WO2005051436A2 - Method for tooth organ reconstruction - Google Patents

Abstract

A method of cultivation of two kind of mammalian cells types for the aim of their tooth organ or tooth germ reconstruction by extraction of a diseased tooth or tissues (cells) of the diseased or normal tooth, isolation of adult multipotent stem cells, epithelial rests of Malassez in particular, from periodontal ligament and undifferentiated pulpocytes from the pulp with following reconstruction of a tooth organ in vitro.

Description

METHOD FOR TOOTH ORGAN RECONSTRUCTION

HELD OF INVENTION

The present invention is related to the field of medicine, biotechnology, and tissue engineering. More particularly, it concerns the dentistry, oral surgery, and regeneration of tooth germ, or tooth organ from adult tooth stem cells using ex vivo co-culture on a structural matrix or without it. The approach described here also could be applied to medicine in general for the regeneration of organs. The tooth organs provided herein may be used in a variety of clinical applications.

BACKGROUND OF THE INVENTION

Dental caries is an enormous, still globally increasing problem of mankind likewise the diseases of the periodontal ligament. Dental care necessitates a considerable part of the scarce resources aimed for the health care in general to be directed to dentistry. The final form of caries demands whole tooth to be extracted with a subsequent insertion of a nonliving artificial tooth implant. Although such implants are useful, they can create a variety of complications. Long-advanced dental diseases are claimed to be connected to other diseases, as well.

A number of animals can regenerate new teeth for three or even more times during their life cycle. For example, some fishes and reptiles can renew teeth constantly during all lifetime. It is common consent that humans can get teeth only twice. However, it has been reported in the literature that some old people could get a third generation of teeth, although there are no evidences in medical literature that humans have a tooth germs for such teeth regeneration. Sometimes a similar tooth regeneration happens in old dogs, too. Therefore, there exists a rare, accidental, but a totally unpredictable mechanism to generate teeth of adult mammals.

Engineering new tooth organ from cultured cells represents a new era to treat patients suffering from any kind of loss of teeth. At present stage of the science, no marked progress has been achieved to develop methods for tooth regeneration. There are little ideas expressed in the literature for even the possibility of induction of regeneration of human or mammalian teeth (Thesleff, I. et al, Bio-implant interface. Improving biomaterials and tissue reactions. Ed. by Jan Eirik Ellingsen, S. Petter Lyngstadaas, CRC Press LLC (2003). It was stated that dental pulp stem cells could be used for tooth tissues regeneration (Bianco, P. et al, Nature, vol. 414, pp. 118-121 (2001); Shi, S. et al., US patent WO 0207679A2 (2002)) and myofibroblastoid pericytes are the stem cells (Murray, P.E. et al., Stem Cells and Dev., vol. 13, pp. 255-262. (2004). It has been described in the literature a tissue engineering method for dental pulp tissue replacement utilizing cultured cells seeded upon synthetic extracellular matrices as described by Bohl, K.S. et al, J. Biomat. Sci. Polymer Edn., vol. 9, pp. 749-764 (1998). It has also been shown that such tissues could be mineralised in vitro (Bouvier, M. et al, Archs Oral Biol., vol. 35, pp. 301-309 (1990)). The latter study contemplated that dental pulp-like tissues could be engineered in vitro, and this may provide the first step to engineering a complete tooth. However, an obvious hindrance for the dental regeneration is that enamel cannot be regenerated. It is, in fact, the only material in mammals that cannot be regenerated because of the lack of proper tissue in adults. Enamel cannot be strictly called a tissue since it lacks alive cells. Thus, the main problem in tooth regeneration is to know which adult stem cell line in mammals is able to differentiate into the enamel-producing tissue. Some researchers claims general ideas that bone marrow and palate could be a source of adult stem cells without specifying the necessity of both epithelial and mesenchymal cell lines to mimic the normal development of the tooth organ (Chai, Y et al., Microsc. Res. and Tech., vol. 60, pp. 469-479 2003).

Another attempt for tooth organ reconstruction from embryonic porcine tooth cells was made by Stashenko P. et al., US patent 2002119180 (2002) and Young, C.S. et al., J Dent.Res., vol. 81, pp. 695-700 (2002), but those result have been already demonstrated by several independent researchers in 60' s. The novelty of the described method was only a use of the biodegradable scaffold.

