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Cells 4 health

HealthTeeth Science pack


Ulternative Therapy

Dental Pulp Stem Cells,

a New Era in Tissue Engineering

Abstract

Stem 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. Introduction

Dr. Ghada A. Karien BDS, JDB (Paed) • Paediatric dentist Jordanian Dental Board in paediatric dentistry • MOH/Al-Basheer hospital ghadakarien@gmail.com

The 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.

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Smile Dental Journal Volume 4, Issue 2 - 2009


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 to regenerate 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 unerupted 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.

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

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 Dentistry

In 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.

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. Smile Dental Journal Volume 4, Issue 2 - 2009

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Ulternative Therapy

Refrences 1. 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 medicine. 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 potential 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 scaffold materials. Biomaterials. 2006 Nov;27(33):5658-68. Epub 2006 Aug 17.


Odontology (2011) 99:1–7 DOI 10.1007/s10266-010-0154-z

© The Society of The Nippon Dental University 2011

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

Dental pulp stem cells in regenerative dentistry

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

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 postnatally. In recent years, stem cell research has grown exponentially 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, osteoblasts, 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 fundamental concepts of stem cell biology and tissue engineering within the context of regenerative dentistry. Key words Tissue engineering · Endodontics · Odontoblasts · Endothelial cells · Dentin

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, USA Tel. +1-734-936-9300 e-mail: jenor@umich.edu J.E. Nör Department of Biomedical Engineering, University of Michigan College of Engineering, Ann Arbor, MI, USA J.E. Nör Department of Otolaryngology, University of Michigan School of Medicine, Ann Arbor, MI, USA

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 identification of appropriate cells, the development of conducive 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 conceptual approach to therapy, named “regenerative dentistry,” 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 engineering, and presents a prospectus for the field 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 generating complex tissues and organs.2 These cells can be classified 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 cellular source for cell-based regenerative therapies. However, the use of embryonic stem cells is controversial. Indeed, legal and ethical issues have significantly impaired the feasibility of their use in the laboratory and in the clinic.5 Therefore, this review will not discuss the use of embryonic


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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 finally, 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 first tooth-related stem cell types was found in the pulp of permanent 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 potential, and accessibility. For example, when bone marrow stem cells were compared with DPSCs, DPSCs presented favorable 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

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 immunodeficient mice. After 21 days, the tooth slices were retrieved, fixed, demineralized, and prepared for standard histology. Photomicrographs of hematoxylin/eosinstained 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

the plasticity and the differentiation potential of stem cells of dental origin. And finally, 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 specific 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 regeneration 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 physiological tissue regeneration can also be used therapeutically to guide stem cell differentiation toward specific 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 specifically, there are many similarities between morphogenic factors regulating dentinogenesis and the factors that regulate reparative dentinogenesis.29 One rapidly concludes that the field of dental tissue engineering can benefit 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


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growth factor, fibroblast growth factor, and vascular endothelial growth factor (VEGF) are incorporated into the dentin matrix during dentinogenesis and are retained there as “fossilized” molecules.31–33 Interestingly, when these molecules 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 solubilize dentin and allow the release of bioactive molecules that can potentially regenerate dentin.35 Such events involve the recruitment of DPSCs, their differentiation into odontoblasts, and the secretion of mineralizable matrices.36–38 Collectively, the release of growth factors from the dentin appears to constitute an important mechanism of defense against injuries, allowing for a finite 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 formation.41,42 Moreover, bone sialoprotein (BSP) can also stimulate 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 BMPinduced dentin). Such results raise the intriguing possibility that it might be possible to select a specific 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 sialophosphoprotein, 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 hypochlorite, lost their ability to differentiate into odontoblasts. In search of the specific 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 odontoblasts.22 Such results from cell and molecular biology experiments can provide guidance for translational experiments 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 microenvironment to survive, proliferate, and function. In tissue physiology, 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 biocompatible 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 manipulation of their physicochemical properties such as degradation 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 first examples of successful replacement of scaffold by dental tissues was the use of copolymers (PGA/PLLA and PLGA) that allowed for the engineering 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 subcutaneous space of immunodeficient 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 differentiation 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 significant role in the differentiation of DPSCs. From a translational standpoint, it would be beneficial for scaffolds designed for dental pulp tissue engineering


4 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 magnification image depicts a pore containing multiple SHED cells

purposes to be made of injectable materials. The goal of these injectable scaffolds is to allow for stem cell transplantation 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 generated and characterized as highly biocompatible and injectable. Interestingly, the addition of a matrix metalloprotease 2 specific 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 characterization is quickly becoming a critically important new area in the field of dental materials. We firmly believe that this is an emerging area that will play a critical role in the translation of laboratory findings to the application of stem cell-based tissue engineering approaches in the dental clinic.

