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Cells health 4

Dental Pulp Stem Cells, a New Era in Tissue Engineering

AbstractStem cells are primitive cells that can differentiate and regenerate deteriorating cells in different parts of the body such as heart, bones, muscles and nervous system.For years scientists all over the world have been working on possibilities of using these stem cells to regenerate human cells which are damaged due to illness, developmental defects and accidents. This article is to give an overall idea about stem cells in general, history and future, and where does dentistry stand in that field.

Key words: Stem cell, Embryonic stem cells, Adult stem cells, SHED, Chondrocytes, Osteoblasts, Adipocytes, Mesenchymal stem cells.

IntroductionThe term stem cell was proposed for scientific use by Russian histologist Alexander Maksimov in 1908. While research on stem cells grew out of findings by Canadian scientists in the 1960s.1, 2

In general there are two broad types of stem cells which are: Embryonic stem cells, and Adult stem cells. Embryonic stem cells were harvested from embryos, they are cells derived from the inner cell mass of the blastocyst (early stage embryo, 4-5 days old, consist of 50-150 cells) of earlier morula stage embryo.3 In other words these are the cells that form the three germ layers, and are capable of developing more than 200 cell types. In 1998 the first human embryonic stem cell line was derived at university of Wisconsin-Madison.4

Embryonic stem cells have both moral and technical problems, because these cells will later develop into a human being, taking these cells will require destruction of an embryo. Technically these cells are difficult to control and grow and they might as well form tumors after their injection.

Differentiating embryonic stem cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face.5 And after ten years of research6, there are no approved treatments or human trials using embryonic stem cells; but because of the combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source of regenerative medicine and tissue replacement after injury or disease.

Dr. Ghada A. KarienBDS, JDB (Paed)

• Paediatric dentist Jordanian Dental Board in paediatric dentistry• MOH/Al-Basheer hospital

[email protected]

Ulternative Therapy

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Ulternative Therapy

Tissue engineering and regenerative medicine seek to replace lost or damaged tissues due to any reason, and this needs three major ingredients which are:

1- Morphogenic signals such as growth factors and differentiation factors, these factors play an important role in the multiplication and differentiation of stem cells into the specifically needed type of cells.

BMPs (bone morphogenic proteins) and cytokines play a major role in organogenesis, and in the dental aspect specifically GDf-11 (growth/differentiation factor 11) which is a novel member of BMP/TGF B family is expressed in differentiating odontoblasts and plays a major role in differentiation of dental pulp stem cells into odontoblasts which is the corner stone in teeth tissue engineering.13

2- Responding stem cells which are originally harvested from the patient and preserved under good conditions to maintain their special ability to differentiate into a wide range of cells. 3- Scaffold of extra cellular matrix, which provide these cells with the environment and mold to grow into what we want them to become and function.

One of the major advantages one gets from harvesting stem cells from his own body and then using them later in his tissue regeneration if he has an illness is that there will be no refusal of these cells as they are already body parts, in other words the patient will not need to go through the process of immunosuppressant and that will spare him lots of suffering and time.

In the future, medical researchers anticipate being able to use technologies derived from stem cell research to treat a wider variety of diseases including cancer, Parkinson’s, Alzhimer, spinal cord injuries, diabetes, heart diseases, liver disease,

blindness, multiple sclerosis, muscle damage and many other diseases.14,15,16,17

Specifically talking about the dental field, years from now dental stem cells will hopefully be able to correct cleft palate sparing children from multiple surgeries, stem cells will also have the potential to save injured teeth and jaw bones, correct periodontal defects, and most strikingly regenerating entire teeth structures is the horizon.

Many people will ask themselves, how can the scientists be able to use dental stem cells in regenerating dental tissues?

Well, there are three approaches which were investigated by different labs to implant stem cells from teeth in humans and these are:

1- Placing the stem cell into a mold of tooth crown which is made of Enamel-like substance with a scaffold material, and then they will start to loop blood vessels through this scaffold, after that this will be implanted elsewhere in the body and wait until it is mature, then these teeth will be extracted and implanted in the oral cavity.

2- Harvesting a wisdom tooth of a person and releasing stem cells from their pulp tissue, the stem cells are then implanted in a severely injured tooth, for example in cases of car accidents or falling down, and these implanted stem cells will help toregenerate the pulp of the injured teeth sparing them root canal treatments.

3- If there are no teeth present in the oral cavity from which stem cells can be harvested, we can take stem cell from un-erupted wisdom tooth, organize them into three dimensional structures and give proper cues to them before putting them back into the socket; this is like planting a seed and waiting for it to grow.18

Discovery that human mature pulp tissue contains a population of multi-potent mesenchymal dental pulp stem cells with high proliferative potential for self renewal and the ability to differentiate into functional odontoblast has revolutionized dental research and opened new avenues in particular for reparative and reconstructive dentistry and tissue engineering in general.

Stem cell therapy which was once a science fiction is now becoming more towards reality, and it might make the dream of many people come true. So parents taking the decision to bank their children’s milk teeth might be the best gift they could ever give to their child.Milk teeth which were kept by children under their pillows to be collected by the tooth fairy might have a greater meaning; the tooth fairy might be able one day to save their life.

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Ulternative Therapy

That opened the window wide for the so called adult stem cells, which are cells found in a developed organism and they have two properties: first the ability to divide and create another cell like itself. Second they divide to create a more differentiated cell than itself. They can be found in both children and adult.7

Adult stem cells can in general be found in umbilical cord blood, blood and bone marrow. Pluripotent stem cells can be found in cord blood but are small in Number.8 These adult stem cells have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants. The first one marrow transplant between two siblings was done successfully in 1968.

Most adult stem cells are lineage restricted and are generally referred by their tissue of origin e.g. mesenchymal stem cells, adipose derived stem cells, endothelial stem cells...etc.9,10

Stem Cells in DentistryIn the year 2003 Dr. Songtao Shi who is a paediatric dentist discovered baby tooth stem cells by using the deciduous teeth of his six year old daughter, he was luckily able to isolate, grow and preserve these stem cells’ regenerative ability, and he named them as SHED (Stem cells from Human Exfoliated Deciduous teeth).11

After the scientists studied the dental pulp looking for stem cells they found that the dental pulp was rich in different stem cell types such as:

Chondrocytes: which are stem cells that have the ability to regenerate cartilage and these cells play an important role in the treatment of arthritis and joint diseases.

Osteoblasts: They are stem cells that have the ability to regenerate bone.

Adipocytes: Another type of stem cells that have the ability to repair damaged cardiac tissues following a heart attack.

Mesenchymal Stem Cells: Those are the most potent among all tissue stem cells and have the ability to differentiate into various types of reparative cells.

In general Mesenchymal Stem Cells MSC are non-haematopoietic stromal cells capable of differentiating into a range of cells, those cells were first discovered in bone marrow and they were noticed to have the ability to double into many populations without loss of function, they also have the so called homing property which means that when they are delivered systemically they migrate to the site of injury.So it is to say that MSC are more promising for therapeutic applications than other types of stem cells.12

Mesenchymal stem cells

Recently stem cell banks are present, and even some of these banks do not only freeze cord stem cells but also dental stem cells of baby teeth. This can be done easily when a child’s anterior milk tooth is shedding, the tooth is extracted by the dentist and preserved in a special kit provided from the stem cell bank company who then in their turn transfer the tooth to their special labs to harvest the dental stem cells and store them in their bank for each child confidentially until they are needed later for the child himself or a member of his family.

