An important basic concept in this proposal is that dentin structural characteristics and properties are the key considerations for future development of dentin bonding systems. The most promising approach thus far is the formation of the resin-dentin interdiffusion zone (also known as resin hybrid layer or resin reinforced layer). The fundamental concept of the resin hybrid layer is that it provides bonding to dentin through establishment of intimate mechanical entanglement between partially demineralized dentin and dentin bonding agents that penetrate this structure and are polymerized in situ. The resin component of this newly formed composite structure can then be chemically bonded to other polymeric agents and composites. This concept has provided significant improvements in dentin bonding. Many key questions remain to be answered concerning dentin associated variables which impact the formation of the interdiffusion zone. In this project the impact of the following variables on structure and properties of this zone will be addressed: 1) nature of adhesive system; 2) dentin depth; 3) alterations of collagen fibril structure at each bonding step; 4) altered and clinically important forms of dentin; and 5) moist vs dry bonding. The substructure and properties of the resin-dentin interdiffusion zone of a modern dentin bonding system and an advanced single component bonding system will be compared as a function of intratooth location in aim 1 using a novel reverse sandwich method allowing study of superficial and deep dentin in the same sample. Methods will include SEM, AFM nano-hardness, and TEM. The hypothesis that the resin-dentin interdiffusion zones prepared in transparent dentin of coronal caries and sclerotic dentin of cervical lesions exhibit altered substructure and properties as compared to normal dentin will be tested (aim 2) using similar methods. Alterations in the collagen fibril network at each of the steps associated with formation of the resin-dentin interdiffusion zone will be determined using high resolution AFM (aim 3). The structure and properties of the interdiffusion zones formed when bonding to wet and dry dentin will be determined using AFM nano-hardness, TEN and SEM methods (aim 4). Micro- punch shear strength (aim 5) will be used as a method to detect differences in the strength of altered forms of dentin and interdiffusion zones. These comparisons will evaluate the different adhesive systems, using moist and dry bonding methods, and the role of superficial normal dentin, deep normal dentin, the transparent caries layer and sclerotic cervical dentin.