Department of Oral Biology

Research Overview

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The research efforts in UMKC-CRISP are directed towards the identification, characterization and synthesis of materials that can be used to replace skeletal or oral tissues lost because of trauma, disease, or age. The development of tissue-engineered materials that could serve as natural tissue replacements is one of the most exciting areas of investigation in both medicine and dentistry. In the exploration of these new materials, one area that has been largely overlooked is chemical and mechanical characterization of the material/tissue interface. This is a particularly challenging area of investigation since many of the current analytical techniques do not offer the required spatial resolution to study reactions occurring at the interface or the conditions (i.e., temperature, vacuum, etc.) under which the sample must be analyzed destroy or significantly damage/alter the biological tissue. To address these problems, we have developed nondestructive techniques to characterize and quantify reactions at the material/tissue interface.

The efforts in the research center are closely aligned with clinical problems associated with the application and utilization of synthetic materials in the reconstruction of damaged and/or diseased calcified tissues. We have a strong working relationship with several clinical programs including periodontics, pediatric and restorative dentistry. This strong collaborative and cooperative relationship provides the necessary catalyst to effectively and efficiently move our research from the lab bench to the clinic.

Research Highlights
UMKC-CRISP activities are focused in the following areas:

1) understanding the micro- and nano-structure/properties relationships of natural tissues including bone, dentin, enamel and other connective tissues and using this knowledge to design new biomaterials;

2) development of new biomaterials that can be used to replace skeletal or oral tissues lost because of age, trauma or disease;

3) defining the fundamental phenomena that control reactions occurring at the interface of biological tissues with synthetic and tissue-engineered materials.

The instrumentation in the Research Center provides structure/property imaging capabilities at the cellular and tissue levels. These resources offer investigators the opportunity to explore the new frontiers where imaging interfaces with bioengineering.

Spectroscopic imaging of biointerfaces

Work from the UMKC-CRISP laboratories has provided the first molecular structural analysis of acid-etched smear layers (Spencer et al. 2001; Wang and Spencer 2002). This work also represents the first study to quantitate dentin demineralization under conditions that permit hydration of the specimen throughout the analysis. Hydration is critical to these efforts, since it is widely accepted that the collagen within the demineralized dentin will collapse if it is allowed to dry; such collapse would lead to inaccurate characterization of the extent or degree of dentin demineralization. The micro-Raman spectral data indicate that 15 sec of acid-etching with 35% phosphoric acid gel demineralized dentin to a depth of ~ 10 micrometers. The spectral results presented in this study indicate that collagen within the smear layer is disorganized but not denatured. This disorganized collagen is denatured by the 15-second acid-treatment used in this study.

Microphysicochemical characterization of synthetic material/biological tissue interfaces;
Based on the results of both in vivo and in vitro studies, the hybrid layer does not form an impervious 3-dimensional collagen/polymer network throughout the breadth of the demineralized dentin. Instead, as recorded in a recent study from the UMKC-CRISP laboratories, the adhesive may undergo a physical "oil and water" separation as it interacts with the wet demineralized dentin matrix. Under these conditions, the critical dimethacrylate component (BisGMA), which contributes the most to the crosslinked polymeric adhesive, would infiltrate a fraction of the total demineralized, intertubular dentin layer (Spencer and Wang 2002). Dr. Spencer's laboratory is exploring the use of surfactants to decrease phase separation in dentin adhesives. The adjacent image is a reflected light micrograph of the same adhesive (62 wt% BisGMA/ 38 wt% HEMA) in the presence of 25 vol% water with/without surfactant. Without surfactant the adhesive separates such that distinct particles are obvious in the mixture.

Development of non-destructive techniques for analyzing material/tissue interfaces
Scanning acoustic microscopy (SAM) was used in the burst mode at 400 MHz, nominal lateral resolution 2.5 micrometers, to study the micromechanical properties of the dentin/adhesive interface. Corresponding specimens from the same tooth were investigated using micro-Raman spectroscopy (µRS), light microscopy, and scanning electron microscopy (Katz et al. 2001, 2002, 2003; Spencer et al. 2004). The elastic moduli of the components of the dentin/adhesive interface were determined by comparing the recorded acoustic impedance values to a calibration curve generated on standard materials.

The primary focus of this work was to analyze the micromechanical properties of the unprotected protein interface formed between a biocompatible adhesive and biological tissue. These results underline the unique capabilities of this instrumentation; an investigator can trace the micro-mechanical properties through an interface at a resolution comparable to that of optical microscopy. This type of data acquisition is not possible with conventional mechanical testing. In contrast to conventional mechanical testing, this technique provides direct in situ measurement of the micro-mechanical properties at the interface.

Specimens were available from optical and Raman spectroscopic studies (Spencer et al. 2000, 1999) that indicated an unprotected protein layer, ~3-5 micrometers, at the d/a interface. Based on the SAM analysis of these specimens the modulus of elasticity at the d/a interface ranged from 13 to <2 GPa. The 13 GPa was recorded from the zone of partially demineralized dentin. Images obtained from micro-Raman spectroscopy, scanning acoustic microscopy and optical microscope of the same small region of the dentin/adhesive interface are presented in Figure 1.

