d e n t a l m a t e r i a l s 2 8 ( 2 0 1 2 ) 341–348
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Review
Tissue engineering: From research to dental clinics
Vinicius Rosaa, Alvaro Della Bonaa,!, Bruno Neves Cavalcantib, Jacques Eduardo Nörc
a Post-graduate Program in Dentistry, Dental School, University of Passo Fundo, Brazilb Dental School, Department of Dentistry, University of Taubaté, Brazilc Angiogenesis Research Laboratory, Department of Cariology, Restorative Sciences and Endodontics, University of Michigan School ofDentistry, USA
a r t i c l e i n f o
Article history:Received 23 August 2010Received in revised form23 August 2011Accepted 29 November 2011
Tissue engineering is an interdisciplinary field that combines the principles of engineer-ing, material and biological sciences toward the development of therapeutic strategies andbiological substitutes that restore, maintain, replace or improve biological functions. Theassociation of biomaterials, stem cells, growth and differentiation factors has yielded thedevelopment of new treatment opportunities in most of the biomedical areas, includingDentistry. The objective of this paper is to present the principles underlying tissue engineer-ing and the current scenario, the challenges and the perspectives of this area in Dentistry.Significance. The growth of tissue engineering as a research field has provided a novel set oftherapeutic strategies for biomedical applications. Indeed, tissue engineering may lead tonew strategies for the clinical management of patients with dental and craniofacial needsin the future.
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1. Introduction
The use of synthetic restorative materials as substitutes fordental structures is a practice nearly as old as Dentistryitself [1]. To date, most of the procedures performed in Den-tistry are limited to the replacement of damaged tissuesfor biocompatible synthetic materials that may not presentchemical, biological, or physical characteristics and behav-iors similar to the host tissues. These discrepancies, togetherwith the hostile environment of the oral cavity, result in rel-atively short-lived successful outcomes and frequent needfor re-treatment. Tissue engineering is a multi-disciplinaryfield focused on the development of materials and strategiesto replace damaged or lost tissues for biological materialsby merging principles, methods and knowledge of chem-istry, physics, engineering and biology [2]. The achievementsobtained by tissue engineering in the past few years haveresulted in new therapies such as the production of skin totreat burns [3], bone grafts to replace large bone defects [4],small-caliber arteries to treat atherosclerotic vascular disease[5] and cartilage for plastic and reconstructive surgeries [6].Important advances have been reported in Dentistry aimingthe regeneration of temporo-mandibular joint [7], periodon-tal ligament [8,9], dentin [10], enamel [11,12], pulp [10,13] andintegrated tooth tissues [14,15].
The concept underlying tissue engineering was first pro-posed in the United States in the mid-1980s in order to reducethe donor scarcity to organ transplantation [16]. The classicalcell-based tissue engineering approach involves the seedingof biodegradable scaffolds with cells and/or growth factors,then, implanting it in order to induce and conduct the tis-sue growth [17]. Obtaining good responses from this modeldemands the fine orchestration of the three tissue engineer-ing fundamental elements: cells, scaffold and cell signaling.The objective of this review is to present the fundaments ofthe tissue engineering components and their application inDentistry.
2. Tissue engineering
2.1. Cells
Stem cells are clonogenic cells capable of self-renewal andcapable of generating differentiated progenies. These cells areresponsible for normal tissue renewal as well as for heal-ing and regeneration after injuries [18]. Some stem cells aresaid to be pluripotent, i.e. have the ability to differentiate intomany different cell types while the range of others are morerestricted. The most pluripotent cells are found in the innercell mass of blastocyst during the early stages of embryo devel-opment [19]. They can differentiate into each of the morethan 200 cell types of the adult body [20] when exposed toappropriate stimuli. Along with the potential applications oftotipotent cells lies a strong ethical discussion regarding theuse of human embryos. This issue has strengthened the ratio-nal for the use of adult stem cells, which have been identifiedin every tissue formed after embryonic development and canbe used to the same purpose of embryonic stem cells.
Fig. 1 – The principles of tissue engineering using dentalstem cells may allow the regeneration of osseous, neuraland tooth-related tissues.
