RICERCA ITALIANA     

ISOLATION, CHARACTERIZATION AND DIFFERENTIATION OF STEM CELLS FROM HUMAN DENTAL PULP.

Seconda Università degli Studi di Napoli

Abstract

The ability to regenerate tissues in the human body is due to a pool of non differentiated cells which are capable to substitute damaged cells.
We know that these cells are known as stem cells, which are present both in embyonic and adult tissues and are able to differentiate into various cytotypes.
The aim of our proposed research is to isolate, characterize, differentiate and reconstruct a 3D tissue from human dental pulp stem cells from permanent(DPSCs)and deciduous (SHED) teeth.
We know that at present only one research group, which is in the USA (Dr. Gronthos, NIH), is studying these cells with significant results.
Our main goal, taking into consideration international literature as well as our preliminary results (submitted for publication) will be:
- to obtain a sufficient, selected number of DPSCs belonging to human dental pulp of deciduous and permanent teeth and use these cells for culture and differentation;
- to characterize these cells by means of monoclonal antibodies and FACscan; in addition we will try to obtain a purified lineage using a cloning method;
- to make a stem cell bank of DPSCs when the number will be sufficient to store them in liquid nitrogen;
- to obtain an in vitro osteogenic "coversion" into bone cells tissue, by means of the use of growth factors and a polymer (polylactic-glycolic), placed in a roller apparatus (at slow speed) in order to achieve a 3D structure;
- to obtain an >>>

Principal Investigator

Fernando GOMBOS Seconda Università degli Studi di NAPOLI

Research Objectives

The specific aim of the proposed research, taking into consideration both the international literature on this topic and our preliminar results (submitted for publication), includes the following:

a) isolate and characterize cultured stem cells from pulpar tissue belonging to both deciduous and permanent teeth and observe eventual differencies; then observe their capability to differentiate into bone tissue using a polymer and a roller apparatus at a slow speed. This will be done in order to obtain a suitable bone tissue for future reimplantation;
b) utilization of stem cells for reconstructing periodontal elements based on their potential ability to differentiate into mesenchimal cells;
c) realization of a bank of SHEDs as a reservoir of stem cells available for therapeutic use.

Aim a) The purpose will be done, after isolation and cloning of human stem cells belonging to human dental pulp, by characterization of those elements using specific monoclonal antibodies in a FACscan. Then, after a subsequent further proliferation, cells will be cultured with a polymer in a roller apparatus, in order to obtain a 3D of the tissue. In particular, the latter will be particularly suitable for clinical use.

Aim b) Stem cells isolated from the pulp of SHEDs utilized for the reconstruction of periodontal structures

The loss of dental anchorage involves alterations affecting all the periodontal components >>>

First Results

At the end of the first step of the proposed research, we predict:
-to standardize the technique of dental pulp withdrawal;
-to standardize the separation and culture methods to obtain an efficient cellular spreading and long term culture;
-to obtain an adeguate number of proliferant stem cells by clonal selection;
-to ascertain the positivity to stem cell markers and obtain purified cells using the FACsorter;
-to evaluate the differencies between DPSCs and SHED in order to make a stem cell bank;
-to evalute the production of specific fibroblast protein (like type I and type III collagen) and protein characteristic of the cells of the periodontal ligamen present in vivo (osteopontin and osteocalcin);
-to evaluate the great mitogenic effects of the treatment with growth factors and hormones of isolated cells;
-to induce to formation of cementum and the insertion of Sharpey fibers
-to improve the efficiency of the implantation of a dental element in the bone cavityAt the end of the second phase of the proposed research:
-in order to realize a stem cell bank from human pulp, we will predict to have a sufficient number of cells.
Therefore, a sufficient number of positive stem cells to the adopted markers we will obtain and we will store them in N2 liquid.
A quantitative other than qualitative method will allow us to understand the ability of those elements to convert in different cytotypes. >>>

Timescale

24 months

National and international background

Stem cells can be obtained from embryos as well as from adult organisms. It has long been known the totipotent stem cells can be isolated from the inner cell mass of a blastocyst. Such embryonic stem (ES) cells display an almost unlimited replicative potential and the capacity of giving rise to virtually all cell lineages [Potten & Loeffer, 1990]. ES cells could therefore be a source of different types of mesenchimal cells, however their use is limited by ethical considerations. In addition they can be used only for heterologous transplants.
Segregation of embryonic cells to specific cell layers during embryogenesis has long been considered as a final, irreversible event. It was thought that all cells of a given layer, including mature cells, their progenitors and the stem cells were, with few exceptions as in the neural crests, to maintain in the adult life the lineage specificity acquired in the early phases of embryonic development. Furthermore it has long been thought that cell substitution and tissue regeneration in continuously renewing tissues in the adult was only due to the limited precursor cell complement of that tissue, which were considered to be unipotent, id est unable to give rise to cells of a different lineage.
However recent evidence has modified these views and allowed us to determine that adult tissues accomodate stem cell populations possessing a differentiation potential not limited to the tissue they belong to [Bianco & Cossu, 1999 >>>