Scaffolds for regeneration of the pulp–dentine complex

Scaffolds for regeneration of the pulp–dentine complex

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Lavanya Ajay Sharma, Robert M. Love School of Oral Health and Dentistry, Griffith University, Gold Coast, Australia

22.1  Introduction Dental caries remains one of the major public health concerns worldwide [1]. This multifactorial disease results in demineralization of enamel and dentine with an associated inflammatory reaction in the pulpal tissue [2,3]. Infection of dental pulp, by direct invasion of cariogenic bacteria or as a consequence of trauma, often necessitates pulpal amputation. Removal of diseased tissues followed by restorative and endodontic root canal procedures with inert synthetic material has been a routine clinical practice for decades. Vital pulp therapy/pulp capping procedures to induce dentinal bridge formation with the use of cements such as calcium hydroxide or mineral trioxide aggregate (MTA) are considered to promote dentinogenesis [4–6]. However, these materials fail to replace the biological function, vitality, and mechanical properties of the original tissue and show variable treatment outcomes [7,8]. Despite the high clinical success rate of traditional root canal therapy following removal of a dental pulp (pulpectomy) [9–12], other consequences need to be considered. For instance: 1. In a root canal–treated tooth, the esthetics are of the tooth crown, which are affected through structural loss of tissues or discoloration of the crown by staining from the endodontic filling material [13,14]. 2. The structural integrity of root canal–treated teeth may also be undermined because of loss of tooth structure and subsequent restorative procedures [15–17]. 3. Pulpless teeth lose some of their ability to sense environmental changes, which may lead to the progression of caries being unnoticed by patients. 4. Long-term studies have shown that tooth loss is higher for root canal–treated teeth than nontreated because of secondary caries and associated complex restoration problems [18–21].

The desire to develop a biological alternative to root canal therapy to encourage regeneration of pulp–dentinal tissues to revitalize teeth has gained interest [4,22–24]. Advances in tissue engineering and biotechnology have opened new directions for designing biological methods for pulp treatment that are aimed at in situ regeneration of partial pulp or de novo synthesis of total pulp replacement [25,26]. The approaches include (1) an effort to harness the pulp’s own regenerative capacity, i.e., induce host cells from the apical region of the pulp to migrate toward the interior of the root canal [27] or (2) the replacement of the entire pulp tissue by transplantation of in vitro engineered pulp tissue [28,29]. The idea of pulp regeneration is not new. It emerged as early as 1963 when Ostby demonstrated tissue ingrowth into an empty pulp space following the introduction Handbook of Tissue Engineering Scaffolds: Volume One. https://doi.org/10.1016/B978-0-08-102563-5.00022-8 Copyright © 2019 Elsevier Ltd. All rights reserved.

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of a blood clot [30]. However, the term “pulp regeneration” is justified only if new and functional odontoblast-like cells line the dentine walls of the pulp chamber and a vascularized connective tissue is present. After early attempts at pulp regeneration, the scene was set for a number of new studies based on the tissue engineering concept, as delineated by Langer and Vacanti in 1993, which involves the triad of stem/progenitor cells, scaffolds for cell growth, and growth factors [31]. This chapter will initially highlight pulpal biology, its reaction to trauma or caries, and existing treatment strategies. Later sections will present an informative summary on scaffolds used for pulp–dentine regeneration and recent updates regarding the newer advanced biomaterials investigated for the same. This review would be of importance to future researchers in terms of the present status and future potential for the clinical application of regenerative biomaterials in dentistry.

