THE ANATOMICAL RECORD 297:6–15 (2014)

Periodontal Tissue Engineering Strategies Based on Nonoral Stem Cells ~ FILIPE REQUICHA,1,2,3 CARLOS ALBERTO VIEGAS,1,2,3 JOAO 4 ~ FERNANDO MUNOZ, RUI LUIS REIS,1,3 AND MANUELA ESTIMA GOMES1,3* 1 3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Guimar~ aes, Portugal 2 Department of Veterinary Sciences, University of Tr as-os-Montes e Alto Douro, Vila Real, Portugal 3 ICVS/3B’s—PT Government Associated Laboratory, Braga/Guimar~ aes, Portugal 4 Department of Veterinary Clinical Sciences, Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain

ABSTRACT Periodontal disease is an inflammatory disease which constitutes an important health problem in humans due to its enormous prevalence and life threatening implications on systemic health. Routine standard periodontal treatments include gingival flaps, root planning, application of growth/differentiation factors or filler materials and guided tissue regeneration. However, these treatments have come short on achieving regeneration ad integrum of the periodontium, mainly due to the presence of tissues from different embryonic origins and their complex interactions along the regenerative process. Tissue engineering (TE) aims to regenerate damaged tissue by providing the repair site with a suitable scaffold seeded with sufficient undifferentiated cells and, thus, constitutes a valuable alternative to current therapies for the treatment of periodontal defects. Stem cells from oral and dental origin are known to have potential to regenerate these tissues. Nevertheless, harvesting cells from these sites implies a significant local tissue morbidity and low cell yield, as compared to other anatomical sources of adult multipotent stem cells. This manuscript reviews studies describing the use of non-oral stem cells in tissue engineering strategies, highlighting the importance and potential of these alternative stem cells sources in the development of advanced therapies for periodontal regeneraC 2013 Wiley Periodicals, Inc. tion. Anat Rec, 297:6–15, 2014. V

Key words: periodontium; periodontal regeneration; cellbased therapies; tissue engineering; stem cells; non-oral stem cells; preclinical models

Grant sponsor: Jo~ ao Filipe Requicha PhD Scholarship; Grant number: SFRH/BD/44143/2008; Grant sponsor: Portuguese Foundation for Science and Technology (FCT). *Correspondence to: M.E. Gomes, 3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and C 2013 WILEY PERIODICALS, INC. V

Regenerative Medicine, AvePark, Guimar~ aes, Portugal. Fax: 1351-253-510. E-mail: [email protected] Received 13 September 2013; Accepted 13 September 2013. DOI 10.1002/ar.22797 Published online 2 December 2013 in Wiley Online Library (wileyonlinelibrary.com).

PERIODONTAL TISSUE ENGINEERING STRATEGIES

Periodontium is an organ constituted by various tissues namely the alveolar bone, which forms the dental alveolus, the cementum which surrounds the root, the periodontal ligament (PDL) which is sustained by the bone and the cementum, bonding them, and finally the gingiva, which involves all the above (Melcher, 1976; Polimeni et al., 2006) (Fig. 1). The periodontium is often affected by the periodontal disease (PD), an inflammatory disease which includes two different inflammatory stages: an initial form called gingivitis and an advanced form called periodontitis (Fig. 2), which usually progresses with bone resorption, cementum necrosis, and gingival recession or hyperplasia. Ultimately, when left untreated, this leads to the

