| Tooth lesions and tooth loss caused by dental caries, periodontal diseases and trauma are common and frequently-occurring diseases, which may affect masticator efficiency, language function, facial aesthetics and even psychological health. Current treatment methods depend on restorative materials, such as amalgam, resin and gutta-percha, few of which shared the same physical or chemical characteristics of the natural tooth and may lead to a compromised clinical outcome. The emergence of tissue engineering techniques presents a prospect for the development of natural tooth substitutes. In general, dental tissue engineering requires three basic elements:an appropriate cell source, a biodegradable scaffold and optimal growth factors.Tissue engineering approaches often benefit from advanced cell carriers. Various carriers have been designed to provide a three-dimensional physicochemical and biological microenvironment for cell attachment, proliferation, differentiation and neo tissue genesis. However, the repair of irregularly-shaped tissues or defects often require complex design or fabrication of a monolithic scaffold to achieve an accurate fi Injectable cell carriers can fill the entire defect site through injection, simplifying the surgical procedure. Recently, novel injectable nanofibrous microspheres (NF-MS) were developed in our lab which will be applied to repair irregularly shaped defects caused by dental caries by promoting development of dentin-like tissue. In addition, researchers in our lab encapsulated BMP-2into PLGA (lactic-co-glycolic acid) microspheres and applied these microspheres in combination with NF-MS to evaluate whether this system could better promote odontogenic differentiation of stem cells of apical papilla (SCAP) and dentin-like tissue regeneration. Thus far, NF-MS have not been applied to dentin regeneration and the role of BMP-2in SCAP regulation is largely unknown. Effective controlled release of BMP-2is key to sustain the activity of BMP-2, prevent degradation of BMP-2and sustain the biological concentration of BMP-2in vivo. This is very important for clinical application.In the present study, firstly, the capability of odontogenic differentiation of SCAP cultured on novel injectable NF-MS was compared with traditional solid-interior microspheres (SI-MS) in vitro and in vivo. Comparisons were made by SEM, DNA content assay, RT-PCR, immunofluorescence assay, immunohistochemistry assay and calcium content assay. Secondly, the effect of BMP-2on odontogenic differentiation of SCAP and the formation of dentin-like issue in monolayer culture in vitro and3D NF-MS culture system in vitro and in vivo was evaluate by several measures, including by ALP activity assay, calcium content assay, RT-PCR and immunohistochemistry assay. Lastly, the effect of NF-MS combined with BMP-2control release on odontogenic differentiation of SCAP and the formation of dentin-like tissue was evaluated by SEM, ELISA, Von Kossa and immunohistochemistry assay. The main results and conclusions are:1.Compared to SI-MS, cells on the NF-MS displayed more rounded morphology and more extracellular matrix deposition as well as faster proliferation rate. The ALP activity of SCAP on NF-MS was higher than SI-MS after odotogenic induction for3and7days. The expression levels of Col I, BSP, OCN and DSPP genes were up-regulated on bot SI-MS and NF-MS after odotogenic induction for2and4weeks. The expression levels DSPP and BSP genes on NF-MS were significantly higher than SI-MS at weeks2and4, whereas the difference on the expression levels of Col I and OCN genes between the NF-MS and SI-MS groups was only significant at week4. There was a higher expression level of DSPP protein on the NF-MS group than that on SI-MS group at week4. No DSPP protein expression was detected on SI-MS group at week2. The samples in vivo showed that denser tissue were present in the NF-MS group than that in the SI-MS group. NF-MS were more uniformly distributed inside the new tissue and surrounded by large number of cells and collagen. Degradation of some NF-MS was observed in new tissues, whereas SI-MS clustered together and were surrounded by fewer cells and a small amount of collagen which resulted in poor tissue formation. Moreover, more calcium deposition and positive DSPP protein expression existed on the NF-MS group. In conclusion, our results demonstrated that NF-MS better promoted odontogenic differentiation and mineralization of SCAP than SI-MS both in vitro and in vivo.2.Compared to the control group, the proliferation of SCAP in monolayer culture was affected to some extent by BMP-2at100ng/ml. But the ALP activity and mineralization ability of SCAP were remarkably enhanced by BMP-2. In NF-MS3D system, ALP activity at days7and14as well as calcium content at week4were remarkably enhanced by BMP-2compared to the control group. There was a significant difference of Col I expression at week2, OCN expression at week4as well as BSP and DSPP expression at weeks2and4between the BMP-2group and the control group. The expression of DSPP protein on the NF-MS was enhanced by BMP-2at weeks2and4. The in vivo results demonstrated that larger amount of collagen and dentin-like tissue existed in the BMP-2group, as well as more mineralized nodules and positive DSPP protein expression, compared to the control group. In conclusion, the odontogenetic differentiation of SCAP was enhanced by BMP-2both in monolayer culture and3D culture.3.Although BMP-2in soluble form promoted human SCAP ondontogenic differentiation both in vitro and in vivo, its potential application in this form is limited in clinic due to its rapid diffusion away from the injection site and rapid loss of bioactivity i vivo. BMP-2was encapsulated into PLGA MS successfully using a double emulsio method to address these issues. PLGA MS released BMP-2in a multi-phasic release pattern. After an initial burst release of16%of encapsulated BMP-2at days1and2, it was subsequently released from fast pattern to slow pattern from day10to day35. From day42to day49, there was a second rapid release of22%BMP-2. After4weeks of subcutaneous implantation, no obvious mineralization was found in the BMP-2encapsulated PLGA MS group. The obvious mineralization in the BMP-2encapsulated PLGA MS group occurred at week8. PLGA MS containing BMP-2at dosage of25μg yielded areas of mineralization with embedded cells that resemble osteodentin structure, whereas small amounts of osteodentin was found in the PLGA MS containing BMP-2at dosage of5μg. Positive DSPP protein staining existed in both25μg and5μg BMP-2dosage groups. The expression levels of Col I, BSP, OCN and DSPP genes were up-regulated in both BMP-2groups. Significant statistical differences were found between BMP-2groups and the corresponding BSA groups. The calcium content quantification in vivo further confirmed the above results. There was significant difference between BMP-2MS groups and the corresponding BSA MS groups as well as between BMP-2at dosage of25μg and BMP-2at dosage of5μg groups. In conclusion, the release of BMP-2encapsulated into PLGA MS was time and dose dependent. The odotogenic differentiation of SCAP was effectively enhanced by BMP-2controlled release system. |
PDF Full Text Request