Structure, Property and Function of Tissues, Materials and Tissue/Material Interfaces
The research within the UMKC Center for Research on Interfacial Structure & Properties (UMKC-CRISP) is directed towards understanding the fundamental phenomena controlling biological interactions at interfaces and the development of non-destructive techniques for analyzing solid-liquid interfacial interactions in biological systems. For example, one area of investigation that is largely overlooked during the development of new biomaterials is chemical characterization of reactions occurring at the material/cell and/or tissue interface. This is a particularly challenging area of investigation since many of the current analytical techniques do not offer the required spatial resolution to study reactions occurring at the interface or the sample analysis conditions (i.e. temperature, vacuum, etc.) destroy, damage or alter the biological tissue. To address these problems, UMKC-CRISP investigators have developed a novel technique using confocal Raman microspectroscopy to characterize and quantify reactions at the biomaterial/tissue interface (Spencer et al. 2000, Spencer et al. 2001, Spencer and Wang 2002, Wang and Spencer 2002a, 2002b, Wang and Spencer 2003). Raman microspectroscopy is an exceptional tool for investigating the chemistry of biomaterial/cell and tissue interfaces because it does not rely on homogenization, extraction or dilution, but rather each structure is analyzed in situ. The analysis is completed under normal atmospheric conditions and the tissue is maintained in a “wet” environment throughout the process. By combining spectroscopy with microscopy, Raman microspectroscopy can be used to detect and quantify the molecular chemistry of microscopic samples. The technique can be used to study changes in the chemistry or molecular structure of the biomaterial at its interface with cells and/or tissue. It is the only nondestructive technique that allows the identification of chemical bonds at the micrometer level.

UMKC-CRISP investigators currently use Raman microspectroscopy in combination with nondestructive micro-mechanical measurement (scanning acoustic microscopy) to determine the interfacial chemistry and mechanics of synthetic materials with natural tissues, e.g. bone, dentin, enamel, and collagen. Micromechanical property measurements by scanning acoustic microscopy can be performed on the same fresh in vitro specimens as those analyzed by Raman microspectroscopy and environmental SEM. All three analytical approaches are mandatory for an integrated analysis of the chemical, mechanical, and morphologic relationships at the biomaterials/tissue interface. The basic objective of this integrated analysis is to relate the biomaterial mechanical properties to its structure (microstructure). An understanding of the fundamental relationship between structure and property provides the means for establishing a rationale approach for modifying the properties to match the intended function. The collaborative issues to be addressed include the desirable material properties for specific biological applications and how the mechanical response of the biomaterial can be matched to the appropriate living system.

Multi-Scale Computational Modeling of Hierarachical Biological Tissues

The function aspect of the structure/property/function paradigm requires the development of mathematical models to explain the behavior of hierarchical biological tissues and material/tissue interfacial behavior. Finite element models of the behavior at the mineral/matrix interface in biological tissues such as bone and dentin are generated using the exact geometrical and mechanical parameters determined from the characterization studies described above. A fundamental goal of these mathematical models is to establish the relationship between the ultra-structural morphology, and the multi-scale mechanics of collagen/biomineral interfaces with the view of bridging the behavior at nano- to micro-scales. 

In general, the material constitutive behavior critically depends upon the mechanisms that occur at scales smaller than the material-scale. Therefore, modeling methodologies that account for these underlying mechanisms are expected to provide better insight to the material stress-strain behavior. In the proposed approach, we consider that the material is composed of interacting grains (representing molecular bonds) whose centroids represent material points. Similar granular or discrete microstructure models have been considered in the past for developing constitutive relations, such as the virtual internal bond model developed by Gao and Klein (1998), the higher order constitutive relationships developed by Chang and co-workers (Chang et al. 2002), and the micromechanics models developed by Misra and colleagues (Misra and Chang 1993, Misra 1999, Misra et al. 2004, Thiagarajan and Misra 2004).

Since no single imaging modality or experimental technique provides a complete picture of the physico-chemico-mechanical characteristics, properties measured with diverse imaging techniques of different modalities and spatial resolution are simulated through FE computations. The computational results are expected to provide insight to the likely response of the different phases composing the domain. These results may then be utilized to verify the data interpretation of a particular imaging modality or experimental technique and to refine the imaging or experimental procedure. Since FE method is a continuum mechanics approach, FE models that implement scale bridging stress-strain relationships, which bridge the discrete molecular scale to the continuum material scale, are likely to capture the nano-scale phenomena. Thus, the proposed multi-scale modeling approach will provide the link between the molecular mechanisms and continuum mechanics models; models that are necessary to understand the mechanical behavior at these collagen/biomineral interfaces at a variety of scales and under a wide variety of loading conditions. The combination of experimental measurements with the modeling provides additional insight beyond what could be accomplished if either of the approaches were applied independently (Misra et al. 2004).