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Matichescu, A.; Ardelean, L.C.; Rusu, L.; Craciun, D.; Bratu, E.A.; Babucea, M.; Leretter, M. Oral Tissue Engineering and Regeneration. Encyclopedia. Available online: https://encyclopedia.pub/entry/3333 (accessed on 12 April 2024).
Matichescu A, Ardelean LC, Rusu L, Craciun D, Bratu EA, Babucea M, et al. Oral Tissue Engineering and Regeneration. Encyclopedia. Available at: https://encyclopedia.pub/entry/3333. Accessed April 12, 2024.
Matichescu, Anamaria, Lavinia Cosmina Ardelean, Laura-Cristina Rusu, Dragos Craciun, Emanuel Adrian Bratu, Marius Babucea, Marius Leretter. "Oral Tissue Engineering and Regeneration" Encyclopedia, https://encyclopedia.pub/entry/3333 (accessed April 12, 2024).
Matichescu, A., Ardelean, L.C., Rusu, L., Craciun, D., Bratu, E.A., Babucea, M., & Leretter, M. (2020, December 03). Oral Tissue Engineering and Regeneration. In Encyclopedia. https://encyclopedia.pub/entry/3333
Matichescu, Anamaria, et al. "Oral Tissue Engineering and Regeneration." Encyclopedia. Web. 03 December, 2020.
Oral Tissue Engineering and Regeneration
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The reconstruction or repair of oral and maxillofacial functionalities and aesthetics is a priority for patients affected by tooth loss, congenital defects, trauma deformities, or various dental diseases. Therefore, in dental medicine, tissue reconstruction represents a major interest in oral and maxillofacial surgery, periodontics, orthodontics, endodontics, and even daily clinical practice. The current clinical approaches involve a vast array of techniques ranging from the traditional use of tissue grafts to the most innovative regenerative procedures, such as tissue engineering. In recent decades, a wide range of both artificial and natural biomaterials and scaffolds, genes, stem cells isolated from the mouth area (dental follicle, deciduous teeth, periodontal ligament, dental pulp, salivary glands, and adipose tissue), and various growth factors have been tested in tissue engineering approaches in dentistry, with many being proven successful. However, to fully eliminate the problems of traditional bone and tissue reconstruction in dentistry, continuous research is needed.

regenerative medicine regenerative dentistry tissue engineering stem cells biomaterials scaffolds

1. Introduction

The traditional standard techniques based on replacing missing or deteriorated tissue with autologous grafts from living donors or even cadavers are still used in dentistry as well as in other medical fields, despite their disadvantages, such as risk of infections and rejection following the transplantation procedure. An innovative alternative is provided by regenerative medicine, which aims to regenerate, repair, or replace tissues and to ensure restoration of their impaired function by combining tissue engineering with the self-healing ability of humans. In vitro engineering of tissues and organs involves the emerging field of biotechnology in a multidisciplinary approach together with medicine, materials science, cell and molecular biology, bioengineering, and genetics [1].

Tissue engineering is a term associated with regenerative medicine and is distinct in its focus on aspects regarding the engineering and manufacturing of replacement tissue, but regenerative medicine and tissue engineering are often treated as a single field of interest in the literature. Tissue engineering aims to create functional tissue or even organs using patients’ own cells, offering an alternative method to grafts or transplants. This approach is being increasingly used in dental and maxillofacial reconstruction medicine, providing a new option for the reconstruction of teeth, periodontium, bones, oral mucosa, conjunctiva, skin, temporomandibular joint, both bone and cartilage as well as nerves, muscles, tendons, and blood vessels of the oral and maxillofacial area [2].

Tissue engineering can be used to regenerate tissue for specific defects, which represents a major advantage compared with other current treatments which have numerous disadvantages for patients like loss of sensorial and motor functionalities of craniofacial structures due to prosthetic alloplastic materials, high risk of infection, inflammation, requirement for lifelong immunosuppression, or unpredictable compatibility with the donor in the case of autologous grafts. Additionally, the unlimited available bioengineered resources do not require immunosuppression [3]. Tissue engineering is classically based on three pillars: (a) the cells (stem cells/progenitor cells), responsible for synthesizing the new tissue matrix; (b) the signaling/growth factors necessary to promote and facilitate the functionalities; (c) the biomaterial scaffolds, necessary for cell differentiation, multiplication, and biosynthesis, that act as an extracellular matrix (ECM) (Figure 1).

