Isolation, Identification Of Dental Pulp Stem Cells And Their Application For Tissue-engineered Dentin-pulp Complex

Stem cells are generally defined as undifferentiated cells capable of both self-renewal and multi-linage differentiation. According to their origins, these cells can be divided into embryonic stem cells originated from the inner cell mass of blastocyst and adult stem cells that exist in various adult tissues. Recently, much attention has been paid on the post-natal adult stem cells with their advantages of reliable source, plasticity and lack of immunologic rejection for clinical application. As a result, many adult stem cells have been identified and successfully isolated in almost all tissues of the body and this may not only provided a new plateau for the mechanism study of repair and regeneration in damaged tissue or organ, but also may bring about a revolution in the treatment of many thorny diseases through the application of stem-cell-based therapy or tissue engineering approach to create tissues or organs.Dental pulp stem cells(DPSCs), which is confirmed as one of adult stem cells were firstly isolated from adult dental pulp by Dr Gronthos on year 2000.The most striking feature of DPSCs is their ability to regenerate a dental-pulp-like complex. Thus may provide a potential source of seeding cells and theory basis for regeneration of dentin-pulp complex by tissue-engineering method. It is reported that DPSCs derived from various species including human being, mice, rat and dog have been successfully isolated in recent years and other studies on DPSCs are limit to several aspects such as phenotype of the cells,differentiation to odontoblasts and dentinogenesis of it. But these researches may be initial stage for betterunderstanding of them as adult stem cells, regulators in homeostasis of physiological and pathological conditions, tools for clinical treatment. Therefore, the aim of the present study was to generate dentin-pulp-like complex through tissue engineering method using DPSCs as a cell source based on successfully isolation and identification from human and rat dental pulp, respectively. Furthermore, we investigate DPSCs-based tissue engineering in vivo for dentin-pulp regeneration therapy during pulpotomy of rat teeth.Part I. Isolation and identification of human and rat dental pulp stem cellsSingle cell clones of human and rat dental pulp cells were obtained by cell-clone cultural methods,respectively. And human dental pulp stem cells were used as main line in the whole study for cells identification and below other aspects. It was showed that cells originated from single-clone cells of human dental pulp shared the characteristic ultrastructure and cell cycle with adult stem cells by transmission electron microscopy (TEM) and flow cytometry(FCM), respectively. The cells were also examined the origin and the state by Immunocytochemistry assay. It was found that the cells positively expressed CD44, which is the main marker of undifferentiated mesenchymal progenitor; vimentin is also positive express, ON, one of mineralized related protein expressed weakly and the cells negatively expressed DSP. The evidences suggested that the cells isolated from dental pulp were undifferentiated mesenchymal stem cell.When cultured the stem cells with mineralization solution for 14 days, their morphology changed greatly and characterized by a polarized shape with nucleus at one side of cell body and one needle-like cell process at the other side. Von Kossa positive staining reflected formation of mineralized nodes by the continue-cultured cells on day 21 .Meanwhile, the ultrastructure and cell cycle changed in responde to synthesizing and secreting protein, and expressed OC and DSPP which were putative marker of functional odontoblasts. All of these suggested that the mineralization induced cells might change into odontoblast-like cells.To confirm the capacity of the trans-differentiation, the cells expended from single-clone were cultured with neural-inductive cocktail. Results showed that the inductive cells differentiated into neurons-like cells in morphology after four-days culture and expressed GFAP(glial fibrillary acid protein, GFAP) and NSE(neuron specific enolase,NSE) ,markers of glial cells and neurons, respectively. GFAP mRNA was also detected in this cells by RT-PCR.Based on the findings above mentioned, the cells originated from single-clone of dental pulp,similar to other stem cell populations,can be proved to be dental pulp stem cells possessing the ability both to self-renew and to multi-lineage differentiate. These cells can serve as a tool for the study of adult stem cell differentiation in vitro and a source for dentin-pulp tissue-engineering and regeneration in vivo. Part II Effects of different growth factors on the proliferation and differentiation of dental pulp stem cells(DPSCs) in vitroGrowth factors of bFGF,TGF- β ,EGFand BMP were examined on the proliferation by MTT assay and alkaline phosphatase activity of human DPSCs at different concentrations and different time points as well. Certain concentrations of bFGF and TGF-βwere selected as the strongest effect