Scaffold-based developmental tissue engineering strategies for ectodermal organ regeneration.

Tissue engineering (TE) is a multidisciplinary research field aiming at the regeneration, restoration, or replacement of damaged tissues and organs. Classical TE approaches combine scaffolds, cells and soluble factors to fabricate constructs mimicking the native tissue to be regenerated. However, to date, limited success in clinical translations has been achieved by classical TE approaches, because of the lack of satisfactory biomorphological and biofunctional features of the obtained constructs. Developmental TE has emerged as a novel TE paradigm to obtain tissues and organs with correct biomorphology and biofunctionality by mimicking the morphogenetic processes leading to the tissue/organ generation in the embryo. Ectodermal appendages, for instance, develop in vivo by sequential interactions between epithelium and mesenchyme, in a process known as secondary induction. A fine artificial replication of these complex interactions can potentially lead to the fabrication of the tissues/organs to be regenerated. Successful developmental TE applications have been reported, in vitro and in vivo, for ectodermal appendages such as teeth, hair follicles and glands. Developmental TE strategies require an accurate selection of cell sources, scaffolds and cell culture configurations to allow for the correct replication of the in vivo morphogenetic cues. Herein, we describe and discuss the emergence of this TE paradigm by reviewing the achievements obtained so far in developmental TE 3D scaffolds for teeth, hair follicles, and salivary and lacrimal glands, with particular focus on the selection of biomaterials and cell culture configurations.

Overview of the different methods used in the primary culture of oral mucosa cells.

The culture of primary cells in vitro has enabled to gain knowledge in the field of cell biology, disease mechanisms and to offer great potential in drug testing. To date, two main techniques of isolating and culturing oral mucosal cells, the direct explant method and the enzymatic method, dominate the literature and practice. In the present study, both techniques are discussed in detail, comparing the advantages and disadvantages of the two approaches in setting up a primary culture of oral mucosal cell. The direct explant technique is well-established and has been commonly used for the past 20-30 years. Although the method of setting up the cultures did not show much variations in the methodology described by authors, the culturing conditions varied according to the aims of the projects.

Mesenchymal Cell Community Effect in Whole Tooth Bioengineering.

In vitro expanded cell populations can contribute to bioengineered tooth formation but only as cells that respond to tooth-inductive signals. Since the success of whole tooth bioengineering is predicated on the availability of large numbers of cells, in vitro cell expansion of tooth-inducing cell populations is an essential requirement for further development of this approach. We set out to investigate if the failure of cultured mesenchyme cells to form bioengineered teeth might be rescued by the presence of uncultured cells. To test this, we deployed a cell-mixing approach to evaluate the contributions of cell populations to bioengineered tooth formation. Using genetically labeled cells, we are able to identify the formation of tooth pulp cells and odontoblasts in bioengineered teeth. We show that although cultured embryonic dental mesenchyme cells are unable to induce tooth formation, they can contribute to tooth induction and formation if combined with noncultured cells. Moreover, we show that teeth can form from cell mixtures that include embryonic cells and populations of postnatal dental pulp cells; however, these cells are unable to contribute to the formation of pulp cells or odontoblasts, and at ratios of 1:1, they inhibit tooth formation. These results indicate that although in vitro cell expansion of embryonic tooth mesenchymal cells renders them unable to induce tooth formation, they do not lose their ability to contribute to tooth formation and differentiate into odontoblasts. Postnatal pulp cells, however, lose all tooth-inducing and tooth-forming capacity following in vitro expansion, and at ratios >1:3 postnatal:embryonic cells, they inhibit the ability of embryonic dental mesenchyme cells to induce tooth formation.

Dental pulp in mature replanted human teeth: morphological alterations and metalloproteineses-2 and -9, Annexin-5, BCL-2 and iNOS modulation.

