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Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Shinjuku-ku, Tokyo, Japan
Correspondence to: Teruo Okano, Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Fax: +81333596046. E-mail: email@example.com
Periodontitis is a global disease that destroys the tooth-supporting attachment apparatus, which consists of alveolar bone, cementum, and periodontal ligament. Recent studies have reported numerous associations between periodontitis and systemic diseases, such as cardiovascular disease (de Oliveira et al., 2010) and diabetes mellitus (Lalla and Papapanou, 2011), as well as a higher risk of preterm low birth-weight babies (Offenbacher et al., 1996). Furthermore, research has recently shown that Bisphosphonate-Related Osteonecrosis of the Jaws (BRONJ) is associated with severe periodontitis (Vescovi et al., 2011). Consequently, periodontal treatment may not only contribute to oral hygiene but also improve systemic conditions (Seymour et al., 2007).
Conventional treatments such as scaling, root-planing, and surgical cleaning are currently performed to remove the bacteria and contaminated tissue. However, these procedures frequently result in the formation of weak attachment called a “long junctional epithelium,” LJE, (Caton et al., 1980). In addition, these conventional procedures cannot restore the periodontal tissue to its original form, and the decrease in bone height and gingival recession usually cause aesthetic and functional problems. Several regenerative procedures have already been introduced to clinical practice to overcome these problems, including bone grafts, guided tissue regeneration, and enamel matrix derivative. However, the outcomes of these procedures have limited success and have poor clinical predictability (Esposito et al., 2009). Therefore, research has turned to investigating stem cell-based approaches for periodontal regeneration [see in review (Ishikawa et al., 2009)].
“Tissue engineering,” a concept proposed by Langer and Vacanti, is widely accepted in the field of regenerative medicine to create replacement tissues using a combination of cells, scaffolds, and growth factors (Langer and Vacanti, 1993). However, there are limitations to this concept; grafts may be rejected as a result of inflammation caused by scaffold degradation, necrosis, or unstable transplanted cells (Yang et al., 2005). Several technologies using new biomaterials are being introduced to overcome these limitations (Williams, 2009). In this review, we focus on recent studies involving tissue engineering and regenerative medicine using biomaterials in the periodontal field.
Periodontal tissue consists of four component structures: alveolar bone, periodontal ligament, cementum, and gingiva (Figs. 1-3). Specifically, alveolar bone, periodontal ligament, and cementum are called the “attachment apparatus” and are the target tissues for periodontal regeneration (Fig. 2).
Pathogenesis and Treatment of Periodontal Disease
“Gingivitis” or gingival inflammation is initiated and promoted by the accumulation of dental plaque (Fig. 4a). If the inflammation is localized within the gingiva, proper plaque control can restore a healthy condition. However, once the inflammation and the host immune response expand to the adjacent periodontal tissues (alveolar bone, cementum, and periodontal ligament), connective tissues, and bone become permanently damaged. This spread of inflammation is called “periodontitis,” and spontaneous healing is not expected (Fig. 4b). Although decontaminating procedures can be performed, the height of bone will not improve. Moreover, gingival recessions create aesthetic issues, especially in anterior teeth (Fig. 4c). If periodontitis is left untreated, the attachment apparatus is completely destroyed resulting in tooth mobility and loss.
The purpose of conventional periodontal therapies, such as scaling, root planing, and periodontal surgery, is to remove the bacterial pathogens and infected granulation tissues surrounding the teeth. These procedures can halt the disease process, but they cannot restore the tissue to its original form. Additionally, the treatments may result in the formation of an LJE; thus, patients tend to relapse without maintenance therapies (Axelsson and Lindhe, 1981). The final goal of periodontal regeneration is to reform the lost tissue and restore to its original form (bone structures with well-oriented periodontal ligament anchoring to dental cementum).
