General

STEM CELLS – A BIOENGINEERING MARVEL

Introduction
The term “Stem cells” is increasingly becoming linked to the word “CURE”. Stem cells are not science fiction but something that one day will become a part of each dentist’s clinical practice. 1961 Till & McCulloch identified hematopoitic stem cells in mice.
Mesenchymal stem cells were originally identified from bone marrow stroma by Fridenstein et al in 1966. Ever since then, research has been directed towards attempts to isolate stem cells from various sources, investigate their characteristics and use them in repair and regeneration of living tissues, including those in the oral cavity.
The term Regenerative medicine is used nowadays to describe medical acts, treatments & research that use stem cells (embryonic or adult) to restore the function of organs or tissues. This can be achieved by (a) administering stem cells or specific cells derived from stem cells in the lab. (b) by administering drugs that coax stem cells that are already present in tissues to more efficiently repair the involved tissue. Currently, the only routinely applied medical practice using stem cells is for bone marrow transplantation.
In fact, this field of stem cells and tissue engineering has been identified with a new field of “Regenerative Dentistry” by Prof. Paul Sharpe of the Dental institute, King’s College, London. According to Prof. Paul Sharpe – “Tooth rudiments can be formed from in vitro cultures of non dental stem cell population and complete teeth & associated bone can be obtained on implantation in jaw bone.”

Basic background and treatment
Stem cells are the foundation cells for every organ, tissue & cell in the body. Stem cells are undifferentiated “Blank” cells that do not yet have a specific function.
All human beings start their lives from a single cell called as zygote. The zygote divides & forms two cells; each of those cells divides again, so on. After approx 5 days after fertilization, the “Blastocyst” is formed that is a hollow ball of approximately 150 cells. The blastocyst contains two types of cells – the trophoblast cells along the periphery develop into the embryonic membranes and placenta, which the inner cell mass develops into the foetus. Beyond the blastocyst stage, development is characterized by cell migration in addition to cell division.
Embryonic stem cells are those which make up the inner cell mass of the Blastocyst. They are “Pluripotent” because they can form all the cell types in an adult. Adult stem cells are typically programmed to form different cell types of their own tissue & so are called as mutipotent stem cells.
Adult stem cells are distinct from cells isolated from embryos or foetus and are found in tissue that have already developed, as in animals or humans after birth. The most common place to obtain these cells if from the bone marrow e.g. iliace crest. There are different types of stem cells found in the bone marrow, including hematopoitic stem cells, endothelial stem cells & mesenchymal stem cells. Hematpoitic stem cells form blood, endothelial stem cells form the vascular system & mesenchymal stem cells form bone, cartilage, muscle, fat & fibroblast.
Stem cell plasticity denotes that some adult stem cells may have a broader potential to form different cell types e.g. stem cells from the bone marrow may contribute to regeneration of damaged livers, kidney, heart, lungs etc.
Therapeutic cloning is the process by which an embryo is created through nuclear transfer in order to obtain stem cells from it for therapeutic purposes. The goal of therapeutic cloning is to produce human stem cells & subsequent tissues and organs which can be used to replace damaged tissue.
Unique characteristics of stem cells :-
Stem cells are unspecialized cells with 2 main characteristics
1. The ability of differential into other cells
2. The ability to self regenerate.
The ability to differentiate is the potential to develop into other cell types. A totipotent stem cell (e.g. fertilized egg) can develop into all cell types including the embryonic membranes.
Organ regeneration is through organ specific and tissue specific stem cells. However stem cell plasticity has been demonstrated, i.e. stem cells of one tissue can be used to multiply and repair other damaged tissues.
Tissue regeneration is achieved by two mechanism (1) circulating stem cells divide and differentiate under appropriate signaling by cytokines & growth factors. Differentiated cells which are capable of division, can also self repair e.g. hepatocytes, endothelial cells, smooth muscle cells, keratinocytes & fibroblasts. These fully differentiated cells are limited to local repair. (2) For more extensive repair, stem cells are maintained in the quiescent state and can then be activated & mobilized to the required site.
Plasticity is a newly recognized ability of stem cells to expand their potential beyond the tissue from which they are derived. E.g. dental pulp sacs develop into tissues of teeth but can also be induced to develop into neural tissue.
ISOLATION OF STEM CELLS AND CELL MARKERS
Typically, stem cells generate intermediate cell types before they achieve their fully differentiated state. The intermediate cell is called as precursor / progenitor cell. These are partly differentiated cells that divide and give rise to differentiated cells. Such cells are usually regarded as “Committed” to differentiating along a particular cellular development pathway. Identification of cell surface markers that are specific to mesenchymal stem cells is important for their isolation.
STRO – 1 : a mouse raised immunoglobin M antibody has been shown to identify a cell surface antigen expressed by stromal cell precurson in human bone marrow.
No single antigenic marker has been shown to be specific for mesenchymal stem cells and therefore combination of antibodies must be utilized. e.g. 1) cell surface antigens like CD 146, MUC18, 2) Pericyte associated antigen CD106 (3G5), CD 44 and V CAM – 1, 3) Alkaline phosphatase and α – smooth muscle actins
Stem cells have also been isolated from the pulp of human exfoliated desiduous teeth (SHED) – acronym from (Stem cells from human exfoliated deciduous teeth) Miura et al (2003)
SHED were positive for STRO – 1 & CD 146 / MUC – 18.
After transplantation into immunocompromised mice, they induced bone formation, generate dentin, survive in the mouse brain and express neural tissue markers. It has been suggested that SHED represent a population of multipotent stem cells that are more immature in the cell hierarchy than previously examined post natal stem cell populations.
STRO – 1 and CD 146 antigen markers were used to isolate periodontal ligament stem cells – Seo et al (2004). Under defined culture conditions, periodontal ligament stem cells differentiated into cementoblast like cells, adopocytes & collagen forming cells.