The present invention is based on a surprising discovery that epithelial rests of Malassez are adult stem cells and could be used for tooth organ reconstruction and enamel regeneration once they could be combined with dental pulp stem cells, or get in contact with the surface of the dentin, containing alive odontoblasts. Contact of epithelial rests of Malassez with mesenchymal cells is essential for that process.

The known techniques in tissue engineering describes the regeneration of certain tissues of the tooth, e.g. periodontal ligament with guided periodontal tissue regeneration, pulp regeneration, and dentin regeneration as illustrated by Bianco, P. et al, Nature, vol. 414, pp. 118-121 (2001) and by Mooney, D.J. et al, US patent 5,885,829 (1999). Publication WO 0207679A2 describes only a cultivation of adult dental pulp stem cells in vitro and in vivo. It however, represent a totally different approach than in the present invention, because the referred publications describe only a regeneration of a single tissue. The regeneration of a single tissue should be presently considered as a trivial technique. Instead, the regeneration of the whole tooth organ including all tissues by the present invention has been thus-far completely unsolved problem. As indicated by numerous investigations on many organs, productive interaction of two different tissues, i.e. epithelial and mesenchymal tissues, cannot be achieved by the techniques in the prior art.

Evidently, the technological significance of the present invention describing tooth reconstruction is of an extreme importance. Such a technology can be applied not only to tooth organs but also for reconstruction of other organs, exemplified by eye, lung, and kidney. The art of the present invention therefore represents a novel level of biotechnology applied for medicine with a high commercial significance.

SUMMARY

A method of cultivation of two kind of mammalian cells types for the aim of their tooth organ or tooth germ reconstruction by extraction of a diseased tooth or tissues (cells) of the diseased or normal tooth, isolation of adult multipotent stem cells, epithelial rests of Malassez in particular, from periodontal ligament and undifferentiated pulpocytes from the pulp with following reconstruction of a tooth organ in vitro. The methods can be also applied for reconstruction of other organs like eye, kidney and lung. BRIEF DESCRIPTION OF THE DRAWINGS

With reference to the attached drawings that illustrate in schematic form an example of how the invention can be realized.

FIG. 1 is a cross section of a human tooth, showing its all major components.

FIG. 2 is a view of a human tooth with caries decay or gum disease.

FIG. 3 shows an extracted tooth with cleaned areas of decay.

FIG. 4 represents an isolation of epithelial rests of Malassez from the periodontal ligament and obtaining of primary culture of those cells.

FIG. 5 represents an isolation of pulpocytes from the pulp and obtaining of primary culture of those cells.

FIG. 6 shows a process of tooth organ reconstruction in vitro.

DETAILED DESCRIPTION OF THE INVENTION

A major goal of dental research is the development of effective clinical approaches to promote the teeth restoration following various diseases. While synthetic materials have been successfully utilised as restorative materials for teeth for a number of years, they do not replace the normal tooth structure and function of the lost tissues. Tooth is a result of evolution for millions of years and for the present moment there is no better substitute for that unique organ than a natural tooth. Many people suffer from the adontia due to the caries, periodontal diseases or accidental injury. Modern dentistry tries to help them by using metal implants, but they cannot functionally substitute the natural teeth. The described here approach will help to obtain new teeth in patients who need them. Engineering of the dental tissues and the whole tooth organ using cultured cells will provide an alternative to the traditional therapy. The present invention describes a fundamental approach for tooth organ reconstruction in mammals. The invention generally concerns the ex vivo culture of viable epithelial and mesenchymal cells in co-culture with combination of structural matrix or without it, that results in organization of a new tooth organ.

There has been only one serious attempt of successful bioengineering of complex tooth crowns closely resembling those of naturally developing from embryonic tooth cells (Young, C.S. et al., J Dent.Res., vol. 81, pp. 695-700 (2002)). In the present invention, we describe usage of the adult stem cells and surprisingly found that a combination of two or more of different adult stem cell lines is required. It is an object of the present invention to provide novel means for human tooth reconstruction with methods of tissue engineering and oral surgery. For these and other ethical reasons, the experiments were not yet performed with human patients but allowed experimental animals closest to humans. It is considered that the results of the present invention are therefore applicable to humans.

