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Oral Pathology

Authors: Dr. N.N. Singh, (Professor and Head),Dr. (Mrs) V.R.Brave (Professor), Dr. G. Sreedhar (Associate Professor),

Introduction :
Dental and medical treatment for loss of tissue or end stage organ failure is required by all1. The field of tissue engineering has developed over the past decade to recreate functional, healthy tissues and organs in order to replace diseased, dying or dead tissues1 .The amalgamation of bioengineering & dentistry has resulted in explosion of knowledge that has enhanced our understanding and started a new era of dentistry, enabling us to restore the lost tissue function.
Tooth loss commonly accompanies a variety of oral diseases and physiological causes, including dental caries, periodontal disease, trauma, genetic disorders and aging, and can lead to physical and mental suffering that markedly lowers the individual’s quality of life2.
Keeping one’s own teeth throughout life is not only beneficial for enjoying food and maintaining quality of life, but also helps to prevent dementia as mastication stimulates brain3. Henceforth, tooth regeneration is of particular relevance to field of regenerative medicine4.
The current strategies to tissue replacement and reconstruction include the utilization of autogenous grafts, allografts  and synthetic materials, but each of them has limitations, which may include lack of significant stores for excess tissue transplantation, possibility of eliciting an immunologic response due to genetic differences or inducing transmissible diseases1.
The gold standard to replace an individual’s  lost or damaged tissues is the one made from patient’s own tissue and grown in its intended location5. This standard has also lead to the concept of engineering or regenerating new tissue from pre existing tissue.
Tissue engineering is a multidisciplinary field which applies the principles of life science, engineering and basic science to the development of viable substitutes which restore, maintain or improve the function of human tissues. Modern isolation and culturing technique of any types of human cells provides the basis of tissue engineering. Naturally derived or synthetic biomaterials are fashioned into scaffolds, which when cultured and implanted in combination with cells, provide a template that allows such constructs to form new soft and hard tissues, during which time the scaffold degrades and is finally metabolized6.


STRATEGIES TO ENGINEER TOOTH
Currently, there are two major approaches to tooth regeneration. The first is based on tissue engineering and aims to regenerate teeth by seeding cells on scaffolding biomaterials 7,8. This technique has already been applied to the regeneration of periodontium and holds great future promise for predictable periodontal regeneration9. The second approach involves reproducing the developmental processes of embryonic tooth formation and this requires an understanding of the basic principles that regulate early tooth development and uses embryonic tissues (dental epithelium and dental mesenchyme) harvested from a mouse fetus10. Artificial tooth germs bioengineered through both approaches are transplanted into the bodies of animal hosts, where there is sufficient blood flow to provide the necessary nutrients and oxygen for tissue formation2.


TOOTH REGENERATION USING TISSUE ENGINEERING
In the late 1980’s, organ transplant surgeon Joseph P. Vacanti of Harward Medical School and polymer chemist Robert S. Langer of  the  Massachusetts Institute of Technology conceived the idea of placing the cells of an organ or tissue on a prefabricated biodegradable scaffold with the goal of generating tissues and organs for transplantation.
The approach is based on the fact that living tissues are made of cells constantly signaling to one another and often moving around within a three dimensional community of sorts. Each cell knows its place and role in the larger collective. Therefore, if the right mix of dissociated cells is reaggregated with a scaffold that replicates their natural 3-D environment, the cells should instinctively reform the tissue or organ to which they belong. 5
Vacanti and Langer’s successful technique of bioengineering neonatal intestine11 and stomach using the scaffold based strategy has led to experimentation to produce other complex tissues like heart muscle, intestine, mineralised bone and teeth by Pamela C. Yelick and John D. Bartlett of Forsyth Institute in Boston.11
Yelick’s group enzymatically dissolved dental epithelial and pulpal mesenchymal tissues form the unerupted third molars of a six month old pig10 and seeded the mixture of the heterogeneous single cells onto a tooth shaped biodegradable polymer scaffold constituting of polyglycolic acid (PGA) and poly-co-glycolide copolymer (PLGA).The cells, scaffolds constructs were implanted into the intestine of rat hosts to receive sufficient blood supply, nutrients and oxygen. By 2-30 weeks, after the implantation, tiny tooth like tissues (like enamel, dentin and pulp)7 were observed within the implants, which resembled the crowns of natural teeth, with rudimentary tooth root structures. Although most dental tissues were regenerated with cells from an adult source and scaffold materials, with success rate for achieving the correct arrangement of the natural tooth is only 15% -20%5. Further studies are therefore required to consistently achieve reconstituted and structurally sound teeth2.