22.2  Pulp–dentine biology and response to current treatment therapies Dental pulp is highly specialized, loose connective tissue in the central portion of the tooth. It contains heterogeneous cell populations responsible for its maintenance, defense, and repair. The cell types identified within the pulp include fibroblasts, which are the predominant cell type, as well as inflammatory and immune system cells, including dendritic cells, neutrophils, histiocytes/macrophages, T/B lymphocytes, and odontoblasts [32]. Several niche environments for latent or dormant pulpal stem cells (progenitors), necessary for repair and regenerative processes, have been identified, and these include perivascular regions and the cell-rich layer of Hohl [33]. Also, key to successful regenerative responses are the complex and rich neuronal and vascular networks, which exist within the pulp. Anatomically and phsiologically the term “pulp–dentine complex” refers to the dental pulp encased by dentine along its periphery. The primary function of pulp is to produce dentine (dentinogenic), whereas it also provides nutrition, sensory, and defensive functions for a tooth. During tooth development, a series of complex epithelial–­ mesenchymal interactions within the developing tooth germ initiate differentiation of the ectomesenchymal cells in the dental papilla into odontoblasts. Odontoblast cells are specialized postmitotic cells responsible for the secretion of primary dentine. Dentine is composed of inorganic (hydroxyapatite) material, an organic matrix, and water. About 90% of the organic matrix is type 1 collagen, and the remaining 10% is composed of noncollagenous proteins (NCPs) and proteoglycans. The series of NCPs associated with the collagen fibrils help in dentine mineralization. There are three types of dentine: primary, secondary, and tertiary. Dentine formed throughout tooth development before tooth eruption is termed primary dentine; after complete root formation and normally after tooth eruption, it is called secondary dentine; and later as a response to various stimuli or injury to the pulp–dentine complex, an inflammatory response triggers the formation of tertiary dentine. An ambiguous relationship between pulp inflammation and dentine repair/regeneration exists i.e., inflammation may impair or support

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dentinogenesis [34]. Low-grade inflammation, potentially induced by mechanical trauma and tissue necrosis, may promote regenerative mechanisms including angiogenic and stem cell processes. Notably, locally derived growth factors, neuropeptides, cytokines, and chemokines released from the host dentine matrix and by resident pulpal cells, immune cells, neurons, and/or dying cells, will modulate defense and repair processes within the tissue [35]. In the case of a rapidly progressing disease, the relatively high levels of immune and inflammatory molecular and cellular activity will likely result in impairment of the healing process including induction of cell death. Formation of tertiary dentine (tertiary dentinogenesis) is of particular interest in relation to the process of repair and regeneration (Fig. 22.1). Any stimulus to the dentine, whether it is from disease, trauma, iatrogenic, and/ or of restorative materials, will rapidly trigger a response in odontoblasts and other pulpal cells. Accordingly, the tertiary dentine formed can be classified into either reactionary or reparative type [36,37]. Reactionary dentinogenesis is a response to mild injury (initial caries/mild trauma) in which the existing odontoblasts respond and secrete tertiary dentine. Any stimulus, be it mechanical, e.g., from cavity preparation or chemical from the material being applied to exposed dentine surface, may be capable of initiating the process of reactionary dentine formation. In contrast, reparative dentinogenesis is a response to sustained insult, greater stimuli, or severe injury to the pulp resulting in death of local odontoblasts. As a result, this requires the recruitment of progenitor/stem cells from the pulp to induce differentiation of odontoblast-like cells and secrete dentine [38,39]. The resulting reparative dentine differs in many aspects from reactionary dentine [40]. Histologically, reparative dentine has more matrix, often containing cell inclusions, and fewer irregular dentinal tubules [8,41,42]. Inductive signaling molecules, called growth factors, mediate this entire mechanism of stimulation and differentiation. These are small protein molecules, which have a major influence on cell division, differentiation, and migration. Among them, TGF-β (subclass of transforming growth factor family), secreted by inner enamel epithelial Tertiary dentinogenesis Reactionary Odontoblast up regulation

Mild injury Reactionary dentinogenesis

Reparative Extensive injury Reparative dentinogenesis

Progenitor cell recruitment Induction of odontoblast-like cell differentiation Odontoblast-like cell up regulation

Tertiary dentine secretion Figure 22.1  Illustration of pulp healing reaction to injury.

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cells, plays a vital role in signaling odontoblast differentiation during tooth development [36]. Once differentiated, the odontoblasts synthesize and secrete TGF-β, as well as other growth factors such as BMPs, insulin-like growth factor-1 and 2 (IGF-1 and IGF-2), fibroblast growth factor-2 (FGF-2), and various angiogenic growth factors within the dentine matrix. These growth factors are essentially “fossilized” within the dentine matrix and remain protected in their bioactive state until the matrix is degraded/solubilized during carious or other injurious episodes. When released, these molecules diffuse to the cells of the pulp and thus are available to participate in the reparative events [42]. Most of the dental materials used presently in the above procedures induce reparative dentine formation most likely through an indirect mechanism, which causes the release of growth factors from the dentine matrix [40,43–45]. The major problem with inorganic restorative materials is their inability to induce cell differentiation [46].

