Experimental Approaches for Pulp Tissue Regeneration

Abstract

The aim of this PhD thesis was to study experimental approaches for revitalization of necrotic teeth. Revitalization, also known as regenerative endodontic procedures (REPs), is a relatively new treatment for necrotic teeth which tries to regenerate the dentine-pulp complex instead of obturating the root canal with biologically inert materials (root canal treatment). Until very recently, the most reliable option for the treatment of immature necrotic teeth was apexification followed by root canal treatment. However, endodontically treated teeth remain devitalized throughout the patient's lifetime and therefore defenceless to new caries lesions, as the absence of pulp implies the lack of tooth immune mechanisms. On the contrary, the regeneration of the dentine-pulp complex allows further root development and aims to recover the natural immune and secretory system of the pulp, making teeth more resistant to future lesions or traumatisms. The therapy was developed to treat necrotic immature teeth (i.e. those that have not completed their root development). Clinically, the outcomes can be considered successful since there is a resolution of the symptomatology, healing of the apical pathosis and further root development in most cases. However, histological analysis has demonstrated that the tissues formed after the therapy are reparative tissues – such as cementum-like tissue – instead of dentine, as well as an unorganized connective tissue, instead of pulp with its characteristic odontoblast layer. Currently, numerous efforts are being made to shed light on the clinical and biological aspects involved in the regeneration of pulp. Chapter 1: As previously said, evidence shows that no dentine but reparative tissues (cementum-like tissue) are responsible for the root development after regenerative endodontics. As cementum is less hard and less elastic than dentine, the question arises whether a root with apposition of cementum can endure mechanical stress similarly to roots completed by dentine. Thus, one of the objectives of this thesis was to compare the biomechanical performance of cementum- and dentine-reinforced teeth, and therefore to evaluate the biomechanical advantages of dentine regeneration after regenerative endodontics. We developted a finite element model of cementum- and dentinereinformed teeth and studied the stress distribution after the simulation of biting, trauma and orthodontic movement. The results showed that apposition of hard tissue (whether cementum or dentine) after REPs reduces mechanical stress on 17 immature teeth and, more important, that the formation of dentine is advantageous because it, unlike cementum, facilitates an even stress distribution throughout the root. As far as we know, ours was the first study showing the biomechanical advantages of dentine regeneration. Chapter 2: Odontoblasts are post-mitotic cells that secrete dentine. The isolation and culture of odontoblasts may open numerous possibilities to study this cell type under standardized conditions, shedding light on their roles in dentine formation, immune defence and transmission of external stimuli. We evaluated different protocols of enzymatic treatment to isolate primary odontoblasts from human molars. The results showed that, regardless of the enzymatic solution used, odontoblasts in culture did not remain viable after 24 h. Additionally, we identified increased expression of nestin (NE), bone sialoprotein (BSP) and dentine matrix acidic phosphoprotein 1 (DMP1) in the odontoblast layer compared to pulp fibroblasts. Though primary odontoblasts can still not be cultivated after isolation, characteristic genes were identified to differentiate odontoblasts from pulp fibroblasts. Chapter 3: We analysed the effects of autologous platelet concentrates (APCs) in the clinical and histological outcomes of the therapy and the different clinical protocols clinically used through systematic reviews. The results indicated that APCs improve the clinical and radiographic outcomes

