Method of preparing an artificial tooth primordium in vitro and artificialtooth primordium derived therefrom

Abstract

The present invention is directed to a method of preparing an artificial tooth primordium in vitro, comprising the steps: a) providing isolated mesenchymal dental pulp cells; and b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium; as well as to an artificial tooth primordium derived therefrom.

1 . A method of preparing an artificial tooth primordium in vitro, comprising the steps:
 a) providing isolated mesenchymal dental pulp cells; and   b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium.   
     
     
         2 . The method of  claim 1 , wherein the mesenchymal dental pulp cells comprise or consist of primary adult mesenchymal dental pulp cells. 
     
     
         3 . The method of  claim 1 , wherein the mesenchymal dental pulp cells are derived from dental pulp tissue of a tooth, preferably from pulp tissue of a third molar. 
     
     
         4 . The method of  claim 1 , wherein the mesenchymal dental pulp cells are subjected to non-adherent culture conditions at a concentration of 5×10 4  to 5×10 7  per ml. 
     
     
         5 . The method of  claim 1 , wherein the mesenchymal dental pulp cells are cultured under non-adherent conditions for at least 48 hours. 
     
     
         6 . The method of  claim 1 , wherein the mesenchymal dental pulp cells are cultured under non-adherent conditions at least until a cell aggregate is formed with an average diameter of 0.3 mm to 2 mm. 
     
     
         7 . The method of  claim 1 , wherein the mesenchymal dental pulp cells are cultured under non-adherent conditions at least until the cell aggregate formed exhibits upregulated expression of BMP4, HGF, PAX9, MSX1, Collagen type I, DSPP and/or predentin compared to isolated mesenchymal dental pulp cells immediately prior to subjecting to non-adherent culturing conditions. 
     
     
         8 . The method of  claim 1 , wherein no artificial biological or non-biological scaffold is used in the formation of the cell aggregate. 
     
     
         9 . A method of preparing an artificial tooth primordium in vitro, comprising the steps:
 a) providing isolated mesenchymal dental pulp cells; and   b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium, wherein, after culturing of the mesenchymal dental pulp cells under non-adherent conditions for at least 2 days, epithelial cells are added and co-cultured under non-adherent conditions.   
     
     
         10 . The method of  claim 9 , wherein the epithelial cells are keratinocytes, preferably keratinocytes derived from skin or gingiva. 
     
     
         11 . An artificial tooth primordium produced by a method of preparing an artificial tooth primordium in vitro, comprising the steps:
 a) providing isolated mesenchymal dental pulp cells; and   b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium.   
     
     
         12 . A transplant comprising an artificial tooth primordium produced by a method of preparing an artificial tooth primordium in vitro, comprising the steps:
 a) providing isolated mesenchymal dental pulp cells; and   b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium.   
     
     
         13 . A pharmaceutical composition comprising an artificial tooth primordium produced by a method of preparing an artificial tooth primordium in vitro, comprising the steps:
 a) providing isolated mesenchymal dental pulp cells; and   b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium and at least one pharmaceutically acceptable excipient.   
     
     
         14 . A method for screening substances in vitro, which modulate properties of tooth tissue, comprising the steps of:
 providing a sample of an artificial tooth primordium or a cell aggregate or tissue derived therefrom; wherein the artificial tooth primordium is produced by a method of preparing an artificial tooth primordium in vitro, comprising the steps:   a) providing isolated mesenchymal dental pulp cells; and   b) culturing the mesenchymal dental pulp cells under non-adherent conditions to form a cell aggregate representing an artificial tooth primordium;   dividing the respective sample into portions;   incubating at least one portion with a substance to be screened; and   comparing parameters measured for the treated portion with another portion that was not incubated with the substance to be screened.   
     
     
         15 . The method of  claim 14 , wherein the portion is subjected to a self-contained circulation system prior to incubating the portion with a substance to be screened.