22.3  Role of tissue engineering in regenerative endodontics Current advances in tissue engineering and biotechnology have greatly changed the concept of medical treatment. A biological approach for the replacement of lost tissues appears to be the major goal of the present research. During the last decade, the field of dental tissue regeneration has also emerged as an exciting area of research. Two approaches were popularly tried. The first involves an effort to harness the pulp’s own regenerative capacity to form new tissue, i.e., host cells from the apical region are induced to migrate toward the interior of the root canal. The second approach includes replacement of the entire pulp tissue by either transplantation of in vitro engineered pulp tissue or the transplantation of a matrix loaded with stem cells into the root canal for differentiation [28,29]. The concept of tissue engineering, which includes the triad of stem cells, scaffold, and growth factors, seems applicable for pulp tissue engineering (Fig. 22.2). Growth factors

Stem cells

Scaffold

Figure 22.2  Triad of tissue engineering.

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22.4  Scaffolds 22.4.1  Definition, ideal requirements, and biomaterial selection Equally as important as the stem cell source in the classic tissue engineering paradigm is the scaffold. According to the American Society for Testing Materials (ASTM), a scaffold is defined as “the support, delivery vehicle, or matrix for facilitating the migration, binding, or transport of cells or bioactive molecules used to replace, repair, or regenerate tissues” [47]. A biological scaffold acts as a platform that provides a three-dimensional substrate/temporary matrix for developing cells and, therefore, plays an important role in tissue engineering [48]. Moreover, an ideal tissue engineering scaffold material would be able to mimic the natural extracellular matrix (ECM) [49]. The following are the general requirements for biomaterial scaffolds in pulp regeneration [50–53]: (1) biocompatible, (2) undergo biodegradation at a rate that is in synchrony with constructive remodeling of new tissue, (3) an injectable system considering the anatomy of tooth/root canal systems, (4) provide encapsulation or surface adhesion for cells to regenerate/differentiate into different dental tissues, (5) help in spatial organization of cells and eventual replacement by appropriate tissues, and (6) ability to modify mechanical, physical, and biological properties to suit specific applications. The different type of materials used to make scaffolds include synthetic biodegradable polymers (e.g., polylactic acid [PLA], polyglycolic acid [PGA], and their copolymer polylactic-co-glycolic acid [PLGA]), natural biodegradable polymers from biological sources (e.g., collagen, gelatin, fibrin, albumin chitin, hyaluronic acid, cellulose), and inorganic materials (e.g., hydroxyapatite–[HA] and tricalcium phosphate [TCP]) and composites (Table 22.1). They can be processed into porous scaffolds, nanofibrous materials, microparticles, and hydrogels. The use of synthetic polymers provides advantages such as ease of manufacture and excellent mechanical properties with modifiable physicochemical characteristics, whereas the disadvantages include lack of bioactivity and chronic or acute host inflammatory response [53]. Considerable focus on the selection of biomaterials over the last decade has been fundamental to the understanding of complex pulpal cellular responses and their application in tissue regeneration. Table 22.2 gives a summary of the biomaterials used for pulpodentinal regeneration.

22.4.2  Scaffolds derived from biological sources Natural polymers mimic the ECM and offer advantages of good structural strength, bioactivity, and biodegradability but are often difficult to process and have the associated risk of immune reaction. Natural polymers (proteins and polysaccharides) such as fibrin, collagen, alginate, chitosan, hyaluronan, keratin, and fibronectin have the advantages of good cytocompatibility and bioactivity [85–88]. Collagen hydrogels are popular scaffolds used for pulp–dentine regenerative purpose [66,67,83,84,89], and their use in tooth tissue engineering in sponge or gel form has been well demonstrated [83,84,86,90]. They have been shown to improve cell proliferation and differentiation with formation of mineralized tissues. Following

Table 22.1  Types of biomaterials as scaffolds. TYPE of scaffold

Classification

Examples

Advantages

Disadvantages

Natural (derived from plants, animals, microbes)

1. Materials inspired by the extracellular matrix 2. Based on structure a. proteins b. polysaccharids c. polyesters 1. Thermoplastic aliphatic polyesters 2. Bioceramics/inorganic materials 3. Composites