Blood vessels and tooth tissue regeneration Vasculogenesis is defined 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 lethality.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 differentiate themselves into endothelial cells that make functional, blood-carrying blood vessels.19,20 These findings raise the intriguing possibility that stem cells of dental pulp origin might be useful in the treatment of severe ischemic conditions of the heart, brain, or limbs. Furthermore, it is unquestionable that the success of tissue engineering relies heavily upon the rapid establishment of local microvascular networks to provide blood and nutrients for cells that are engaged in tissue regeneration processes. More specifically, one of the critical challenges of dental pulp tissue engineering is the generation of a functional vascular network, con-

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

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 formation from preexisting vasculature. Therefore, it is fundamentally different from the process of vasculogenesis. While vasculogenesis is critically important in the early stages of embryonic development, angiogenesis allows for the remodeling of the vascular networks later in embryonic development 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


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response. During physiological wound healing, cells from wounded sites release chemotactic factors that contribute to the organization of a transient inflammatory process.58–60 Notably, local cells release angiogenic factors that quickly organize a robust proangiogenic response that allows for the influx of inflammatory cells and provides the oxygen and nutrients that are required to maintain the high metabolic 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 subcutaneous space of immunodeficient 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 flow 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 beneficial for stem cell-mediated regeneration of dentin and pulp. Notably, dentin matrices contain VEGF,33 which likely contributes to the angiogenic responses mediated 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 efficient blood vessel networks that allow for the survival of transplanted cells and provide the influx of oxygen and nutrients required to maintain 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 development 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 findings to the clinic. These studies should provide the knowledge to determine the nature of biological modifiers 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 benefited from such therapies. However, the decision to incorporate stem cell-based therapies into routine clinical dental practice requires careful analysis of the risks and benefits associated with the procedure. For example, while the potential benefits 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 procedures. 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 procedures 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 increasingly 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 toothrelated 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 field 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 demarcated by an in-depth knowledge of the potential risks and likely benefits associated with each regenerative procedure. The field of stem cell-based regenerative dentistry is complex and multidisciplinary by nature. Progress will depend on the collaboration between clinicians and researchers from diverse fields (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 technical help during the execution of these studies. We also would like to acknowledge the support received from CAPES (Brazilian Government) for the pursuit of the studies presented in this review.

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Human Dental Pulp Stem Cells: From Biology to 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 1 Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, TESLab, Secondo Ateneo di Napoli, Napoli, Italy 2 Dipartimento di Scienze Odontostomatologiche, Ortodontiche e Chirurgiche, Secondo Ateneo di Napoli, Napoli, Italy 3 Dipartimento di Discipline Odontostomatologiche, Universita` degli Studi di Bologna, Alma Mater Studiorum, Bologna, Italy

ABSTRACT Dental pulp stem cells (DPSCs) can be found within the ‘‘cell rich zone’’ of dental pulp. Their embryonic origin, from neural crests, explains their multipotency. Up to now, two groups have studied these cells extensively, albeit with different results. One group claims that these cells produce a ‘‘dentin-like tissue’’, whereas the other research group has demonstrated that these cells are capable of producing bone, both in vitro and in vivo. In addition, it has been reported that these cells can be easily cryopreserved and stored for long periods of time and still retain their multipotency and bone-producing capacity. Moreover, recent attention has been focused on tissue engineering and on the properties of these cells: several scaffolds have been used to promote 3-D tissue formation and studies have demonstrated that DPSCs show good adherence and bone tissue formation on microconcavity surface textures. In addition, adult bone tissue with good vascularization has been obtained in grafts. These results enforce the notion that DPSCs can be used successfully 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: from biology to clinical applications. J. Exp. Zool. (Mol. Dev. Evol.) 310B:[page range].