The present invention refers to an approach how to obtain in vitro reconstruction of the tooth, which is a complex craniofacial structure and includes enamel, dentin, cement, pulp (odontoblasts and undifferentiated pulp cells), nerves, blood vessels (perivascular cells, endothelial cells), oral epithelium (oral keratinocytes) and gingival tissues (gingival fibroblasts), periodontal ligament (epithelial rests of Malassez, periodontal ligament fibroblasts, myofibroblasts, cementoblasts, cementocytes, cementoclasts, defence cells), and alveolar bone (osteoblasts, osteocytes and osteoclasts) attached to the tooth root by the means of methods of tissue engineering.

The term "biodegradable" means that the polymer and/or polymer matrix of the reconstructed tooth organ will degrade over time by the action of enzymes, by hydrolytic action and/or by other similar mechanisms in the human body or in vitro. "Bioerodible" means that the implant matrix will erode or degrade over time due, at least in part, to contact with substances found in the surrounding tissue fluids, cellular action, and the like. "Bioabsorbable" means that the polymer matri will be broken down and absorbed within the human body, for example, by a cell, a tissue, and the like. In this context, the term "tooth stem cells" means that the cells are capable of proliferating and have a potential to form a developing tooth organ. Examples for such cells are epithelial rests of Malassez and undifferentiated pulpocytes.

The term "tooth germ" means an aggregate of tooth stem cells on the structural matrix or without it, which is able to grow into a functional tooth over a certain period of time. Tooth organ reconstruction is defined here as a process of formation of non-mineralized tooth germ or a completely mineralized tooth developed in vitro. Transdifferentiation is defined here as a change of a cell from one differentiated state to another.

The present invention form a principally new approach to the problem of tooth regeneration. The basic embodiment of the present invention is to use at least two biological living cell components for tooth organ reconstruction. In particular, epithelial and mesenchymal cell lines, epithelial rests of Malassez and undifferentiated pulp cells, respectively are used in such a combination. Although these cells are preferably used, the present invention is not limited to use only these cells grown together in a combination. The invention does not also limit into any special relative amounts of the cells grown in vitro. The undifferentiated pulpocytes and epithelial rests of Malassez are multipotent adult stem cells and are able to form a new tooth organ in adults by the methods of tissue engineering as described in the present invention.

Tooth development, like development of other epidermal organs (lung, kidney, eye lens, mammary gland, hair, skin, feathers, etc.), is mediated by reciprocal interdependent epithelial-mesenchymal interactions resulting in the differentiation of mesenchymal cells into odontoblast cells and epithelial cells into ameloblast cells (Thesleff, I. et al, Differentiation, vol. 18, pp. 75-88 (1981); Ruch, J.V. Cell Biol. Rev., vol. .14, pp. 1-112 (1988)).

The first embodiment of the present invention is that two major components are involved in tooth regeneration process in adults, an epithelial rests of Malassez representing an epithelial part and undifferentiated pulpocytes representing a mesenchymal part for organ development. These rests of Malassez first appears as network of epithelial cords derived from Hertwig's epithelial root sheath. Such reticular arrays usually further disintegrate into isolated cell clusters located in the periodontal ligament surrounding the dental root. Epithelial rests of Malassez can persist life-long although their number may decrease with age. The unusual location and longevity of these epithelial cells are also of importance for the present invention. Another of our discovery was that epithelial rests of Malassez are multipotent adult stem cells and could be used for regeneration of numerous organs which needs an epithelial part.

It is suggested that the periodontal ligament is a fetal connective tissue (see Moxham, B.J. et al, Tooth morphogenesis and differentiation, L SERM, vol. 125, pp. 557-564 (1984)). It is known in the prior art that the human pulp cells obtained from adult teeth are able to grow and to form a mineralized extracellular matrix in vitro and in vivo (see Tsukamoto, Y. et al, Archs Oral Biol., vol. 37, pp. 1045-1055 (1992); Gronthos, S. et al, Proc. Natl. Acad. Sci., vol. 97, pp. 13625-13630, (2000)). Pulp cells are also able to show an odontoblasts-like cytodifferentiation in vitro in the presence of calcium hydroxide- containing cement (see Seux, D. et al, Archs Oral Biol., vol. 36, pp. 117-128 (1991)). It was shown that epithelial rests of Malassez are able to secrete a bone resorbing factor what could be the first step in root resorbtion of the old tooth, their contact to undifferentiated pulp cells and a new tooth organ formation (see Birek, C. et al, J. Period. Res., vol. 18, pp. 75-81 (1983). Hertwig's root sheath which results in epithelial rests of Malassez during the development is known to express amelogenin (Hamamoto, Y. et al, Oral Surg., Oral Path., Oral Rad., & Endod., vol. 81, pp. 703-709 (1996)). Those cells could be precursors for ameloblasts. Fibroblasts from the periodontal ligament also have an ability to produce a mineralized nodules induced by bioactive glass and vitamin D3 (see Kubo, K. et al, J. Biomed. Mat. Res., vol. 29, pp. 503-509 (1995)). These cited references describe technical level of certain individual steps of the present invention and the cell cultivation and other methods described in them can exploited in the accomplishing the present invention.