Collagen (Tp-1), fibronectin, glycosaminoglycans, fibrin, dentine chips Collagen, elastin, fibrin, silk Starch, cellulose, alginate, chitin, hyaluronic acid, dextran Polyhydroxyalkanoates (PHAs) Polylactic acid (PLA), polyglycolic acid (PGA), and their copolymers—poly(lactic-coglycolic acid) (PLGA) Low- and high-temperature calcium orthophosphates, tricalcium phosphate, hydroxyapatite, bioactive glasses Combination of natural, artificial materials Fibrin, hyaluronic acid, collagen, alginate Poly(ethylene glycol) (PEG), polyfumarate Collagen with poly(ethylene glycol) SAP (self-assembling peptide hydrogel)

Good biocompatibility; can be processed into different forms; safe degradation; good cytocompatibility and bioactivity

Limited supply; possible immune reaction; pathogen transmission; difficult to manufacture; poor mechanical properties Does not mimic ECM; variable rates of degradation

Synthetic (artificial materials)

Hydrogels

1. Natural 2. Synthetic 3. Hybrid 4. Biomimetic

Abundant supply; low immune reaction; can be tailored to possess a broad range of structural, mechanical, and chemical properties; degradation rate; and ease of manufacturing process

Biocompatible; mimic the viscoelastic properties of soft connective tissue; help in transport of nutrients and waste; enable cell encapsulation; possibly exist to inject and cause gelation in situ; option to incorporate bioactive cues by chemical or physical cross-linking

Limited supply; low mechanical stiffness

Table 22.2  Summary of scaffolds used for pulpodentinal regeneration. Biomaterial

References

Type of study

Experimental materials

Results

PGA PGA, collagen type 1, alginate HA/TCP

[54] [55]

* *

Human pulp fibroblasts + PGA Human pulp fibroblasts + scaffolds

Formation of pulp tissue PGA—formation of pulp tissue

[56] [57] [58]

** ** **

hDPSCs and BMSCs + ha/tcp DPSCs + ha/tcp hDPSCs and BMSCs + ha/tcp

[59] [60] [29] [61] [62]

** *; ** *; ** * *; **

SHED + ha/tcp DPSCs + PLG hDPSCs and SCAP + PLGA into root MSCs + HA/CS hDPSCs + PLLA/tooth slice (ts)

[28,63] [64]

** *; **

[65]

*; **

SHED + PLLA/tooth slice (ts) hDPSCs + NF-PLLA + dxm/ bmp-7+dxm NF-PLLA and SW-PLLA compared

[66]

**

[67]

**

[68]

*

[69]

*

Collagen + hDPSCs andand DMP-1 in dentine Collagen + hDPSCs andand DMP-1 in dentine wafer SHED + 3D porous scaffold + growth factors into root canal of human teeth SHED + hDPSCs onto SAP

Formation of a pulp–dentine-like structure Ectopic dentine formation BMSCs-elevated expression of bfgf&mmp-9,dpscs-elevated expression of DSP Formation of dentinelike structure Formation of pulplike tissue, oosteodentine, tubular dentine Formation of vascularized pulp–dentine-like structure Positive for cell adhesion and proliferation Expression of odontoblastic markers—PLLA/Ts > PLLA; cell proliferation of PLLA/Ts < PLLA; formation of vascularized pulplike tissue after 28 days with PLLA/Ts Formation of vascularized pulplike tissue andand dentine NF-plla + BMP-7 + DXM effective odontogenic differentiation then NF-PLLA + DXM NF-PLLA shows ↑odontogenic differentiation and hard tissue formation Formation of vascularized pulplike tissue

[70]

*; **

hDPSCs + growth factors onto SAP

PLGA HA,CS PLLA

Collagen type 1

OPLA and collagen SAP (hydrogel)

Formation of highly cellular, vascular, and mineralized matrix after 12 wk Positive for cell adhesion and activity irrespective to inclusion of growth factors SHED onto SAP: ↑proliferation, collagen formation, soft tissue formation; hDPSCs onto SAP: ↑ expression of osteoblast marker, mineral deposition Formation of a vascularized pulplike tissue Continued

Table 22.2  Summary of scaffolds used for pulpodentinal regeneration.—cont’d Biomaterial

References

Type of study

Experimental materials

Results

Collagen [1,3], chitosan, gelatin Chitosan Alginate KH

[71]

*

hDPSCs + scaffold materials

[72] [73,74] [75,76]

* *; ** *; **

Chitosan + simvastatin + hDPSCs Alginate + TGF-β KH injected after partial pulpotomy