DENTAL PULP EMBRYOGENESIS Embryonic cells migrate from the neural crests to reinforce head and neck mesenchyme strongly determining the development of this area of the human body. During the sixth week of embryogenesis, ectoderm covering the stomodeum begins to proliferate, giving rise to the dental laminae. Reciprocal interactions between ectoderm and mesoderm layers lead to placode formation. One of these thick, ovoid ectodermal structures develops into tooth germs, where cells, belonging to the neural crest, will differentiate into the dental germ, containing both dental papilla and follicle. Therefore, dental pulp is made of ecto-mesenchymal components, containing neural crest-derived cells, which disr 2008 WILEY-LISS, INC.

play plasticity and multipotential capabilities (Sinanan et al., 2004). Pulp is externally separated from dentin by odontoblasts and by Ho¨hl’s subodontoblastic cells, that are pre-odontoblasts (Goldberg and Smith, 2004). Adjacent to this layer the pulp is rich in collagen fibers and poor in cells. Then, another, more internal layer, contains progenitor cells and undifferentiated cells, some of which are

Grant sponsor: MIUR; Grant numbers:PRIN 2005–2007; SIRIOFIRB 2007. Correspondence to: Gianpaolo Papaccio, Department of Experimental Medicine, Section of Histology and Embryology, 5 via L. Armanni, Second University of Naples, 80138 Naples, Italy. E-mail: gianpaolo.papaccio@unina2.it Received 13 November 2008; Accepted 14 November 2008 Published online in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/jez.b.21263


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considered stem cells. (Jo et al., 2007). From this layer, undifferentiated cells migrate to various districts where they can differentiate under different stimuli and make new differentiated cells and tissues. The final, innermost layer is the core of the pulp; this area comprises the vascular plexus and nerves. Up to the more recent discoveries (Gronthos et al., 2000; D’Aquino et al., 2007), researchers hypothesized that Dental pulp stem cells (DPSCs) were present in this layer (Fitzgerald et al., 1990). Actually, only undifferentiated perivascular cells can be found in it. The third molar tooth germ begins development around the sixth year of life. Until this time, embryonic tissues of dental lamina remain quiescent and undifferentiated within the jaw of the child. Although crown mineralization begins during the eighth year of life, often third molar roots are still incomplete at the age of 18. This means that the structure of those teeth is still immature at this age and a conspicuous pool of undifferentiated cells, resident within the ‘‘cell rich zone’’ of the dental germ pulp, are needed for development. STUDIES ON DPSCS Several studies have been carried out to verify whether stem cells could become a source of stable differentiated cells, capable of inducing tissue formation: during the embryonic development these cells proliferate and differentiate to generate all tissues. Postnatal stem cells have an extraordinary plasticity. The individual cells, when expanded into colonies, retain their multilineage potential. Although their number is higher before the birth, there are several ‘‘loci’’ or ‘‘niches’’ inhabited by a significant number of stem cells within the adult human body (Laino et al., 2005). Among the adult tissues, dental pulp, the soft connective tissue entrapped within the dental crown, is an extremely rich site for stem cell collection: owing to its peculiar formation, the pulp chamber is a sort of ‘‘sealed niche’’ and may explain why it is possible to find a rather large number of stem cell there. During the sixth week of embryogenesis, the ectoderm covering the stomodeum begins to proliferate, giving rise to the dental laminae. Ectoderm–mesoderm interactions then lead to placode formation. One of these ovoidal ectodermal structures develops into tooth germs, where neural crest cells differentiate into the dental organ, dental papilla and dental follicle. Therefore, dental pulp is made of both ectodermic and J. Exp. Zool. (Mol. Dev. Evol.)