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formation of a periodontal pocket with permanent loss of tooth support, and consequently increasing mobility and teeth loss (Kuo et al., 2008; Bosshardt and Sculean, 2009; Albuquerque et al., 2012). Periodontal disease has a multifactorial etiology involving microbial, behavioral (absence of oral hygiene and diet), local (malocclusions and overcrowding), systemic (immunodeficiency, infections, metabolic disease and nutritional disturbances), and genetic factors (Pihlstrom et al., 2005; Tatakis and Kumar, 2005; Stabholz et al., 2010). The PD is an important health problem in Medicine, due to its enormous prevalence and life threatening implications on systemic health. It has been reported a correlation between PD and preterm birth, low weight at birth and neonatal morbidity, diabetes, respiratory, osteoarticular and cardiovascular diseases (Kuo et al., 2008). Chronic periodontitis affects about 30% of the adult population and 7–13% of them have the severe form of the disease (Nares, 2003). Periodontal regeneration consists of a complex and orchestrated sequence of biological events at a molecular and cellular level, accomplishing success when the following goals are met: formation of new cementum in the radicular surface, restoration of the alveolar crest till the cementum–enamel junction, reestablishment of the junctional epithelium and formation of a dense set of periodontal ligament fibers obliquely oriented assuring its functionality (Bartold et al., 2000; Polimeni et al., 2006; Lin et al., 2008). This phenomenon involves the pool of progenitor cells present in the periodontium, clustered near the periodontal ligament blood vessels, which form the fibroblasts, osteoblasts, and cementoblasts (Melcher, 1976) helped by progenitor cells existent in the bone endosteal spaces which migrate to the PDL (McCulloch et al., 1987).

PERSPECTIVE ON THE CURRENT PERIODONTAL THERAPIES

Fig. 1. Schematic representation of the dental and periodontal anatomy.

The main aims of the current periodontal therapies include the infection control and the re-establishment of a periodontal tissue apparatus. As the dental plaque is the primary cause of PD, their removal from the tooth crown, gingival sulcus and root surfaces is essential for the prevention and/or control of periodontal disease. Plaque removal can be accomplished by a combination of home care procedures that include mechanical and chemical plaque reduction techniques. To control the microbial population, clindamycin hydrochloride, amoxicillin/clavulanate and metronidazole seem to be

Fig. 2. Periodontal disease aspect in humans. A: healthy gingiva; B: gingivitis; and C: periodontitis.

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REQUICHA ET AL.

Fig. 3. Schematic image of the several stem cells sources for application in periodontal regeneration.

particularly effective systemic antimicrobials. Locally delivered antimicrobials (perioceutics), such as doxycycline gel, can be applied to teeth that have been cleaned and polished (Pihlstrom et al., 2005; Chen and Jin, 2010; Stabholz et al., 2010). However, when the disease progresses, advanced periodontal surgery becomes necessary. To treat patients with deep pockets and bone loss, mucogingival surgery (flap exposure), open curettage (Lin et al., 2008; Bosshardt and Sculean, 2009) and the root conditioning with demineralizing agents (Wikesjo and Nilveus, 1991; Bartold et al., 2000; Lin et al., 2008; Bosshardt and Sculean, 2009) are required. To stimulate the periodontal regeneration, the clinicians sometimes inject into the defects cocktails of growth factors, such as enamel matrix derivatives (EMD) or platelet-rich plasma (PRP) (Viegas et al., 2006; Tobita et al., 2008) or specific growth/differentiation factors (e.g., PDGF, IGF-1, BMP-2, and BMP-7) (Bartold et al., 2000; Bosshardt and Sculean, 2009). In some cases, depending on the dimensions of the defect and the degree of osteolysis and/or tooth root exposition, it is advised the application of filler materials such as autografts, allografts, and alloplastic materials (most frequently, hydroxyapatite-HA and tricalcium phosphate-TCP) (Wikesjo and Nilveus, 1991; Lin et al., 2008; Bosshardt and Sculean, 2009) and/ or guided tissue regeneration (GTR) membranes. GTR techniques were firstly proposed in the 80s (Wikesjo and Nilveus, 1991; Bartold et al., 2000) and

have the objective to guide selectively the cell proliferation in different compartments, namely the alveolar bone, cementum and PDL, using ePTFE, PLA, PGLA, or collagen membranes as a physical barrier (Wikesjo et al., 2003; Lin et al., 2008; Chen and Jin, 2010). In fact, this was one of the first techniques which considered the physiopathology of the periodontal regeneration, avoiding some of the major drawbacks reported for the other existing therapies, namely, the gingival epithelium and connective tissue expansion, ankylosis and radicular resorption phenomenon and the difficulty to avoid the collapse of the periodontal defect (Bartold et al., 2000; Wikesjo et al., 2003; Polimeni et al., 2006; Bosshardt and Sculean, 2009). However, none of these procedures, even when used in combination (e.g., simultaneous application of filler materials with GTR membranes) have revealed enough efficacies to achieve full periodontal regeneration.