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Refrences1. BECKER AJ, McCULLOCH EA, TILL JE. Cytological demonstration of the clonal nature

of spleen colonies derived from transplanted mouse marrow cells. Nature. 1963 Feb 2;197:452-4.

2. SIMINOVITCH L, MCCULLOCH EA, TILL JE. The distribution of colony-Forming cells among spleen colonies. J Cell Physiol. 1963 Dec;62:327-36.

3. Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6;282(5391):1145-7.

4. “New Stem-Cell Procedure Doesn’t Harm Embryos, Company Claims”. Fox News. http://www.foxnews.com/story/0,2933,210078,00.html.

5. Wu DC, Boyd AS, Wood KJ. Embryonic stem cell transplantation: potential applicability in cell replacement therapy and regenerative medicine. Front Biosci.

2007 May 1;12:4525-35.6. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones

JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6;282(5391):1145-7.

7. JJiang Y, Jahagirdar BN, Reinhardt RL, et al. (2002). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002 Jul 4;418(6893):41-9. Epub 2002 Jun 20.

8. Ratajczak MZ, Machalinski B, Wojakowski W, Ratajczak J, Kucia M. A hypothesis for an embryonic origin of pluripotent Oct-4(+) stem cells in adult bone marrow and other tissues. Leukemia. 2007 May;21(5):860-7. Epub 2007 Mar 8.

9. Barrilleaux B, Phinney DG, Prockop DJ, O’Connor KC. Review: ex vivo engineering of living tissues with adult stem cells. Tissue Eng. 2006 Nov;12(11):3007-19.

10. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medi-cine. Circ Res. 2007 May 11;100(9):1249-60.

11. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003 May 13;100(10):5807-12. Epub 2003 Apr 25.

12. Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and poten-tial for homing. Stem Cells. 2007 Nov;25(11):2739-49. Epub 2007 Jul 26.

13. Nakashima M, Mizunuma K, Murakami T, Akamine A. Induction of dental pulp stem cell differentiation into odontoblasts by electroporation-mediated gene delivery of growth/differentiation factor 11 (Gdf11). Gene Ther. 2002 Jun;9(12):814-8.

14. Fiegel HC, Lange C, Kneser U, Lambrecht W, Zander AR, Rogiers X, Kluth D. Fetal and adult liver stem cells for liver regeneration and tissue engineering. J Cell Mol Med. 2006 Jul-Sep;10(3):577-87.

15. Timper K, Seboek D, Eberhardt M, Linscheid P, Christ-Crain M, Keller U, Müller B, Zulewski H. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem Biophys Res Commun. 2006 Mar 24;341(4):1135-40. Epub 2006 Jan 26.

16. Lindvall O. Stem cells for cell therapy in Parkinson’s disease. Pharmacol Res. 2003 Apr;47(4):279-87.

17. Goldman SA, Windrem MS. Cell replacement therapy in neurological disease. Philos Trans R Soc Lond B Biol Sci. 2006 Sep 29;361(1473):1463-75.

18. Zhang W, Walboomers XF, van Kuppevelt TH, Daamen WF, Bian Z, Jansen JA. The performance of human dental pulp stem cells on different three-dimensional scaf-fold materials. Biomaterials. 2006 Nov;27(33):5658-68. Epub 2006 Aug 17.

Abstract Stem cells constitute the source of differentiated cells for the generation of tissues during development, and for regeneration of tissues that are diseased or injured post-natally. In recent years, stem cell research has grown expo-nentially owing to the recognition that stem cell-based therapies have the potential to improve the life of patients with conditions that span from Alzheimer’s disease to cardiac ischemia to bone or tooth loss. Growing evidence demonstrates that stem cells are primarily found in niches and that certain tissues contain more stem cells than others. Among these tissues, the dental pulp is considered a rich source of mesenchymal stem cells that are suitable for tissue engineering applications. It is known that dental pulp stem cells have the potential to differentiate into several cell types, including odontoblasts, neural progenitors, osteo-blasts, chondrocytes, and adipocytes. The dental pulp stem cells are highly proliferative. This characteristic facilitates ex vivo expansion and enhances the translational potential of these cells. Notably, the dental pulp is arguably the most accessible source of postnatal stem cells. Collectively, the multipotency, high proliferation rates, and accessibility make the dental pulp an attractive source of mesenchymal stem cells for tissue regeneration. This review discusses fun-damental concepts of stem cell biology and tissue engineer-ing within the context of regenerative dentistry.

Key words Tissue engineering · Endodontics · Odontoblasts · Endothelial cells · Dentin

Introduction

The discovery of dental stem cells and recent advances in cellular and molecular biology have led to the development of novel therapeutic strategies that aim at the regeneration of oral tissues that were injured by disease or trauma. Tissue engineering is multidisciplinary by nature, bringing together biology, engineering, and clinical sciences with the goal of generating new tissues and organs.1 Tissue engineering is a science based on fundamental principles that involves the identifi cation of appropriate cells, the development of con-ducive scaffolds, and the understanding of the morphogenic signals required to induce cells to regenerate a tissue or organ. Over the last few years, dentistry has begun to explore the potential application of stem cells and tissue engineering towards the repair and regeneration of dental structures. It is becoming increasingly clearer that this con-ceptual approach to therapy, named “regenerative den-tistry,” will have its place in the clinical practice of dentistry in the future. This review discusses the state-of-the-science with regard to dental pulp stem cells in tooth tissue engi-neering, and presents a prospectus for the fi eld of stem cell-based regenerative dentistry.

Stem cells of dental origin

Stem cells are nonspecialized cells that continuously divide, have the ability of self-renewal, and are capable of generat-ing complex tissues and organs.2 These cells can be classifi ed as either embryonic or postnatal.3 Embryonic stem cells are found in the inner cell mass of the blastocyst during the early stages of embryo development.4 Their self-renewal potential and unrestricted ability to generate new tissues and organs (totipotency) make these cells an attractive cel-lular source for cell-based regenerative therapies. However, the use of embryonic stem cells is controversial. Indeed, legal and ethical issues have signifi cantly impaired the fea-sibility of their use in the laboratory and in the clinic.5 Therefore, this review will not discuss the use of embryonic

Odontology (2011) 99:1–7 © The Society of The Nippon Dental University 2011DOI 10.1007/s10266-010-0154-z

Luciano Casagrande · Mabel M. Cordeiro · Silvia A. Nör Jacques E. Nör

Dental pulp stem cells in regenerative dentistry

L. Casagrande · M.M. Cordeiro · S.A. Nör · J.E. Nör (*)Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School of Dentistry, 1011 N. University, Rm. 2309, Ann Arbor, MI 48109-1078, USATel. +1-734-936-9300e-mail: [email protected]

J.E. NörDepartment of Biomedical Engineering, University of Michigan College of Engineering, Ann Arbor, MI, USA

J.E. NörDepartment of Otolaryngology, University of Michigan School of Medicine, Ann Arbor, MI, USA

Received: November 9, 2010 / Accepted: November 11, 2010

REVIEW ARTICLE

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stem cells in dentistry. Similarly to embryonic stem cells, postnatal stem cells are capable of self-renewal. However, postnatal cells are multipotent; that is, they have a more limited capacity for differentiating into other cell types than the totipotent embryonic stem cells. Notably, postnatal stem cells present the obvious advantage of being a source of cells for autologous transplants, minimizing risks related with immune rejection. And fi nally, postnatal stem cells can, at least in theory, be obtained from individuals at any stage in life.