mesenchymal components, containing neural crest cells that display plasticity and multipotential capability. These adult stem cells have been called DPSCs, when found in permanent teeth, and SHEDs (Stem Cells from Human Exfoliated Deciduous), when found in deciduous teeth. DPSCs have been isolated for the first time in 2000 by Gronthos et al.; these cells exhibited a differentiation potential for odontoblastic, adipogenic and neural cytotypes. The same group isolated SHEDs from deciduous teeth and compared DPSCs with bone marrow stem cells (BMSCs) (Miura et al., 2003). They reported a superimposable ability of these cells to form calcified tissue although different lineages were observed: DPSCs seemed to undergo odontoblastic differentiation, whereas BMSCs became osteoblasts after loading on HA-TCP scaffold. The same authors reported that SHEDs were different from DPSCs, affirming that they were ‘‘more immature’’: this probably because they were able to differentiate into a variety of cell types, to an extent greater than than DPSCs. Actually, they demonstrated that SHEDs were able to differentiate into a variety of cell types. They called some of these cytotypes ‘‘osteoblast-like’’ and ‘‘odontoblast-like’’ cells; because of their immaturity (Miura et al., 2003). The main commitment of these cells seemed to be the formation of a mineralized tissue (Luisi et al., 2007; Wei et al., 2007), similar to dentin, (Kitagawa et al., 2007), as shown by in vivo transplantation of these cells into immunodeficient mice (Miura et al., 2003). These cells are involved in the development of several but different hard tissues, including crown and root dentin, cementum and alveolar bone; a role in root reabsorption of deciduous teeth has been hypothesized for DPSCs (Yildirim et al., 2008). In vitro DPSCs have been shown to produce sporadic but densely calcified nodules (Gronthos et al., 2000). In addition, the same group (Gronthos et al., 2002) found that these cells are capable of forming ectopic mineralized tissue, similar to dentin, but only when grafted in vivo or when placed on the surface of human dentin in vivo (Batouli et al., 2003) and when exposed to tooth germ conditioned medium (Yu et al., 2006) or similar differentiation factors (Tonomura et al., 2007). These studies, performed in deciduous and permanent teeth, were done without specifically selecting stem cells (Gronthos et al., 2000, 2002; Batouli et al., 2003), which under specific stimuli, differentiate into several cell types, including neurons and adipocytes. Sporadic dense nodules


DENTAL PULP STEM CELLS AND CLINICAL USE

were found to be formed in vitro, but they underwent mineralization and bone or dentin-like formation only when grafted in vivo. These data, regarding a multipotential differentiative ability of those cells were further confirmed (Iohara et al., 2006; Srisawasdi and Pavasant, 2007) although the main commitment remains to form mineralized tissues, as evidenced in several papers (Ueno et al., 2006; Otaki et al., 2007; Hosoya et al., 2007); however, this is what usually happens during dental tissues development. (Bosshardt, 2005). Moreover, other researchers have investigated another aspect of DPDCs, i.e. their putative immunosuppressive activity (Pierdomenico et al., 2005). This would be fascinating, if confirmed. STROMAL BONE-PRODUCING DPSCS AND STROMAL BONE-PRODUCING SHEDS: MAIN PROPERTIES AND PERFORMANCIES Laino et al. (2005) isolated a selected subpopulation of DPSC called Stromal Bone Producing Dental Pulp Stem Cells (SBP-DPSCs), multipotential cells that could give rise to a variety of cell types and tissues including adipocytes, neural cell progenitors and myotubes (Laino et al., 2005, 2006; Papaccio et al., 2006). Previous experiments demonstrated that stem cells, isolated from the pulp of human exfoliated deciduous teeth and expanded in vitro, showed a9% positivity for STRO-1, considered an early marker of mesenchymal stem cells (Gronthos et al., 2002). This antibody identified a cell surface antigen expressed by the osteogenic fraction of stromal precursors in human bone marrow as well as in erythroid precursors. In particular, the STRO-1 antigen is extremely important in selecting dental pulp cells (Yang et al., 2007a,b). SBP-DPSCs, representing roughly 10% of dental pulp cells display their multipotency (Laino et al., 2005,

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2006); in fact they differentiate into smooth muscle cells, adipocytes, neurons and osteoblasts. The latter is also substantiated by the RUNX-2 expression, a transcription factor essential for inducing osteoblast differentiation. These cells, selected for c-kit, CD34 and STRO-1 positivity (Fig. 1), were found to be able to produce an autologous woven bone tissue in vitro (Fig. 2). Head and neck hard tissues of the body have, other than a mesodermal origin, a neural crest source, and this has been demonstrated by the expression of the c-kit antigen, which is usually expressed in neural crest-derived cells, such as melanocyte precursors. The latter is compatible with the presence of c-kit expressing cells in the area of developing teeth. Therefore, CD34 and c-kit co-expression was used to isolate a population of stromal stem cells of neural crest origin. PASSAGES AND SENESCENCE OF DPSCS DPSCs can survive for long periods and can be passaged several times. It is possible to obtain more 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 under a confocal microscopy. The green fluorescence stains the cell cytoskeleton (revealed by phalloidin); DAPI stains the nucleus. Original magnification  400.

