Elsevier

Acta Biomaterialia

Volume 50, 1 March 2017, Pages 493-501
Acta Biomaterialia

Full length article
Promoting bioengineered tooth innervation using nanostructured and hybrid scaffolds

https://doi.org/10.1016/j.actbio.2017.01.001Get rights and content

Abstract

The innervation of teeth mediated by axons originating from the trigeminal ganglia is essential for their function and protection. Immunosuppressive therapy using Cyclosporine A (CsA) was found to accelerate the innervation of transplanted tissues and particularly that of bioengineered teeth. To avoid the CsA side effects, we report in this study the preparation of CsA loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles, their embedding on polycaprolactone (PCL)-based scaffolds and their possible use as templates for the innervation of bioengineered teeth. This PCL scaffold, approved by the FDA and capable of mimicking the extracellular matrix, was obtained by electrospinning and decorated with CsA-loaded PLGA nanoparticles to allow a local sustained action of this immunosuppressive drug. Dental re-associations were co-implanted with a trigeminal ganglion on functionalized scaffolds containing PLGA and PLGA/cyclosporine in adult ICR mice during 2 weeks. Histological analyses showed that the designed scaffolds did not alter the teeth development after in vivo implantation. The study of the innervation of the dental re-associations by indirect immunofluorescence and transmission electron microscopy (TEM), showed that 88.4% of the regenerated teeth were innervated when using the CsA-loaded PLGA scaffold. The development of active implants thus allows their potential use in the context of dental engineering.

Statement of Significance

Tooth innervation is essential for their function and protection and this can be promoted in vivo using polymeric scaffolds functionalized with immunosuppressive drug-loaded nanoparticles. Immunosuppressive therapy using biodegradable nanoparticles loaded with Cyclosporine A was found to accelerate the innervation of bioengineered teeth after two weeks of implantation.

Introduction

Dental caries, periodontal diseases and oropharyngeal cancers are the most prevalent oral diseases. Dental caries and tooth loss are important oral health indicators for adults and are key measures for monitoring progress of the disease [1]. The National Health and Nutrition Examination Survey, 2011–2012, revealed that in adults aged 20–64, 91% had dental caries and 27% had untreated tooth decay. Only 48% of adults aged 20–64 had a full set of permanent teeth (excluding third molars) and nearly 19% of adults aged 65 and over were edentulous [1]. Current dentures and implants used to replace missing teeth do not remodel and show a reduced integration with the host. Therefore, there is a need for new biomaterials to promote regeneration.

Regenerative nanomedicine is a rapidly expanding domain which has as objective the development of compatible biomaterials accepted by the body able to interact with cells/tissues present in the site of implantation. Such biomaterials can be combined with nanoparticles allowing a controlled or sustained release of active molecules. On the other hand, tissue engineering aims at replacing or repairing damaged tissues.

Keller et al. [2] showed that cultured re-associations between dissociated mesenchymal cells and intact epithelium from Embryonic Day (ED) 14 mouse molars gave well formed teeth after implantation under the skin of adult ICR mice. The vascularization of the dental pulp occurred while the innervation was never observed [2]. Siemionow et al. [3] showed that, immunosuppressive therapy with tacrolimus, a calcineurin inhibitor, accelerated nerve regeneration in the case of face transplantation. Cyclosporine A (CsA), another calcineurin inhibitor, widely used in organ transplantation [4] has also direct effect on nerve growth [5], [6]. When cultured re-associations were co-implanted with trigeminal ganglia in CsA-treated ICR mice, the innervation of the dental mesenchyme occurred after one week of implantation. After two weeks, the axons coming from the trigeminal ganglia reached the odontoblasts. These results demonstrated that the innervation of the dental pulp can be obtained in immunosuppressive conditions [7]. However, the oral availability of CsA is slow and highly variable owing to its biopharmaceutical properties. The use of this molecule is controversial because it can induce different forms of kidney dysfunction, cancers and lymphomas [8], [9]. Different approaches have been investigated to reduce its nephrotoxicity by developing CsA-loaded PLGA nanoparticles as delivery vehicles because of their excellent biocompatibility and sustained release [10].

On the other hand, the development of compatible biomaterials is also an essential step in the regeneration of a functional tooth as we previously showed by using a FDA approved nanofibrous polycaprolactone (PCL) scaffold functionalized with nerve growth factor (NGF) for a local release of this neurotrophic factor [11]. Indeed, when the scaffold was functionalized with nanoparticles containing NGF the innervation occurred in the dental pulp indicating the capability of the NGF nanoreservoirs to direct axon formation from the trigeminal ganglion into the bioengineered tooth [11]. So, following this strategy it should be possible to fabricate a combination cell-therapy implant capable of regeneration of a vascularized and innervated tooth as tooth replacement during regenerative therapy.

The aim of this study was to combine a local sustained effect of CsA with the use of a PCL-based scaffold, using reduced doses of the immunosuppressant molecule and so avoiding the unwanted side effects attributed to the ingestion or burst release of this drug. For this purpose, we functionalized PCL scaffolds with CsA-loaded nanoparticles and studied the innervation in the bioengineered teeth pulp by immunofluorescence and transmission electron microscopy (TEM).

Section snippets

Materials

Poly (D, L-lactic acid/glycolic acid) 50/50 polymer (PLGA; MW 24–38 KDa), under the commercial name Resomer® RG 503 was purchased from Evonik Industries AG (Darmstadt, Germany). Polycaprolactone (PCL; MW 80 KDa) analytical grade, Cyclosporine A, Dexamethasone (used as HPLC internal standard), Pluronic® F-68 surfactant, ethyl acetate (Class 3 solvent according to the pharmacopeia), acetonitrile and methanol (HPLC grade), were all purchased from Sigma Aldrich (St. Louis (MO), USA) and used as

Characterization of the nanoparticles and scaffolds

The experimental set up for the continuous synthesis of the CsA-loaded PLGA nanoparticles is depicted in Fig. 1. The synthesis is based on the use of microfluidic platforms (PEEK-based interdigital static micromixer) to achieve a narrow nanoparticle size-distribution with a high throughput and avoiding batch-to-batch product variations. Those nanoparticles were prepared by an oil/water (o/w) microchannel emulsification process and solvent evaporation method.

It is important to point out that by

Discussion

The PLGA nanoparticles, widely used in tissue engineering applications, were prepared in a continuous manner by an O/W emulsification process and solvent evaporation method in a microfluidic reactor. It is important to point out that the emulsion was formed without the aid of external mechanical forces (i.e., ultrasonic sources or homogenizers), but only using the shear stress caused by the fluid flow through microfluidic interdigital channels [14]. The polyester PLGA, approved by the FDA in

Conclusions

An implant consisting of an electrospun nanofibrous scaffold functionalized with CsA-loaded PLGA nanoparticles produced by a continuous emulsification process was developed. By using microfluidic reactors it is possible to produce CsA-loaded PLGA nanoparticles with a narrow particle-size distribution and elevated encapsulation efficiency. The layer-by-layer technique allows the deposition of PLGA nanoparticles on electrospun PCL nanofibers in a controlled manner. The proposed system has

Acknowledgments

The authors thank Hervé Gegout for histology and Nadia Messaddeq for the TEM observations. This work was funded by the Inserm (France). Financial support from the EU thanks to the ERC Consolidator Grant program (ERC-2013-CoG-614715) is gratefully acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III (Spain) with assistance from the European Regional

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