HYA DDM, CBB, SIS, PLGA, Co-Cs-HA Silk PLGA, PLGA/ tcp,plga/ ha,plga/CDHA PLLA, collagen, CaP CaP

[77] [78]

** *; **

HYA pulp capping DPSCs (rat) + different scaffolds

[79] [78]

*; ** *; **

hDPSCs + HFIP ssilk/aqueous silk hDPSCs/rat tooth bud cells + different scaffold materials

Collagen type 1 higher cell adhesion, proliferation, positive for DSPP, DMP-1, OCN Increased chemotaxis and differentiation of hDPSCs Secretion of tubular dentin matrix Reparative dentine formation with positive expression of DMP Reparative dentine formation DDM and CBB showed formation of organized pulp tissue including dentine, predentine, odontoblasts,↑ ALP, mRNA expression of DSPP, DMP-1,↑ MAPK l HFIP silk scaffold—formation of pulplike tissue PLGA/TCP superior generation of dentine pulplike tissues in comparison with other composite scaffolds

[80]

*

[81]

*

Different scaffolds placed onto dental pulp of tooth slice (ts) hDPSCs + 3D CaP granules

Fibrin, PEG

[82]

*; **

DPSCs + PEGylated fibrin

Collagen [1,3] (hydrogel)

[83,84]

*; **

Injection of scaffold + pulp-derived SP cells + SDF-1 to root canal of canine

PLLA—increased cell proliferation of fibroblast phenotype Support odontogenic differentiation, express ↑ DSPP, DMP-1, OCN Positive for odontoblast-specific markers, formation of pulplike tissue Effective in pulp regeneration, with a continuous layer of dentinelike tissue along the dentinal wall

*, in vitro; **, in vivo; bFGF, basic fibroblast-derived growth factor; BMP7, bone morphogenic protein 7; CaP, calcium phosphate; CBB, ceramic bovine bone; CDHA, calcium carbonate hydroxyapatite; Co-Cs-HA, collagen, chondroitin sulfate, hydroxyapatite; DDM, demineralized dentine matrix; DMP, dentine matrix protein; DSPP, dentine sialophosphoprotein; DXM, dexamethasone; hDPSCs, human dental pulp stem cells; HFIP, hexafluoro-2-propanol; HYA, hyaluronic acid; KH, keratin hydrogel; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloprotein 9; MSCs, mesenchymal stem cells; MTA, mineral trioxide aggregate; NF-PLLA, nanofibrous poly-l-lactic acid; OCN, osteocalcin; OPLA, open-cell polylactic acid; PEG, polyethylene glycol; PGA, polyglycolic acid; PLG, polylactic-co-glycolic acid; PLLA, poly-l-lactic acid; SAP, self-assembling polypeptide; SCAP, stem cells derived from apical papilla; SDF, stromal-derived factor; SHED, stem cells from human exfoliated deciduous teeth; SIS, small intestinal submucosa; SP, side population cells; SW, solid welled poly-l-lactic acid; TCP, tricalcium phosphate. This table is adapted from L. Ajay Sharma, A. Sharma, G.J. Dias, Advances in regeneration of dental pulp–a literature review, J Investig Clin Dent 6 (2) (2015) 85–98 (after permission).

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subcutaneous implantation in mice, collagen scaffolds loaded with dental pulp stem cells were able to form pulplike tissues [66]. Among the polysaccharides tested, chitosan, the deacetylated form of chitin, has been used as root canal dressing material because of its antibacterial and wound healing properties [91]. The chitosan monomer when exposed to osteoblasts enhanced the ALP activity and BMP-2 expression significantly in vitro and promoted proliferation of pulp fibroblasts with mineralization [91]. Alginate along with TGF-β induced differentiation of dental pulp cells into ­odontoblast-like cells and resulted in tubular dentin matrix secretion following subcutaneous transplantation of loaded scaffold in mice [73,74]. High-molecular-weight hyaluronic acid when exposed to amputated rat pulp resulted in formation of reparative dentin formation [77]. Similarly, keratin hydrogel has shown to enhance odontogenic proliferation and differentiation by in vitro and in vivo study [75,76].