Focus on teeth for research
Teeth are an ectodermal organ & as such, like other ectodermal organ e.g. hair, skin, - glands, are located close to the extremity of the body. Teeth develop in the embryo via interactions between the ectoderm and the underlying mesenchyme. Teeth are thus easily accessible organs that can be visualized in the mouth. Their removal & replacement does not involve major surgery.
Teeth, thus, have important advantages for providing a test case for proof of concept of organ engineering. By carrying out research on laboratory engineering of teeth, in which the final stage of clinical trials will be simple, the whole field of organ engineering can benefit in the long term.
In human dentition the teeth are essentially of the following shapes – incisors, caniners, & multicuspids. Tooth shape is determined very early in development by expression of different genes in different regions of the mesenchyme in the primordial jaw. These genes especially, Homoeobox genes e.g. BARX – 1, act to provide cells with positional information which will direct the cells down specific pathways of morphogenesis (Tucker & Sharpe, 2004).
Homeobox genes e.g. Barx -1 act upon mesenchymal cells prior to tooth initiation and this positional information must be interpreted by cells & used to direct epithelial folding to produce tooth shape. The enamel host is a transient epithelial signaling centre observed at the Cap Stage of tooth development that is involved in regulating crown morphogenesis.
One of the mechanisms used by cells to interpret this information generated by homoepoitic proteins is the nuclear Factor – kB pathway (NF – kB).

Possible Applications of Stem Cells in Dentistry
Damaged or missing teeth are a large & significant problem – both esthetic and economic. Present day treatment modalities are removable prosthesis, fixed prosthesis or implants. Dental researchers are now increasingly in favour of tissue engineered teeth.
In 2002, Forysth lab researchers demonstrated development of mammalian teeth by using stem cells from deciduous teeth of pigs, seeding them onto polymer scaffolds & transplanting them into rat hosts. Within 30 weeks, small rudimentary tooth crown had formed. These crown contained dentin, odontoblasts , a well defined pulp chamber, Hertwig’s root sheath, cementoblasts and a morphologically correct enamel.
The term “Bio teeth” or biological replacement teeth was coined by Prof. Paul Sharpe.

There are basically 4 ways to make a BioTooth -
1. Try and reconstruct the mature tooth as it appears in the mouth.
2. Try and reproduce the embryonic development of a tooth in the mouth.
3. Try & induce a third dentition
4. Create a matrix in the shape of a tooth and seed it with stem cells.
Robey et. al(2005) proposed that constituent parts of an adult tooth be made individually. Bone marrow stromal cells & HA / TCP matrix could be used to engineer the alveolar bone.
The dental pulp & enamel could be constructed using dental pulp cells & HA/TCP in a enamel – like mould. The periodontal ligament could be engineered using periodontal ligament stem cells.
One of the most successful techniques of tissue engineering is the use of biodegradable scaffolds into which stem cells can be seeded. Classic example ear on the back of a mouse – Vacanti et al (1997).
The optimal properties of a scaffold or carries for use in cell based tissue engineering are –
1. The ability of the carrier to maintain the important characteristics of a stem cell and yet allow for the appropriate differentiation of its progeny.
2. To provide adequate support for the developing tissue and then
3. To be resorbed without generation of toxic by produce
Commonly used scaffolds are :- Hydroxyapatite, tri calcium phosphate (HA/TCP) .
• Collagen based sponges
• Dimineralized bone matrix
• Poly α – lactic acid fibres
• Poly α – glycolic acid fibers
Maintain alveolar ridge height is critical to prosthodontic procedures like complete dentures and implants & preventing bone destruction induced by periodontal disease.
Researchers demonstrated reconstruction of a part of mandible in a patient who had undergone extensive tumor resection. In this case, the workers used CT scan and model the mandible. They fabricated a custom made titanium mesh cage to reconstruct the mandible.

Conclusions :
In view of the ageing population and shortage of donor tissue and ethical issues, the field of biomimetics and regenerative medicine and dentistry is expected to become a major field in modern biomedicine.
Products in this area include scaffolds that guide and encourage stem cells to form specific tissues, expanded & engineered cell population, biological agents like DNA or recombinant proteins, or regulatory genes.
The immediate challenge is for dentists not only to be better able to address the issues concerning stem cell – based therapy but also to familiarize themselves with the spectrum of tools they may have in the future to restore form andfunction effectively.

REFERENCES

1. Sartaj Rachel and Sharpe Paul, Biological tooth replacement, Journal of Anatomy (2006),209, 503-509.
2. Robey Pamela G, Bianco Pailo, The use of adult stem cells in rebuilding the human face, Journal of Americal Dental Association(2006),137,961-971.
3. Ivanovski S, Gronthos S, Shi S, Bartold PM, Stem cells in the periodontal ligament, Oral diseases(2006), 12,358-363.
4. Luyten Frank P, Accio F.D, Bari CD, Skeletal tissue engineering:opportunities and challenges, Best Practice and Research Clinical Rheumatology (2001),15(5),759-770.
5. Zhang W, Walboomers XF, Van Kuppevelt TH, Daamen WF, Bian Z, Jansen JA, the performance of human dental pulp stem celss on different three dimensional scaffold materials, Biomaterials, 2006,27, 5658-5668.
6. George T.-J. Huang, A paradigm shift in endodontic management of immature teeth: Conservation of stem cells for regeneration, Journal of dentistry (2008), 36, 379 – 386.