Tooth replantation, as a treatment concept, has been subject to controversies regarding the mechanism as well as the various parameters underlying this process. This work aimed to study time-related changes in the pulp of replanted mature human premolars through the changes in the levels of certain factors involved in the underlying mechanisms of pulpal tissue healing after replantation. Eleven experimental mature teeth were extracted, immediately replanted in the original socket and left without any other intervention for 1, 2, 3 and 12 weeks before re-extraction. Three premolars served as control. All specimens were subject to histological analysis and the levels of MMP-2, MMP-9, Annexin V, iNOS and BCL-2 (anti-apoptotic family) were analyzed employing immunohistochemistry. The results showed degradation of the extracellular matrix (ECM), inflammatory cell infiltrate, loss in pulpo-dentine interface and loss of odontoblasts in the dental pulp tissue. This was accompanied by increase over time of MMP-9, Annexin V, iNOS and a decrease of BCL-2 and MMP-2, suggesting that apoptosis increased throughout the experimental period.

Adult human gingival epithelial cells as a source for whole-tooth bioengineering.

Teeth develop from interactions between embryonic oral epithelium and neural-crest-derived mesenchyme. These cells can be separated into single-cell populations and recombined to form normal teeth, providing a basis for bioengineering new teeth if suitable, non-embryonic cell sources can be identified. We show here that cells can be isolated from adult human gingival tissue that can be expanded in vitro and, when combined with mouse embryonic tooth mesenchyme cells, form teeth. Teeth with developing roots can be produced from this cell combination following transplantation into renal capsules. These bioengineered teeth contain dentin and enamel with ameloblast-like cells and rests of Malassez of human origin.

Human dental pulp cell apoptosis: immunohistochemical study after applying orthodontic traction.

The aim of this study was to compare human dental pulp stress and programmed cell death after 3 and 6 months of orthodontic treatments by assessing the degree of apoptosis and related proteins. Human dental pulps were collected from twenty young patients orthodontically treated by Straight Wire technique. Samples were fixed, paraffin-embedded and processed for histology and immunohistochemistry using anti-heat shock protein 60 kDa (Hsp60), -caspase 3, -caspase 9, and -PCNA antibodies, as well as TUNEL reactions. Moreover, we performed immunoprecipitation for Hsp60 and caspase 3, and for Hsp60 and caspase 9, from paraffin extracted tissues. Increased levels of both caspases and Hsp60 occurred in 6-months treated samples; at the same time, we found increased levels of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells. Immunoprecipitation showed that Hsp60 forms a complex with both Pro-caspase 3 and Caspase 3, and this may accelerate Pro-caspase 3 activation, especially in the 6-months treated group. On the contrary, no complex between Hsp60 and Pro-caspase 9 was detected. The orthodontic tractions may be a cause of stress, apoptosis and proliferation in pulp tissue. These results suggest the need of further studies about the effects of long term orthodontic treatments on the dental pulp.

Dental caries in children with asthma undergoing treatment with short-acting beta2-agonists.

AIM: This study sought to evaluate possible higher risk for dental caries among asthmatic children undergoing treatment with short-acting beta2-agonists. METHODS: Dental clinical assessments, saliva analysis and a questionnaire survey were carried out on 60 children aged 6-12, of whom 30 were asthmatic subjects undergoing treatment with short-acting beta2-agonists and 30 were used as controls. The obtained data for DMFT/dmft scores, Silness-Löe plaque index, buffer capacity and bacteria counts for Streptococcus mutans and Lactobacillus in the saliva, oral hygiene and dietary habits were compared using Student t-test and Pearson chi-square test. RESULTS: We registered a higher DMFT score among asthmatics of 1.2-/+1.8 (SD) and 0.3-/+0.8 among non-asthmatic patients (p<0.05), while comparison of dmft scores between the examined groups showed not significant (Student t-test). Saliva analysis revealed lower buffer capacity in 43.3% of the asthmatic children, followed by higher cariogenic bacteria counts in their saliva (p<0.05 Student t-test). These results show the lower plaque index in the asthmatic group (1.6+/-0.4) compared with the control (2.1+/-0.3). Asthmatic children expressed better oral-health habits with more frequent tooth- brushing and usage of fluorides. CONCLUSION: The results from our study suggest a higher caries-susceptibility among asthmatic children undergoing treatment with short-acting beta2-agonists, but a clear association between these drugs, salivary changes and dental caries among children, still remains to be demonstrated.