TISSUE ENGINEERING Recently, tissue engineering (TE) and other cell based therapies have emerged as an alternative approach for the regeneration of several tissues damaged by disease or trauma, including the periodontium. TE involves the use of a support material—membrane or scaffold—where cells are seeded and cultured in order to obtain hybrid

PERIODONTAL TISSUE ENGINEERING STRATEGIES

materials which can induce the regeneration of the target tissue. Several materials have been proposed to be used in TE, mostly biodegradable polymers of synthetic and natural origin (Wikesjo et al., 2003; Salgado et al., 2004; Rai et al., 2007) aimed at acting as a support for cells and new tissue ingrowth until complete regeneration of the tissue defect is accomplished (Bartold et al., 2000; Salgado et al., 2004; Gomes et al., 2008; Martins et al., 2009). Tissue engineering approaches usually rely on the use of adult mesenchymal stem cells (MSCs) (Fig. 3), that have the capacity to differentiate in various types of cells, as for example, osteoblasts, chondroblasts, adipocytes, cardiomyocytes, neuronal cells, or periodontal ligament cells (Caplan, 1991; Lin et al., 2008; Requicha et al., 2012). Periodontium is a highly specialized and complex organ and is derived from the dental follicle and the neural crest cells. These cells constitute a multipotent cell population in vertebrates and participate in the embryonic development of most dental tissues including the gingiva, the dental follicle, the periodontal ligament and the alveolar bone (Tapadia et al., 2005; Coura et al., 2008). From the embryonary dental structures, Gronthos et al. identified for the first time, in 2000, the dental pulp stem cells (DPSCs) (Gronthos et al., 2000). Stem cells have also been isolated from human exfoliated deciduous teeth (SHEDs) (Miura et al., 2003). In the periodontal ligament have been described the PDL fibroblasts (Murakami et al., 2003) and the PDL stem cells (PDLSCs) (Seo et al., 2004), which are the most studied cell source from periodontal origin. From the gingiva, it is possible to obtain distinct cell populations, such as the gingival fibroblasts and gingival precursor cells (Schor et al., 1996; Phipps et al., 1997). Other undifferentiated dental stem cells have been referred in the literature along the years, namely, the dental follicle cells (DFCs) (Morsczeck et al., 2005), the stem cells from the apical papilla (SCAP) (Sonoyama et al., 2008) and the cementum-derived cells (CDCs) (Grzesik et al., 2000). Regarding the osseous component of the periodontium, McCulloch et al. proposed, in 1987, that paravascular cells from the endosteal spaces of alveolar bone communicate with the PDL and may contribute to its cell populations (McCulloch et al., 1987). Recently, undifferentiated stem cells were isolated from the alveolar bone margin (El-Sayed et al., 2012). However, the development of new strategies for periodontal regeneration requires significant amounts of cells with regenerative potential to have therapeutic efficacy, combined or not with a biodegradable supportive matrices. Furthermore, autologous approaches are usually preferred for safety reasons and, for this purpose, it is very important to collect these cells without sacrifice of the tissues that we intent to regenerate, namely, the periodontium or even the teeth. Thus, it is of utmost relevance to consider the use of adult MSCs from other origins. Among those cells, the bone marrow stem cells and the adipose-derived stem cells assume an important role in the present and future research in this field. Therefore, this review will focus on the application of undifferentiated stem cells from non-dental and nonperiodontal origin in tissue engineering approaches targeting the regeneration of periodontal defects.

