Tissue engineering: From research to dental clinics

Abstract

Tissue engineering is an interdisciplinary field that combines the principles of engineering, material and biological sciences toward the development of therapeutic strategies and biological substitutes that restore, maintain, replace or improve biological functions. The association of biomaterials, stem cells, growth and differentiation factors has yielded the development of new treatment opportunities in most of the biomedical areas, including Dentistry. The objective of this paper is to present the principles underlying tissue engineering 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 of therapeutic strategies for biomedical applications. Indeed, tissue engineering may lead to new strategies for the clinical management of patients with dental and craniofacial needs in the future.

Introduction

The use of synthetic restorative materials as substitutes for dental structures is a practice nearly as old as Dentistry itself . To date, most of the procedures performed in Dentistry are limited to the replacement of damaged tissues for biocompatible synthetic materials that may not present chemical, biological, or physical characteristics and behaviors similar to the host tissues. These discrepancies, together with the hostile environment of the oral cavity, result in relatively short-lived successful outcomes and frequent need for re-treatment. Tissue engineering is a multi-disciplinary field focused on the development of materials and strategies to replace damaged or lost tissues for biological materials by merging principles, methods and knowledge of chemistry, physics, engineering and biology . The achievements obtained by tissue engineering in the past few years have resulted in new therapies such as the production of skin to treat burns , bone grafts to replace large bone defects , small-caliber arteries to treat atherosclerotic vascular disease and cartilage for plastic and reconstructive surgeries . Important advances have been reported in Dentistry aiming the regeneration of temporo-mandibular joint , periodontal ligament , dentin , enamel , pulp and integrated tooth tissues .

The concept underlying tissue engineering was first proposed in the United States in the mid-1980s in order to reduce the donor scarcity to organ transplantation . The classical cell-based tissue engineering approach involves the seeding of biodegradable scaffolds with cells and/or growth factors, then, implanting it in order to induce and conduct the tissue growth . Obtaining good responses from this model demands the fine orchestration of the three tissue engineering fundamental elements: cells, scaffold and cell signaling. The objective of this review is to present the fundaments of the tissue engineering components and their application in Dentistry.

Tissue engineering

Cells

Stem cells are clonogenic cells capable of self-renewal and capable of generating differentiated progenies. These cells are responsible for normal tissue renewal as well as for healing and regeneration after injuries . Some stem cells are said to be pluripotent, i.e. have the ability to differentiate into many different cell types while the range of others are more restricted. The most pluripotent cells are found in the inner cell mass of blastocyst during the early stages of embryo development . They can differentiate into each of the more than 200 cell types of the adult body when exposed to appropriate stimuli. Along with the potential applications of totipotent cells lies a strong ethical discussion regarding the use of human embryos. This issue has strengthened the rational for the use of adult stem cells, which have been identified in every tissue formed after embryonic development and can be used to the same purpose of embryonic stem cells.

Studies have showed that it is possible to isolate clonogenic and highly proliferative cells from dental pulp using similar research protocol to isolate and characterize bone marrow stem cells . Dental pulp stem cells (DPSC) can differentiate into multiple cell lineages, such as adipocytes, chrondocytes, neurons and odontoblasts . Stem cells from human exfoliated deciduous teeth (SHED) were also identified and isolated . SHED has the advantage of being retrievable from naturally exfoliated teeth, which are one of the only disposable post-natal human tissues. As primary teeth are clearly a feasible source of post-natal stem cells, the interest toward the differentiation power of SHED cells has increased. Indeed, today we know that SHED can undergo adipogenic, chondrogenic, osteogenic, endothelial and odontoblastic differentiation . The ability that these cells have to cross lineage boundaries expands the potential use of SHED for therapies involving a large number of tissues ( Fig. 1 ).

Fig. 1
The principles of tissue engineering using dental stem cells may allow the regeneration of osseous, neural and tooth-related tissues.

