Engineering oral tissues

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Disclosed are methods for regenerating dental and oral tissues from viable cells using ex vivo culture on a structural matrix. The regenerated oral tissues and tissue-matrix preparations thus provided have both clinical applications in dentistry and oral medicine and are also useful in in vitro toxicity and biocompatibility testing.

The present application claims the priority of U.S. Provisional Patent Application Serial No. 60/018,450 filed May 28, 1996, abandoned, the entire text of which is specifically incorporated by reference herein without disclaimer.

1. Field of the Invention

The present invention relates generally to the fields of dentistry and oral biology. More particularly, it concerns the generation of oral tissues from viable cells using ex vivo culture on a structural matrix. The regenerated oral tissues provided herein may be used in a variety of clinical applications and also in in vitro biocompatibility testing.

2. Description of Related Art

A major goal of dental research is the development of effective clinical approaches to promote the regeneration of oral tissues following various insults or diseases. While synthetic materials have been successfully utilized as restorative materials for dental tissues for a number of years (Craig, 1989), these materials do not replace the normal structure and function of the lost tissue and are incapable of remodeling/repairing in the face of ongoing insult or stimulation.

One such example is that of root canal therapy. Approximately 15 million patients in the U.S. require a root canal each year. Here, necrotic pulp tissue resulting from trauma or bacterial infection is removed and replaced with a non-viable synthetic material. The synthetic material is clearly unable to provide the biological functions of pulp tissue, and failure leads to tooth loss.

Engineering new tissues from cultured cells represents a new approach to treat patients suffering from the loss or malfunction of certain tissues (Langer and Vacanti, 1994). However, with the limited exception of oral mucosa, the techniques of cell and tissue culture have not been successfully applied in the engineering of oral tissues. Current cell culture techniques, such as those used in the regeneration of skin, and even oral mucosa, are not transferable to the regeneration of other oral tissues as the existing techniques produce epithelia which require an appropriate connective tissue bed in vivo for successful grafting. The art therefore lacks appropriate techniques for the production of tissues ex vivo that will repair and regenerate specific oral connective tissues in vivo.

Dental pulp is a loose connective tissue that provides dentinogenic, nutritive, sensory and defensive functions to the tooth (Chiego, 1994). Dentin is produced by specialized cells, odontoblasts, which reside in dental pulp. Tertiary dentinogenesis is often initiated following injury to or loss of dentin by the original odontoblasts, or if lost, by unidentified cells which differentiate into odontoblasts (Lesot et. al., 1993).

Dental pulp and other oral tissues may be capable of regenerating following injury, but the specific mechanisms underlying pulp regeneration and reparative dentinogenesis have not been identified. As there is little known about such processes, the ability to culture or regenerate dental pulp and other oral tissues has been severely hampered. The development of methodology by which to culture oral tissues would thus represent a significant breakthrough in this field.

The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing methods and compositions for use in culturing, engineering and reconstructing oral tissues. The invention generally concerns the ex vivo culture of viable oral tissue cells in combination with a structural matrix, or scaffold, that results in the proliferation of the cells, the production of extracellular matrix, and their organization into a new tissue structure. The regenerated oral tissue or matrix-tissue structure may then be implanted back in the body to form a new functional oral tissue. Methods of using such tissues or matrix-tissue preparations for in vivo drug delivery and for in vitro toxicity and biocompatibility testing are also provided.

In certain embodiments, the present invention provides methods for culturing oral tissue cells, which methods generally comprise growing viable oral tissue cells, or "starter cells", on a matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable oral tissue cells.

The method may comprise growing viable oral tissue cells on a three dimensional matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable oral tissue cells to form a three dimensional structure comprising viable oral tissue cells. Alternatively, the method may comprise growing viable oral tissue cells on a three dimensional matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable oral tissue cells to form a three dimensional biological structure of viable oral tissue cells.

Further, the method may comprise growing viable oral tissue cells on a three dimensional matrix or framework in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable oral tissue cells to form a three dimensional structure comprising viable oral tissue cells on or around said matrix or framework. Or the method may comprise growing viable oral tissue cells on a matrix, preferably a three dimensional matrix or scaffold, in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable cells that express at least one marker indicative of oral tissue cells.

The method may comprise growing viable oral tissue cells on a matrix, preferably a three dimensional matrix, in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable cells that express one or more markers indicative of oral tissue cells. Alternatively, the method may comprise growing viable oral tissue cells on a matrix, preferably a three dimensional matrix, in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable cells that express a plurality of markers indicative of oral tissue cells.

The invention also provides a method for culturing oral tissue cells, comprising growing viable cells obtained from an oral tissue on a three dimensional matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable oral tissue cells. The method may alternatively comprise growing viable oral tissue cells on a preferably three dimensional matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of viable oral tissue cells in functional association with said matrix or said three dimensional matrix.