J Med Microbiol. 2016 Nov;65(11):1332-1340. doi: 10.1099/jmm.0.000353.

Structural and recovery mechanisms of 3D dental pulp cell microtissues challenged with Streptococcusmutans in extracellular matrix environment.

Pubmed

 

Abstract:

Appropriate biomimetic modeling of the impact of oral pathogens on dental pulp tissue requires an adequate environment inclusive of physiological conditions similar to the native tissue microenvironment. Our recently developed organotypic 3D dental pulp stem cells (DPSCs) microtissue platform was utilized to determine the effect of the caries-causing agent, Streptococcus mutans (S. mutans), on structural configuration and recovery potential of pathogen-challenged microtissues. The bacterial biofilm was found to interact with individual areas on the surface of the microtissue and increase its thickness. However, important cellular functions, such as the self-renewal and differentiation/mineralization capabilities, were maintained leading to the creation of new tissue and deposit of dentin. S. mutans attached to individual cells without invading them or modifying the external structure of the microtissue. However, the internal architecture was changed as indicated by the appearance of micro-canal-like structures that penetrated the tissue. Besides providing a unique platform for determining the impact of oral pathogens on structural and functional properties of dental pulp microtissue in a bio-relevant milieu, this study also demonstrated the potential of S. mutans to disrupt the internal structure of pulp microtissue thus making it potentially vulnerable to multi-pathogen infections.

 

Supplementary Material:

Dental pulp, a loose connective tissue, is basically composed of cells and cell-secreted extracellular matrix (ECM) that surrounds them (1,2). We have designed an organotypic (3D) dental pulp platform that includes spontaneously assembled dental pulp stem cells (DPSCs) and yields micro-tissues in ECM environment that closely resemble physiological conditions.

This 3D ECM/DPSCs microtissue infection platform assimilates various aspects of host-pathogen interactions in vivo. ECM provides more than just the mechanical and structural support to the cells; it also functions as a framework of signaling events (3). Basement membrane matrix (Matrigel), used in this study, is considered a good niche for stem cells to maintain their self-renewal capacity (4) and differentiation (5) capabilities to regenerate dentin.

Bacterial adhesion and entry into the tissue is accompanied by a change from a less-rigid environment, such as oral/tissue fluids, to a stiffer one, representative of tissue matrix (6). Rather than spreading over the entire cell layer (typical feature of the conventional 2D cultures), bacteria, influenced by the matrix rigidity, perfused to form elongated trails and interacted with isolated spots on the surface of the microtissue. Bacterial passage through the matrix is likely to involve interaction(s) with ECM components, which are speculated to play a major role in enhancing the attachment of the bacteria and invasion into the host tissue (7). ECM also regulates biofilm development and, therefore, contributes directly to the virulence of the pathogen (8). The reduced oxygen and nutrient conditions in the matrix recapitulate similar state in vivo and possibly induce the formation of more resistant biofilms to antibiotics (9,10).

In contrast to normal cell cultures, which expose only one side of the layer, matrix allows spatial assembly of cells into 3D microtissue and exposure of its entire surface area to bacterial challenges (11,12). The organotypic cultures facilitate architectural development at higher complexity level, allowing us to determine the impact of the pathogen on the structural properties. Creation of micro canal-like structures described in our study indicates that 3D cultures also allow invasion into assembled cell-cell and cell-matrix structures.

S.mutants is considered a caries-causing agent which mostly colonizes at tooth enamel surface. However, plethora of papers reports its existence in samples isolated from deeper layers, such as infected pulp canals, and considers it to be among the primary causes of endodontic infection (13,14). Other reports link S. mutans to infections of heart tissues (15). Bacterium is disseminated from dental tissues, such as dental pulp, via infiltration of blood vessels through the blood stream (16). We have utilized our 3D infection platform to determine whether S. mutans indeed alters structural properties and basic functions of dental pulp cell layers. We found that surface architecture of the dental pulp microtissues was not changed and basic functions, such as self-renewal capacity and differentiation/dentin formation capabilities, were recovered. Although the invasion into individual cells was not demonstrated, formation of micro-canals partially colonized by bacteria may put the tissue in risk and diminish the integrity of internal structure thus making microtissues susceptible to other odonto-pathogens. These observations may be explained by previous studies reporting the presence of bacterium-secreted enzymes that stimulate the formation of these channels. In conclusion, our organotypic model studies suggest that, while possibly contributing to the infection process, S. mutants doesn’t play a major role in pulp infection. The proposed 3D ECM/DPSC microtissue infection platform is an efficient tool for studying interactions between oral mono and/or multispecies biofilms with pulp tissues. Its utility can easily be extended to include validating antimicrobial agents in a biomimetic environment.

 

 

The pathogen interacts with the extracellular matrix components and the microtissue. The arrows, labeled in different color represent the unique relationships between individual platform components. Our 3D ECM/DPSC platform is an attractive biomimetic model for studying host-pathogen interactions.

 

 

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