Figure 1. Classical pillars of tissue engineering: (a) the cells (stem cells/progenitor cells), (b) the signaling/growth factors, (c) the biomaterial scaffolds/extracellular matrix.

Cells communicate with their environment using different components to regenerate tissues by combining human cells with specific scaffold biomaterials. The biomaterial scaffolds provide templates for tissue regeneration and guide new tissues in their growth [4][5]. A successful approach in tissue engineering and regeneration implies that the combination of these three principles must be able to replace the damaged tissue and enable its function similarly to the original tissue or must be able to stimulate regeneration of the original tissue [6][7]. Several kinds of cells have been used in tissue engineering and regenerative medicine as reported in clinical studies, including stem cells, fibroblasts, chondrocytes, and keratinocytes originating from the same patient, another human, or animals [8].

The aim of this narrative review article is to approach this broad-spectrum subject in view of the literature from recent years specifically on the topic of potential orofacial stem cell usage in regenerative dentistry, both for hard and soft tissues. A large literature survey was performed on this topic in free-access digital archives of full-text articles (PubMed, Medline, Web of Science, and Google Scholar), with articles published between 2010–2020 being considered. More than 300 articles were referenced, with over 50% published in the last five years. The keywords used for searching were “regenerative dentistry”, “tissue engineering”, and “orofacial stem cells”. A specific search was performed to identify clinical studies involving the application of dental stem cells for tissue regeneration in endodontics, periodontics, and maxillofacial surgery.

2. Stem Cells, Biomaterials, and Scaffolds for Oral Tissue Engineering and Regeneration—Types, Sources, and Technologies

2.1. Orofacial Stem Cells

Stem cells (SCs) are defined as primitive, unspecialized, and pluripotent cells of the human body characterized by two major properties: production of other new stem cells and multidirectional differentiation into cells with a specific functionality, such as bone cells, skin cells, and blood cells [8][9]. Their presence was first reported in bone marrow [10].

SCs have powerful potential in medicine; their study has revealed important information about the complex processes of human body development. Due to these abilities, SCs have attracted interest regarding their use in the regeneration, repair, and functionality improvement of degenerated or injured tissue using implants of engineered tissue as well as biohybrid organs. The strategies involving the use of stem cells for tissue regeneration can be optimized using bioactive scaffolds or by adding various growth factors. [11].

Considering their origin, physiological stem cells include embryonic stem cells (ESCs) from embryos and adult stem cells (ASCs) from adult tissue. Other types of stem cells are the perinatal stem cells, from amniotic fluid, and induced pluripotent stem cells (iPSCs) [12], obtained by transforming regular ASCs under genetic reprogramming. iPSCs, which are generated directly from a somatic cell, were pioneered by Yamanaka, in 2006. Shinya Yamanaka’s discovery was awarded with the 2012 Nobel Prize, jointly with Sir John Gurdon, who, in 1962, demonstrated that the specialization of cells is reversible. The immature cell nucleus in an egg cell of a frog was replaced with the nucleus from a mature intestinal cell. This modified egg cell developed into a normal tadpole, proving that the DNA of the mature cell still had all the information needed to develop all cells in the frog [13]. More than 40 years later, Shinya Yamanaka discovered how intact mature cells in mice could be reprogrammed to become PSCs, able to develop into all types of cells in the body, by introducing only a few genes [14][15][16].

The ESCs are present in the blastocyst and can be differentiated into all types of cells, and are therefore pluripotent. Various postnatal tissues present ASCs for their normal renewal as well as regeneration or injury healing. Recent research in tissue engineering and regenerative medicine has demonstrated that SCs can be widely used in dentistry, more so than synthetic materials because teeth are a rich source of SCs [17]. Mesenchymal stem cells (MSCs) are a type of ASC of great importance in regenerative medicine due to their responsibilities in tissue repair and growth, cell substitution, and wound healing due to physiological or pathological causes. MSCs can be isolated especially from bone marrow and adipose tissue, but also from other various human tissues like the placenta, amniotic fluid, liver, umbilical cord, synovial membrane, skin, muscle, and dental tissues [18].