Several, if not all, adult tissues have a subpopulation of stem cells. Examples of such tissues are the bone marrow, brain, skin, muscle, and adipose tissue.6–9 Stem cells have also been found in several dental tissues. One of the fi rst tooth-related stem cell types was found in the pulp of per-manent teeth and was named dental pulp stem cells (DPSCs).10 In addition, stem cells from human exfoliated deciduous teeth (SHED), stem cells from the apical papilla, dental follicle progenitor cells, and periodontal ligament stem cells have also been characterized.11–15 Mechanistic studies focused on these cells are certainly improving our understanding of tooth development. In addition, this knowledge has been applied in translational studies that aim at the use of these stem cells in clinical settings where the regeneration of dental and craniofacial tissues is indicated.

The usefulness of stem cells in clinical applications depends on their proliferation rate, differentiation poten-tial, and accessibility. For example, when bone marrow stem cells were compared with DPSCs, DPSCs presented favor-able results with regard to odontogenic capability.16 Stem cells of dental origin can certainly generate dental tissues.10,11,13,17,18 We have shown that SHED and DPSCs are capable of generating a tissue that has morphological and functional characteristics that closely resemble those of human dental pulp (Fig. 1).19–22 Other studies have expanded the potential of these cells in the treatment of diseases and conditions such as muscular dystrophies, critical size bone defects, corneal alterations, spinal cord injury, and systemic lupus erythematosus.23–28 Such studies clearly demonstrate

the plasticity and the differentiation potential of stem cells of dental origin. And fi nally, SHED cells have the unique advantage of being retrievable from naturally exfoliated teeth, which can be considered a “disposable” source of postnatal human tissue. Collectively, these studies suggest that the tooth constitutes an attractive source of stem cells that can potentially be useful in a wide spectrum of clinical scenarios.

Signaling molecules and dental pulp stem cell differentiation

Growth factors and morphogenic factors are proteins that bind to specifi c membrane receptors and trigger a series of signaling pathways that coordinate all cellular functions. These molecules play a critical role during development, guiding processes that determine the fate of stem cells and regulate the generation of all tissues and organs in the developing embryo. Similarly, these morphogenic molecules play a critical role in physiological processes of tissue regen-eration as, for example, wound healing in the skin or dental pulp responses to the progression of dentinal caries. The same growth factors that guide embryogenesis and physio-logical tissue regeneration can also be used therapeutically to guide stem cell differentiation toward specifi c cell fates and to coordinate cellular processes that result ultimately in the generation of a new tissue or organ via tissue engineering-based approaches. More specifi cally, there are many similarities between morphogenic factors regulating dentinogenesis and the factors that regulate reparative den-tinogenesis.29 One rapidly concludes that the fi eld of dental tissue engineering can benefi t tremendously from studies focused on the cellular and molecular mechanisms of odontogenesis.

Growth factors have an important role in signaling reparative processes in dentin and pulp.29,30 Indeed, it is known that factors such as transforming growth factor β, bone morphogenic proteins (BMPs), platelet-derived

Fig. 1. Dental pulp engineered with stem cells from human exfoliated deciduous teeth (SHED). SHED were seeded into tooth slice/scaffolds and transplanted into the subcutaneous space of immunodefi cient mice. After 21 days, the tooth slices were retrieved, fi xed, demineralized, and prepared for standard histology. Photomicrographs of hematoxylin/eosin-stained tissue sections (×400) depicting the periphery and the central portion of the dental pulp-like tissue formed in the pulp chamber of these tooth slices

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growth factor, fi broblast growth factor, and vascular endo-thelial growth factor (VEGF) are incorporated into the dentin matrix during dentinogenesis and are retained there as “fossilized” molecules.31–33 Interestingly, when these mol-ecules are released from the dentin, they are bioactive and fully capable of inducing cellular responses, as for example those that lead to the generation of tertiary dentin and to dental pulp repair.30,34 The tubular arrangement of the dentin facilitates the movement of growth factors released from dentin matrix that has been demineralized by caries, acidic tooth conditioning agents, or pulp capping materials. Interestingly, calcium hydroxide has been shown to solubi-lize dentin and allow the release of bioactive molecules that can potentially regenerate dentin.35 Such events involve the recruitment of DPSCs, their differentiation into odonto-blasts, and the secretion of mineralizable matrices.36–38 Col-lectively, the release of growth factors from the dentin appears to constitute an important mechanism of defense against injuries, allowing for a fi nite level of dental tissue regeneration.

Studies from the early 1990s demonstrated that BMPs (e.g., BMP-2, BMP-4, BMP-7) trigger signaling events that induce the generation of dentin in animal models.39,40 However, the ability to induce the formation of dentin is not limited to BMPs. Dentin matrix protein (DMP)-1 has been shown to nucleate apatite crystals and to induce dentin for-mation.41,42 Moreover, bone sialoprotein (BSP) can also stim-ulate the differentiation of pulp cells into cells that are capable of secreting mineralizable matrices in pulp exposure sites.43,44 Interestingly, different morphologic characteristics are observed when dentin is induced by different factors (e.g., BSP-induced dentin appears to be different from BMP-induced dentin). Such results raise the intriguing possibility that it might be possible to select a specifi c type of biological inducer of dentin repair according to the patient’s dentin needs. Notably, all these morphogenic factors can be found in dentin matrices and are presumptive inducers of DPSC differentiation into odontoblast-like cells.43,45,46

Recent studies from our laboratory have added evidence for the important role of the bioactive molecules that are present in dentin as inducers of differentiation of pulp stem cells into odontoblasts.22 We have observed that SHED seeded in scaffolds surrounded by dentin differentiated into odontoblasts, as demonstrated by the acquisition of markers of differentiation such as DMP-1, dentin sialophosphopro-tein, and matrix extracellular phosphoglycoprotein. In contrast, SHED seeded in scaffolds without dentin, or in scaffolds surrounded by dentin that had been previously deproteinized by long-term treatment with sodium hypo-chlorite, lost their ability to differentiate into odontoblasts. In search of the specifi c dentin proteins that were mediating the odontoblastic differentiation of SHED, we performed a series of neutralizing antibody experiments. These studies demonstrated that dentin-derived BMP-2, but not BMP-7, is necessary for the differentiation of stem cells into odon-toblasts.22 Such results from cell and molecular biology experiments can provide guidance for translational experi-ments aimed at the regeneration of dentin via targeted induction of odontoblastic differentiation of DPSCs.