CONVENTIONAL APPROACHES FOR PERIODONTAL REGENERATION BONE GRAFT MATERIALS
Bone graft regeneration is still somewhat controversial; bone grafts may not always regenerate true periodontal tissue with well-oriented fibers anchoring the cementum to the alveolar bone (Mellonig, 1992). Histological analyses have revealed that LJE between the alveolar bone and root surface often migrate apically. In addition, root resorption and ankylosis are occasionally observed (Table 1).
Table 1. Bone graft materials
Tissue engineering in periodical tissue
Osteogenic, osteoinductive and osteoconductive no immunological rejection living cells and matrices
Morbidity at donor sites amount of bone volume is limited rapid absorption
Demineralized freeze-dried bone allograft (DFDBA)
Osteoinudctive and osteoconductive
Potential of infection and immunological rejection
Freeze-dried bone allograft (DFDBA)
Osteoinudctive and osteoconductive
Potential of infection and immunological rejection
Potential of infection and immunological rejection Slow resorption or non-resorbable
Sintered hydroxyapatite (HA)
Slow resorption or non-resorbable
β-tricalcium phosphate (TCP)
Natural products (coral, citosan etc.)
Osteoconductive, low immunological rejection
Slow resorption or non-resorbable
Clinical studies have shown that the gain of bone height is ∼3.0 mm irrespective of the type of bone graft material (Mellonig, 1992). The gold standard for bone grafting is autologous bone, which is capable of osteogenesis, osteoinduction, and osteoconduction. For these grafts, a portion of bone is taken from a healthy site of the patient receiving the graft. The advantage of autologous bone is that it inherently has the vital structures of bone tissue and contains cells, minerals, and proteins. The disadvantages of autologous bone grafts are the patient morbidities at the removal sites and limited amount of bone that can be harvested.
As a result of these disadvantages, many allogeneic bone grafts and xenograft materials have been investigated and approved and are commercially available (Miron and Zhang, 2012). However, clinicians must consider the potential risks of disease transmission, rejection, and resorption when using these grafts. There are two types of allografts: demineralized freeze-dried bone allografts and freeze-dried bone allografts. Both of these types of allografts are chemically pretreated, retaining osteoinductive proteins such as BMPs and TGF-βs, to enhance the osteoinduction of endogenous mesenchymal stromal cells. It is thought that the process of demineralization enhances the release of inherent growth factors, such as BMPs (Urist, 1965).
Xenografts are derived from non-human species, such as bovine, porcine, or equine animals. They are usually sintered and chemically pretreated to remove their antigenicity. The advantage of these products is that the shape of the gragt can be retained, because the resorption of these materials is relatively slow. Although they are only capable of osteoconductivity, the safety and efficacy of these products have been proved clinically (Camelo et al., 1998; Richardson et al., 1999).
Because of the risks associated with allograft and xenograft materials, researchers have also investigated synthesized graft materials, including polymer and inorganic composite grafts, to regenerate bone [see in review (Rezwan et al., 2006)]. Specifically hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) have been studied in human periodontal defect models. Although synthesized materials are only osteoconductive (not osteogenic or osteoinductive), they can successfully restore bone defects if the environment is favorable. Recently, moldable in situ hardening polylactide-coated TCP has been developed and applied to repair bone defects in rabbits; the results showed good biocompatibility (Schmidlin et al., 2013).
Natural products, such as coral, have also been investigated for bone grafting because their structures are similar to those of bone (Damien and Revell, 2004). Like synthetic materials, natural products are only osteoconductive, but they are cost-effective and considered to pose no risk to human health.
Physical Barriers for Epithelial Tissue
In the past, it was predicted that the periodontal ligament tissue contains the cells responsible for periodontal regeneration (Melcher, 1976); recently, such multipotent cells have been identified in periodontal tissue (Seo et al., 2004; Gay et al., 2007; Iwata et al., 2010). In the 1980's, physical barriers were introduced into periodontal surgeries to eliminate epithelial downgrowth, and selective proliferation of mesenchyme derived cells was enhanced (Nyman et al., 1982). Physical barriers have been widely employed in clinical practices, but this procedure involves technically difficult steps and potential complications, such as exposure of the barrier or infection. Various barrier membranes have been developed and are commercially available.