22.4.3  Scaffolds of synthetic polymers, bioceramics, and composites Synthetic polymers in particular have generated substantial interest because of their favorable properties such as good biocompatibility, biodegradability, and mechanical strength. In addition, these polymers are easy to formulate into different devices for carrying a variety of drug classes such as vaccines, peptides, proteins, and other micromolecules. The processing of scaffolds into various forms like highly porous three-­ dimensional scaffolds, linearly oriented scaffolds, fibrous meshes, sheets, hydrogels, and pellets enables to support cell and tissue ingrowth/penetration. Among these, PLA has found many applications where structural strength is important. PGA used as an artificial scaffold for cell transplantation undergoes degradation as the ECM is secreted. Most importantly, the US Food and Drug Administration (FDA) has approved them for biomedical applications [51,92,93]. However, the inherent disadvantage lies with their lack of biological reactivity because of absence of reactive functional sites, reported chronic or acute inflammatory response in the host, and localized pH decrease because of relative acidity of hydrolytically degraded by-products [94,95]. Initially, pulp regeneration by in vitro analyses was performed using dental pulp fibroblast cells on various scaffolds. Comparative studies showed culturing cells on PGA resulted in a very high cell density tissue with significant collagen deposition after 45 days compared with type one collagen and alginate [54,55]. The use of PLGA with different stem cell sources (DPSCs, SCAP) supported formation of soft pulp-like tissue and deposition of new dentin [29,60]. Similarly, a comparative study of four different composite PLGA scaffolds by Zheng et al. [96] showed superior generation of dentin–pulp-like tissues with PLGA/TCP. In a study by Huang et al. (2010), PLG scaffolds were seeded with stem cells from the apical papilla (SCAP), or dental pulp (hDPSC) was inserted into root fragments and implanted into mice subcutaneously. The results of the study demonstrated regeneration of pulplike tissue and dentinelike tissue along the dentinal wall [29]. Another synthetic polymer with promising results was that with PLLA. In a series of in vitro and in vivo studies revealed formation of vascularized pulp-like tissue with tubular dentin formation with PLLA/tooth slice discs seeded with SHED [28,97].

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Nanofibrous PLLA with additional growth factors such as bone morphogenetic ­protein 7 (BMP-7) and dexamethasone (DXM) showed effective odontogenic differentiation [64,65]. Calcium phosphate compounds such as hydroxyapatite (HA) and tricalcium phosphate (TCP) have been widely used as bone substitutes and bone-filling materials in dental and orthopedic fields [98]. Calcium phosphate cement is also used as a root canal filling material [99]. Several studies reported the isolation and characterization of dental stem cells (DPSCs, SHED) and their ability to generate reparative dentinlike tissue on the surface of transplants when mixed with bioceramics (HA/TCP) as carrier [33,56–59,100]. Nevertheless, the disadvantages such as difficulty of shaping, poor mechanical strength, brittleness, and slow degradation rate have been the hurdles. Composite scaffolds of HA and other biomaterials have the potential to overcome these disadvantages [96]. Various hybrid combinations (natural and synthetic) have also shown positive results for odontoblast-specific markers [68,82].

22.4.4  Cell-laden versus cell-free scaffolds The use of stem/progenitor cells has been a major approach to in vivo preclinical pulp tissue regeneration. It is hypothesized that the transplanted stem/progenitor cells participate in repair/regenerative process by supplying cells per se and providing biomolecules or growth factors released from cells as trophic factors to enhance the cellular activity [101]. Pulp regeneration by use of patient-specific stem cells dictates a pointof-care (POC) therapy. Potential difficulties with cell-based therapies such as excessive costs, including cell isolation, handling, storage, shipping, ex vivo manipulation, and immune rejection (for allogeneic cells) have raised expectations of this alternative cell homing technique [102,103]. Moreover, cell-laden scaffolds often need to be expanded in vitro before transplantation. Alternatively, the concept of cell homing and use of exogenous biomolecule-loaded scaffolds are proposed to circumvent many of the challenges associated with cell-based therapies. Cell homing is regarded as an active means of recruitment of endogenous cells, including stem/progenitor cells, into an anatomical location [103]. The regenerative technique by chemotactic cell homing represents an alternative approach for pulp regeneration compared with mainstream cell transplantation. Such an approach was also proposed for in situ pulp and periodontal tissue regeneration [89,104]. In this concept, different bioactive cues were adsorbed or encapsulated into biomaterial scaffolds. Upon release of these bioactive cues into endodontically treated root canals, local and/or systemic cells, including stem/progenitor cells, migrated and homed in vivo into the root canal, leading to subsequent pulp regeneration [105] (Fig. 22.3). In vivo studies of recombinant human BMP-2 and 4 have shown that they induce reparative dentine formation [23,25,106]. The application of recombinant BMP-7 and TGF (transforming growth factor-β1 and β3) on rat incisor tooth slices caused stimulation of the dentine–pulp complex formation [107,108]. A study by Kim et al. [89] demonstrated the regeneration of pulplike tissue integrated into the root canal dentinal wall following implantation of an endodontically treated human tooth in a mouse for 3 weeks. Addition of other growth factors such as fibroblast growth factor (FGF2),