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

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senescence (Laino et al., 2005, 2006). Remarkably, after several passages, DPSCs still exhibit plasticity and capacity for nodule formation and are capable of forming bone chips in vitro. Osteoblastderived 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 bone chips when detached from their substrates, because they lose cell to cell contacts, which are of primary relevance for extracellular matrix secretion (D’Aquino et al., 2007). IN VIVO STUDIES The woven bone tissue generated in vitro by SBPDPSCs, called living autologous bone (LAB), is remodeled into a lamellar bone when transplanted in vivo (Laino et al., 2005, 2006; d’Aquino et al., 2007). Actually, after transplantation in vivo, the tissue is remodeled to form a lamellar bone through co-differentiation of SBP-DPSC into osteoblasts and endotheliocytes (d’Aquino et al., 2007). In fact, SBPDPSCs produce bone but not dentin, as shown by mRNA transcript that express all markers of bone including osteocalcin, Runx-2, collagen I, but not dentin sialo phospoho protein (DSPP), which is specific for dentin, by the high expression of alkaline phosphatase (Laino et al., 2005, 2006) and by in vivo histomorphometry (d’Aquino et al., 2007). In the latter paper it was shown that SBP-DPSCs change their antigenic surface expression during differentiation. Moreover, after in vivo transplantation, a complete integration of vessels within bone chips takes place, leading to the formation of a vascularized bone tissue (Fig. 3). During their differentiating process, SBP-DPSCs were observed to change their surface antigen

expression, as they differentiated. At day 40, starting from 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 the remaining 30% became Flk-11/STRO-11/CD441/ CD541 endothelial cells. Interestingly, these cells were always negative for DSPP, a marker of dentin, demonstrating that the hard tissue they produce is bone and not dentin: the production of dentin actually needs a pool of factors present in dental papilla (Lesot et al., 2001; Yuasa et al., 2004). This observation indicates that vasculogenesis takes place in vitro within the newly synthesized tissue. The formation of vessels explains the vitality of bone chips when transplanted in immunosuppressed rats. CO-DIFFERENTIATION AND VESSEL FORMATION Transplantation results obtained with SBPDPSCs are of extreme interest for the development of novel therapies. After transplantation in immunosuppressed rats, both woven chips and stem cells challenged with a scaffold become adult bone (d’Aquino et al., 2007). In addition, the dimensions of the obtained bone are the same as the grafted chips or the scaffolds (Trubiani et al., 2003). In particular, complete Haver’s channels, containing blood vessels, and surrounded by bone arranged in a lamellar configuration have been obtained. This is the first demonstration of a complete bone obtained from stem cells. During the ossification process, SBP-DPSCs give rise to both osteoblasts and endotheliocytes, leading to the formation of an adult bone tissue after in vivo transplantation. The presence of vessels and their complete integration with host (D’Aquino et al., 2007), other than being the first demonstration of a complete tissue growth from stem cells, is of great importance for therapy. This is a model of synergic differentiation, whose key aspect is the expression of flk-1, which is pivotal for the coupling of osteogenesis and vasculogenesis. This also represents an interesting aspect for development and a complete and efficient three-dimensional tissue reconstruction therapy. CRYOPRESERVATION OF DPSCS

Fig. 3. In vivo transplantation results. Figure showing a bone sample stained with hematoxylin–eosin staining obtained after in vivo transplantation of LAB. Original magnification  100.

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Cryopreservation of cells and tissue, mainly of the reproductive system, has been significantly improved recently, but to date prevailingly hematopoietic stem cells have been cryopreserved and