Scaffolds for dental pulp stem cells

Mammalian cells require interactions with their microenvi-ronment to survive, proliferate, and function. In tissue phys-iology, these three-dimensional (3-D) environments are largely composed of extracellular matrix proteins. In tissue engineering, these 3-D structures are initially provided to the cells through the use of biodegradable and biocompat-ible scaffolds.1 They provide an environment that allows for the adhesion of cells and their proliferation, migration, and differentiation until these cells and the host cells begin to secrete and shape their own microenvironment. Therefore, scaffolds are considered a critical component of tissue engineering.47,48

Scaffolds made of synthetic polymers allow for the manip-ulation of their physicochemical properties such as degrada-tion rate, pore size, and mechanical resistance. The most common synthetic polymers in tissue engineering are likely poly-(l-lactic acid) (PLLA), poly-(glycolic acid) (PGA), and the copolymer poly-(lactic-co-glycolic acid) (PLGA). These scaffolds are biodegradable and biocompatible and allow for cell growth and differentiation, making them highly suitable for tissue engineering applications.17,45,49 The degradation rate can be controlled by the proportion of PLLA/PGA used in the manufacturing of these scaffolds. Notably, it is important for the rate of scaffold degradation to be compatible with the rate of tissue formation. In other words, the scaffold should be designed to provide structural integrity for the cells used in tissue engineering until the newly formed tissue becomes autosustainable.50 One of the fi rst examples of successful replacement of scaffold by dental tissues was the use of copo-lymers (PGA/PLLA and PLGA) that allowed for the engi-neering of complex dental structures with characteristics similar to the crowns of natural teeth.17

We have used PLLA scaffolds extensively in dental pulp tissue engineering.19–22 The PLLA scaffolds are cast inside the tooth slices prepared in the cervical area of extracted sound human third molars (Fig. 2). Stem cells are seeded in the tooth slice/scaffolds and transplanted into the subcuta-neous space of immunodefi cient mice. We have named this experimental approach the tooth slice/scaffold model of dental pulp tissue engineering.19–22,51

Our studies have demonstrated that 21–28 days after transplantation, DPSCs seeded in tooth slice/scaffolds and transplanted into mice generate a tissue with morphological characteristics similar to those of human dental pulp (Fig. 1).19–22 We have also investigated the effect of the method for the creation of pores in the scaffolds on the differentia-tion of DPSCs into odontoblasts and on the generation of pulp-like tissues. These experiments revealed that scaffolds generated with gelatin or salt porogens resulted in similar proliferation of DPSCs, but the expression of odontoblastic markers (e.g., DMP-1) was higher in gelatin-based scaffolds in vitro.21 These initial experiments demonstrated that the structural characteristics of the scaffold may play a signifi -cant role in the differentiation of DPSCs.

From a translational standpoint, it would be benefi cial for scaffolds designed for dental pulp tissue engineering

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purposes to be made of injectable materials. The goal of these injectable scaffolds is to allow for stem cell transplan-tation throughout the full extent of the root canal and pulp chamber. An excellent example of such an approach was recently described by Galler and colleagues.52 In this case, self-assembling multidomain peptide hydrogels were gener-ated and characterized as highly biocompatible and inject-able. Interestingly, the addition of a matrix metalloprotease 2 specifi c cleavage site and a cell adhesion motif (i.e., RGD) enhanced cell survival and induced cell motility within these hydrogels. In summary, scaffold development and charac-terization is quickly becoming a critically important new area in the fi eld of dental materials. We fi rmly believe that this is an emerging area that will play a critical role in the translation of laboratory fi ndings to the application of stem cell-based tissue engineering approaches in the dental clinic.

Blood vessels and tooth tissue regeneration

Vasculogenesis is defi ned as de novo formation of blood vessels. Temporal and spatial regulation of vasculogenesis is required for normal embryogenesis. Indeed, loss of a single allele of the VEGF gene causes early embryonic lethal-ity.53,54 There is solid evidence for a functional link between vasculogenesis and bone development.55,56 It is also well known that the teratogenic effect of thalidomide is caused by aberrant vasculogenesis leading to impaired long bone development and limb truncation.57 We have recently observed that SHED have the potential to differentiate into functional vascular endothelial cells via a process that closely resembles that of vasculogenesis,19,20 as depicted graphically (Fig. 3). We have reported that DPSCs differen-tiate themselves into endothelial cells that make functional, blood-carrying blood vessels.19,20 These fi ndings raise the intriguing possibility that stem cells of dental pulp origin might be useful in the treatment of severe ischemic condi-tions of the heart, brain, or limbs. Furthermore, it is unques-tionable that the success of tissue engineering relies heavily upon the rapid establishment of local microvascular net-works to provide blood and nutrients for cells that are engaged in tissue regeneration processes. More specifi cally, one of the critical challenges of dental pulp tissue engineer-ing is the generation of a functional vascular network, con-

sidering the anatomical constraints imposed by the fact that all vascularization must access the root canal through the apical foramen. Therefore, much research is needed in the area of induction of vasculogenesis accompanying efforts of dental pulp tissue engineering. Notably, the use of stem cells as a single cellular source for blood vessels and for “tissue making” is obviously very attractive from a translational standpoint.

Angiogenesis is the process of new blood vessel forma-tion from preexisting vasculature. Therefore, it is fundamen-tally different from the process of vasculogenesis. While vasculogenesis is critically important in the early stages of embryonic development, angiogenesis allows for the remod-eling of the vascular networks later in embryonic develop-ment and plays a major role in postnatal physiological responses (e.g., wound healing). In the context of the dental pulp, it is well known that conservative pulp treatments such as direct pulp capping trigger wound healing events that are orchestrated by an exquisitely regulated angiogenic

Fig. 2. Tooth slice/scaffold model of dental pulp tissue engineering. Highly porous biodegradable poly-l-lactic acid scaffolds were cast in the pulp chamber of 1.5-mm-thick human tooth slices. The porogen used here was NaCl particles. The higher magnifi cation image depicts a pore containing multiple SHED cells

Fig. 3. Schematic representation of the multipotency of dental pulp stem cells. In this hypothetical example, factor A induces vasculogenic differentiation of the dental pulp stem cells, while factor B induces odontoblastic differentiation

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response. During physiological wound healing, cells from wounded sites release chemotactic factors that contribute to the organization of a transient infl ammatory process.58–60 Notably, local cells release angiogenic factors that quickly organize a robust proangiogenic response that allows for the infl ux of infl ammatory cells and provides the oxygen and nutrients that are required to maintain the high meta-bolic demands of cells actively engaged in tissue repair.60 In the dental pulp, elegant work has recently shown that endothelial cell injury is involved in the recruitment of odontoblastic-like cells.61

VEGF is considered the most important regulator of vasculogenesis and angiogenesis in physiological as well pathological conditions.62,63 VEGF induces endothelial cells to form capillary structures when seeded in 3-D collagen gels.64 In vivo, VEGF enhances permeability and induces potent proangiogenic responses.65,66 Notably, VEGF plays a critical role in the regulation of angiogenesis by enhancing endothelial cell survival.64 We have observed that VEGF induces angiogenesis and enhances the survival of dental pulp cells from human tooth slices transplanted in the sub-cutaneous space of immunodefi cient mice.67 We have also demonstrated that VEGF induces the differentiation of DPSCs (i.e., SHED) into endothelial cells.20 The Nakashima group has elegantly shown that porcine pulp stem cells enhance local blood fl ow in experimental ischemic sites by secreting angiogenic factors (e.g., VEGF) and inducing an angiogenic response by host endothelial cells.68 Collectively, these data suggest that a local increase in VEGF availability is highly benefi cial for stem cell-mediated regeneration of dentin and pulp. Notably, dentin matrices contain VEGF,33 which likely contributes to the angiogenic responses medi-ated by dentin extracts.69 We have recently begun to explore the possibility of incorporating VEGF in the scaffolds that are used for transplantation of stem cells for dental pulp tissue engineering purposes.