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Figure 22.3  Cell homing: (a) infected root canal, (b) cleaned and prepared root canal, (c) transplantation of a scaffold with growth factors, (d) (enlarged) attraction of stem cells from the perivascular niche - Growth factor - Stem cells. This figure is obtained from L. Ajay Sharma, A. Sharma, G.J. Dias, Advances in regeneration of dental pulp–a literature review, J Investig Clin Dent 6 (2) (2015) 85–98 (after permission).

vascular endothelial growth factor (VEGF), and platelet-derived growth ­factor (PDGF) with nerve growth factor (NGF) and BMP-7 led to new blood vessels formation and dentin regeneration [89].

22.4.5  Partial pulp regeneration and complete regeneration of pulp–dentine complex As discussed in the previous section, the potential of pulp tissue to regenerate lost dentin in mild injuries due to disease and trauma is well known. However, regeneration of whole pulp tissue following pulpectomy has always been a major challenge. The issues being (1) revascularization—the pulpal tissue is encased in dentine without a collateral blood supply except from the root apical end and (2) induction of differentiation of odontoblasts and spatial arrangement. The blood clot revascularization method was proposed for nonvital immature teeth where the tooth apex is opened (1 mm in diameter or more) by producing systemic bleeding into the canal [109,110]. These studies show radiographic evidence of continued development and formation of the root end; however, histologically the regenerated tissue is not dentin but rather cementum-like or osseos-like tissue. Therefore, for mature and immature teeth, the tissue engineering concept provides potential strategies for successful pulp regeneration.

22.4.6  Advanced scaffolds for pulp–dentine regeneration The use of nanofibrous scaffolds processed via electrospinning, self-assembly, and phase separation–has been developed to support the proliferation and differentiation of dental pulp stem cells toward the functional regeneration of the pulp–dentine

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complex [111,112]. Nanofibrous polymer scaffolds are becoming increasingly popular with demonstrated positive cell–ECM interactions, higher cell proliferation, differentiation of stem cells, and activation of cell signaling pathways by providing physical and chemical stimuli [111,113,114]. Most importantly, nanofibers assembled in solution resulted in gels following in situ injection that can be used in cell encapsulation [112,115,116]. In addition to the use of molecular self-assembly to fabricate nanofibrous scaffolds, thermally induced phase separation method has been used to construct macro-micropore networks within such 3D nanoscaffolds [111]. Recently, electrospinning has been successfully used to generate scaffolds that deliver bioactive agents (e.g., antimicrobial drugs and angiogenic factors) for pulp–dentine complex regeneration [117–119]. The use of nanofibrous spongy microspheres (NF-SMS) that combined NF architecture and interconnected microsized pores into injectable microspheres was also investigated. The NF-SMS was made of block copolymer, poly(l-lactic acid)block-poly (l–lysine) capable of self-assembling. These scaffolds enhanced DPSC proliferation in vitro and resulted in regenerated pulplike tissue with higher vascularity when NF-SMS was combined with hypoxia-primed DPSCs compared with normal conditions [120,121]. A copolymeric micelle‐in‐microsphere platform to control inflammation and promote dentine regeneration was investigated with chitosan–graft– poly(lactic acid) copolymer. The concurrent release of fluocinolone acetonide (FA) and BMP-2 showed reduced inflammation of DPSCs and enhanced odontogenesis [101]. A recent study has demonstrated that the combination of low-dosage simvastatin and NF-PLLA scaffolds enhanced regenerative potential of resident stem cells [122]. Another promising hydrogel-based nanofibrous scaffold called Puramatrix was proposed [112]. Puramatrix, a self-assembling peptide hydrogel, polymerizes and forms a biodegradable nanofiber hydrogel scaffold on interaction with physiological conditions [112] and supports dental pulp stem cell survival and proliferation in vitro [115]. A mixture composed of stem cells from exfoliated deciduous teeth (SHED) and Puramatrix was able to generate a pulp–dentine-like tissue when injected into root canals of teeth implanted subcutaneously into immunodeficient mice [112]. Galler et al. [69,70,123,124] have worked extensively on multidomain peptides (MDPs) made of short sequences of aminoacids that self-assemble to form fibers in aqueous solution. MDPs primarily consist of cell adhesion motif and enzyme cleavable sites such as arginine–glycine–aspartic acid (RGD) and matrix metalloproteinase (MMP) that facilitated growth factor conjugation and controlled and slow release of the growth factor. MDPs loaded on dentin cylinders led to formation of vascularized pulplike tissue with tubular dentin formation following 5 weeks’ implantation in immunocompromised mice [69,70,125]. Similarly, another hydrogel gelatin methacrylate (GelMA), which is mainly composed of denatured collagen with RGD and MMP sites to facilitate cell adhesion and scaffold degradation, was investigated by Khayat et al. [126] showed that tooth root segments injected with GelMA hydrogel combined with dental pulp stem cells and human umbilical vein endothelial cells promoted formation of organized vasculature. Nonetheless, it is worth mentioning that most of the preclinical research that reported pulp–dentin-like tissue regeneration involved transplantation of “tooth-root or dentin slice models” loaded with ex vivo expanded scaffolds with stem cells and/or

