering. The postmitotic nature of odontoblast, the unidentified progenitors of odontoblast make it of pivotal importance to recruit and induce the differentiation of odontoblast in a predictable manner. what kind strategy should be utilized to control the shape and size of tissue engineered dentin? What kind of effect would be caused to the dentinogenesis process with the replacement of natural extra cellular matrix with scaffold material? Does the dentin own the capacity to induce the cementogenesis and anchorage of periodontal ligament?For the discussion listed above, we did the following research work: Part One: In vitro inducing odontoblast-like cell differentiation with different scaffold designAiming to explore the features of the scaffold material suitable for inducing dentin tissue engineering, we isolated dental papillae from 4 dpn SD rat molars and cultured them on the porous biodegradable polylactic acid(PLA) or filter combining with or not with transforming growth factor-β1(TGF-β1). The cell response contacting with the scaffold was characterized with Haematoxylin-Eosin(HE), transmission electron microscopy examination (TEM), immunohistochemical staining and reverse transcription-PCR(RT-PCR). The results showed that cells contacting with the filter polarized, and polar secreted matrix, which was significantly different from those cultured on the porous PLA. The polarized cells stained positive for dentin sialoprotein(DSP), osteopotin(OPN) and typeⅠcollagen(Col I). The gene expression pattern was further confirmed by RT-PCR for dentin sialophosphoprotein(DSPP) and dentin matrix protein 1(DMP1). The results implied that growth factors and their presentation pattern, a substrate to anchor the progenitors all are important to the odontoblast differentiation. Furthermore, the results confirmed that the polarization of the odontoblast morphology and expression of specific gene phenotype should be separate events. It was concluded that specific consideration according to the postmitotic feature and differentiation mechanism of odontoblast should be taken when designing scaffold material for dentin tissue engineering.Part Two: It is possile to control the shape and size of tissue engineered dentin with a distinct strategyA distinct strategy was proposed to control the shape and size of tissue engineered dentin based on the differentiation mechanism of odontoblast. We proposed that if a scaffold could reliably recruit and induce the progenitors of odontoblast into fuctional ones on its surface, while the whole process of cell differentiation and dentinogenesis could happen in a homogenous manner, then tissue engineered dentin blocks with predictable shape and size could be made. To confirm to the feasibility of the above proposal, we inserted the transfilter combining with TGF-β1 into the tooth papillae of postnatal 4 d SD rat, and transplanted it under the renal capsule for 4 d, 7 d, 20 d and 30 d. HE examination showed that the filter could induce odontoblast differentiation after 4 d in vivo culture; the TEM confirmed that the polarized cell was of active secretary activities. 7 d later the polarized cell elongated further. Tubular dentin formed after 20 d's transplantation. Immunohistochemistry analysis showed that the induced odontoblast expressed specific markers like DSP,OPN,Col I. The homogenousity of the cell response and dentinogenesis was confirmed with statistical analysis. The results supported the proposed strategy with experimental evidence.Part Three: Tissue engineer dentin-pulp complexTo examine the effect of replacing the extracellular matrix of tissue engineered tooth pulp, dental papillae cell pellet or collagen seeding with dental papillae cell were transplanted under the renal capsule for 30 d, and the tooth papillae was used as control. Results showed that in the papillae group, tubular dentin formed on the outer surface of papillae, while the inner soft tissue was scattered with few mineralization; in the cell pellet group in which the extracellular matrix was deprived of, the tissue was scattered with tubular dentin and osteoid matrix; in the collagen seeding the primary cultured dental papillae cell group, the tissue was full of osteoid mineralized matrix and undegradable scaffold. It was concluded that extracellular matrix played an important role in mantaining the phetotype of pulp cells。To examine the potential of millipore transfilter combining with TGF-β1 in inducing dentinogenesis from tissue engineered pulp, transfilter combining with TGF-β1 were combined with different type of pulp tissue and transplanted under the renal capsule of SD rat for 7d and 30d. In the transfilter combining with papillae group, fibrodentine formed along the transfilter after 7 days'transplantation, and regular layer of odontoblast formed along the surface of fibrodentine; 30 d later tubular dentin formed with few mineralizations scattered in the pulp. In the transfilter combining with centrifuged papillae cell pellet, scattered odontoblasts were induced along the transfilter after 7 days, and tubular dentin formed after 30 days with amount of mineralization scattered in the pulp tissue. In the transfilter combining with collagen seeding with papillae cells, scattered odontoblast formed along the transfilter after 7 days, and 30 days later the pulp tissue was full of mineralization composed of osteoid tissue and undegradable collagen. It was concluded that transfilter combining with growth factors could induce the dentinogenesis from tissue engineered pulp, while the cell response was influenced by the scaffold, and the scaffold material might had an effect on the migration and differentiation of progenitors of odontoblast.Part Four: Tissue engineer tooth root like structureTo tissue engineer tooth root like structure and investigate the inductive effect of dentin on the periodontal tissue regeneration, bioengineered dentin was combined with primary cultured periodontal ligament cells and transplanted under the renal capsule of immunocompromised mice for 20 days. The results showed that dense collagen fibers expressing Col I arranged perpendicularly on the dentin surface, and the SEM revealed a layer cementum-like tissue in-between the periodontal ligament and dentin. It was concluded that tooth root like stucture was tissue engineered composing of bioengineered dentin, cementum and periodontal ligament. The dentin showed inductive potential in recruit and stimulate periodontal tissue generation.In summary, growth factor and their presentation pattern, a suitable substrate for the progenitors to anchor all play important roles in odontoblast differentiation. A suitable scaffold that take all the factors listet would be of impotance in dentin tissue engineering ; in vitro results showed that transfilter combining with TGF-β1 could reliably recruit progenitors onto its surface and induce their differentiation. Furthermore, in vivo results showed that the cell response and dentinogenesis could happen in a homogenous manner, thus it supplied experimental evidence to support the strategy for controlling the shape and size of tissue engineered dentin. To explain the verse effect that might be caused by the replacement of extracellular matrix with scaffold, dental pulp was tissue engineered with different design, and the results showed that unsuitable replacement of the ECM with artificial scaffold would cause difficulties to migration and differentiation of progenitors of odontoblast. Finally, with bioengineered dentin and primary cultured periodontal ligament cells, tooth root structure composed of dentin and cementum, with periodontal ligament arranged perpendicularly on the dentin surface was tissue engineered.