A major challenge for stem cell-based tissue regeneration is to ensure rapid establishment of effi cient blood vessel net-works that allow for the survival of transplanted cells and provide the infl ux of oxygen and nutrients required to main-tain the high metabolic demands of cells participating in tissue regeneration. Our laboratory has made the novel and unexpected observation that SHED have the potential to differentiate into functional blood vessels in vivo.19,20 This raises the exciting possibility that dental pulp stem cells may constitute a single cell source for regenerating the tissue in question (e.g., dental pulp, dentin), while providing at the same time the required vascular network that will support the newly formed tissue. Notably, during embryonic develop-ment the molecular cues required for timely differentiation of stem cells are inherently regulated. However, the same is not necessarily true when stem cells are used therapeutically in foreign microenvironments. Therefore, studies focused on the cellular and molecular signaling events regulating the determination of stem cell fate will be critically important for the translation of laboratory fi ndings to the clinic. These studies should provide the knowledge to determine the nature of biological modifi ers that can guide stem cells toward the desired differentiation paths.

Prospectus for stem cell-based dental tissue engineering

Stem cell-based regenerative therapies certainly hold much potential in the treatment of medical and dental conditions. Indeed, many patients around the world have already ben-efi ted from such therapies. However, the decision to incorpo-rate stem cell-based therapies into routine clinical dental practice requires careful analysis of the risks and benefi ts associated with the procedure. For example, while the poten-tial benefi ts of stem cell transplantation for patients with hematological cancer tend to outweigh the risks, the same may not be necessarily true for their use in dental proce-dures. It is unquestionable that the processes of storage and expansion of stem cells in laboratory settings, as well as the transplantation of these cells back to the patient, carry certain risks. There is a risk of transformation of the stem cells, and there is also a risk of unwanted contamination of these cells with pathogens during these procedures.70 While these risks are relatively small, they exist and cannot be ignored. Indeed, it is certainly imperative that patients undergoing such pro-cedures with stem cells in investigative or clinical settings are made fully aware of such risks.

Based on the analysis of the existing literature, and our own clinical and research judgment, it is becoming increas-ingly clear that dentistry will embrace new concepts of tissue regeneration. It is likely that such approaches will involve the use of stem cell-based therapies combined (or not) with biomimetic approaches. While the use of stem cells brings many new therapeutic opportunities, and perhaps will allow for the treatment of dental conditions that are untreatable with today’s materials and procedures, one must proceed with caution. It is imperative for clinical procedures with stem cells to be supported by solid basic and translational research. It will be only through rigorous research that the full extent of the potential risks involved in the use of these cells will be understood, and the means to prevent (or overcome) them will be discovered. It will also be only through research that the biology of tooth-related stem cells and the therapeutic potential of these cells will be better understood.

In conclusion, stem cell-based dental tissue regeneration is a new and exciting fi eld that has the potential to transform the way that we practice dentistry. Its future will depend on the understanding of the biology of the cells that will be used to regenerate tissues, and its boundaries will be demar-cated by an in-depth knowledge of the potential risks and likely benefi ts associated with each regenerative procedure. The fi eld of stem cell-based regenerative dentistry is complex and multidisciplinary by nature. Progress will depend on the collaboration between clinicians and researchers from diverse fi elds (e.g., biomaterials, stem cell biology, endodontics) working together toward the goal of developing biological approaches to regenerate dental and craniofacial tissues.

Acknowledgments The authors would like to thank Chris Jung for his contribution with the schematic drawing presented here, and the

6

members of the Angiogenesis Research Laboratory at the University of Michigan School of Dentistry for their invaluable input and techni-cal help during the execution of these studies. We also would like to acknowledge the support received from CAPES (Brazilian Govern-ment) for the pursuit of the studies presented in this review.

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Human Dental Pulp Stem Cells: From Biologyto Clinical Applications

RICCARDO D’AQUINO1, ALFREDO DE ROSA2, GREGORIO LAINO2,FILIPPO CARUSO2, LUIGI GUIDA2, ROSARIO RULLO2, VITTORIO CHECCHI3,LUIGI LAINO1, VIRGINIA TIRINO1, AND GIANPAOLO PAPACCIO1�1Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia,TESLab, Secondo Ateneo di Napoli, Napoli, Italy2Dipartimento di Scienze Odontostomatologiche, Ortodontiche e Chirurgiche,Secondo Ateneo di Napoli, Napoli, Italy3Dipartimento di Discipline Odontostomatologiche, Universita degli Studi diBologna, Alma Mater Studiorum, Bologna, Italy

ABSTRACT Dental pulp stem cells (DPSCs) can be found within the ‘‘cell rich zone’’ of dentalpulp. Their embryonic origin, from neural crests, explains their multipotency. Up to now, two groupshave studied these cells extensively, albeit with different results. One group claims that these cellsproduce a ‘‘dentin-like tissue’’, whereas the other research group has demonstrated that these cellsare capable of producing bone, both in vitro and in vivo. In addition, it has been reported that thesecells can be easily cryopreserved and stored for long periods of time and still retain theirmultipotency and bone-producing capacity. Moreover, recent attention has been focused on tissueengineering and on the properties of these cells: several scaffolds have been used to promote 3-Dtissue formation and studies have demonstrated that DPSCs show good adherence and bone tissueformation on microconcavity surface textures. In addition, adult bone tissue with good vasculariza-tion has been obtained in grafts. These results enforce the notion that DPSCs can be usedsuccessfully for tissue engineering. J. Exp. Zool. (Mol. Dev. Evol.) 310B, 2008. r 2008 Wiley-Liss, Inc.

How to cite this article: D’aquino R, De Rosa A, Laino G, Caruso F, Guida L, Rullo R,Checchi V, Laino L, Tirino V, Papaccio G. 2008. Human dental pulp stem cells: frombiology to clinical applications. J. Exp. Zool. (Mol. Dev. Evol.) 310B:[page range].

DENTAL PULP EMBRYOGENESIS

Embryonic cells migrate from the neural creststo reinforce head and neck mesenchyme stronglydetermining the development of this area of thehuman body. During the sixth week of embry-ogenesis, ectoderm covering the stomodeumbegins to proliferate, giving rise to the dentallaminae. Reciprocal interactions betweenectoderm and mesoderm layers lead to placodeformation. One of these thick, ovoid ectodermalstructures develops into tooth germs, wherecells, belonging to the neural crest, will differ-entiate into the dental germ, containing bothdental papilla and follicle. Therefore, dentalpulp is made of ecto-mesenchymal components,containing neural crest-derived cells, which dis-

play plasticity and multipotential capabilities(Sinanan et al., 2004).

Pulp is externally separated from dentin byodontoblasts and by Hohl’s subodontoblastic cells,that are pre-odontoblasts (Goldberg and Smith,2004). Adjacent to this layer the pulp is rich incollagen fibers and poor in cells. Then, another,more internal layer, contains progenitor cellsand undifferentiated cells, some of which are

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jez.b.21263

Received 13 November 2008; Accepted 14 November 2008

Grant sponsor: MIUR; Grant numbers:PRIN 2005–2007; SIRIO-FIRB 2007.�Correspondence to: Gianpaolo Papaccio, Department of Experi-

mental Medicine, Section of Histology and Embryology, 5 via L.Armanni, Second University of Naples, 80138 Naples, Italy.E-mail: [email protected]

r 2008 WILEY-LISS, INC.

JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 310B (2008)

considered stem cells. (Jo et al., 2007). From thislayer, undifferentiated cells migrate to variousdistricts where they can differentiate underdifferent stimuli and make new differentiatedcells and tissues. The final, innermost layer isthe core of the pulp; this area comprises thevascular plexus and nerves. Up to the more recentdiscoveries (Gronthos et al., 2000; D’Aquino et al.,2007), researchers hypothesized that Dental pulpstem cells (DPSCs) were present in this layer(Fitzgerald et al., 1990). Actually, only undiffer-entiated perivascular cells can be found in it.

The third molar tooth germ begins developmentaround the sixth year of life. Until this time,embryonic tissues of dental lamina remain quies-cent and undifferentiated within the jaw of thechild. Although crown mineralization begins duringthe eighth year of life, often third molar roots arestill incomplete at the age of 18. This means thatthe structure of those teeth is still immature at thisage and a conspicuous pool of undifferentiated cells,resident within the ‘‘cell rich zone’’ of the dentalgerm pulp, are needed for development.

STUDIES ON DPSCS

Several studies have been carried out to verifywhether stem cells could become a source of stabledifferentiated cells, capable of inducing tissueformation: during the embryonic developmentthese cells proliferate and differentiate to generateall tissues. Postnatal stem cells have an extra-ordinary plasticity. The individual cells, whenexpanded into colonies, retain their multilineagepotential. Although their number is higher beforethe birth, there are several ‘‘loci’’ or ‘‘niches’’inhabited by a significant number of stem cellswithin the adult human body (Laino et al., 2005).Among the adult tissues, dental pulp, the softconnective tissue entrapped within the dentalcrown, is an extremely rich site for stem cellcollection: owing to its peculiar formation, thepulp chamber is a sort of ‘‘sealed niche’’ and mayexplain why it is possible to find a rather largenumber of stem cell there.

During the sixth week of embryogenesis, theectoderm covering the stomodeum begins toproliferate, giving rise to the dental laminae.Ectoderm–mesoderm interactions then lead toplacode formation. One of these ovoidal ectoder-mal structures develops into tooth germs, whereneural crest cells differentiate into the dentalorgan, dental papilla and dental follicle. Therefore,dental pulp is made of both ectodermic and

mesenchymal components, containing neuralcrest cells that display plasticity and multipoten-tial capability. These adult stem cells have beencalled DPSCs, when found in permanent teeth,and SHEDs (Stem Cells from Human ExfoliatedDeciduous), when found in deciduous teeth.DPSCs have been isolated for the first time in2000 by Gronthos et al.; these cells exhibited adifferentiation potential for odontoblastic, adipo-genic and neural cytotypes. The same groupisolated SHEDs from deciduous teeth and com-pared DPSCs with bone marrow stem cells(BMSCs) (Miura et al., 2003). They reported asuperimposable ability of these cells to formcalcified tissue although different lineages wereobserved: DPSCs seemed to undergo odontoblasticdifferentiation, whereas BMSCs became osteo-blasts after loading on HA-TCP scaffold. Thesame authors reported that SHEDs were differentfrom DPSCs, affirming that they were ‘‘moreimmature’’: this probably because they were ableto differentiate into a variety of cell types, to anextent greater than than DPSCs. Actually, theydemonstrated that SHEDs were able to differenti-ate into a variety of cell types. They called some ofthese cytotypes ‘‘osteoblast-like’’ and ‘‘odonto-blast-like’’ cells; because of their immaturity(Miura et al., 2003). The main commitment ofthese cells seemed to be the formation of amineralized tissue (Luisi et al., 2007; Wei et al.,2007), similar to dentin, (Kitagawa et al., 2007), asshown by in vivo transplantation of these cells intoimmunodeficient mice (Miura et al., 2003). Thesecells are involved in the development of severalbut different hard tissues, including crown androot dentin, cementum and alveolar bone; a role inroot reabsorption of deciduous teeth has beenhypothesized for DPSCs (Yildirim et al., 2008). Invitro DPSCs have been shown to produce sporadicbut densely calcified nodules (Gronthos et al.,2000). In addition, the same group (Gronthoset al., 2002) found that these cells are capable offorming ectopic mineralized tissue, similar todentin, but only when grafted in vivo or whenplaced on the surface of human dentin in vivo(Batouli et al., 2003) and when exposed to toothgerm conditioned medium (Yu et al., 2006) orsimilar differentiation factors (Tonomura et al.,2007). These studies, performed in deciduous andpermanent teeth, were done without specificallyselecting stem cells (Gronthos et al., 2000, 2002;Batouli et al., 2003), which under specific stimuli,differentiate into several cell types, includingneurons and adipocytes. Sporadic dense nodules

R. D’AQUINO ET AL.2

J. Exp. Zool. (Mol. Dev. Evol.)

were found to be formed in vitro, but theyunderwent mineralization and bone or dentin-likeformation only when grafted in vivo. These data,regarding a multipotential differentiative abilityof those cells were further confirmed (Iohara et al.,2006; Srisawasdi and Pavasant, 2007) althoughthe main commitment remains to form miner-alized tissues, as evidenced in several papers (Uenoet al., 2006; Otaki et al., 2007; Hosoya et al., 2007);however, this is what usually happens duringdental tissues development. (Bosshardt, 2005).

Moreover, other researchers have investigatedanother aspect of DPDCs, i.e. their putativeimmunosuppressive activity (Pierdomenico et al.,2005). This would be fascinating, if confirmed.

STROMAL BONE-PRODUCING DPSCSAND STROMAL BONE-PRODUCINGSHEDS: MAIN PROPERTIES AND

PERFORMANCIES

Laino et al. (2005) isolated a selected subpopula-tion of DPSC called Stromal Bone ProducingDental Pulp Stem Cells (SBP-DPSCs), multipo-tential cells that could give rise to a variety of celltypes and tissues including adipocytes, neural cellprogenitors and myotubes (Laino et al., 2005,2006; Papaccio et al., 2006). Previous experimentsdemonstrated that stem cells, isolated from thepulp of human exfoliated deciduous teeth andexpanded in vitro, showed a�9% positivity forSTRO-1, considered an early marker of mesen-chymal stem cells (Gronthos et al., 2002). Thisantibody identified a cell surface antigen ex-pressed by the osteogenic fraction of stromalprecursors in human bone marrow as well as inerythroid precursors. In particular, the STRO-1antigen is extremely important in selecting dentalpulp cells (Yang et al., 2007a,b). SBP-DPSCs,representing roughly 10% of dental pulp cellsdisplay their multipotency (Laino et al., 2005,

2006); in fact they differentiate into smoothmuscle cells, adipocytes, neurons and osteoblasts.The latter is also substantiated by the RUNX-2expression, a transcription factor essential forinducing osteoblast differentiation.

These cells, selected for c-kit, CD34 and STRO-1positivity (Fig. 1), were found to be able to producean autologous woven bone tissue in vitro (Fig. 2).Head and neck hard tissues of the body have, otherthan a mesodermal origin, a neural crest source, andthis has been demonstrated by the expression of thec-kit antigen, which is usually expressed in neuralcrest-derived cells, such as melanocyte precursors.The latter is compatible with the presence of c-kitexpressing cells in the area of developing teeth.Therefore, CD34 and c-kit co-expression was used toisolate a population of stromal stem cells of neuralcrest origin.