Figure1.

TOOTH REGENERATION USING DEVELOPMENTAL APPROACH

Rather than attempting to build adult teeth from their constituent cells, Sharpe’s group replicated the natural processes involved in embryonic tooth development, focusing on the reciprocal interactions between the epithelium and mesenchyme10.Recombinations between mesenchyme created in vitro(by aggregation of non dental cultured cells from different  cell sources) and embryonic oral epithelium collected from mouse embryos at 10th embryonic day(E10) , stimulated an odontogenic response in the mesenchyme. When such explants were transferred intact into adult renal capsules, they developed into teeth (crowns) with associated bone and soft tissues5.
The results offer important insights into tooth development and suggest that stem cells derived from adult bone marrow can take place of dental mesenchyme2 and the odontogenic process can be initiated in non dental cells of different origins10. Bone and soft tissues can be formed from non dental cell populations consisting entirely of purified stem cells or from heterogeneous population such as bone marrow derived cells10. But no suitable source of epithelial components has yet been found to replace the embryonic oral epithelium2.
Many years of experiments have established that embryonic epithelium12 contains a unique set of signals for odontogenesis that disappear from the mouth after birth. Sharpe’s group is continuing to seek an effective population of substitute cells that could be derived from an adult source5.

The teeth obtained from Sharpe’s group were in the normal size range for mouse teeth and showed earliest signs of root formation. Though it was doubtable whether such explants could also form teeth in the mouth. In embryonic jaw, soft tissues, teeth and bone, all are developing together without external stresses such as chewing and talking, where as it was not the case in adult jaws.

Sharpe group extracted tooth buds from embryonic mice (E14.5), then transplanted them into the diastema pockets between molars and incisors of adult mice. The mice were fed on soft diet and the transplants were monitored.5 The decalcified sections of the diastema, clearly revealed ectopic tooth formed at the site of transplantation. The teeth were in correct orientation, of appropriate size and attached to underlying bone by soft connective tissue. Thus, adult mouth could provide a suitable environment for tooth development10.
These approaches to tooth reconstitution using developing tissues are far from ready for patient application because it would be impractical to use human embryonic tissue. Strategic improvements are needed prior to clinical application to prevent immune rejection and to overcome ethical issues.

Though identification of stem cells in dental pulp and from exfoliated deciduous teeth also raises the possibility that a patient’s own tooth cells could be used to generate new tooth primordia13.

FUTURE TRENDS
Although in its infancy, tissue engineering approach can be used to bioengineer highly mineralized, anatomically correct replacement tooth tissues, reflecting its need for alternative therapies to treat variety of dental repair needs14. It is eventually possible to device clinically relevant therapies to replace damaged or lost dental tissues with biologic dental materials as a viable alternative to synthetic dental materials.
The research also provides intermediate products that can be used to augment existing synthetic dental repair materials eg, it is possible to use bioengineered dental materials to improve the function and duration of currently used titanium implants to underlying alveolar bone via autologous bioengineered periodontal ligament would help transmit mechanical forces of mastication from implants  to underlying bone and might also help perform orthodontic treatments11.
Post natal stem cells isolated from developing wisdom teeth can regenerate functional tooth roots and periodontal ligaments that support synthetic crowns2.