Different types of SCs obtained from oral and maxillofacial tissues, with similar in vitro properties as bone marrow-derived MSCs, are being defined as multipotent stromal cells. They are able to differentiate into different types of cells like chondrocytes, myocytes, osteoblasts, and adipocytes. Recently, the immunomodulatory properties of MSCs have been reported, which enable their clinical use in the treatment of inflammatory conditions [19]. Considering their location in the oral and maxillofacial region, the ASCs are grouped in two major categories: dental and non-dental [20] (Figure 2).

Figure 2. Types of human SCs in the oral and maxillofacial region.

The easy access, proliferation capacity, and multidirectional in vivo/in vitro differentiation makes orofacial SCs an important source of SCs for use in regenerative dentistry and medicine. Therefore, their potential clinical application in dentistry or other medical fields is diverse.

  • Dental pulp stem cells (DPSCs), the first human dental MSCs found inside teeth, are considered a significant source for future regenerative procedures both in dental and general medical applications [21]. DPSCs are isolated from the dental pulp of primary or permanent teeth. Their high capacity for in vitro differentiation includes odontoblast, osteoblast, myoblast, adipocyte, dentin–pulp, cardiomyocyte, neuron-like cell, and hepatocyte-like cells, whereas in vivo, they are limited to only adipocytes, endotheliocytes, and myofibers [22][23].
  • Periodontal ligament stem cells (PDLSCs), present on alveolar bone surfaces and the root, play a specific role in cementum or periodontal ligament (PDL) tissue regeneration. They are capable of giving rise to mesenchymal cell lineages to produce in vitro osteoblast-like cells, cementum tissue, Sharpey’s fibers, adipocytes, and collagen-forming cells [24].
  • Stem cells from apical papilla (SCAPs) are mesenchymal They can be found within immature roots and isolated from the immature permanent apical papilla. SCAPs are good sources of and cause apexogenesis. They have a higher capacity to proliferate than DPSCs, being the first option for tissue regeneration. SCAPs represent a promising source of SCs, as they can differentiate into various lineages of cells, such as odontogenic, chondrogenic, osteogenic, adipogenic, neurogenic, and hepatogenic cells [25].
  • Dental follicle stem cells (DFCs) are sourced from the dental follicle, which is loose connective tissue surrounding the developing tooth germ [17]. DFCs can differentiate osteoblast, cementoblast, alveolar bone, dentin-like tissues, PDL, cementum, adipocyte, chondrocyte, cardiomyocyte, and neuron-like cell. Their regenerative potential is highlighted by clinical applications in periodontal and neural tissue regeneration, tooth root regeneration, and bone defects [26][27].
  • Tooth germ progenitor cells (TGPCs) are obtained from the dental mesenchyme of the human third molar germ in the late bell stage of tooth development. Studies on TGPCs have demonstrated their high proliferation activity and capacity to differentiation into adipogenic, chondrogenic, osteogenic, odontogenic, and neurogenic tissue [28][29]. In addition, TGPCs can differentiate into hepatocytes in vitro [30] and are able to form tube-like structures, possibly evidence of vascularization [31].
  • Stem cells of human exfoliated deciduous teeth (SHEDs), obtained from exfoliated deciduous teeth, have higher proliferation capacity than DPSCs and the capability to differentiate into many more different body tissues than other types of SCs, including into adipocytes, osteoblasts, odontoblasts, neural cells, hepatocytes, and endothelial cells. SHEDs have a high proliferation capacity, high multipotency, immunosuppressive ability, and minimal risk of oncogenesis. [32]. The major disadvantage of SHEDs is that an incomplete pulp-dentin-like complex is formed in vivo.
  • Alveolar bone-derived mesenchymal stem cells (ABMSCs), isolated from the human alveolar bone, are a more convenient tissue source of MSCs and have the ability of multipotent differentiation into osteoblasts, adipocytes, and chondroblasts. In addition, they can induce ectopic bone formation in vivo.
  • Salivary gland-derived stem cells (SGDSCs) are isolated from human salivary glands. The regeneration of salivary gland function with SGDSCs is still being investigated, though certain studies have already concluded that progenitor cells isolated from stromal tissue can be guided to differentiate into osteoblasts, chondrocytes, and adipocytes [33].
  • Oral mucosa-derived mesenchymal stem cells (OMSCs), include oral epithelial stem cells (OESCs), gingiva-derived mesenchymal stem cells (GMSCs), and periosteum-derived stem cells (PSCs). SCs within the mucosa lining the oral cavity can be isolated from normal or inflamed gingiva, from attached and free gingiva, and from hyperplastic gingiva. OMSCs can differentiate into different mesenchymal lineages and have immunomodulatory properties [33]