PASSAGES AND SENESCENCE OF DPSCS

DPSCs can survive for long periods and can bepassaged several times. It is possible to obtainmore than 80 passages without clear signs of

Fig. 1. Cytoarchitecture of a dental pulp stem cell. Cells,selected for c-kit1, CD341 and STRO-11 were observed undera confocal microscopy. The green fluorescence stains thecell cytoskeleton (revealed by phalloidin); DAPI stains thenucleus. Original magnification � 400.

Fig. 2. Living autologous bone nodules. (A) Calcified nodule within cultured dental pulp stem cells after 40 days. Originalmagnification � 100. (B) Living autologous bone (LAB) chip obtained after 60 days of culture of dental pulp stem cells.

DENTAL PULP STEM CELLS AND CLINICAL USE 3


senescence (Laino et al., 2005, 2006). Remarkably,after several passages, DPSCs still exhibit plasti-city and capacity for nodule formation and arecapable of forming bone chips in vitro. Osteoblast-derived cells produce a large-scale woven bone,which was observed in at least 100 25 cm2 flasks.They can lose their capability to form woven bonechips when detached from their substrates, be-cause they lose cell to cell contacts, which are ofprimary relevance for extracellular matrix secre-tion (D’Aquino et al., 2007).

IN VIVO STUDIES

The woven bone tissue generated in vitro by SBP-DPSCs, called living autologous bone (LAB), isremodeled into a lamellar bone when transplantedin vivo (Laino et al., 2005, 2006; d’Aquino et al.,2007). Actually, after transplantation in vivo, thetissue is remodeled to form a lamellar bone throughco-differentiation of SBP-DPSC into osteoblasts andendotheliocytes (d’Aquino et al., 2007). In fact, SBP-DPSCs produce bone but not dentin, as shown bymRNA transcript that express all markers of boneincluding osteocalcin, Runx-2, collagen I, but notdentin sialo phospoho protein (DSPP), which isspecific for dentin, by the high expression of alkalinephosphatase (Laino et al., 2005, 2006) and by in vivohistomorphometry (d’Aquino et al., 2007). In thelatter paper it was shown that SBP-DPSCs changetheir antigenic surface expression during differentia-tion. Moreover, after in vivo transplantation, acomplete integration of vessels within bone chipstakes place, leading to the formation of a vascular-ized bone tissue (Fig. 3).

During their differentiating process, SBP-DPSCswere observed to change their surface antigen

expression, as they differentiated. At day 40, startingfrom a common Flk-11/STRO-11/CD441 progenitor,stem cells start to differentiate in two cytotypes:about 70% of them became Flk-11/STRO-1�/CD441/RUNX-21 osteogenic progenitor cells, whereas theremaining 30% became Flk-11/STRO-11/CD441/CD541 endothelial cells. Interestingly, these cellswere always negative for DSPP, a marker of dentin,demonstrating that the hard tissue they produce isbone and not dentin: the production of dentinactually needs a pool of factors present in dentalpapilla (Lesot et al., 2001; Yuasa et al., 2004).

This observation indicates that vasculogenesistakes place in vitro within the newly synthesizedtissue. The formation of vessels explains the vitalityof bone chips when transplanted in immunosup-pressed rats.

CO-DIFFERENTIATION AND VESSELFORMATION

Transplantation results obtained with SBP-DPSCs are of extreme interest for the develop-ment of novel therapies. After transplantation inimmunosuppressed rats, both woven chips andstem cells challenged with a scaffold become adultbone (d’Aquino et al., 2007). In addition, thedimensions of the obtained bone are the same asthe grafted chips or the scaffolds (Trubiani et al.,2003). In particular, complete Haver’s channels,containing blood vessels, and surrounded by bonearranged in a lamellar configuration have beenobtained. This is the first demonstration of acomplete bone obtained from stem cells.

During the ossification process, SBP-DPSCs giverise to both osteoblasts and endotheliocytes,leading to the formation of an adult bone tissueafter in vivo transplantation. The presence ofvessels and their complete integration with host(D’Aquino et al., 2007), other than being the firstdemonstration of a complete tissue growth fromstem cells, is of great importance for therapy. Thisis a model of synergic differentiation, whose keyaspect is the expression of flk-1, which is pivotalfor the coupling of osteogenesis and vasculo-genesis. This also represents an interesting aspectfor development and a complete and efficientthree-dimensional tissue reconstruction therapy.

CRYOPRESERVATION OF DPSCS

Cryopreservation of cells and tissue, mainly ofthe reproductive system, has been significantlyimproved recently, but to date prevailingly hema-topoietic stem cells have been cryopreserved and

Fig. 3. In vivo transplantation results. Figure showing abone sample stained with hematoxylin–eosin staining ob-tained after in vivo transplantation of LAB. Original magni-fication � 100.



then successfully utilized for transplantation.Moreover, to date there are no reports on theability of either stem cells or already differentiatedcells to re-start proliferation, differentiation andnew tissue formation for therapeutic use.

After long-term cryopreservation (2 years),osteoblasts differentiated from SBP-DPSCs, arestill capable of quickly re-starting proliferationand the production of mineralized matrix, in amanner similar to what we have already demon-strated for fresh cells (Laino et al., 2005, 2006;Papaccio, 2006). The differences in percentagesregarding STRO-1 and flk-1 with respect to thepercentages observed for the other stem cellantigens are owing to the fact that we performedmulti-parametric cell sorting using both morpho-logical and antigenic criteria, and sorted first forCD117 and CD34 together and then sequentiallyfor STRO-1 and flk-1 antigens, before and aftercryopreservation. Thus, pre-endothelial cells, suchas pericytes positive for both CD117 and CD34,could have altered the overall percentages. Thesecells would be responsible for the differentiation ofendothelium, which occurs in parallel with osteo-blast differentiation, as demonstrated in theembryo during the ossification process. Moreover,both osteoblasts and endotheliocytes express theVEGF-2 receptor (flk-1).

Furthermore, after thawing, no apoptotic deathwas observed, and cells retained their differentia-tion multipotency, all of which are of interestwhen assessing the suitability of stem cells for useafter cryopreservation. Moreover, osteoblasts pro-duced a large-scale woven bone, which wasobserved in at least 100 25 cm2 flasks. Samples ofthis bone, when transplanted into immunosup-pressed rats, were remodeled into lamellar bone,further demonstrating their vitality.

Ultrastructurally, osteoblasts were cuboidal inshape, forming a layer along the border of theextracellular matrix, as observed in vivo duringosteogenesis. These differentiated cells containedan extremely diffuse RER as well as matrixmembrane vesicles, containing crystal-like struc-tures. These ultrastructural observations con-firmed that cells were unaltered.

A study was performed on cryopreserved tissuesamples of minced periodontal ligament (Seo et al.,2005). Conversely, another group (Papaccio et al.,2006) obtained completely negative results cryo-preserving whole pulps. This is probably owing tothe fact that dental pulp is a loose connective or,more appropriately a ‘‘mucous connective’’tissue with high water content. In any case,

cryopreservation of whole dental pulp does leadto safe recovery (Zhang et al., 2006a). Thesefeatures and abilities make these cells attractivefor therapeutic three-dimensional tissue recon-struction, with the potential of tailoring storageand recovery to the needs of the patient.

BONE TISSUE ENGINEERING STUDIES

DPSCs showed differentiation profiles similar tothose showed during bone differentiation (Hwanget al., 2008) and this event make them veryinteresting as a model to study osteogenesis (Liuet al., 2007) and the relationship with scaffolds(Zhang et al., 2006a,b).