We have entered an exciting era where the diverse fields of tissue engineering, material science, nano technology and stem cell biology have converged synergistically to provide unprecedented opportunities to characterize and manipulate signaling cascades, regulating tissue and organ regeneration.
The future for regenerative and tissue engineering applications to dentistry is one with immense potential, capable of bringing quantum advances in treatment for patients. The need for high quality research in the basic sciences is paramount to ensuring that the development of novel clinical treatment modalities is underpinned by robust mechanistic data and that such approaches are effective. This translational model epitomizes how dentistry should evolve and highlight the needs for close partnership between basic and clinical scientists.
Apart from the potential benefit to people who need new teeth, this research also offers two significant advantages for testing the concept of organ replacement15. Teeth are easily accessible and whereas our quality of life is greatly improved if we have them, we do not need our teeth to live. These may seem trivial points, but as the first wave of replacement organs start to make their way towards the clinic, teeth will serve as a crucial test of feasibility of different tissue engineering techniques. With organs essential to life, doctors will have no leeway to make mistakes, but mistakes with teeth would not be life threatening and could be corrected.

CHALLENGES
Tooth regeneration has also identified certain challenges. First, there are limits to the traditional principles of tissue engineering, related to whole tooth regeneration with correct morphology. Secondly, adult bone marrow cells can though alternate dental mesenchymal cells but no suitable substitute for embryonic epithelial compartment has yet been recognized.

Moreover, the techniques have not yet been established to control tooth size, shape and colour, particularly full human tooth size. Problems concerning the cell numbers obtained, host immune rejection and ethical issues of the use of human embryos still remains. Relevant ethical issues include the source of cells (patient’s own vs donated cells) and type (adult donor vs fetal cells).

CONCLUSION
The control of morphogenesis and cytodifferentiation is a challenge that necessitates a thorough understanding of the cellular and molecular events involved in development, repair and regeneration of teeth. The identification of several types of epithelial and mesenchymal stem cells in the tooth and the knowledge of molecules involved in stem cell fate is a significant achievement. Though, many problems remain to be addressed before considering the clinical use of  these technologies. The use of animal cells for human diseases is restricted by immune rejection risks. It is possible to replace dental mesenchymal stem cells with stem cells of another origin, but not so is the case with epithelial stem cells.
A reliable source of epithelial stem cells remains to be determined. Alternative solutions such as use of artificial crowns are considered. The engineering of tridimensional matrices (either PLA polymers or collagen sponge) with a composition more or less similar to that of  the organs to reconstruct and the addition of growth factors such as FGF or BMP might facilitate transplantation and differentiation of stem cells. However, engineering of tooth substitutes is hard to scale up, costly, time consuming and incompatible with the treatment of extensive tooth loss.
The field of tooth tissue engineering is one of the many areas likely to see significant progress in the next decade.

REFERENCES

  1. Kaigler Darnell, Mooney David. Tissue engineering’s impact on dentistry: Journal of Dental Education 2001;65(5):456-462
  2. Nakahara T, Yoshiaki DE. Tooth regeneration: Implications for the use of bioengineered organs in first wave organ replacement. Human Cell 2007;20:63-70
  3. www.sciencedaily.com/releases/2007/10/071010111807.htm
  4. Smith AJ. Tooth tissue engineering and regeneration: a Translational vision! J  Dent Res 2004;83:517
  5. Sharpe PT, Young CS. Test tube teeth.  Sci Am 2005;293:34-41
  6. http://www.lumrix.net/medical/bioengineering/tissue_engineering.html
  7. Young CS, Terada S, Vacanti JP, Yelick PC. Tissue engineering of complex tooth structures on biodegradable polymer scaffolds. J Dent Res 2002;81:695-700
  8. Duailibi MT, Duailibi SE ,Young CS, Bartlett JD, Vacanti JP, Yelick PC. Bioengineered teeth from cultured rat tooth bud cells. J Dent Res 2004;83:523-28
  9. Nakahara T.A review of new developments in tissue engineering therapy for periodontics. Dent Clin North Am 2006;50:265-76
  10. Ohazama A, Modino SA, Miletich I, Sharpe PT. Stem cell based tissue engineering in murine teeth.J Dent Res 2004;83:518-22

References are available on request

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