Although both DPSC and SHED cells are originated from the dental pulp, they present differences regarding the odontogenic differentiation and osteogenic induction. For example, the levels of alkaline phosphatase activity and osteocalcin production during osteogenic differentiation are higher for SHED than for DPSC . However, the ability to regenerate a dentin-pulp-like complex found in DPSC is also observed in SHED cells . Furthermore, SHED may present an osteoinductive potential once they were able to induce differentiation from recipient murine cells into bone-forming cells .

The periodontal ligament was found to be a source of que a novel population of dental stem cell (PDLSC – periodontal ligament stem cell). This cell express high levels of telomerase , a key molecule in mediating cell proliferation and have the capacity to develop adipocytes and cementoblast/osteoblastic-like cells in vitro . In addition they also form collagen fibers, similar to Sharpey’s fibers, and cementum/periodontal ligament-like tissue when transplanted into immunocompromised mice using hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold .

The stem cells from the apical papilla (SCAP) were recently isolated from the apical papilla of immature human permanent teeth . The population seems to be the source of odontoblasts responsible for the formation of root dentin . These may be the reason why SCAP present similarities to DPSC regarding osteo/dentinogenic and growth factor receptor gene profiles. The in vivo implantation of SCAP with HA/TCP scaffold allowed the differentiation into odontoblast-like cells capable o regenerate a mineralized structure having a layer of dentin tissue formed over the surface of the HA/TCP besides connective tissue .

Additionally, the ability of SCAP to regenerate the periodontal ligament and alveolar bone in vivo . Analogous to DPSC and SHED, SCAP express a wide variety of neurogenic markers attesting its neurogenic potential .

Scaffolds

Scaffolds are temporary frameworks used to provide a three-dimensional microenvironment where cells can proliferate, differentiate and generate the desired tissue . The design of the ideal scaffold for each tissue to be formed is a challenging task. Ideally, a scaffold must allow cell attachment and migration, permit the localized and sustained delivery of growth factors, and enable the influx of oxygen to maintain the high metabolic demands of cells engaged in tissue regeneration.

Scaffolds are usually made from ceramics , natural or synthetic polymers , or composites from these materials . The choice of scaffold material depends on the desired outcome thus physical ( e.g. rheological behavior, mechanical properties, surface roughness and porosity) as well as chemical characteristics ( e.g. mode, velocity and products of degradation) must be considered.

The scaffold’s physical properties have to attend the needs of the target environment. It must present proper mechanical resistance to support in vivo stresses, and it should be mechanically compatible with the surrounding tissues . The scaffold’s mechanical properties have a direct impact in tissue formation by affecting cell differentiation into the desired phenotype through mechanotransduction . Therefore, linear elastic scaffolds are preferred when one attempts to generate bone, and nonlinear elastic or viscoelastic models are more suitable for soft tissues . Scaffold porosity is also critical to tissue generation. The quantity and extension of pores change the specific scaffold surface modifying its permeability and mechanical properties, having strong impact in cell seeding, nutrient diffusion and tissue ingrowth . Notably, higher number and extension of pores allows for enhanced cellularity but reduces scaffold strength . A study suggested pore size ranging from 50 to 400 μm for the optimum bone growth into porous-surfaced metallic implants . 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 μm . It has been proposed that pore interconnectivity is even more important to sustain bone growth than size of the pore size itself .

The scaffold degradation is fundamental to achieve success in tissue engineering therapies . The scaffold should ideally reabsorb once it has served its purpose of providing a template for tissue regeneration. Importantly, the degradation must occur at a rate compatible with the new tissue formation . For example, the implantation of fast degrading scaffolds decreases the in vitro viability of primary smooth muscle cells resulting in less cell population and lower angiogenesis levels . Furthermore, the degradation products should not be toxic and must be easily cleared or resorbed to minimize the risk of inflammatory response . It must be emphasized that during the scaffold degradation, the local pH should not be significantly lower than the physiological pH , otherwise cell death and protein degradation may occur.

Cell signaling

Cell signaling is part of a complex system of communication that governs cell activities and organizes their interactions ( Fig. 2 ).