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BONE MARROW STEM CELLS Bone marrow stem cells (BMSCs) were firstly identified in 1968 (Friedens Aj et al., 1968) and since then they have been extensively studied, being considered the gold standard in cell-base therapies and other regenerative medicine approaches. In 2009, Kramer et al. after inject male DiI-labeled BMSCs into a female rat periodontal pocket, observed for the first time that these cells obtain PDL morphology and PDL protein markers in vivo over a 6-week period (Kramer et al., 2009). Along the years, as described below, different tissue engineering approaches combining distinct types of supportive matrices (gels or scaffolds) with undifferentiated BMSCs were developed and assessed in preclinical models, as summarized in Table 1. One of the first reports on the use of BMSC in periodontal regeneration therapies refers to a study by Kawaguchi et al. (2004) that proposed the autotransplantation of canine BMSCs on an atelocollagen gel into dog furcation defects and observed that cementum with extrinsic fibers was formed along almost denuded root surfaces (Kawaguchi et al., 2004), providing us one of the first data on periodontal regeneration in a superior animal model with resemblances to human. In a subsequent study, the same researchers transplanted BMSCs labeled with green fluorescence proteins (GFP), using the same gel and experimental model, and identified cementoblasts, osteoblasts, osteocytes, and fibroblast derived from those cells, concluding that they participate effectively in the periodontal regeneration (Hasegawa et al., 2006). This data was corroborated by Wei et al. who used BMSCs labeled with bromodeoxyuridine (BrdU) mixed with alginate gel in the same model (Wei et al., 2010) and observed new bone, PDL, cementum and blood vessels formation and expression of typical surface markers of osteoblasts and fibroblasts. Another approach (Chen et al., 2008) described the implantation of BMSCs engineered to express BMP-2 in a pluronic F-127 hydrogel into rabbit periodontal defects where it was demonstrated that this growth/differentiation factor enhances the regeneration of periodontal tissues comparing to the use of cells or polymeric gel alone (Chen et al., 2008). A distinct study reported the use of goat BMSCs seeded onto a PLG scaffold that was fitted around a titanium fixture immediately after tooth removal and the results showed the formation of new bone and periodontal tissue–like bundles of fibers extending from the periphery of the wound toward the surface of implant, suggesting that BMSCs-PLG construct promoted osteointegration (Marei et al., 2009). In the same year, Zhang and his team implanted a tooth/bone construct seeded with BMSCs into mandible defects in suines and observed the formation of small tooth-like structures consisting of organized dentin, enamel, pulp, cementum, periodontal ligament resembling Sharpey’s fibers and surrounded by regenerated alveolar bone (Zhang et al., 2009). Rat BMSCs (GFP1) have also been loaded into gelatin microbeads and transplanted into a surgically created rat periodontal defect (Yang et al., 2010). After 3 weeks of implantation, it was found higher evidence of bone, cementum and PDL regeneration in defects filled with

Collagen membranes Calcium alginate gel Microcarrier gelatin bead Collagen membrane

Femur

Tibia

Iliac crest

Femur

Goat (6m–3.5y)

Canine (6–10m, Beagle)

Pig (3.6m, female, Yucatan mini pigs) Canine (adult, Beagle) Canine (adult, Beagle) Rat (8w, male, Sprague-Dawley) Canine (adult, Beagle)

Femur

Iliac crest

Human

Canine (adult, mongrel)

Bone ceramic particles PRP

Cells transfected with BMP-2 EMD

Iliac crest

Pluronic F 127 gel

PPP and PRP

Cells transfected with BMP-7 Cells expressing

Cells transfected with human bFGF

Cells expressing BMP-2

Molecular signal

Iliac crest

PGA membrane

Canine (adult, Beagle, male) Canine (12–18m, mongrel) Canine (adult, Beagle, male)

Iliac crest

PLGA scaffold

Calcium alginate gel

Canine (adult, Beagle)

Tibia

PLGA 3D scaffold

Iliac crest

Rabbits (adult, New Zealand) PLG scaffold

Iliac crest

Canine (12–20m, Beagle)

Atelocollagen (2% type I collagen) gel Atelocollagen (2% type I collagen) gel Pluronic F 127 gel

Material

Iliac crest

Source

Canine (2y, Beagle)

Animal origin

Mouse (6w, NOD/ SCID) Canine (autologous)

Canine (autologous)

Canine (autologous)

osteoprogesterin

Canine (autologous)

Rat (allogenic)

Canine (autologous)

Canine (autologous)

Pig (autologous)

Canine

Periodontal defect

Rabbits (autologous)

Subcutaneous implantation Class II furcation defect

Extracted incisors replantation Periodontal defect

One-wall defect

Canine (autologous)