Studies have showed that it is possible to isolate clonogenicand highly proliferative cells from dental pulp using similarresearch protocol to isolate and characterize bone marrowstem cells [21]. Dental pulp stem cells (DPSC) can differentiateinto multiple cell lineages, such as adipocytes, chrondocytes,neurons and odontoblasts [22–24]. Stem cells from humanexfoliated deciduous teeth (SHED) were also identified andisolated [24]. SHED has the advantage of being retrievablefrom naturally exfoliated teeth, which are one of the onlydisposable post-natal human tissues. As primary teeth areclearly a feasible source of post-natal stem cells, the interesttoward the differentiation power of SHED cells has increased.Indeed, today we know that SHED can undergo adipogenic,chondrogenic, osteogenic, endothelial and odontoblastic dif-ferentiation [10,25,26]. The ability that these cells have to crosslineage boundaries expands the potential use of SHED for ther-apies involving a large number of tissues (Fig. 1).
Although both DPSC and SHED cells are originated fromthe dental pulp, they present differences regarding the odon-togenic differentiation and osteogenic induction. For example,the levels of alkaline phosphatase activity and osteocalcin pro-duction during osteogenic differentiation are higher for SHEDthan for DPSC [23]. However, the ability to regenerate a dentin-pulp-like complex found in DPSC [21,24] is also observed inSHED cells [25]. Furthermore, SHED may present an osteoin-ductive potential once they were able to induce differentiationfrom recipient murine cells into bone-forming cells [24].
The periodontal ligament was found to be a sourceof que a novel population of dental stem cell (PDLSC –periodontal ligament stem cell). This cell express high lev-els of telomerase [27], a key molecule in mediating cell
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proliferation [28] and have the capacity to develop adipocytesand cementoblast/osteoblastic-like cells in vitro. In addi-tion they also form collagen fibers, similar to Sharpey’sfibers, and cementum/periodontal ligament-like tissue whentransplanted into immunocompromised mice using hydrox-yapatite/tricalcium phosphate (HA/TCP) scaffold [29,28].
The stem cells from the apical papilla (SCAP) were recentlyisolated from the apical papilla of immature human perma-nent teeth [29]. The population seems to be the source ofodontoblasts responsible for the formation of root dentin [30].These may be the reason why SCAP present similarities toDPSC regarding osteo/dentinogenic and growth factor receptorgene profiles. The in vivo implantation of SCAP with HA/TCPscaffold allowed the differentiation into odontoblast-like cellscapable o regenerate a mineralized structure having a layer ofdentin tissue formed over the surface of the HA/TCP besidesconnective tissue [31].
Additionally, the ability of SCAP to regenerate the periodon-tal ligament and alveolar bone in vivo [28]. Analogous to DPSCand SHED, SCAP express a wide variety of neurogenic markersattesting its neurogenic potential [31].
2.2. Scaffolds
Scaffolds are temporary frameworks used to provide a three-dimensional microenvironment where cells can proliferate,differentiate and generate the desired tissue [32]. The design ofthe ideal scaffold for each tissue to be formed is a challengingtask. Ideally, a scaffold must allow cell attachment and migra-tion, permit the localized and sustained delivery of growthfactors, and enable the influx of oxygen to maintain the highmetabolic demands of cells engaged in tissue regeneration.
Scaffolds are usually made from ceramics [33], natural orsynthetic polymers [14], or composites from these materials[34]. The choice of scaffold material depends on the desiredoutcome thus physical (e.g. rheological behavior, mechani-cal properties, surface roughness and porosity) as well aschemical characteristics (e.g. mode, velocity and products ofdegradation) must be considered.