A diseased or normal (a third molar or supernumeral) human tooth from the certain patient could be a source of a cell lines such as pulpocytes, odontoblasts, epithelial rests of Malassez, cementocytes, periodontal ligament fibroblasts, oral keratinocytes, gingival fibroblasts which could be used to create a tooth organ in vitro which after that could be transplanted back to the patient. The above-mentioned tissues could be obtained also by local oral surgery starting with an incision through the mucosa and periosteum of the jaw, raising a mucoperiosteal flap, taking a biopsy of periodontal ligament, reaching the pulp chamber with taking the sample of the pulp tissue from the apical part of the pulp chamber.

Tooth stem cells may be also obtained at the earlier period of life time in advance and preserved in cryostorage to the moment when they will be needed for tooth organ reconstruction. Tooth organs also may be reconstructed and preserved in cryostorage to the moment when it would be needed for the patient.

The adult dental stem cells could be isolated with cell sorter for specific stem cell markers according to the methods known in the prior art. For epithelial cells of Malassez an epidermal growth factor receptor could be such marker or cell-specific (see Gottlier, A.B. et al, J. Invest. Derm., vol. 85, pp. 299-303 (1985)) or ordinary monoclonal antibodies could be used (see Thesleff, I., J Period. Res., vol. 22, pp. 419-421 (1987); Nordlund, L., J Period. Res., vol. 26, pp. 333-338 (1991)).

If preferable, while manipulating the cell lines in vitro, they could be genetically modified by the methods of gene therapy (for example, by recombinant vectors carrying the transcription factors) to knock-out the mutant genes and to substitute them with normal genes. Also other necessary manipulations may be needed, for example, if a patient carries a genetic disease affecting the tooth hard tissue structure and mineralization.

The obtained cell lines also could be treated with hormones, peptides, vitamins (especially with vitamin D and/or its derivatives, retinoic acid and its synthetic analogue and derivatives) and/or with growth factors (for example, bone morphogenetic proteins) or other biologically active agents including dioxin and its derivatives or biological analogues or the cell lines could be genetically modified to get in those cells an expression of certain genes to promote the proliferation, differentiation of the cells followed by a tooth regeneration. Basically methods for their application are described in the following publications: Nakashima, M., Archs Oral Biol., vol. 35, pp. 493-497 (1990); Nakashima, M. Archs Oral Biol., vol. 37, pp. 231-236 (1992); Nakashima, M. Archs Oral Biol., vol. 39, pp. 1085-1089 (1994); Onishi, T. et al, Archs Oral Biol., vol. 44, pp. 361-371 (1999). Vitamin D and its derivatives are involved in regulation of amelogenin gene expression and are important in treating the epithelial rests of Malassez as shown by Papagerakis, P. et al, J. Cell. Biochem., vol. 76, pp. 194-205 (1999).

Co-culture of epithelial rests of Malassez and undifferentiated pulp cells are essential for tooth organ reconstruction. Co-culture of human fetal lens epithelial cells and fibroblasts resulted in lens fiber cell differentiation and crystallins expression as shown by Nagineni, C.N. et al, Exp. Eye Res, vol. 54, pp. 193-200 (1992). Recombination of Hertwig's epithelial root sheath (HERS) cells with dental papilla resulted in calcification of HERS cells (Arzate, H, et al, Archs Med. Res., vol 27, pp. 573-577 (1996)). Co-culture of mesenchymal and epithelial cells from human dental root apical explants have shown a mineralization of the epithelial cells. Mesenchymal cells synthesized an abundant collagenous matrix. Epithelial cells deposited a structured basement membrane when they were in contact with mesenchymal cells as shown by Farges, J-C. et al, Con. Tiss. Res., vol. 33, pp. 37-46 (1995). In particular, the role of basement membrane in odontoblast terminal differentiation is critical, as well as that of predentine in ameloblast differentiation as shown by Ruch, J.V. Cell Biol. Rev., vol. 14, pp. 1-112 (1988). According to the present invention it is very important indication that such a morphogenetic process (basement membrane formation in epithelial - mesenchymal co- culture) occurs in vitro conditions.