Class II furcation defect

Class III furcation defect Periodontal defect

Periodontal defect

Alveolar bone defect

Canine fresh extraction socket Class III furcation defect

Class III furcation defect

Class III furcation defect

Experimental defect

Canine

Canine (autologous)

Animal model

8 weeks

6–8 weeks

8 weeks

120 days

8 weeks

One-wall defect

12 weeks

3 weeks

6 weeks

12 and 20 weeks 8 weeks

10 days and 4 weeks 6 weeks

6 weeks

4 weeks

4 weeks

Implantation time

(Mrozik et al., 2012) (Simsek et al., 2012)

(Zhou et al., 2010) (Tsumanuma et al., 2011) (Assunc¸~ ao et al., 2011) (Chung et al., 2011)

6 weeks

(Yang et al., 2010) (Li et al., 2010)

(Wei et al., 2010)

(Zhang et al., 2009) (Li et al., 2009)

(Marei et al., 2009) (Tan et al., 2009)

(Chen et al., 2008)

(Hasegawa et al., 2006)

(Kawaguchi et al., 2004)

Reference

TABLE 1. Summary table describing tissue engineered approaches for periodontal regeneration based on the use of BMSCs

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PERIODONTAL TISSUE ENGINEERING STRATEGIES

TABLE 2. Summary table describing tissue engineered approaches for periodontal regeneration based on the use of ASCs Animal origin

Source

Material

Rat (10 w, male, Inguinal Wistar) region Rabbit (2–6 m female, New Zealand)

Abdomen

Porcine type I collagen gel

Molecular signal

Animal model

PRP

Rat

BMP-2

Rabbit (autologous)

microbeads-BMSCs than in empty defects or defects filled with the beads alone. Recently, a study performed by Tsumanuma et al compared the performance of BMSCs with PDLSCs and alveolar periosteal cells (APCs), when applied as cell sheets in a biodegradable polymer membrane for periodontal regeneration. The obtained histological data showed that the alveolar bone regeneration ratio was higher when using the PDLSCs, as well as the cementum thickness and the quality of PDL fibers orientation (Tsumanuma et al., 2011). Moreover, Wei et al. (2012) reported that the formation of human PDLSCs sheets was promoted by vitamin C induction and extrapolated this finding to both human BMSCs and UCSCs by culturing them with 20 mg mL21 of vitamin C (Wei et al., 2012). Nowdays, several molecular factors have been proposed to promote the regenerative mechanism of the periodontium, for example, the PRP (Tobita et al., 2008), the EMD (Saito et al., 2011; Mrozik et al., 2012) or other specific growth/differentiation factors (e.g., PDGF, IGF1, BMP-2, and BMP-7) (Bosshardt and Sculean, 2009; Ripamonti et al., 2009; Li et al., 2010; Chung et al., 2011). For examples, Assunc¸~ ao et al. evaluated the effect of platelet-poor plasma (PPP), calcium chloride-activated platelet-rich plasma (PRP/Ca), calcium chloride- and thrombin-activated PRP (PRP/Thr/Ca), as well as BMSCs with PRP/Ca (BMSCs/PRP/Ca) on the healing of replanted dog teeth. The obtained outcomes suggest that thrombin activation is more effective in the healing and that the use PRP/Ca with BMSCs show advantages in comparison to PRP/Ca alone (Assunc¸~ ao et al., 2011). A study comparing the use BMSCs plus PRP and PRP or autogenous cortical bone alone concluded that no differences were observed between the treatments (Simsek et al., 2012). Zhou et al. described a study in which canine BMSCs modified by osteoprogesterin gene were seeded onto a PLGA scaffold and implanted in an autologous periodontal defect, leading to enhanced formation of new alveolar bone and cementum as well as new connective tissue, as compared to the controls (without cells and material). This study shows that gene therapy utilizing osteoprogesterin may be used as an adjuvant in periodontal TE therapies/strategies (Zhou et al., 2010). In the same year, it was reported the use of a collagen membrane as a vehicle for canine BMSCs transfected with BMP-7, revealing higher bone formation and new cementum length upon implantation in a canine furcation defect, comparing to the material with nontransfected cells (Li et al., 2010). Chung et al. engineered these cells with BMP-2 and obtained promising results

