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Dentin Regeneration by Dental Pulp Stem Cell Therapy with Recombinant Human Bone Morphogenetic Protein 2

¹ Department of Clinical Oral Molecular Biology, Division of Oral Rehabilitation,
² Department of Orthodontics, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan; and
³ Department of First Anatomy, National Defense Medical College, Tokorozawa 359-8513, Japan;

View larger version (79K): [in a new window]   Figure 1. The three-dimensional pellet culture for porcine primary pulp cells compared with the monolayer culture. (A) Morphological changes in the pellet during culture on day 7, day 14, and day 21 (H–E stain). Note the decrease of cell number and increased matrix on day 21. (B) The changes in cell numbers during pellet cultures of porcine pulp cells compared with monolayer culture in 35-mm dish. Each point is expressed as the mean ± SD of 6 determinations. Note the lack of proliferation of cells in pellet cultures. (C) The changes in alkaline phosphatase activity. Each point is expressed as the mean ± SD of 6 determinations. (D) Real-time RT-PCR analysis of 1(I)collagen, Dmp1, Dspp, enamelysin/MMP20, and Phex expression in pellet cultures compared with monolayer cultures. The experiment was repeated 3 times, and 1 representative experiment is presented. The relative percentage of expression was shown in relation to the highest value as 100% after normalization against ß-actin. Solid lines represent pellet cultures and dashed lines monolayer cultures.

View larger version (60K): [in a new window]   Figure 2. The effect of rhBMP2 treatment in the pellet culture. (A) The changes in cell numbers in the pellet culture with and without rhBMP2 (100 ng/mL). (B) Dose-dependent changes in alkaline phosphatase activity supplemented with rhBMP2 (0 to 200 ng/mL). Each point is expressed as the mean ± SD of 5 determinations. rhBMP2 treatment at the final concentration of 25 ng to 200 ng/mL significantly (p < 0.001) increased alkaline phosphatase activity compared with controls. (C) Morphological changes of the rhBMP2 (100 ng/mL)-treated pellet compared with the non-treated pellet on day 21 (H-E stain). (D) Picrosirius red stain showing collagenous matrix formation of rhBMP2-supplemented pellet on day 21. (E) The histomorphometric evaluation of surface area of collagen fibers stained by Picrosirius red. Digital pictures of the stained sections were converted into gray scale in Adobe Photoshop ver. 5.01. From 9 to 12 non-overlapped square areas of 100 µm2 from sections of the pellets of each group were analyzed. The pixels of the bright areas of collagen fibers were counted, and the surface areas were calculated. The values are expressed as average ± standard deviations. Statistical analysis was performed by the non-paired t test. Note significant increase by rhBMP2 treatment. (F) Real-time RT-PCR analysis of 1(I)collagen, Dmp1, Dspp, enamelysin/MMP20, Phex, Osterix, Cbfa1, and Cbfa3 expression in the pellet culture with rhBMP2 (100 ng) compared with cultures without rhBMP2. The experiment was repeated 4 times, and 1 representative experiment is presented. Solid lines represent pellet cultures and dashed lines monolayer cultures. (G) Alizarin Red staining showing mineralization of rhBMP2-supplemented pellet on day 35.

View larger version (98K): [in a new window]   Figure 3. The autogenous transplantation of the pellet culture on the canine amputated pulp. (A) Canine pulp cells transduced with adenovirus lacZ before the pellet culture began and stained by ß-galactosidase on day 14. (B) The implanted pellet on the amputated pulp showing lacZ transgene expression on day 28. The amputated site (arrow). (C) The amputated pulp without transplantation of the pulp cell pellet. Note no osteodentin matrix formation after 4 wks. (D) The in vivo transplantation of the pellet without rhBMP2. (E) The transplantation with rhBMP2. Note the formation of the thicker osteodentin matrix (OD) beneath the amputated site (arrows) in response to cell therapy with rhBMP2 compared with cultures without rhBMP2. (F–H) Higher magnification of osteodentin. Fewer cells and more homogenous matrix surrounding cells in rhBMP2-supplemented implantation (G) compared with that without rhBMP2 (F). Note the dentinal tubes (arrows) in the matrix (H).