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growth factors into the subcutaneous tissue of immunocompromised rats/mice [127]. An average time period of 28–35 days was reported for pulplike tissue regeneration and up to 180 days for regenerated dentine. However, these studies did not completely simulate clinical conditions. A number of papers published by Nakashima and Iohara et al. [128–132] demonstrated the use of a subpopulation of dental pulp stem cells with additional growth factors such as stromal derived factor (SDF) to stimulate pulp–dentin regeneration following pulpotomy/pulpectomy in a dog animal model. Transplantation of pulp CD31− (SP) cells induced higher vasculogenesis/angiogenesis, neurogenesis, and pulp regeneration in experimental models with ectopic tooth root transplantation compared with that of bone marrow and adipose CD31− SP cells [129], suggesting that DPSC subfractions may be superior for cell-based regenerative endodontics. Another technique to isolate DPSC subsets has recently been devised by the use of an optimized granulocyte colony–stimulating factor (G-CSF)–induced mobilization [131]. The mobilized DPSCs (MDPSCs) in collagen scaffolds resulted in complete pulp regeneration and coronal dentin formation in the pulpectomized root canal of dog canines after 180 days [128]. Thus, based on the results of these preclinical trials, scientific evidence of the safety and efficacy critical for clinical applications has been presented [132]. However, the uses of such reprogrammed stem cells are widely speculated in terms of their safety, complex technology involved, and feasibility.

22.5  Summary and future perspectives To summarize, the ultimate priority of pulp therapies is to maintain vitality of pulp tissue, which is fundamental to the functional life of the tooth. Ideally, the goal is to regenerate vital dental pulp and dentin to reestablish the functions of the pulp–dentin complex to prevent reinfiltration by pathogens. For years, operative dentistry techniques have attempted regenerative treatment, with the use of materials such as MTA and Ca(OH)2 to stimulate reparative/reactionary dentine formation with variable treatment outcomes. Presently, the advent of tissue engineering, combining as it does the principles of molecular biology, developmental embryology, and biomaterial science, will make the concept of pulp regeneration a reality within the next few years. The knowledge gained from adult stem cell biology provides excellent platform for eventual, successful pulpogenesis [133]. To date, collagen type one and synthetic polymers (PLGA and PLA) have shown the most favorable results in the application for endodontic tissue regeneration. The use of hydrogels seems to be a viable option considering clinical applications. Sophisticated scaffolds introducing growth factor delivery systems are currently a major focus of research in the field of pulp tissue engineering. The selection criteria for an appropriate biomaterial depend on which properties are required for a specific application, ease of obtaining and fabrication of the material and, most importantly, whether it is a cost-effective alternative to the existing treatment modalities. In addition, several issues such as clinical handling and feasibility, tests/instruments to confirm the vitality of regenerated pulp, and possible retreatment strategies are still to be addressed. As a consequence of the large number of research

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projects currently occurring in this field globally, a new generation of biological treatment modalities to regenerate lost dental tissues is undoubtedly feasible. Given their promise, they are certainly worthy of this intensive, ongoing exploration.

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