Enamel Matrix Derivatives (EMD)
EMD (commercially available as EMDOGAIN®), a mixture of porcine immature enamel extracts, has been showed to effectively treat infrabony periodontal defects (Heijl et al., 1997). A meta-analysis (eight trials) showed that EMD-treated sites displayed statistically significant improvements compared with sites treated with flap surgery (Esposito et al., 2009). The main component of EMD is amelogenin, a majority of enamel proteins, and amelogenin is believed to possess the properties needed to regenerate periodontal tissue because the expression of amelogenin was observed in developing dental root (Hammarstrom, 1997). In addition to enamel matrix proteins, several studies have demonstrated that EMD contains small amount of growth factors, such as BMPs (Iwata et al., 2002; Johnson et al., 2009) and TGF-β1 (Suzuki et al., 2005), explaining the complex effects of EMD. A particularly interesting study found that propylene glycol alginate (PGA), the vehicle of EMD, has antibacterial properties, suggesting that the combination of EMD and PGA enhances not only periodontal (Sculean et al., 2001) but also epithelial wound healing (Mirastschijski et al., 2004; Al-Hezaimi et al., 2012).
Tissue Engineering Approaches for Periodontal Regeneration Growth Factors used for Periodontal Regeneration
Combining growth factors and carriers (scaffolds) for periodontal regeneration has also been investigated. This concept of using a drug delivery system (DDS) was introduced to prolong the therapeutic effects of growth factors (Chen et al., 2009). In this section, the application of growth factors to periodontal defect models in large animals is reviewed.
Platelet-Derived Growth Factor (PDGF)-BB and Insulin-Like Growth Factor One (IGF-1)
The combination of PDGF-BB and IGF-1 was the first reported use of growth factors for periodontal regeneration, and it was tested in a canine model (Lynch et al., 1989). A subsequent clinical trial also showed that this combination of growth factors in a methylcellulose vehicle was safe and that it enhanced periodontal regeneration (Howell et al., 1997). Giannobile et al. demonstrated that PDGF-BB alone significantly stimulates periodontal regeneration, but that IGF-1 alone does not alter periodontal wound healing in a nonhuman primate model (Giannobile et al., 1996). A multicenter randomized controlled trial with 180 subjects enrolled in 11 clinical centers showed the safety and efficacy of PDGF-BB soaked in β-TCP matrix for the treatment of advanced periodontal osseous defects (Nevins et al., 2005, 2013). This product is commercially available as GEM 21 S® (Osteohealth, Shirley, NY).
Several types of carriers for sustained-releasing of PDGF-BB, including porous poly(l-lactide) membranes (Park et al., 1998), chitosan sponges (Park et al., 2000), chitosan/TCP sponges (Lee et al., 2000), and nanofibrous scaffolds (Wei et al., 2006), have been investigated, and their efficacies were confirmed.
Bone Morphogenetic Proteins (BMPs) and Transforming Growth Factor-βs (TGF-βs)
The BMP/TGF-β signaling pathway mediates osteoblastic differentiation and in vivo bone formation; BMP2 in particular has been thoroughly studied for periodontal regeneration. Several carriers, including autologous blood and poly(d,l-lactide-co-glycolide) (PLGA) particles (Sigurdsson et al., 1995), PLGA and gelatin sponges (Kinoshita et al., 1997), and absorbable collagen sponges (ACS; Selvig et al., 2002), have been investigated. The combination of BMP2/ACS is commercially available as INFUSE® bone graft (Medtronic, Minneapolis, MN); it is approved for bone augmentation for sinus lifting and implant dentistry, but not for periodontal osseous defects at this time. Other members of the BMP family, such as BMP-7/OP-1 (Giannobile et al., 1998; Ripamonti et al., 2001), BMP-12/GDF-7 (Wikesjo et al., 2004), BMP-14/GDF-5 (Kwon et al., 2010; Lee et al., 2010), have also been investigated in large animal models, and their efficacies for periodontal regeneration were observed in all studies. A recent study compared the carriers, β-TCP and ACS, for BMP14/GDF5 delivery in a canine periodontal defect model (Kim et al., 2013). The authors observed that β-TCP was superior for new bone formation.