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then successfully utilized for transplantation. Moreover, to date there are no reports on the ability of either stem cells or already differentiated cells to re-start proliferation, differentiation and new tissue formation for therapeutic use. After long-term cryopreservation (2 years), osteoblasts differentiated from SBP-DPSCs, are still capable of quickly re-starting proliferation and the production of mineralized matrix, in a manner similar to what we have already demonstrated for fresh cells (Laino et al., 2005, 2006; Papaccio, 2006). The differences in percentages regarding STRO-1 and flk-1 with respect to the percentages observed for the other stem cell antigens are owing to the fact that we performed multi-parametric cell sorting using both morphological and antigenic criteria, and sorted first for CD117 and CD34 together and then sequentially for STRO-1 and flk-1 antigens, before and after cryopreservation. Thus, pre-endothelial cells, such as pericytes positive for both CD117 and CD34, could have altered the overall percentages. These cells would be responsible for the differentiation of endothelium, which occurs in parallel with osteoblast differentiation, as demonstrated in the embryo during the ossification process. Moreover, both osteoblasts and endotheliocytes express the VEGF-2 receptor (flk-1). Furthermore, after thawing, no apoptotic death was observed, and cells retained their differentiation multipotency, all of which are of interest when assessing the suitability of stem cells for use after cryopreservation. Moreover, osteoblasts produced a large-scale woven bone, which was observed in at least 100 25 cm2 flasks. Samples of this bone, when transplanted into immunosuppressed rats, were remodeled into lamellar bone, further demonstrating their vitality. Ultrastructurally, osteoblasts were cuboidal in shape, forming a layer along the border of the extracellular matrix, as observed in vivo during osteogenesis. These differentiated cells contained an extremely diffuse RER as well as matrix membrane vesicles, containing crystal-like structures. These ultrastructural observations confirmed that cells were unaltered. A study was performed on cryopreserved tissue samples of minced periodontal ligament (Seo et al., 2005). Conversely, another group (Papaccio et al., 2006) obtained completely negative results cryopreserving whole pulps. This is probably owing to the 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 lead to safe recovery (Zhang et al., 2006a). These features and abilities make these cells attractive for therapeutic three-dimensional tissue reconstruction, with the potential of tailoring storage and recovery to the needs of the patient. BONE TISSUE ENGINEERING STUDIES DPSCs showed differentiation profiles similar to those showed during bone differentiation (Hwang et al., 2008) and this event make them very interesting as a model to study osteogenesis (Liu et al., 2007) and the relationship with scaffolds (Zhang et al., 2006a,b). SBP-DPSCs, when undergoing differentiation into pre-osteoblasts, deposit an extracellular matrix that becomes a calcified woven bone tissue called LAB (Laino et al., 2005). Calcein staining positivity, in addition to the other markers, strongly confirmed the presence of calcium deposits within this tissue, and stresses the effectiveness of the mineralization process. In addition, ALP activity increases significantly in parallel to cell differentiation. In addition, it has been shown that there are no differences regarding expansion rate, number of calcification centers and LAB nodules obtained per well of cells selected from younger (up to 29 years) and older (30–45 years) subjects (Laino et al., 2005). Interestingly, positivity for CD44, seen in inflammatory processes of the pulp (Pisterna and Siragusa, 2007), osteocalcin and RUNX-2, evidences that cells were differentiating toward the osteoblastic lineage. The involvement of Runxrelated gene in dental pulp mineralization processes has been intensely investigated (Zheng et al., 2007). Strong expression of RUNX-2, a transcription factor essential for osteoblast differentiation, is of primary importance, because it is closely related to the promotion of ossification; in addition, targeted disruption of RUNX-2 results in complete lack of woven bone formation by osteoblasts. Other than forming woven bone in vitro it has been demonstrated that this tissue undergoes remodeling, when transplanted in immunocompromised rats, and becomes a lamellar bone with entrapped osteocytes (Laino et al., 2006; D’Aquino et al., 2007). The latter confirms that SHEDs differentiate in osteoblasts and then osteocytes, differently to what has been previously reported (Miura et al., 2003) but similarly to DPSCs (Laino et al., 2005). Actually, it has been speculated that SHEDs possess the ability to differentiate in J. Exp. Zool. (Mol. Dev. Evol.)