Fig. 2
Flow of events leading to protein synthesis. (A) Binding of ligand to its specific cell membrane receptor triggers intracellular signaling and activation of gene expression. (B) Synthesis of new RNA template from transcription of the original strand of DNA. (C) Assembling of the ribosome complex at the initiation codon of the mRNA molecule. (D) Decoding of mRNA in sets of nucleotides forming a polypeptide chain. (E) Folding in the Golgi apparatus to complete protein synthesis. This can be followed by protein secretion to the extracellular environment or “sequestration” within the cell itself.

Many extracellular molecules have been described in the literature. It has been shown that a pool of these extracellular molecules has a major role than a single protein in the differentiation of cells into a functional tissue. This could be observed when proteins present in dentin disks , dentin extract in EDTA or a tooth-germ conditioned extract were found to supplement the scaffolds as a mechanism of cellular induction. Yet, there still is missing information on how each factor acts in isolation. Among the different factors, the TGFβ-1 (Transforming Growth Factor 1) and BMP (Bone Morphogenetic Protein), seem to have an important role in the odontoblastic differentiation . Moreover, there is evidence that the TGFβ-1 is released from the dentin after any injury and that BMPs, more specifically the BMP-2, have dentin induction ability .

The understanding of intracellular events triggered by extracellular proteins is critical for tissue engineering. In general, it is described that BMPs act in the canonical TGF-β pathway modulating smads, leading to odontoblastic differentiation and inducing dentin formation . However, it is important to keep in mind that TGF-β proteins, including BMPs, have also well established roles in cancer progression . Among these pathways, the Wnt pathway appears to be important for stem cell self-renewal and cell differentiation . The interactions and crosstalk between the canonical smad pathway and the Wnt pathway is probably one of the most studied, but there is little information on how these interactions affect DPSC. Additionally, it has been described that Wnt proteins are not able to induce DPSC differentiation . On the other hand, there is a connection between BMPs with β-catenin, an intracellular protein that is part of the Wnt pathway, which has an important role in differentiation processes for other cell types .

Tissue engineering

Cells

Stem cells are clonogenic cells capable of self-renewal and capable of generating differentiated progenies. These cells are responsible for normal tissue renewal as well as for healing and regeneration after injuries . Some stem cells are said to be pluripotent, i.e. have the ability to differentiate into many different cell types while the range of others are more restricted. The most pluripotent cells are found in the inner cell mass of blastocyst during the early stages of embryo development . They can differentiate into each of the more than 200 cell types of the adult body when exposed to appropriate stimuli. Along with the potential applications of totipotent cells lies a strong ethical discussion regarding the use of human embryos. This issue has strengthened the rational for the use of adult stem cells, which have been identified in every tissue formed after embryonic development and can be used to the same purpose of embryonic stem cells.

Studies have showed that it is possible to isolate clonogenic and highly proliferative cells from dental pulp using similar research protocol to isolate and characterize bone marrow stem cells . Dental pulp stem cells (DPSC) can differentiate into multiple cell lineages, such as adipocytes, chrondocytes, neurons and odontoblasts . Stem cells from human exfoliated deciduous teeth (SHED) were also identified and isolated . SHED has the advantage of being retrievable from naturally exfoliated teeth, which are one of the only disposable post-natal human tissues. As primary teeth are clearly a feasible source of post-natal stem cells, the interest toward the differentiation power of SHED cells has increased. Indeed, today we know that SHED can undergo adipogenic, chondrogenic, osteogenic, endothelial and odontoblastic differentiation . The ability that these cells have to cross lineage boundaries expands the potential use of SHED for therapies involving a large number of tissues ( Fig. 1 ).

Fig. 1
The principles of tissue engineering using dental stem cells may allow the regeneration of osseous, neural and tooth-related tissues.