For tooth organ reconstruction, cell lines such as oral keratinocytes and gingival or periodontal fibroblasts can be also used, which could be transdifferentiated in vitro to get a potential for tooth development. Using a gingival tissue sample means that a tooth does not need to be extracted in order to obtain a sample of pulp and periodontal ligament as a source of epithelial rests of Malassez. Gingival biopsies are obtainable by routine dental procedure. Oral keratinocytes can represent an epithelial component (instead of epithelial rests of Malassez) and gingival fibroblasts, a mesenchymal part (instead of undifferentiated pulpocytes) for tooth reconstruction as described herein. Transdifferentiation of those cell lines could be achieved by cultivation in the presence of odontogenic factors, or expression of various genes with odontogenic potential in those cells. This approach is especially fruitful for those patients completely missing teeth due to genetic diseases. It would be possible to repair genetic defects in their own cells in vitro by the methods of genetic engineering to form a tooth organ and to transplant it back to the patient.

Reconstruction of the tooth germ or tooth organ could be achieved by the incorporation of the isolate from the donor tooth cell lines into biodegradable, bioerodible or bioabsorbable polymer with following cultivation in vitro in organotypic culture. It could be also achieved by simple mixing of the cells into pellet with following cultivation in vitro of the resulted tooth germ. Or, the mixture of the undifferentiated pulp cells and epithelial rests of Malassez can be aggregated in vivo and in vitro in appropriate conditions or be injected into the socket of the extracted tooth of the patient. The tooth organ reconstruction also could be achieved by ink-jet organ printing. The reconstructed tooth organ can be grown in vitro to obtain hard tissues mineralization.

The methods of present invention can be also illustrated as a combination of methods for generating tooth germ or tooth organ with calcified tissues which generally comprise isolating a tooth and oral tissue compositions that involve two major components (epithelial and mesenchymal), obtaining and culturing the viable adult multipotent stem cells, preferably in the presence of antimicrobal compounds or antibiotics inhibiting or eliminating the growth of microbes for a period of time sufficient to allow reconstruction of the tooth germ or completely mineralized tooth organ.

The present invention illustrates methods for generating tooth germ or tooth organ with calcified tissues which generally comprise isolating a tooth and oral tissues composition that comprises three or even more of major components, obtaining and culturing viable adult multipotent stem cells, which could be not only epithelial rest of Malassez and undifferentiated pulpocytes but also mesenchymal adult stem cells from the periodontal ligament which were found in paravascular sites and have potential for the formation of the cement and for a period of time sufficient to allow reconstruction of the tooth germ or formation of completely calcified tooth organ, containing enamel (deposited by epithelial rests of Malassez), dentin (deposited by undifferentiated pulpocytes) and cementum (deposited by stem cell from the periodontal ligament).

After obtaining the cell cultures, tooth reconstruction may involve the use of a structural matrix. The matrix can act as a scaffold for the cells to guide the process of tooth germ development and tissues formation. The materials utilized to fabricate a matrix for use in the present invention can generally be categorized into three types: naturally derived materials, including extracellular matrix (ECM) molecules, such as collagens and hyaluronic acid, and polysaccharides, such as alginate; synthetic materials, including any one of a variety of polymers; and relatively newly developed known materials that incorporate specific cell recognition signals found in ECM molecules.

Any one of a variety of naturally-derived matrix-like materials may be used to provide a framework for cell growth and development in accordance with the present invention, including those matrices fabricated from animal or plant tissues (e.g. calcified tissues of corals, mollusks, teeth or bones). By using a biocompatible matrix significant immune responses and inflammatory reactions will be avoided. Potential advantages of these types of materials are their biocompatibility and their biological activity.