2.2. Biomaterials and Scaffolds for Oral Tissue Engineering

In dental tissue regeneration, scaffolds and biomaterials are essential elements. They are used as attachment sites for regenerative cells from the surrounding tissues, as a template for tissue regeneration, as a source of implantable odontogenic cells with the capability to differentiate required cell type, and as bioactive molecules, especially growth factors that intensify the regenerative capability [34][35].

Biomaterials, natural or synthetic, alive or lifeless, are being defined as materials that interact with biological systems. They are often used in medical applications to augment or replace a natural

function. Based on their biocompatibility, biomaterials are classified as bioactive, biotolerant, biodegradable, and bioinert [36].

Bioactive materials, by stimulating the biological response, may lead to osteogenesis by making strong chemical bonds. They are being classified into osteoconductive (hydroxyapatite and β-tricalcium phosphate), which stimulate bone growth along the surface, and osteoproductive (bioactive glasses), which are capable of stimulating the growth of new bone away from the bone/implant interface [36].

Biotolerant materials (polymers and most metals) are being well accepted by the host, but separated from the host tissue by the formation of a fibrous tissue, which is induced by the release of ions, corrosion products, and chemical compounds from the implant.

Biodegradable materials (polymers, such as polyglycolic and polylactic acids, and their co-polymers [37], ceramics as calcium phosphates [38], and magnesium) as biodegradable metal dissolve in contact with body fluids, the dissolution products being eliminated via the kidneys, without noticeable effects to the host. Biodegradable materials are used commonly used for surgical sutures, tissues in growth materials, and controlled drug release .

Bioinert materials (titanium and its alloys) are stable in the human body, and do not react with body fluids or tissues. Generally, bioinert materials are encapsulated by fibrous tissues, similar to biotolerant materials; however, in certain situations, they can develop structural and functional connection with the adjacent bone [39].

The most common approach in tissue engineering involves seeding cells onto a biomaterial matrix using a scaffold.

A wide variety of biomaterials, such as natural organic, synthetic organic, or even inorganic materials, is used for regeneration in oral and maxillofacial area, each of them having advantages and disadvantages. The natural organic materials include peptides (collagen or gelatin) and polysaccharides (alginate, chitosan, agarose). Frequently used synthetic organic materials include poly(lactic acid) (PLA), poly(caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), and poly(glycolic acid) (PGA).

The most commonly used inorganic materials are bioactive ceramics which include glasses or calcium phosphates (hydroxyapatite, β-tricalciumphosphate), which have been extensively studied as bone replacement materials, and cementitious systems of calcium phosphate or calcium silicate [40].

Bioactive ceramics are strongly chemically bonded with bone tissues via chemical reactions [40]. Hydroxyapatite (HA), bioactive and non-degradable, is characterized by chemical and structural similarity to bone minerals. β-tricalcium phosphate also has a chemical composition similar to bone,

and has higher in vivo rates of biodegradation compared to hydroxyapatite. The degradable bioactive ceramics are characterized by gradually degradation, in order to assist as scaffolds or replace the host tissue [40].

Polymers have been widely studied for medical applications, including bone tissue engineering [41]. From a biomedical perspective, polymers and co-polymers can be divided into two classes, biodegradable and biotolerant.

Biodegradable polymers, synthetic and natural, are suitable for additive manufacturing of scaffolds for tissue engineering [42]. The degradation of polymers, enzymatical or hydrolytical, is of most importance for this application. Natural polymers (chitosan, alginate, collagen, gelatin), frequently used as bioinks, are subject to enzymatic degradation, due to the microorganisms present in the biological environment [43].

The rate of enzymatic degradation varies upon the availability and concentration of respective enzymes. Hydrolytical degradation is related to synthetic polymers, and involves cleavage of hydrolytically sensitive bonds in the polymer, with consequent bulk or surface erosion, important in determining the best choice for a certain application [44].

Surface erosion offers several benefits for bone tissue engineering, such as retention of mechanical integrity, enhanced bone ingrowth, and ensures that the scaffold is gradually replaced by bone tissue [45].