The scaffold’s physical properties have to attend the needsof the target environment. It must present proper mechanicalresistance to support in vivo stresses, and it should be mechan-ically compatible with the surrounding tissues [32,35–37]. Thescaffold’s mechanical properties have a direct impact in tissueformation by affecting cell differentiation into the desired phe-notype through mechanotransduction [32]. Therefore, linearelastic scaffolds are preferred when one attempts to gen-erate bone, and nonlinear elastic or viscoelastic models aremore suitable for soft tissues [36,38]. Scaffold porosity is alsocritical to tissue generation. The quantity and extension ofpores change the specific scaffold surface modifying its per-meability and mechanical properties, having strong impact incell seeding, nutrient diffusion and tissue ingrowth [35,37,39].Notably, higher number and extension of pores allows forenhanced cellularity but reduces scaffold strength [40,41]. Astudy suggested pore size ranging from 50 to 400 !m for theoptimum bone growth into porous-surfaced metallic implants[42]. However, it has been described up to 80% of bone in-growth after 2 months from implanting scaffolds in mice,regardless the pore sizes, which ranged from 300 to 1200 !m
Fig. 2 – Flow of events leading to protein synthesis. (A)Binding of ligand to its specific cell membrane receptortriggers intracellular signaling and activation of geneexpression. (B) Synthesis of new RNA template fromtranscription of the original strand of DNA. (C) Assemblingof the ribosome complex at the initiation codon of themRNA molecule. (D) Decoding of mRNA in sets ofnucleotides forming a polypeptide chain. (E) Folding in theGolgi apparatus to complete protein synthesis. This can befollowed by protein secretion to the extracellularenvironment or “sequestration” within the cell itself.
[43,44]. It has been proposed that pore interconnectivity iseven more important to sustain bone growth than size of thepore size itself [45].
The scaffold degradation is fundamental to achieve suc-cess in tissue engineering therapies [37]. The scaffold shouldideally reabsorb once it has served its purpose of providing atemplate for tissue regeneration. Importantly, the degradationmust occur at a rate compatible with the new tissue formation[46]. For example, the implantation of fast degrading scaffoldsdecreases the in vitro viability of primary smooth muscle cellsresulting in less cell population and lower angiogenesis lev-els [47]. Furthermore, the degradation products should not betoxic and must be easily cleared or resorbed to minimize therisk of inflammatory response [38,48]. It must be emphasizedthat during the scaffold degradation, the local pH should notbe significantly lower than the physiological pH [38], otherwisecell death and protein degradation may occur.
2.3. Cell signaling
Cell signaling is part of a complex system of communicationthat governs cell activities and organizes their interactions(Fig. 2).
Many extracellular molecules have been described in theliterature. It has been shown that a pool of these extracellu-lar molecules has a major role than a single protein in thedifferentiation of cells into a functional tissue. This could beobserved when proteins present in dentin disks [49], dentinextract in EDTA [50] or a tooth-germ conditioned extract [51]were found to supplement the scaffolds as a mechanism ofcellular induction. Yet, there still is missing information on
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how each factor acts in isolation. Among the different factors,the TGF"-1 (Transforming Growth Factor 1) and BMP (BoneMorphogenetic Protein), seem to have an important role in theodontoblastic differentiation [52]. Moreover, there is evidencethat the TGF"-1 is released from the dentin after any injury[53] and that BMPs, more specifically the BMP-2, have dentininduction ability [54].
The understanding of intracellular events triggered byextracellular proteins is critical for tissue engineering. In gen-eral, it is described that BMPs act in the canonical TGF-"
pathway modulating smads, leading to odontoblastic differ-entiation and inducing dentin formation [54,55]. However, itis important to keep in mind that TGF-" proteins, includingBMPs, have also well established roles in cancer progression[56]. Among these pathways, the Wnt pathway appears to beimportant for stem cell self-renewal [57] and cell differentia-tion [58]. The interactions and crosstalk between the canonicalsmad pathway and the Wnt pathway is probably one of themost studied, but there is little information on how theseinteractions affect DPSC. Additionally, it has been describedthat Wnt proteins are not able to induce DPSC differentia-tion [59]. On the other hand, there is a connection betweenBMPs with "-catenin, an intracellular protein that is part of theWnt pathway, which has an important role in differentiationprocesses for other cell types [60].