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functional odontoblast-like cells and that unlike DPSCs, they did not directly differentiate in osteoblasts (Miura et al., 2003). These authors other than using different methods and markers to select stem cells, which may lead to different results, needed the use of an osteoinductive template for in vivo transplants (Zhang et al., 2006a,b). The findings of Laino et al. (2006) disagree because the cells that they selected are probably different as they differentiate into osteoblasts and produce a woven or fibrous bone, which, without the need of osteoinductive templates, after in vivo transplantation, is remodeled into a lamellar bone. Therefore, these cells appear to be good candidates for bone tissue reconstruction protocols and bone regeneration models, because of good cellular morphology and high BMP-2 and VEGF secretion (Graziano et al., 2007). The role played by BMP2 is crucial and this protein has been hypothesized as biological tool in gene therapy of dentin regeneration (Nakashima et al., 2006). The concave texturing of the substrate elicits cytoarchitectural responses and adaptation in which the cells appear to favor intimate contacts with the secondary microconcavities and cellular polarization in human tissues (Zhang et al., 2003). Such behavior is accompanied by increased release of BMP-2 and VEGF into the culture medium and by higher ALP activity. It is likely that increased release of potent factors such as BMP-2 and VEGF and the higher ALP activity could have significant biological ramifications. By their proven involvement and potency in bone formation and angiogenesis, these factors and enzymatic activity may influence the responses and developmental program of stromal-derived cells via autocrine mechanisms and it is also influenced by surrounding cells via paracrine pathways. In this likely scenario, increased levels of BMP-2 and VEGF could be responsible for the greater amounts of bone tissue they observed in vitro (Graziano et al., 2008) and after transplantation of the colonized microconcavity-rich scaffold. Angiogenesis could become itself a main goal for the clinical application of DPSCs, owing to the importance that angiogenetic factors (Tran-Hung et al., 2007) have in the native tissue (Grando Mattuella et al., 2007a,b); here, the biological events seem to be supported by nonstem population such as fibroblasts (Tran-Hung et al., 2006). Today in vitro bone regeneration studies are limited by the main difficulty to obtain a cytotype capable of forming a complete tissue and not only a monolayer or cells surrounded by a mineralized J. Exp. Zool. (Mol. Dev. Evol.)

matrix. Owing to their high proliferation rate and efficiency in producing bone chips, DPSCs seem to be the best candidates to study bone formation with respect to BMSCs, whose efficiency is limited by the fact that they differentiate into osteoblasts and produce small calcified nodule, but not chips of bone tissue. Scaffold’s structure and its influence upon cells are a breaking point in bone tissue engineering. To study the relationship between biomaterials and stem cells during their osteogenic differentiation process more specifically in bone tissue building, we need that cells would be able to actively proliferate, differentiate and produce a bone tissue as better as they can. Therefore, SBP-DPSCs may be a good standard to study the ossification process on substrates suitable for clinical application in bone reconstruction (Graziano et al., 2007, 2008). In fact, their ability to produce LAB already in vitro is of great interest to further analyze the effects of scaffolds, which are mediated by cell interactions with substrates. Interestingly, these cells show a lifespan and an high and long-lasting osteogenic capacity (Papaccio et al., 2006). These findings have been further substantiated by the results obtained challenging the surface texturing with SBP-DPSCs in a threedimensional cell culture system. In fact, data obtained from engineered tissues, made on different scaffolds in a roller apparatus for 30 days (Graziano et al., 2008) clearly demonstrate that a new-formed bone tissue is obtained and that their different thickness strictly depends on the growing surface and, in particular, on the specific texture that has been adopted. Actually, the obtained new bone is of considerable thickness when the scaffold is made of a micro concave surface; it is of a lesser thickness in the case of a smooth surface and it is almost absent in the case of a convex surface, confirming all the results obtained with plane cultures. At the end of the rotating period (30 days) on convex surfaces, instead of building a bone tissue, cells were found at the bottom of the scaffold. Moreover, concave texturing induces an earlier and quantitatively more enhanced bone differentiation: in fact, it has been observed that BAP expression occurs earlier; cells adopt a polygonal shape with filopodia-like and lamellipodia-like extensions (cells can be regarded as spreading and differentiating), and nuclear polarity is observed, an index of secretion, cell activity and matrix formation (Graziano et al., 2008). These observations open the way to a more extensive application of tissue engineering of craniofacial tissues (Mao et al., 2006).


