Misako Nakashima

The application of bone morphogenetic proteins to dental tissue engineering.

Progress in understanding the role of bone morphogenetic proteins (BMPs) in craniofacial and tooth development, the demonstration of stem cells in dental pulp and accumulating knowledge on biomaterial scaffolds have set the stage for tissue engineering and regenerative therapy of the craniofacial complex. Furthermore, the recent approval by the US Food and Drug Administration (FDA; Rockville, MD, USA) of recombinant human BMPs for accelerating bone fusion in slow-healing fractures indicates that this protein family may prove useful in designing regenerative treatments in dental applications. In the near term, these advances are likely to be applied to endodontics and periodontal surgery; ultimately, they may facilitate approaches to regenerating whole teeth for use in tooth replacement.

Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2

Regenerative medicine is based on stem cells, signals, and scaffolds. Dental pulp tissue has the potential to regenerate dentin in response to noxious stimuli, such as caries. The progenitor/stem cells are responsible for this regeneration. Thus, stem cell therapy has considerable promise in dentin regeneration. Culture of porcine pulp cells, as a three-dimensional pellet, promoted odontoblast differentiation compared with monolayers. The expression of dentin sialophosphoprotein (Dspp) and enamelysin/matrix metalloproteinase 20 (MMP20) mRNA confirmed the differentiation of pulp cells into odontoblasts and was stimulated by the morphogenetic signal, bone morphogenetic protein 2 (BMP2). Based on the in vitro experiments, an in vivo evaluation of pulp progenitor/stem cells in the dog was performed. The autogenous transplantation of the BMP2-treated pellet culture onto the amputated pulp stimulated reparative dentin formation. In conclusion, BMP2 can direct pulp progenitor/stem cell differentiation into odontoblasts and result in dentin formation.

Side Population Cells Isolated from Porcine Dental Pulp Tissue with Self‐Renewal and Multipotency for Dentinogenesis, Chondrogenesis, Adipogenesis, and Neurogenesis

Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self‐renewal capacities and multipotency with non‐SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non‐SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self‐renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator‐activated receptor γ and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three‐dimensional pellet cultures. Autogenous transplantation of the Bmp2‐supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.

Induction of Dentin Formation on Canine Amputated Pulp by Recombinant Human Bone Morphogenetic Proteins (BMP)-2 and -4

Dental pulp cells have the potential to differentiate into odontoblasts. The molecular mechanisms underlying differentiation are not clear. Demineralized dentin matrix is osteoinductive and contains bone morphogenetic protein (BMP) activity. BMPs have been implicated in embryonic odontogenic differentiation and hence may play a role in the differentiation of adult pulp cells into odontoblasts during pulpal healing. This study examined the hypothesis that BMPs induce dentin formation on amputated canine pulp. Recombinant human BMP-2 and BMP-4 were capped with inactivated dentin matrix on amputated pulp. At two months, the amputated pulp was filled with tubular dentin in the lower part and osteodentin in the upper part. The amount of dentin formed was markedly diminished when dentin matrix alone was implanted. These findings imply that recombinant human BMP-2 and BMP-4 induce differentiation of adult pulp cells into odontoblasts. Thus, BMPs may have a role in dentistry as a bioactive pulp-capping agent to induce dentin formation.

A Novel Stem Cell Source for Vasculogenesis in Ischemia: Subfraction of Side Population Cells from Dental Pulp

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31−;CD146− SP cells, demonstrating CD34+ and vascular endothelial growth factor‐2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF‐A, G‐CSF, GM‐CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell‐based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.

Human dental pulp stem cells with highly angiogenic and neurogenic potential for possible use in pulp regeneration

Dental caries is a common public health problem, causing early loss of dental pulp and resultant tooth loss. Dental pulp has important functions to sustain teeth providing nutrient and oxygen supply, innervation, reactionary/reparative dentin formation and immune response. Regeneration of pulp is an unmet need in endodontic therapy, and angiogenesis/vasculogenesis and neurogenesis are critical for pulp regeneration. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. In this review, we introduce some stem cell subfractions, CD31(-)/CD146(-) SP cells and CD105(+) cells with high angiogenic and neurogenic potential, derived from human adult dental pulp tissue. Potential utility of these cells is addressed as a source of cells for treatment of cerebral and limb ischemia and pulp inflammation complete with angiogenesis and vasculogenesis.