3. Current trends and future applications oftissue engineering in Dentistry
Recent advances brought by tissue engineering suggest thatsignificant changes in “traditional” clinical dentistry are begin-ning to occur. For example, the purpose of periodontal tissueengineering is to establish new therapies to manage periodon-tal diseases beyond the traditional approaches that are basedsolely upon infection control [61]. Chronic periodontitis is oneof the most common oral diseases worldwide, after dentaldecay, with a prevalence of 35% within the United States adultpopulation with at least 6 teeth [62]. A periodontal stem cell-based therapy will congregate the inflammation and infectioncontrol to stem cells capable to regenerate new periodontaltissues. This approach can rapidly providing cells for to thedamaged diseased site reducing the temporal gap between thestart of treatment and the arrival of progenitor cells, whichis frequently seen as one of the shortcomings of the tradi-tional procedures [28,63,64]. One problem to be overcome isthat residual PDLSC are limited in patients with periodontitisdue to the long-term inflammation [65]. However the achieve-ments obtained using these cells for periodontal needs areexciting. It has been shown that the association of PDLSC andSCAP mixed in a HA/TCP scaffold allows for reconstruction ofreconstruct a functional tooth with a root periodontal complex[28]. Similar protocol was responsible for regenerating for theregeneration of alveolar bone in dogs after an 8-week evalua-tion period [64]. The potential of this cells to treat periodontitiswas shown by implanting PDLSC in HA/TCP scaffolds in peri-odontal bone defects for 12 weeks, it was possible to observean osseous regeneration near to 50% of total original defectheight of 7 mm [65].
To date, the regeneration of small- to moderate-sizedperiodontal defects using engineered cell-scaffold constructsis technically feasible, and some of the current conceptsmay represent alternatives for selected clinical scenar-ios. However, the predictable reconstruction of the normalstructure and functionality of a tooth-supporting apparatusremains challenging [8,61]. The future possibilities dependon improved understanding of cellular and molecular mech-anisms involved in the regeneration of periodontal tissues,the differentiation potential of stem cells, and the interac-tion between stem cells and scaffold with host tissues. Majorbone reconstructions because of trauma, cancer, or augmen-tation for dental implants are just few examples of how tissueengineering can be also be used for craniofacial applications[33].
In clinical scenarios involving normal bone, the long-term implant success and survival rates are above 90% [66].However, conditions as smoking, diabetes, radiotherapy, andpostmenopausal estrogen therapy may compromise bonequantity and quality leading to scenarios in which bonehealing may become challenging [67]. Through developing bio-materials and strategies that may increase bone cell adhesion,modulate cell signaling, deliver growth factors and promoteosteoblast differentiation followed by matrix deposition andmineralization [68,69], it might be possible to place implantseven under less than ideal anatomical or biological circum-stances [70].
One interesting strategy to increase osteointegration oftitanium implants is to coat them with extracellular matrixcomponents, such as collagen, BMP, TGF-" or chondroitin sul-fate [71,72]. BMPs are known to improve healing, to induceperi-implant bone formation and to enhance osseointegrationof endosseous implants [71]. TGF-" is produced in response tofactors that stimulate osteoclastic bone resorption and plays akey role in osteoblastic bone formation, inhibiting osteoclastformation and activity [73]. Chondroitin sulfate has an anti-inflammatory effect, accelerates bone repair and increasesbone regeneration [74]. The addition of these growth factorson implant surface is certainly an exciting perspective, butmany questions still remain unanswered. It is known thatBMP and TGF-" act synergistically but this action depends onthe temporal sequence and timing of growth factor delivery[75]. Thus, to gain further advantage on implant osseointe-gration using growth factors, it is necessary to understandbeyond the application mechanisms of these proteins and toorchestrate their release pattern, increasing the time that theyare bioactive and maximizing their potential [71]. It is impor-tant to consider the release kinetics and degradation rate tooptimize the amount of extracellular matrix components overthe implant surface [72,76]. As the cost of proteins is high,to achieve maximum bone formation at minimal concentra-tion is crucial to reduce the toxicity and side effects for thisapproach to become affordable and clinically safe.