factors for further studies on differentiation effects of DPSCs. The results displayed that 20 ng/mL bFGF had the maximum effect on proliferation of the cells and 20 ng/mL bFGF in combined with 5 ng/mL TGF- 3 may cause the strongest effect on differentiation of it at day 14.This was confirmed by odontoblast-like changes in morphology, expression for their relatively specific protein of DMP-l(dentin matrix protein-1,DMP-1) and DSP(dentin sialoprotein,DSP) and DSPP mRNA at molecular level by RT-PCR. The above explores may provide microenvironment with growth factors for DPSCs similar to that are in vivo to establish dentin-pulp complex-like organ by tissue engineering in vitro.Part III Evaluation of biocompatibility between bioengineering scaffolds and dental pulp stem cells (DPSCs).Human DPSCs were seeded onto scaffold of collage gel andcollage-chitosan lyophilized membrane respectively to observe the cells growth in them by HE staining. It was showed that the cells were well-distributed and proliferated in the gel, while it were not well-distributed in lyophilized membrane and many of them accumulated on the surface of the membrane, only a few permeated into the polyporous space of the membrane. This may suggested that the two collage-based materials were biocompatible well with DPSCs, but both of them may not be suitable for bio-scaffold materials in tissue-engineering dentin-pulp complex for such disadvantages as weak mechanical properties, rapid biodegradability and poor inner space.Hydroxyapatite/tricalcium phosphate (HA/TCP) and ceramic bovine bone(CBB), two calcium phosphate bioceramics materials, were chosen as scaffold for DPSCs. To improve the adhesive ratio of the cells to them, the surfaces of the two materials were modified with chitosan(CS) and the biocompatibility were evaluated by counting cells number, scanning electron microscope (SEM).It was found that the adhesive ratio of the cells onto the two scaffolds increased significantly after modification .The cells seeded onto both of the modified materials grew very well and proliferated rapidly.The massive matrix particles was seen on the surface of the attached cells at day 5.These indicated that both HA/TCP/CS and CBB/CS are good enough to serve as bioscaffold material for tissue engineering study on dentin-pulp complex.Part IV Establishment and regeneration of dentin-pulp complex using dental pulp stem cells (DPSCs)by tissue engineering method.Based on the above results, two species DPSCs of human and rat, the microenvironment of growth factors and the optimal bioscaffold of HA/TCP/CS and CBB/CS were integrated to establish three-dimension culture system,respectively. And then the culture systems were transplanted subcutaneously into the dorsal surface of 5-week-old immunocompromised mice. At interval of 2,4,6 weeks for human DPSCs and 4,6,8 weeks for rat DPSCs after posttransplantation, the specimens were fixed,dimineralized ,dehydration, embedded in paraffin sectionedand stained with HE in a routine way. Finally,the histological observation showed that the cells culture system formed atypical atubular dentin or dentin-pulp like structure with a highly ordered collagenous matrix deposited perpendicular to the odontoblast-like-cell layer, and the longer the posttransplantation time elongated, the more the formations deposited. To take 6-week formations of human DPSCs for example, examination by modified Mallory trichrome staining on them indicated that the mature dentin-like matrix was stained with laterite-red and the new formations together with odontoblast-like cell layer stained with blue in contrast to the eosin staining fibrous tissue. Importantly, the regenerated pre-dentin and odontoblast-like tissue was immunoreactive for DSPP antibody on human DPSCs' formation or DSP and BMP-1 for rat DPSCs' formations, respectively. These findings suggested that this DPSCs culture system regenerated dentin-pulp-like complex after transplantation in vivo. Part V Pulp dentinal regeneration therapy for rat dental pulpotomy model with dental pulp stem cells(DPSCs) by tissue engineering method.DPSCs of rats labeled either with 5-Bromodeoxyuridine (BrdU) or with Hoechst 33342 were mixed with HA/TCP ceramic tiny particles for an hour dynamic culture process. Then the cell-particles compound was transferred into dental pulp chambers of rats whose incisor and first molar had been amputated pulp. After 3 weeks post-operation, dental pulp tissue necrosis and inflammation were not observed in all groups based on histological observations. In cell-HA/TCP test group, there are dentinal bridge-like formation. The cavities being amputated pulp was filled with numerous fibrous osteodentin and tubular dentin, and the exposed pulp.was sealed. However, in the amputated pulp control group, it showed that no dentin-pulp-like tissue formed, and the cavity space being amputated pulp was still empty except a few fibrous neoplasm.While fibrous osteodentin matrix was observed in cell-free HA/TCP control group, and there was still a large empty space in the amputated pulp cavity. At 4 weeks, no significant histological changes could be observed in the two control...