Regeneration of dental pulp after pulpotomy by transplantation of CD31-/CD146- side population cells from a canine tooth

AIM To achieve complete regeneration of dental pulp in vivo by stem/progenitor cells obtained from a fraction of side population (SP) cells from canine pulp. MATERIALS & METHODS A subfraction of SP cells, CD31(-)/CD146(-) SP cells, were isolated by flow cytometry from canine dental pulp. The efficiency of this subfraction of SP cells was evaluated in an experimental model of pulp injury in the dog. RESULTS The fractionated SP cells formed extensive networks of tube-like structures in vitro. Transplantation of the SP cells into an in vivo model of amputated pulp resulted in complete regeneration of pulp tissue with capillaries and neuronal cells within 14 days. Gene-expression studies demonstrated the expression of pro-angiogenic factors, implying trophic action on endothelial cells. CONCLUSIONS This investigation demonstrates the potential utility of fractionated SP cells as a source of cells for total pulp regeneration complete with angiogenesis and vasculogenesis.

Induction of Reparative Dentin Formation by Ultrasound-Mediated Gene Delivery of Growth/Differentiation Factor 11

Bone morphogenetic proteins (BMPs) are morphogens implicated in embryonic and regenerative odontogenic differentiation. Gene therapy has the potential to induce reparative dentin formation for potential pulp capping. We have optimized the gene transfer of Growth/differentiation factor 11 (Gdf11)/Bmp11 plasmid DNA into dental pulp stem cells by sonoporation in vivo. Dental pulp tissue treated with plasmid pEGFP or CMV-LacZ in 5-10% Optison (Molecular Biosystems Inc., San Diego, CA) and stimulated by ultrasound (1 MHz, 0.5 W/cm(2), 30 sec) showed significant efficiency of gene transfer and high level of protein production selectively in the local region, within 500 microm of the amputated site of the pulp tissue. The Gdf11 cDNA plasmid transferred into dental pulp tissue by sonoporation in vitro, induced the expression of dentin sialoprotein (Dsp), a differentiation marker for odontoblasts. The transfection of Gdf11 by sonoporation stimulated a large amount of reparative dentin formation on the amputated dental pulp in canine teeth in vivo. These results suggest the possible use of BMPs using ultrasound-mediated gene therapy for endodontic dental treatment.

Regeneration of dental pulp following pulpectomy by fractionated stem/progenitor cells from bone marrow and adipose tissue

Pulp stem/progenitor cells can induce complete pulp regeneration. However, due to the limited availability of pulp tissue with age, there is a need to examine other sources for fractions of side population (SP) cells. In the present investigation bone marrow and adipose tissues of the same individual were evaluated as alternate sources. Pulp CD31(-) SP cells have higher migration activity and higher expression of angiogenic/neurotrophic factors than bone marrow and adipose CD31(-) SP cells. Adipose tissue CD31(-) SP cell transplantation yielded the same amount of regenerated tissue as pulp derived cells. However, bone marrow CD31(-) SP cell transplantation yielded significantly less regenerated tissue in pulpectomized root canals in dogs. The rate of matrix formation was much higher in adipose CD31(-) SP cell transplantation compared to pulp CD31(-) SP cell transplantation on day 28. Microarray analysis demonstrated similar qualitative and quantitative patterns of mRNA expression characteristic of pulp in the regenerated tissues from all three cell sources. Expression of many angiogenic/neurotrophic factors in the transplanted cells demonstrated trophic effects. Our results demonstrate that bone marrow and adipose CD31(-) SP cells might be suitable alternative cell sources for pulp regeneration.

A Novel Combinatorial Therapy With Pulp Stem Cells and Granulocyte Colony-Stimulating Factor for Total Pulp Regeneration

Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin‐pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical‐grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte‐colony stimulating factor (G‐CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G‐CSF yielded a significantly larger amount of regenerated dentin‐pulp complex compared with transplantation of G‐CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G‐CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G‐CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G‐CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.