Experimental defect

Implantation time

Reference

Periodontal fenestration defect Incisors extraction alveolar socket

2, 4, and 8 weeks

(Tobita et al., 2008)

12 weeks

(Hung et al., 2011)

in terms of periodontal regeneration (Chung et al., 2011). BMSCs transfected with human b-FGF have also shown to accelerate significantly the periodontal regeneration in dog class III furcation defects (Tan et al., 2009). A work focused on using either human BMSCs or PDLFs seeded onto bone ceramic particles with EMD in a subcutaneous mice model revealed no differences between the two cell sources in terms of ectopic bone formation. In contrast, a large amount of bone/cementumlike tissue and PDL-like fibrous tissue was found when used implants containing PDLFs, as compared to those based on BMSCs (Mrozik et al., 2012). Another comparative study demonstrated that cocultured human PDLSCs and BMSCs subsequently combined with an osteoinduced ceramic bovine bone led to the formation of a neovascularized ectopic cementum/ PDL-like complex resembling the physiologic PDL Sharpey’s fibers (Xie and Liu, 2012) in a subcutaneous mice model. Apart of the effect of molecular stimuli on the cells behavior, other stimuli has been studied, as recently referred by Cmielova et al. who concluded that BMSCs, as well as PDLSCs, respond to ionizing radiation by induction of senescence without affect viability (Cmielova et al., 2012). Bone marrow stem cells have also been proposed in several strategies for regenerate the alveolar bone, a particular component of the periodontium, combined with different materials, namely b-TCP (Neamat et al., 2009), HA/TCP (Kim et al., 2009) and fluorohydroxyapatite (Pieri et al., 2009), nanohydroxyapatite block scaffolds (Lee et al., 2012), chitosan-gelatin scaffold (Miranda et al., 2012) and fibrin glue (Zhang et al., 2012), or even combined with PRP for improve the osteointegration of hydroxyapatite-coated dental implants (Yamada et al., 2010).

ADIPOSE-DERIVED STEM CELLS Adipose-derived stem cells (ASCs) were firstly identified in 2001 by Zuk et al. (2001b) and, since then, they have been the focus of many studies on regenerative medicine (Gimble and Nuttall, 2004; Li et al., 2008). In fact, the adipose tissue exhibits several attractive advantages over other stem cells sources such as the bone marrow, mainly due to its wide availability (Iverson and Pao, 2008), the relative simplicity of the harvesting procedures (Zuk et al., 2001a; Rada et al., 2009) and their great differentiation potential (Fraser et al., 2006; Requicha et al., 2012). In fact, it is possible to obtain a

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large quantity of cells without causing high morbidity in the harvesting site, as for example, from 1 g of adipose tissue it can be isolated about 5,000 stem cells, whereas the yield from BMSCs is 100–1,000 cells mL21 of bone marrow (Strem et al., 2005). Therefore, ASCs are considered a very attractive stem cell type for the development of new tissue engineering and other cell-based therapies, including for periodontal regeneration. Table 2 summarizes recent studies focusing on the use of ASCs in periodontal TE strategies. The use of ASCs in periodontal regeneration strategies was reported for the first time in 2008 (Tobita et al., 2008). In the referred study, rat ASCs were mixed with PRP and implanted into periodontal defects induced in rats. After 2 and 4 weeks of implantation, a small amount of regenerated alveolar bone was observed. Moreover, 8 weeks after implantation, it was observed the formation of a PDL-like structure along with alveolar bone (Tobita et al., 2008). Hung et al. (2011) demonstrated that implants composed of collagen gels with either rabbit ASCs or rabbit DPSCs, were able to promote the growth of selfassembled new teeth in adult rabbit extraction sockets with high success rate. Furthermore, rabbit ASCs demonstrated a higher growth rate and a better senescence resistance in culture, when compared to rabbit DPSCs (Hung et al., 2011). An interesting in vitro study by Wen et al. described the potential of ASCs for periodontal regeneration showing that when incubated in dentin noncollagenous proteins and dental follicle cell conditioned medium adopted flat, cuboidal or polygonal shapes, features typical of a cementoblast-like morphology. Additionally, under these conditions, cells showed in vitro mineralization ability, ALP activity and expression of bone sialoprotein, type I collagen, osteonectin and osteocalcin characteristic of cementoblasts (Wen et al., 2011).