Whether TGF-β1 directly enhances periodontal regeneration is still controversial (Tatakis et al., 2000); however, it has been reported that TGF-β1 induced endochondral bone formation (Serra et al., 1999). Another study suggested that TGF-β3 in Matrigel® matrix (BD Biosciences, San José, CA) in combination with minced muscle tissue significantly enhanced the periodontal regeneration in class III furcation defects in P. ursinus (Ripamonti et al., 2009).
Basic Fibroblast Growth Factor (bFGF, known as FGF2)
The use of bFGF, a growth factor that is already utilized clinically for wound healing in patients with intractable skin ulcers, has been investigated for bone formation and periodontal regeneration. Its efficacy on periodontal regeneration was demonstrated in beagles and nonhuman primates (Murakami et al., 1999, 2003; Takayama et al., 2001). A multicenter, randomized clinical trial was performed using a 3% hydroxypropyl cellulose carrier (Kitamura et al., 2011, 2008); the results showed that bFGF was safe and effective for periodontal regeneration. Several bFGF carriers, including sandwich membranes of collagen sponge with bFGF-incorporated gelatin microsphere (Nakahara et al., 2003), collagen gel (Sato et al., 2004), and β-TCP (Oi et al., 2009; Anzai et al., 2010), have been investigated.
Brain-Derived Neurotrophic Factor (BDNF)
Despite its name, BDNF is found in several tissues, even in PDL cells (Kurihara et al., 2003). The application of BDNF in combination with atelocollagen sponge stimulated the formation of new alveolar bone, cementum, and connective fibers; the fibers were inserted into the newly formed cementum and bone in a canine class III furcation defect model. BDNF also stimulated blood capillary formation (Takeda et al., 2005). A recent study claimed that scaffolds made from high-molecular-weight hyaluronic acid improved the regenerative capacity of BDNF in a canine periodontal defect model (Takeda et al., 2011).
Cytotherapy for Periodontal Regeneration
Several types of mesenchyme-derived cells have been investigated for periodontal regeneration.
Shi and colleagues isolated and investigated a population of multipotent cells from human PDL tissues, called PDLSCs, which possessed the ability to form all components of the attachment apparatus when they were subcutaneously transplanted with β-TCP/HA scaffolds in immunodeficient mice (Seo et al., 2004). When autologous PDLSCs seeded in a HA/TCP scaffold were transplanted into periodontal defects of miniature pigs, complete periodontal regeneration was observed (Liu et al., 2008). Further studies used temperature responsive culture dishes to create PDL-MSC sheets. Three-layered canine PDL-MSC sheets were fabricated using woven polyglycolic acid (PGA) (Neoveil®, PGA Felt-Sheet Type, 0.15 mm in thickness: Gunze, Tokyo, Japan) and transplanted into the denuded root surface autologously, while bone defects were filled with porous β-TCP (Iwata et al., 2009; Tsumanuma et al., 2011). After healing, complete periodontal regeneration was observed in both studies. In addition, PDLSC sheets without bone graft materials can regenerate complete periodontal tissue in a canine furcation defect model (Figs. 5 and 6). A recent study suggested that the allogeneic transplantation of PDLSC sheets with HA/TCP was applicable to periodontal regeneration; its safety and efficacy were confirmed in a swine model (Ding et al., 2010).
Bone Marrow-derived MSCs (BM-MSCs)
Bone marrow-derived MSCs are thought to be trophic and immunomodulatory, as well as multipotent (Caplan and Correa, 2011). In fact, MSCs have been utilized in numerous clinical trials for various diseases, including graft versus host disease, acute myocardial infarction, stroke, acute kidney failure, and tendonitis.