PGA, PLA, PLGA, and PCL are hydrolytically degradable polymers [46].

PGA is usually used for short-term tissue engineering scaffolds and as fillers, because its rapid degradation and insolubility [47].

PLA, when mixed with glycolic acid, forms the copolymer PLGA, which is one of the most investigated degradable polymer for biomedical applications. Its great cell adhesion and proliferation properties recommend it as an excellent choice for tissue engineering [48][49].

Polymers can be processed to offer porous structures capable of facilitating the transportation of growth factors (nutrients as well as anabolites and catabolites) and are of interest due to their controllable degradation [41].

Recently, composite materials are being increasingly used due to their properties that result from the combination of both organic and inorganic elements. The most recent studies on this subject have considered the targeted and scaffold-assisted regeneration of enamel, dentin, and cementum.

An essential factor in tissue engineering is the scaffold. It offers a surface upon which cells adhere, multiply, thrive, and produce the ECM of proteins and saccharides that create the living tissue. Cells are expanded in culture and then transferred to the scaffold. The composition of the scaffold material and its internal architecture (dimensions of the struts, walls, pores, or channels) modulate and control the biological properties of the cells [50].

Generally, the scaffold materials must be biocompatible, biodegradable, porous, and without toxic metabolites. In particular, in dental regeneration, biomaterials must be suitable for the specific environment characteristics of the oral cavity considering pH, temperature, the presence of microorganisms, and the effect of mastication forces. To achieve these properties, most designed scaffolds deliberately mimic the structure of the natural ECM.

The number of suitable materials for fabricating scaffolds is limited by their biocompatibility, as they must accommodate the encapsulated cells and the recipient’s body. Because of poor biocompatibility, scaffolds can generate aggressive in vivo foreign-body reactions, necessitating the development of smart immunomodulatory biomaterials that ensure the tolerance of foreign scaffolds by the host or regulating the immunological microenvironments to ensure cell survival [49].

The behavior of cells after adhesion to the scaffold is affected by pore shape, volume, size, and geometry. Different pore sizes can affect the extracellular matrix. Porosity and interconnectivity are important for the ingrowth of surrounding tissues [51]. Open and interconnected pores allow oxygen and nutrients to be transported into the interior and eliminate the waste generated by cellular metabolism [52].

A wide range of advanced smart biomaterials and constructs with intelligent properties and functions have recently been developed to improve tissue repair and regeneration processes. Smart scaffolds incorporate bioactive molecules and nanoparticles and their physical and chemical properties are tailored as needed [53][54]. Their role is to improve the interactions with cells by enhancing the osteogenic differentiation for bone repair and to generate a better response to the surrounding environment [55] and include:

Smart scaffold constructs with stem cells for bone tissue engineering

  • Biomimetic and bionic smart scaffolds, such as biomimetic porous PLGA microspheres coupled with peptides prepared to mimic the composition and structure of natural tissues [56].
  • Immune-sensitive smart scaffolds, such as an amino-functionalized bioactive glass scaffold developed to investigate its effects on MSCs, bone marrow, and macrophages [57]. β-tricalcium phosphate has been used to coat Mg scaffolds, and modulate its detrimental osteoimmunomodulatory properties [58].
  • Shape-memory smart scaffolds, such as bone morphogenetic protein2-loaded shape-memory porous nanocomposite scaffold, consisting of chemically crosslinked poly(ε-caprolactone) and hydroxyapatite nanoparticles, used for the repair of bone defects, displayed shape-memory recovery [59].
  • Electromechanical-stimulus smart scaffolds. Piezoelectric poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE) was fabricated into flexible, 3D fibrous scaffolds. These have the ability to stimulate MSCs differentiation and tissue formation [60]. An electrospun PVDF-TrFE fiber scaffold containing zinc oxide nanoparticles was able to promote the adhesion and proliferation of human MSCs and also enhance the blood vessel formation [61].