EMBRYONIC STEM CELLS Embryonic stem cells (ESCs) are derived from the inner cell mass of blastocysts and are pluripotent stem cells capable of differentiating into almost all kinds of cells of the adult body (Thomson et al., 1998). Only a few studies have addressed the potential use of ESCs in periodontal regeneration. Inanc¸ et al. (2007) characterized the osteogenic differentiation potential of human embryonic stem cells (hESCs) under the inductive influence of human PDL fibroblast monolayers (Inanc et al., 2007). The same authors have also achieved with success the differentiation oh hESCs (HUES-9) into periodontal ligament fibroblastic progenitors (Inanc et al., 2007, 2008). A study on the differentiation capacity of hESCs toward the periodontal compartment cells and their relationship with tooth root surfaces in vitro, demonstrated that hESCs differentiation is influenced by tooth structures (extracted tooth root slices), PDLFs, and osteogenic medium, resulting in increased propensity toward mesenchymal lineage commitment, and formation of soft-hard tissue relationship in close contact areas (Inanc¸ et al., 2009). These reports suggest that human ESCs may be considered an important cell source for periodontal tissue engineering applications.

INDUCED PLURIPONTENT STEM (iPS) CELLS The reprogramming of somatic cells into induced pluripotent stem (iPS) cells by forced expression of a small number of defined factors (e.g., Oct3/4, Sox2, Klf4 and cMyc) (Takahashi and Yamanaka, 2006; Yu et al., 2007) has great potential for tissue-specific regenerative therapies, avoiding ethical issues surrounding the use of ESCs and problems with rejection following implantation of nonautologous cells. So far, the only study on iPS cells focusing in periodontal regeneration showed that iPS cells combined with EMD were found to provide a valuable tool for periodontal tissue engineering by promoting the formation of new cementum, alveolar bone, and normal periodontal ligament when applied in an apatite-coated silk fibroin scaffold into a mouse periodontal defect (Duan et al., 2011).

PERIOSTEAL PROGENITOR CELLS The cultured periosteum was also considered as a suitable cell source for periodontal regeneration because it has the capacity to differentiate into an osteoblastic lineage and expresses periodontal tissue-related genes (Breitbart et al., 1998; Horiuchi et al., 1999). Mizuno et al. (2006) grafted autologous cultured cell membrane derived from periosteum into a class III furcation defect in dogs. Three months after the surgery, periodontal regeneration containing alveolar bone, cementum and periodontal ligament-like connective tissue was observed (Mizuno et al., 2006). In 2008, Yamamiya et al. showed that cultured periosteum combined with PRP and hydroxyapatite induced clinical improvements in human periodontal infrabony defects regeneration (Yamamiya et al., 2008).

CONCLUSION Great advances concerning the use of undifferentiated stem cells in regenerative medicine approaches have been done in the last decade. The regeneration of the periodontium is known to be challenging to the clinicians. Thus, the development of new therapies based on cells and/or tissue engineered scaffolds opened a new era of the periodontal regeneration. The stem cells from dental and oral origin were the firsts to be proposed for this aim, nevertheless, adult mesenchymal stem cells, namely, those from bone marrow and adipose tissue, have revealed promising characteristics to fulfill the regeneration of periodontal damages. Among the stem cells from adult origin, the adipose-derived stem cells assume a particular interest in terms of clinical application, regarding their easiness of harvesting, expansion and differentiation in several cellular lineages. Thus, in the near future it is expected that many efforts on the development of new tissue engineering strategies based in this cell source will be proposed for the regeneration of bone and periodontal damages.

ACKNOWLEDGEMENTS Figure 2’s photographs were a courtesy of Orlando Martins (DDS, MSc; Faculty of Dentistry, University of Coimbra, Portugal).

PERIODONTAL TISSUE ENGINEERING STRATEGIES

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