Collagen of different types may be used as the scaffold. It is the most prevalent ECM molecule in the body and in tooth tissues, is readily isolated from animal tissues and has been extensively utilized to fabricate cell delivery devices as described by Bohl, K.S. et al, J. Biomat. Sci. Polymer Edn., vol. 9, pp. 749-764 (1998) and Bellows, CG. et al, J. Cell Sci., vol. 50, pp. 299-314 (1981). Other ECM molecules such as integrin, fibronectin and laminin are essential in processes of the differentiation of pulp cells into preodontoblasts and adhesion (Zhu, Q. et al, Oral Surg., Oral Med., Oral Path., vol. 85, pp. 314-318 (1998)). A presence of chondroitin sulphate stimulates in vitro mineralization of a three-dimensional matrix by human dental pulp cells (Bouvier, M. et al, Archs Oral Biol., vol. 35, pp. 301-309 (1990)). The synthetic support polymers, or scaffolds for teeth tissue cells, may be used in the tissue regeneration. In certain aspects, synthetic polymers are attractive scaffold materials as they can be readily manufactured with a wide range of reproducible properties and structures. Polymer matrices also provide mechanical support against compressive and tensile forces, thus maintaining the shape and integrity of the scaffold in the aggressive enviroment of the body. They can be either biodegradable, bioabsorbable or bioerodible and selected from the group consisting of polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, polymalic acid, polyamino acids, polymethyl vinyl ether, chitin, chitosan, collagen of all types, proteoglycans, chondroitin sulphate, keratan sulphate, dermatan sulphate, glycosaminoglycans and copolymers, polypropylene glycol alginate, polyglycolic and polylactic acids, terpolymers and any combination thereof.

Within the preparation of the implant precursor, the solvent can be selected from the group consisting of N-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, propylene glycol, propylene carbonate, acetone, acetic acid, ethyl acetate, ethyl lactate, methyl acetate, methyl ethyl ketone, dimethylformamide, dimethyl sulfoxide, dimethyl sulfone, tetrahydrofuran, caprolactam, decylmethylsulfoxide, oleic acid, N,N-diethyl-m-toluamide, and l-dodecylazacycloheptan-2-one, and any combination thereof.

Another group of scaffold is a synthetic matrix that mimics natural materials. The advantage of synthetic polymers can be combined with specific biological activity of ECM molecules.

The reconstructed tooth organ is further comprising a biologically-active agent selected from the group consisting of an antibacterial agent, antifungal, and an antiviral agent. The reconstructed tooth organ is further comprising tricalcium phosphate, calcium sulfate, or hydroxyapatite and any combination thereof. Appropriate synthetic calcium phosphate biomaterials, hydrohyapatite and tricalcium phosphate ceramics, calcium hydroxide and calcium hydroxide-containing cement have been developed as pulp-capping agents as described by Seux, D., et al, Archs Oral Biol., vol. 36, pp. 117-128 (1991) and can be also therefore preferably used in the present invention. These materials are extensively employed in bone repair of their biocompatibility and their ability to promote new bone formation (see for example Alliot-Licht, B., et al, Archs Oral Biol., vol. 39, pp. 481-489 (1994)).

In addition, a number of natural or recombinant growth factors can be used for accelerating the regeneration of teeth can be exploited either in cell culture conditions. Such growth promoting factors may be chosen among the group of extracellular matrix (ECM) molecules, bone morphogenetic proteins (BMPs), neurotransmitters, inflammatory mediators, hormones, cytokines, inorganic minerals alone or in mixture of minerals, plant extracts, deviates of dioxin, and any compound which mimics the biological effect of dioxin.

The epithelial rests of Malassez could be used for enamel regeneration on teeth, which needs another procedure instead of the ordinary cavity filling. This can be achieved by placing the epithelial rests of Malassez into the cavity in the structural matrix, or without it, and covering of the operative area with special bioreactor. It can be designed in such a way that it can take the advantage of the continuous flow method for feeding the epithelial rests of Malassez in the patients' mouth.

Presently, genetic engineering of mammalian cells can be exploited in tissue regeneration in a variety of ways. Expression vectors for such cells include an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcription termination sequences. Once the desired vector construct is obtained, it may be delivered into the desired cells by a number of different transformation techniques.

For DNA delivery into the cells in vitro and in vivo could be used electroporation, particle bombardment and transfection, which includes calcium-phosphate co-precipitation or DEAE-dextran, direct microinjection or sonication loading, liposome mediated transformation, adenoviral assisted transfection and receptor mediated transfection. Viral transformation could be done with adenoviral infection, adeno-associated virus infection, retroviral infection or with any other viral vectors and combinations thereof.