Clinical trials using BM-MSCs with platelet-rich plasma (PRP) have also been conducted, and the results demonstrated that periodontal regeneration was successful (Yamada et al., 2006, 2013). Another group used BM-MSCs combined with atelocollagen sponges; eleven patients received the transplantation in Japan, based on results from previous canine studies (Kawaguchi et al., 2004; Hasegawa et al., 2006).
Periosteum-Derived Cell Sheets
Cultured periosteum is considered a suitable source of cells for periodontal regeneration because it is capable of differentiating into the osteoblast lineage. In one study, periosteum cell sheet samples (5 × 5 mm) were harvested from patients by scraping, and an explant culture was performed in each case. Coagulated PRP and porous HA granules were placed into the infrabony defects, and 6-week-cultured periosteum sheets were autologously transplanted in 15 patients (Yamamiya et al., 2008; Okuda et al., 2009). The results showed that the cultured periosteum sheets transplantation exhibited a statistically significant improvement in clinical attachment gain, vertical relative attachment gain, and radiographic infrabony defect fill as compared with the control.
Adipose-Derived Stem Cells (ADSCs)
ADSCs are a useful source for cell transplantation therapy because they are abundant and easy to obtain. ADSCs mixed with PRP enhanced the periodontal regeneration in rats (Tobita et al., 2008). Another source reported that ADSCs in a fibrin gel scaffold enhanced periodontal regeneration at applied sites (Murakami, 2011).
Gingival Epithelial Cells and Fibroblasts
Gingival epithelial sheets derived from autologous gingival tissues were developed and applied clinically as a treatment for chronic desquamative gingivitis (Okuda et al., 2004). The results showed that human cultured gingival epithelial sheets promoted gingival augmentation. Recently, an allogeneic cellular product named GINTUIT™ (Organogenesis) was approved by the FDA and was expected to be commercially available in the summer of 2012 (http://organogenesis.com/products/oral_regeneration/oral_regeneration.html). This product consists of allogeneic cultured keratinocytes and fibroblasts in bovine collagen. In a pivotal, multicenter, randomized, within-patient, controlled, open-label trial, McGuire et al. concluded that the product was a safe and effective therapy for augmenting the keratinized gingival zone (McGuire et al., 2011).
DISCUSSION AND COMMENTARY
In this review, recent tissue engineering approaches for treating periodontal regeneration were described. Because cells or growth factors alone are not sufficient for periodontal regeneration, great efforts have been made to optimize the biological and biophysical design of carriers or scaffolds. Initially, biodegradable polymers were investigated, but they were not successful because controlling the shape of implants is difficult owing to resorption, and inflammation occasionally occurred. Therefore, more recent clinical studies have often use collagen or β-TCP as a carrier/scaffold to retain the cells/growth factors because of the safety, cost-effectiveness, osteoconductivity, and biocompatibility of these materials. Other studies also suggest that well-designed polymers or synthetic materials can be applicable as carriers/scaffolds for periodontal treatment. For example, a biphasic scaffold composed of a fused deposition modeling scaffold (bone compartment) and an electrospun membrane (periodontal compartment) with PDL cell sheets regenerated true periodontal tissue in an athymic rat subcutaneous model (Vaquette et al., 2012).
In terms of cells, mesenchyme- or neural crest-derived cells are the most common cell source for periodontal regeneration, because periodontal tissue originates from dental cranial neural crest cells (Ishikawa et al., 2009). One recent study suggested that the transplantation of induced pluripotent stem (iPS) cells with combination use of silk scaffold and EMD enhanced periodontal regeneration in mice (Duan et al., 2011). Furthermore, allogeneic embryonic stem (ES) cells in a collagen membrane improved periodontal furcation defects in a porcine model (Yang et al., 2013). Although the safety and efficacy of ES/iPS cells have not yet been proved, they may become an alternative cell source for periodontal regeneration in the future (Egusa et al., 2012).