of regenerative endodontics since the teeth treated with APCs achieved significantly better thickening of the dentine walls and root lengthening. However, true regeneration of pulp was not achieved with the addition of platelet concentrates, which only stimulated tissue repair. Additionally, most of the studies did not follow a standard clinical protocol for regenerative endodontic therapy and used irritant and intracanal medicaments that are cytotoxic and affect the differentiation and adherence of the stem cells. Chapters 4 and 5: As will be mentioned in detail, a small apical foramen acts as a physical barrier that hinders tissue ingrowth into the root canal and therefore reduces the possibility of revitalization of mature teeth. We studied different methods for apical foramen enlargement of mature teeth as a basis to apply it in a further animal study. We analysed manual instrumentation at different working lengths and apicoectomy on extracted human teeth and in situ teeth. We concluded that apicoectomy is not an effective technique for apical foramen enlargement and therefore should not be used for that purpose. Instrumentation 18 0.5mm beyond the apex resulted in the most effective technique. Later, we performed an animal study and evaluated pulp tissue regeneration/repair in mature teeth and the differentiation of the stem cells from the periapical tissues into odontoblast-like cells by adding preameloblast-conditioned medium. Preameloblast-conditioned medium was applied in pulpectomized ferret canines, whose apical foramina were enlarged using the previously developed method. We observed vascularized connective tissue occupying the apical third of the canal space in 50% of the teeth, showing the potential of revascularization of mature teeth. However, no odontoblast-like cells were observed showing that in vivo odontoblast-like differentiation of stem cells is still not possible with the tested technique. Chapter 6: Finally, we present here the preliminary data of characterization and odontoblast-like differentiation of amnion epithelial cells. Human amnion epithelial cells (hAECs) express pluripotent stem cell markers and have been proven to differentiate in cells of the three embryologic layers. However, as far as we know, these are the first experiments that have proved the potential of odontoblast-like differentiation of these cells in vitro. To induce the odontoblast-like differentiation, we seeded hAECs over dentine disks treated with EDTA and evaluated the morphological characteristic of cells. We observed that hAECs present a characteristic odontoblast-like morphology, with cytoplasmic processes located in dentinal tubuli, after 48 h. Further studies will be carried out with known concentrations of dentine matrix proteins and qPCR.

El objetivo de esta tesis doctoral fue estudiar enfoques experimentales para la revitalización de dientes necróticos. La revitalización o endodoncia regenerativa es un tratamiento nuevo para dientes necróticos que busca regenerar el complejo dentino-pulpar, en lugar de obturar el conducto radicular con materiales biológicamente inertes (obturación radicular). Los dientes tratados endodónticamente permanecen desvitalizados durante toda la vida del paciente y, por lo tanto, indefensos ante nuevas lesiones de caries, ya que la ausencia de pulpa implica la falta del mecanismo inmune del diente. Por el contrario, la regeneración del complejo dentino-pulpar permite un mayor desarrollo de la raíz y tiene como objetivo recuperar el sistema inmune y secretor natural de la pulpa, haciendo que los dientes sean más resistentes a futuras lesiones o traumatismos. En esta tesis doctoral se realizó un estudio de elementos finitos que probó por primera vez que la distribución de la tensión mecánica es más desventajosa en dientes inmaduros y maduros con tejido reparativo (como el formado después de la endodoncia regenerativa), que en dientes desarrollados con dentina. Se llevaron a cabo estudios en material cadavérico para analizar métodos de ampliación del foramen apical de dientes maduros y un estudio experimental para evaluar a efectividad de la terapia en dientes maduros de hurón con forámenes ampliados. Los resultados mostraron que es posible la formación de un tejido conectivo vascularizado en el interior del canal de dientes maduros, pero este tejido ocupó solo el tercio apical. Se realizaron revisiones sistemáticas relativas al efecto de concentrados de plaquetas en la terapia, y se concluyó que si bien los dientes tratados con concentrados de plaquetas mostraron mejores resultados clínicos, el tejido neoformado es tejido reparativo, es decir, carente de odontoblastos y dentina. Se llevó a cabo un estudio in vitro para aislar y cultivar odontoblastos y se concluyó que no es posible mantener odontoblastos vitales in vitro, probablemente debido a la disrupción del proceso odontoblástico durante la aislación. Por último, se realizó experimentos in vitro que buscan evaluar la capacidad de diferenciación odontogénica de las células pluripotentes del amnios, investigación que actualmente está en fase de ejecución.