Odontogenic promoting factors may be chosen among the group of transcription factors, extracellular matrix molecules, bone morphogenetic proteins, neurotransmitters, inflammatory mediators, hormones, cytokines and others.

Methods described here for tooth organ regeneration can be also used for regeneration of other organs. In fact, regeneration of the other organs is supposed to be considerably more easy, because of the lack of need of formation of enamel in the other organs like eye, kidney or lung. Recombination of epithelial and mesenchymal stem cells with following transplantation of the resulted organ back to the patient could be used for regeneration of eye, lung, kidney, and all other organs, which develops by reciprocal interdependent epithelial-mesenchymal interactions and needs two components, namely epithelial and mesenchymal ones.

The invention is illustrated further by the following non-limiting examples. Although the investigations described in Examples are made by using dogs and monkeys and not humans, for bioethical reasons, it is to be considered that similar techniques will function also with humans. Monkey is a classic animal model for experiments to study periodontal ligament regeneration and tooth transplantation as illustrated by Hammarstrom, L. et al, J Clin. Periodontol., vol 24, pp. 669-677 (1997) and Andreasen, J.O. et al, Int. J. Oral. Surg., vol. 7, pp. 104-112 (1978)). Tooth formula, structure and shape in monkeys are very similar to humans.

EXAMPLE 1 : Tissue preparations

Tooth tissues were obtained from ten 2-3 year old monkeys of both sexes, weighting 3.5- 4.5 kg. During all surgical procedures the animals were sedated with Nembutal (35 mg kg body weight) for 30-60 minutes before tissue dissection and local anesthesia was given. The upper and lower central and lateral teeth were used. Twenty teeth from 10 young adult dogs weighting 10-14 kg were used. Surgical anaestesia was obtained by intramuscular injection of 35 mg pentobarbital sodium per kg of body weight. The tooth tissues of the upper canine were isolated.Ten third lower molars were removed from one- year-old pigs.Ten wisdom teeth were obtained from the adult patients after they were removed for orthodontical reasons.

Extracted teeth were cleaned of food, debris and calculus, and rinsed with Hanks' balanced salt solution (HBSS), containing an antibacterial-antimycotic solution (penicillin 200 u/ml, streptomycin 200 μg/ml, gentamycin 100 μg/ml and amphotericin B 5 μg/ml). The gingiva was excised and the teeth were extracted using forceps and elevators. The teeth were washed by immersion and gentle shaken in HBSS, containing the antibiotic- antimycotic solution. To avoid contamination by gingival and apical tissues, only the periodontal ligament from the middle third part of the tooth surface was removed by scraping with a scalpel and used to obtain the primary cultures of epithelial rests of Malassez. After that, the tooth was sterilized one more time in alcohol and cut with a diamond saw to remove the pulp. The pulp was used to obtain the primary culture of pulpocytes. The tissues were kept at room temperature in HBSS, containing the antibiotic-antimycotic solution. The total time between excision of the tissues and starting cultures was 4-6 hours.

EXAMPLE 2: Tissue cultures of epithelial rests of Malassez and pulpocytes

The explants were placed on the bottom of the plastic 60-mm Petri dish, and covered by a cover glass held in place by two streaks of sterile silicon grease placed along the two edges of the cover slip. After adding 5 ml Dulbecco's modified Eagle's medium (DMEM), supplemented with 10 % foetal bovine serum (FBS) and antibacterial- antimycotic solution (penicillin 200 u/ml, streptomycin 200 μg/ml, gentamycin 100 μg/ml and amphotericin B 5 μg/ml) the dishes were placed in an incubator at 37 °C in a humidified atmosphere of 95 % air and 5% CO₂. The two cell types were separated using known methods based on the tendency of epithelial population to be more resistant to detachment by trypsin than fibroblasts. Mixed cell cultures were rinsed once and then incubated for 5 minutes with trypsin-citrate saline (0.25 % trypsin, 0.1 % glucose dissolved in citrate saline, pH 7.8). The majority of fibroblastic cells rounded up after 4 to 6 minutes. These cells were removed from the rest of the mixed populations and discarded. Growth medium was then added to the cells that remained attached to the plastic substrate of the culture dish. If periodontal ligament fibroblasts could be identified among the cells that were still attached, the separation procedure was repeated after 3 days.