Although both DPSC and SHED cells are originated from the dental pulp, they present differences regarding the odontogenic differentiation and osteogenic induction. For example, the levels of alkaline phosphatase activity and osteocalcin production during osteogenic differentiation are higher for SHED than for DPSC . However, the ability to regenerate a dentin-pulp-like complex found in DPSC is also observed in SHED cells . Furthermore, SHED may present an osteoinductive potential once they were able to induce differentiation from recipient murine cells into bone-forming cells .

The periodontal ligament was found to be a source of que a novel population of dental stem cell (PDLSC – periodontal ligament stem cell). This cell express high levels of telomerase , a key molecule in mediating cell proliferation and have the capacity to develop adipocytes and cementoblast/osteoblastic-like cells in vitro . In addition they also form collagen fibers, similar to Sharpey’s fibers, and cementum/periodontal ligament-like tissue when transplanted into immunocompromised mice using hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold .

The stem cells from the apical papilla (SCAP) were recently isolated from the apical papilla of immature human permanent teeth . The population seems to be the source of odontoblasts responsible for the formation of root dentin . These may be the reason why SCAP present similarities to DPSC regarding osteo/dentinogenic and growth factor receptor gene profiles. The in vivo implantation of SCAP with HA/TCP scaffold allowed the differentiation into odontoblast-like cells capable o regenerate a mineralized structure having a layer of dentin tissue formed over the surface of the HA/TCP besides connective tissue .

Additionally, the ability of SCAP to regenerate the periodontal ligament and alveolar bone in vivo . Analogous to DPSC and SHED, SCAP express a wide variety of neurogenic markers attesting its neurogenic potential .

Scaffolds

Scaffolds are temporary frameworks used to provide a three-dimensional microenvironment where cells can proliferate, differentiate and generate the desired tissue . The design of the ideal scaffold for each tissue to be formed is a challenging task. Ideally, a scaffold must allow cell attachment and migration, permit the localized and sustained delivery of growth factors, and enable the influx of oxygen to maintain the high metabolic demands of cells engaged in tissue regeneration.

Scaffolds are usually made from ceramics , natural or synthetic polymers , or composites from these materials . The choice of scaffold material depends on the desired outcome thus physical ( e.g. rheological behavior, mechanical properties, surface roughness and porosity) as well as chemical characteristics ( e.g. mode, velocity and products of degradation) must be considered.

The scaffold’s physical properties have to attend the needs of the target environment. It must present proper mechanical resistance to support in vivo stresses, and it should be mechanically compatible with the surrounding tissues . The scaffold’s mechanical properties have a direct impact in tissue formation by affecting cell differentiation into the desired phenotype through mechanotransduction . Therefore, linear elastic scaffolds are preferred when one attempts to generate bone, and nonlinear elastic or viscoelastic models are more suitable for soft tissues . Scaffold porosity is also critical to tissue generation. The quantity and extension of pores change the specific scaffold surface modifying its permeability and mechanical properties, having strong impact in cell seeding, nutrient diffusion and tissue ingrowth . Notably, higher number and extension of pores allows for enhanced cellularity but reduces scaffold strength . A study suggested pore size ranging from 50 to 400 μm for the optimum bone growth into porous-surfaced metallic implants . 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 μm . It has been proposed that pore interconnectivity is even more important to sustain bone growth than size of the pore size itself .

The scaffold degradation is fundamental to achieve success in tissue engineering therapies . The scaffold should ideally reabsorb once it has served its purpose of providing a template for tissue regeneration. Importantly, the degradation must occur at a rate compatible with the new tissue formation . For example, the implantation of fast degrading scaffolds decreases the in vitro viability of primary smooth muscle cells resulting in less cell population and lower angiogenesis levels . Furthermore, the degradation products should not be toxic and must be easily cleared or resorbed to minimize the risk of inflammatory response . It must be emphasized that during the scaffold degradation, the local pH should not be significantly lower than the physiological pH , otherwise cell death and protein degradation may occur.

Cell signaling

Cell signaling is part of a complex system of communication that governs cell activities and organizes their interactions ( Fig. 2 ).

Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Tissue engineering: From research to dental clinics
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