Smart drug delivery for bone tissue engineering

  • Stimuli-responsiveness tunable drug delivery systems. These materials can change their properties as response to an endogenous and/or exogenous stimulus; thus, delivering the required amount of drug on-demand [62]. Polymers and hydrogels are used [63][64]. A highly porous, pH-responsive bacterial cellulose-g-poly(acrylic acidco-acrylamide) hydrogel was developed as an oral controlled-release drug delivery carrier [64]
  • . A poly(ethylene glycol) hydrogel, loaded with drugs by β-eliminative linkers, demonstrated tunable capability in drug release [65]. Farnesol-loaded nanoparticles, composed of 2-(dimethylamino)ethyl methacrylate, butyl methacrylate, and 2-propylacrylic acid are characterized by a pH-responsive drug release capability [66].
  • Smart multifunctional nanoparticle-based drug delivery systems: mesoporous silica nanoparticles, bone-forming peptide-1-laden MSNs encapsulated into arginine-glycine-aspartic acid-treated alginate hydrogel [67].
  • Biomimetic drug delivery systems: hydrogels, liposomes, micelles, dendrimers, polymeric carriers, and nanostructures [68][69].

Smart biomaterials and constructs to promote dental and periodontal regeneration, such as bilayered PLGA/calcium phosphate constructs [70] and tri-layered nanocomposite hydrogel scaffold: alveolar bone phase of chitin-PLGA/nanobioactive glass ceramic (nBGC)/platelet-rich plasma derived growth factors, PDL phase of chitin-PLGA/fibroblast growth factor, and cementum phase of chitin-PLGA/nBGC/cementum protein 1.[71]

Smart dental resins that respond to pH to protect tooth structures, such as dental composites containing nanoparticles of amorphous calcium phosphate and tetracalcium phosphate [72].

Smart pH-sensitive materials selectively inhibit acid-producing bacteria, and include cationic poly(phenylene vinylene) derivative, pH-sensitive quaternary pyridinium salts, for which the antibacterial potency can be controlled by varying the pH [73][74].

Smart resins that modulate the oral biofilm composition: quaternary ammonium methacrylates such as 12-methacryloyloxy dodecyl pyridinium bromide, methacryloxylethyl cetyl dimethyl ammonium chloride, quaternary ammonium polyethylenimine, and dimethylaminododecyl methacrylate [75][76].

Smart tailoring of alkyl chain length in quaternary ammonium methacrylates to avoid drug resistance [77].

SCs are capable to differentiate into various cell phenotypes based on their lineage and exposure to different environmental stimuli, such as ECM, growth factors, hypoxia, etc. [78]. The growth factor, usually a secreted protein or a steroid hormone, stimulates wound healing, cell proliferation, and occasionally cellular differentiation, and regulates various cellular processes. Cytokines and hormones bind to specific receptors on the surface of the target cells. Growth factors typically act as signaling molecules between cells, thus promoting cell differentiation and maturation [79][80].

The authors experience related to the subject includes tetracycline loaded collagen-carboxymethylcellulose/hydroxyapatite ternary composite materials [81], antiseptic composite materials containing silver nanoparticles, based on collagen, hydroxyapatite, and collagen/hydroxyapatite [82], collagen matrices with lidocaine [83], bone regeneration using synthetic HA, with high porosity and surface area for osteointegration [84][85][86][87].

2.3. Additive Manufacturing Technologies for Oral Tissue Engineering

Continuous development of manufacturing technologies enable printing of biofunctional scaffolds similar to the ECM, acting as a microenvironment for cell adhesion, proliferation, and differentiation [88][89].

The additive manufacturing (3D printing) of biomaterials offers promising future perspectives for the field of biomedical engineering [90], especially in regard to patient-specific clinical applications.

Additive manufacturing techniques for medical and tissue engineering purposes can be classified as: techniques which involve printing of live cells along with other materials (3D bioprinting) [91], and non-cellular fabrication techniques.

3D bioprinting, based on the layer-by-layer precise positioning of biological constituents, biochemicals and living cells, facilitates on-demand “printing” of cells, tissues and organs [92][93] for regenerative medicine purposes [94]. Utilizing diverse bioprinting techniques, tissue-engineered constructs can be tailored to obtain desired structures and properties [95][96].

Inkjet bioprinting functions by depositing small ink droplets into a predetermined location. It can be driven by thermal or piezoelectric actuation [97]. In thermal technology, heat-generated, the inflated bubble forces the ink out of the narrow nozzle and onto the substrates. In piezoelectric technology, drops are generated in absence of heat, by the transient pressure from the piezoelectric actuator. The droplets remain directional with regular and equal size [98], but, if used too frequently, this technology can cause damage to the cell membrane and cell lysis.