DENTAL PULP STEM CELLS AND CLINICAL USE

In conclusion: (1) dental pulp is a remarkable site of stem cells; (2) collecting stem cells from dental pulp is a noninvasive practice that can be performed in the adult during life and in the young after surgical extraction of wisdom teeth, a common surgical practice; (3) tissue sacrifice is very low when collecting dental pulp stem cells; (4) several cytotypes can be obtained from dental pulp stem cells owing to their multipotency; (5) transplantation of new-formed bone tissue obtained from dental pulp stem cells leads to the formation of vascularized adult bone and integration between the graft and the surrounding host blood supply; (6) dental pulp stem cells can be cryopreserved and stored for long periods; (7) dental pulp is ideal for tissue engineering and for clinical use in several pathologies requiring bone tissue growth and repair. In addition, tooth extraction is a clinical/therapeutical need. If bone marrow is the site of first choice for hematopoietic stem cell collection, dental pulp must be considered one of the major sites for mesenchymal cell collection. The good results obtained up to now reinforce this thought. LITERATURE CITED Batouli S, Miura M, Brahim J, Tsutsui TW, Fisher LW, Gronthos S, Robey PG, Shi S. 2003. Comparison of stemcell-mediated osteogenesis and dentinogenesis. J Dent Res 82:976–981. Bosshardt DD. 2005. Are cementoblasts a subpopulation of osteoblasts or a unique phenotype? J Dent Res 84:390–406. D’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, Papaccio G. 2007. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ 14:1162–1171. Fitzgerald M, Chiego Jr DJ, Heys DR. 1990. Autoradiographic analysis of odontoblast replacement following pulp exposure in primate teeth. Arch Oral Biol 35:707–715. Goldberg M, Smith AJ. 2004. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med 15:13–27. Grando Mattuella L, de Figueiredo JA, No¨r JE, de Araujo FB, Fossati AC. 2007a. Vascular endothelial growth factor receptor-2 expression in the pulp of human primary and young permanent teeth. J Endod 33:1408–1412. Grando Mattuella L, Westphalen Bento L, de Figueiredo JA, No¨r JE, de Araujo FB, Fossati AC. 2007b. Vascular endothelial growth factor and its relationship with the dental pulp. J Endod 33:524–530. Review. Graziano A, d’Aquino R, Cusella-de Angelis MG, Laino G, Piattelli A, Pacifici M, De Rosa A, Papaccio G. 2007. Concave pit-containing scaffold surfaces improve stem cell-derived osteoblast performance and lead to significant bone tissue formation. PLoS ONE 2:e496. Graziano A, d’Aquino R, Cusella-De Angelis MG, De Francesco F, Giordano A, Laino G, Piattelli A, Traini T,

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when transplanted into immunocompromised mice. Cell Biol Int 31:1191–1197. Papaccio G, Graziano A, d’Aquino R, Graziano MF, Pirozzi G, Menditti D, De Rosa A, Carinci F, Laino G. 2006. Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol 208:319–325. Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, Becchetti E, Marchionni C, Alviano F, Fossati V, Staffolani N, Franchina M, Grossi A, Bagnara GP. 2005. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 80:836–842. Pisterna GV, Siragusa M. 2007. CD44 Presence in inflamed pulp tissue. J Endod 33:1203–1207. Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S. 2005. Recovery of stem cells from cryopreserved periodontal ligament. J Dent Res 84:907–912. Sinanan AC, Hunt NP, Lewis MP. 2004. Human adult craniofacial muscle-derived cells: neural-cell adhesion-molecule (NCAM; CD56)-expressing cells appear to contain multipotential stem cells. Biotechnol Appl Biochem 40:25–34. Srisawasdi S, Pavasant P. 2007. Different roles of dexamethasone on transforming growth factor-beta1-induced fibronectin and nerve growth factor expression in dental pulp cells. J Endod 33:1057–1060. Tonomura A, Sumita Y, Ando Y, Iejima D, Kagami H, Honda MJ, Ueda M. 2007. Differential inducibility of human and porcine dental pulp-derived cells into odontoblasts. Connect Tissue Res 48:229–238. Tran-Hung L, Mathieu S, About I. 2006. Role of human pulp fibroblasts in angiogenesis. J Dent Res 85:819–823. Tran-Hung L, Laurent P, Camps J, About I. 2007. Quantification of angiogenic growth factors released by human dental cells after injury. Arch Oral Biol 53:9–13. Trubiani O, Tripodi D, Delle Fratte T, Caputi S, Di Primio R. 2003. Human dental pulp vasculogenesis evaluated by CD34 antigen expression and morphological arrangement. J Dent Res 82:742–747. Ueno A, Yamashita K, Miyoshi K, Horiguchi T, Ruspita I, Abe K, Noma T. 2006. Soluble matrix from osteoblastic cells induces mineralization by dental pulp cells. J Med Invest 53:297–302.

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