EXAMPLE 3: Tissue cultures of human oral keratinocytes and gingival fibroblasts

Tissue sample was obtained from patients as a biopsy after the procedure of crown lengthening. Tissue sample was stored in Hanks' balanced salt solution solution with penicillin 200 u/ml, streptomycin 200 μg/ml, gentamycin 100 μg/ml and amphotericin B 5 μg/ml. It was cultivated in the solution of dispase (Boeringer Mannheim, grade π, Cat. # 295825) at 4 °C for 18 hours. Epidermis was separated mechanically from the mesenchymal part. After that epidermis was cultivated with the solution of trypsin (0.25%) - EDTA (1 mM), cut with scissors and filtered through the stainer (100 μm). The trypsin was neutralized with 20 ml of keratinocyte growth medium (KGM), containing penicillin 100 u/ml and streptomycin 100 μg/ml. Cells suspension was centrifuged at 2000 rpm for 5 minutes and resuspended in 10 ml of KGM-2 and placed into 100-mm Petri dish and placed in an incubator at 37 °C in a humidified atmosphere of 95 % air and 5% CO₂.

Periodontal fibroblasts were obtained from the separated sample of tissue by incubation with the solution of trypsin (0.25%) - EDTA (1 mM) for 30 minutes, cut with scissors and filtered through the stainer (100 μm). Cells suspension was centrifuged at 2000 rpm for 5 minutes and resuspended in 5 ml of DMEM supplemented with 10% of FBS and placed into 60-mm Petri dish and placed in an incubator at 37 °C in a humidified atmosphere of 95 % air and 5% CO₂.

EXAMPLE 4: Tooth organ reconstruction Monkey, dog, porcine and human pulp cells were incorporated into 3-dimensional collagen gels. Stock collagen solutions comprising the following ingredients were prepared on ice in prechilled sterile bottles: 0.3 ml of 10X DMEM; 0.3 ml 0.26 M NaHCO₃ buffer; 0.3 ml FBS; 0.3 ml of 10X antibacterial-antimycotic mixture in DMEM; 0.12 ml of 0.1 M NaOH; 1.2 ml of collagen solution 3 mg/ml; and 0.48 ml of a cell suspension (1 x 10⁶ cells/ml in DMEM). The first five constituents were mixed well by shaking before the addition of the collagen solution. Aliquots of 0.5 ml of the cold stock solution were pipetted into standard 1.5-ml Eppendorf plastic test tubes kept at room temperature. The plated collagen solutions were immediately incubated at 37 °C in a humidified atmosphere of 95 % air and 5 % CO₂ and were found to gel after 5-10 minutes. After that monkey, dog, porcine and human periodontal ligament epithelial cells were seeded on the surface of the formed gel. An 0.5 ml of 2 x 10⁶ cells/ml in DMEM was added to the test tube containing the gel and placed back to the CO₂-incubator. After incubation for 24 hours the gel contracted to the size of 2-3 millimeters and was solid.

REFERENCES CITED

Alliot-Licht, B., et al, "Comparative effect of calcium hydroxide and hydroxyapatite on the cellular activity of human pulp fibroblasts in vitro." Archs Oral Biol., vol. 39, pp.

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Claims

CLAIMS:

1. A method of cultivation of at least two kind of mammalian cell types together in such a way that these cells, growing together, will acquire properties which differ from those of the parent cells, the properties characterized by differentiation of said cells toward forming of of tooth germ or tooth organ, the method including an isolation of the primary cultures of the epithelial and mesenchymal adult multipotent stem cells with following reconstruction of tooth germ or tooth organ by means of tissue engineering methods.

2. A method according to claim 1, wherein the adult multipotent stem cell are the epithelial rests of Malassez and undifferentiated pulpocytes or dental pulp stem cells.

3. A method according to claim 1, wherein oral keratinocytes and both gingival and periodontal fibroblasts are transdifferentiated in vitro to get a potential means for tooth organ regeneration.

4. A method according to any of the claims 1-3, wherein said cell types are forced to grow and differentiate together by using a biodegradable, bioadsorbable, bioerodible, or non-biodegradable, or non-bioerodible scaffold material.

5. A method according to any of the claims 1-3, wherein said cell types are injected into the socket of the extracted tooth of the patient.

6. A method according to any of the claims 1-3, wherein said cell types are combined into the organ by ink-jet organ printing.

7. Use of the method of claims 1-6 for producing a tooth organ ex vivo.