Laser-based bioprinting consists of a pulsed laser source, a ribbon, and a receiving substrate. The biological material, in liquid form, is irradiated by the laser, evaporates, and reaches the receiving substrate as droplets. Laser-based bioprinting enables high-resolution printing of biological material such as cells, DNA, and peptides [99]. Its drawback is that the use of the pulsed laser source may result in compromised cell viability [100].

Stereolithography bioprinting uses a photo-crosslinking light source to obtain desired patterns. It is highly tunable and prints in a layer-by-layer manner, the bioink from the reservoir being transferred to a movable platform [101].

Pressure-assisted bioprinting uses biomaterials in form of solutions, pastes or dispersions. The material, in form of a filament, is extruded by pressure through a microneedle or a microscale nozzle orifice [102].

Bioink printability has an important role in the fabrication process [103][104]. Besides being biocompatible and biodegradable, bioinks should be deformable and flowable [102]. After printing, the bioink should be stable in order to maintain shape and architecture of the design model [105].

The components of the bioink are polymers, ceramics, hydrogels, and composites, currently used in tissue engineering [106]. Hydrogel inks are much more attractive as bioprinting materials, compared to polymers and ceramics have received much more attention, and novel ink formulations have been designed. [107]. Complex, functional, and biocompatible hydrogels can be fabricated using bioprinting technology. Adding different amounts of HA was attempted to a tunable alginate-gelatin hydrogel composite [108], human MSCs being subsequently mixed. Adding HA to the hydrogel resulted in enhanced mechanical properties, recommending it hard tissue reconstruction. No reduction in cell viability was detected [109]. The freeform reversible embedding of suspended hydrogels, a 3D bioprinting technique which deposits and crosslinks different kind of hydrogel inks, has been proven successfully [110].

An important concern when printing SCs—including ESCs, MSCs, and ASCs—is that their activity, including proliferation and pluripotency, may change during the process [111][112]. MSCs were successfully laser-printed for the construction of scaffold-free autologous grafts. The seed cells survived and maintained their ability to proliferate and continue differentiating into the osteogenic lineage [113].

Non-cellular additive manufacturing techniques include (Table 1):

The powder bed fusion methods which use either electron beam or laser to selectively consolidate material powder. The techniques involve spreading material powder over the previous layers, melting and fusing it [114].

The binder jetting technique is similar to the powder bed fusion technique and utilizes material powder that is spread over previous layers. Unlike powder bed fusion, this technique uses a binder as an adhesive for its consolidation [115][116].

The fused deposition modeling technique is based on the extrusion of heated polymer wires through a nozzle tip. The polymer rods are deposited and arranged in a layer by layer fashion [117].

The material jetting technique uses a liquid photopolymer resin that is light-cured. Similar to the material extrusion technique, the material is deposited from a nozzle and cured, defining a cross section. Individual cross sections are consolidated in a layer by layer fashion as the building platform moves in the vertical direction [118].

The vat polymerization technique uses a vat of liquid photopolymer resin, deposited in a layer by layer fashion. The build platform moves (depending on the position of the light source) to create additional layers on top of the previous [119].

These techniques all have their pros and cons and can process different types of biomaterials [120] (Table 1).

Table 1. Additive manufacturing methods of biomaterials for oral tissue engineering

Biomaterial

Type

Fabrication Method

Application

Reference

Hydroxyapatite

Bioactive/non-degradable ceramic

Vat polymerization;

powder bed fusion;

fused deposition; binder jetting

Bone tissue

engineering

[121][122][123][124][125]

Bio glass

Bioactive ceramic

Vat polymerization

Bone tissue

engineering

[126]

Calcium silicate

Bioactive ceramic

Powder bed fusion

Tissue engineering

[127]

β-tricalcium phosphate

Bioactive/

biodegradable ceramic

Binder jetting;

vat polymerization;

fused deposition

Bone tissue

engineering

[128][129][130][131][132]

Polycaprolactone

Biodegradable polymer

Powder bed fusion; fused deposition

Bone tissue

engineering;

cartilage tissue

engineering

[133][134][135][136]

Poly(lactic acid)

Biodegradable polymer

Fused deposition

Bone

regeneration

[137]

Poly(lactic

acid-co-glycolic acid)

Biodegradable polymer

Material jetting;

fused deposition

Tissue engineering

[138][139][140][141]

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