SBP-DPSCs, when undergoing differentiationinto pre-osteoblasts, deposit an extracellular ma-trix that becomes a calcified woven bone tissuecalled LAB (Laino et al., 2005). Calcein stainingpositivity, in addition to the other markers,strongly confirmed the presence of calcium depos-its within this tissue, and stresses the effective-ness of the mineralization process. In addition,ALP activity increases significantly in parallel tocell differentiation. In addition, it has been shownthat there are no differences regarding expansionrate, number of calcification centers and LABnodules obtained per well of cells selected fromyounger (up to 29 years) and older (30–45 years)subjects (Laino et al., 2005).

Interestingly, positivity for CD44, seen in in-flammatory processes of the pulp (Pisterna andSiragusa, 2007), osteocalcin and RUNX-2, evi-dences that cells were differentiating toward theosteoblastic lineage. The involvement of Runx-related gene in dental pulp mineralization pro-cesses has been intensely investigated (Zhenget al., 2007). Strong expression of RUNX-2, atranscription factor essential for osteoblast differ-entiation, is of primary importance, because it isclosely related to the promotion of ossification; inaddition, targeted disruption of RUNX-2 results incomplete lack of woven bone formation by osteo-blasts. Other than forming woven bone in vitro ithas been demonstrated that this tissue undergoesremodeling, when transplanted in immunocom-promised rats, and becomes a lamellar bone withentrapped osteocytes (Laino et al., 2006; D’Aquinoet al., 2007). The latter confirms that SHEDsdifferentiate in osteoblasts and then osteocytes,differently to what has been previously reported(Miura et al., 2003) but similarly to DPSCs (Lainoet al., 2005). Actually, it has been speculated thatSHEDs possess the ability to differentiate in



functional odontoblast-like cells and that unlikeDPSCs, they did not directly differentiate inosteoblasts (Miura et al., 2003). These authorsother than using different methods and markersto select stem cells, which may lead to differentresults, needed the use of an osteoinductivetemplate for in vivo transplants (Zhang et al.,2006a,b). The findings of Laino et al. (2006)disagree because the cells that they selected areprobably different as they differentiate intoosteoblasts and produce a woven or fibrous bone,which, without the need of osteoinductive tem-plates, after in vivo transplantation, is remodeledinto a lamellar bone. Therefore, these cells appearto be good candidates for bone tissue reconstruc-tion protocols and bone regeneration models,because of good cellular morphology and highBMP-2 and VEGF secretion (Graziano et al.,2007). The role played by BMP2 is crucial andthis protein has been hypothesized as biologicaltool in gene therapy of dentin regeneration(Nakashima et al., 2006). The concave texturingof the substrate elicits cytoarchitectural responsesand adaptation in which the cells appear to favorintimate contacts with the secondary microcon-cavities and cellular polarization in human tissues(Zhang et al., 2003). Such behavior is accompaniedby increased release of BMP-2 and VEGF into theculture medium and by higher ALP activity. It islikely that increased release of potent factors suchas BMP-2 and VEGF and the higher ALP activitycould have significant biological ramifications. Bytheir proven involvement and potency in boneformation and angiogenesis, these factors andenzymatic activity may influence the responsesand developmental program of stromal-derivedcells via autocrine mechanisms and it is alsoinfluenced by surrounding cells via paracrinepathways. In this likely scenario, increased levelsof BMP-2 and VEGF could be responsible for thegreater amounts of bone tissue they observed invitro (Graziano et al., 2008) and after transplanta-tion of the colonized microconcavity-rich scaffold.

Angiogenesis could become itself a main goal forthe clinical application of DPSCs, owing to theimportance that angiogenetic factors (Tran-Hunget al., 2007) have in the native tissue (GrandoMattuella et al., 2007a,b); here, the biologicalevents seem to be supported by nonstem popula-tion such as fibroblasts (Tran-Hung et al., 2006).

Today in vitro bone regeneration studies arelimited by the main difficulty to obtain a cytotypecapable of forming a complete tissue and not onlya monolayer or cells surrounded by a mineralized

matrix. Owing to their high proliferation rate andefficiency in producing bone chips, DPSCs seem tobe the best candidates to study bone formationwith respect to BMSCs, whose efficiency is limitedby the fact that they differentiate into osteoblastsand produce small calcified nodule, but not chipsof bone tissue.

Scaffold’s structure and its influence upon cells area breaking point in bone tissue engineering. To studythe relationship between biomaterials and stem cellsduring their osteogenic differentiation process morespecifically in bone tissue building, we need that cellswould be able to actively proliferate, differentiateand produce a bone tissue as better as they can.Therefore, SBP-DPSCs may be a good standard tostudy the ossification process on substrates suitablefor clinical application in bone reconstruction (Gra-ziano et al., 2007, 2008). In fact, their ability toproduce LAB already in vitro is of great interest tofurther analyze the effects of scaffolds, which aremediated by cell interactions with substrates. Inter-estingly, these cells show a lifespan and an high andlong-lasting osteogenic capacity (Papaccio et al.,2006). These findings have been further substan-tiated by the results obtained challenging thesurface texturing with SBP-DPSCs in a three-dimensional cell culture system. In fact, dataobtained from engineered tissues, made on differ-ent scaffolds in a roller apparatus for 30 days(Graziano et al., 2008) clearly demonstrate that anew-formed bone tissue is obtained and that theirdifferent thickness strictly depends on the grow-ing surface and, in particular, on the specifictexture that has been adopted. Actually, theobtained new bone is of considerable thicknesswhen the scaffold is made of a micro concavesurface; it is of a lesser thickness in the case of asmooth surface and it is almost absent in the caseof a convex surface, confirming all the resultsobtained with plane cultures. At the end of therotating period (30 days) on convex surfaces,instead of building a bone tissue, cells were foundat the bottom of the scaffold. Moreover, concavetexturing induces an earlier and quantitativelymore enhanced bone differentiation: in fact, it hasbeen observed that BAP expression occurs earlier;cells adopt a polygonal shape with filopodia-likeand lamellipodia-like extensions (cells can beregarded as spreading and differentiating), andnuclear polarity is observed, an index of secretion,cell activity and matrix formation (Graziano et al.,2008). These observations open the way to a moreextensive application of tissue engineering ofcraniofacial tissues (Mao et al., 2006).



In conclusion: (1) dental pulp is a remarkablesite of stem cells; (2) collecting stem cells fromdental pulp is a noninvasive practice that can beperformed in the adult during life and in theyoung after surgical extraction of wisdom teeth, acommon surgical practice; (3) tissue sacrifice isvery low when collecting dental pulp stem cells; (4)several cytotypes can be obtained from dental pulpstem cells owing to their multipotency; (5)transplantation of new-formed bone tissue ob-tained from dental pulp stem cells leads to theformation of vascularized adult bone and integra-tion between the graft and the surrounding hostblood supply; (6) dental pulp stem cells can becryopreserved and stored for long periods; (7)dental pulp is ideal for tissue engineering and forclinical use in several pathologies requiring bonetissue growth and repair. In addition, toothextraction is a clinical/therapeutical need. If bonemarrow is the site of first choice for hematopoieticstem cell collection, dental pulp must be consid-ered one of the major sites for mesenchymal cellcollection. The good results obtained up to nowreinforce this thought.

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