Autogenous bone transplantation has been used for repair-ing the bone defects for a long time. Although this techniquemay provide to the patient its own scaffold, growth factorsand cells [77] it also present some shortcomings such as:the sacrifice of healthy tissue, limited availability of donortissue, post-surgical bone resorption, and donor-site mor-bidity [78]. Tissue engineering may circumvent many of the
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Fig. 3 – Dental pulp tissue engineered for 35 days inside root canal using SHED cells (A) and natural dental pulp from ayoung premolar (B). It is possible to observe the formation of a healthy tissue without inflammatory signs and adensification of odontoblast-like cell along dentin walls in the SHED originated tissue similar to the control. The engineeredtissue occupies the whole apical portion (C) and immunohistochemistry with PCNA (proliferating cell nuclear activity) andFactor VIII show a proliferative tissue with well established and mature blood network (D and E).
limitations of the available techniques and its power has beenexplored to minimize the need of surgery to obtain the bonegraft [68,69]. In vivo studies observed stem cell differentia-tion toward osteoblast-like cell producing a 30-mm long repairin a mandibular segmental defect of a dog 32 weeks afterimplantation of scaffolds with bone marrow stem cells [79,80].Other reports showed that mesenchymal stem-cells derivedfrom bone marrow or peripheral adipose tissue were able toregenerate tissue in critical-sized craniofacial defects of micewithout the addition of exogenous growth or morphogeneticfactors [81,82]. These findings allow foreseeing that tissueengineering can become a breakthrough approach to recon-struct bone deformities in a more effective and less traumaticway.
Restorative dentistry is looking for techniques and mate-rials to regenerate the dentin–pulp complex in a biologicalmanner. Tissue engineering-based approaches have thepotential to do it (Fig. 3). It has been reported that DPSCand SHED can be induced to differentiate into odontoblasts[10,26]. SHED seeded in tooth slices containing scaffolds andimplanted in immunodeficient mice are able to produce a den-tal pulp tissue [10,13]. In addition, it has been also shown thatSHED cells can differentiate into odontoblasts that generatetubular dentin in vivo [10,83]. It was shown that DPSC are ableto produce pulp/dentin-like tissues in an emptied human rootcanal with the deposition of a layer of mineralized tissue onthe canal walls [84]. One important step toward regenerativeendodontics was achieved when SHED mixed with nanofiberpeptide scaffold and injected into full-length root canals wereable to generate a dental pulp. The original data presentedhere in Fig. 3 shows the presence of a pulp tissue fulfilling the
hollow passageway of the root canal, with proliferative activityand blood network maturity comparable to the ones observedin a young human dental pulp. The dental pulp engineeredwas able to deposit organized dentin along walls (data notshown). These findings are exciting once SHED may be readilyretrievable from decidous tooth, the only “disposable” tissueand injectable scaffolds will allow dentists to easily deliverstem cells inside root canals disregarding its internal anatomy.
These findings support the concept that the regenera-tion of the dentin–pulp complex with stem cells might beclinically achievable. However, this may not be applicable toall clinical scenarios. For example, older teeth with smallpulp chamber and root canals and with closed apex maypresent a formidable challenge to tissue engineering. How-ever, engineering a dental pulp to deposit dentin and continuethe natural root growth of non-infected, accidentally injuredyoung teeth is apparently more feasible.
4. Future direction in Dentistry
The future of tissue engineering in Dentistry is exciting. It iscertainly possible that, once dentist-scientists bring togetherthe new discoveries in material’s sciences, genetics, molecu-lar and cell biology, new alternatives for regeneration of boneand soft tissues [33,82], management of periodontal disease[8,61,64] and restorative procedures to regenerate enamel,dentin and pulp will become available for clinical application[2,10]. However, one of the important considerations will cer-tainly be the cost of these procedures. It does not only appliesto the cost of treatment itself, but the aggregate costs required
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to introduce such technology to clinicians and students andto build facilities for dental stem cell obtaining and bank-ing and to produce scaffolds at affordable prices. History hasshown that most of the revolutionary technologies becamemore affordable as they have become more popular, and, per-haps, this will be also true for tissue engineering in Dentistry.It is difficult to predict at this time the full impact of tissueengineering to the future of Dentistry.
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