WO2015188125A1 - Method for assessing bioactivity - Google Patents

Abstract

A test method to assess in vitro or in vivo the bioactivity of a material such as dental or medical biomaterial. The method includes forming an assembly (10) having at least one first material (20) and at least one second material (30), wherein a gap space (50) is defined between the least one first material (20) and the at least one second material (30), recording the dimensions of the gap space (50), incubating the assembly (10) in a body fluid or simulated body fluid for a time period, recording the dimensions of the gap space (50) following incubation, and comparing the change in gap space dimensions. This test method uses an approach that is clinically relevant and meaningful in assessing a material's ability to display bioactivity via the formation of some form of bioapatite. Variations of this test method may also be used to assess the ability of a material to display properties of self-reparability or to assess the ability of a biological tissue or substrate to display bioactivity under certain conditions or treatments to that tissue or substrate.

Description

METHOD FOR ASSESSING BIOACTIVITY

A significant number of dental restorations or "fillings" fail due to factors related to marginal interfacial gaps between the restorative material and the tooth, such as i) recurrent caries, ii) marginal discoloration, iii) marginal edge fracture, iv) defective marginal adaptation due to technique inadequacies or polymerization shrinkage, and v) microleakage leading to pulpal sensitivity or inflammation. What is needed is a test method to determine if a particular material is able to provide marginal stability for repair or reconstructive areas, such as the tooth/restorative material interface, among other dental and medical interventions.

A new class of cement-containing dental materials has emerged in the dental field. This group of cement-containing materials contains comparably high levels of calcium, displays a pH in the alkaline range, and are bioactive.

The ability of a restorative material to seal and reseal its marginal interface with a tooth structure would be a significant advancement in dental restoratives and may lead to a new class of dental restorative materials with greater safety and efficacy against secondary caries and prevent bacteria from accessing dentinal tubules and the pulp itself.

The subject test method is able to discriminate bioactive from non-bioactive materials based on new findings regarding the behavior of these materials. The subject test method provides a straightforward and clearly demonstrable approach to understanding the degree of bioactivity of a material. It has been found that bioactive materials appear to have the ability to close or reseal marginal gaps of various dimensions, a property that non-bioactive materials do not apparently possess. As a result, this discovery permits a new and highly efficient method to differentiate bioactive from non-bioactive materials, and to permit a more straightforward and clinically relevant approach to the assessment of bioactivity in a currently available or newly formulated material.

It has been discovered over the course of the last two decades that certain calcium-containing materials have the ability to form hydroxyapatite or some form of calcium phosphate mineral, when immersed in a simulated body fluid such as a physiologic phosphate buffered saline solution. However, current methods utilized to demonstrate whether a material is bioactive involve elaborate and sophisticated equipment employed for surface analysis to detect the presence of some form of apatite formation.

Provided is a test method and method of analysis which permits a more rapid, definitive, clinically relevant and easier determination of whether a material, such as biomaterial, is bioactive. The terms "bioactive" or "bioactivity" refer to the property of a material or substance to form hydroxyapatite or some form of calcium phosphate mineral, when incubated with a physiologic phosphate buffered saline solution. The term "biomaterial" refers to any matter, surface or construct of natural or artificial origin that interacts with biological systems. The nature of the interaction is that a bioactive material demonstrates, in the presence of simulated body fluids that contain physiologic levels of inorganic phosphate, the ability to form surface deposits of apatite-like mineral.

In one embodiment, the subject test method includes creating an "assembly", which may be a fixed assembly, involving a first material and a second material resulting in the formation of a space or gap of pre-selected dimensions between the first and second materials, immersing the assembly in a suitable environment, and then observing and measuring the formation of mineral deposits within the gap by various means over time. The presence and degree of mineral deposits in the space or gap over time, as measured by a variety of analytical methods, indicates the degree of bioactivity of the test material. Brief Description of the Drawings

FIG. 1 is a diagram of an illustrative testing assembly to assess and measure the bioactivity of a sample test material.

FIG. 2 is an illustration of an artificial marginal gap model utilizing a dentin substrate adjacent to the artificial gap.

FIGS. 3A and 3B are microphotographs illustrating a basic test assembly measuring bioactivity of a material lacking bioactivity.

FIGS. 4A and 4B are microphotographs illustrating a basic test assembly measuring bioactivity of a material with a high level of bioactivity.

FIG. 5 is an illustration of a modified test assembly to measure bioactivity using an annular geometry to simulate an actual filling material marginal behavior and potential marginal defects. FIG. 6 is an illustration of a modified test assembly to measure bioactivity using a natural extracted tooth to simulate an actual filling material marginal behavior and potential marginal defects.

FIG. 7 are microscopic photos of artificial gap changes over time during incubation in phosphate buffered saline of five dental cement test materials and dentin tooth substrates in the test assembly of FIG. 2

FIG. 8 are microscopic photos illustrating progressive closure of artificial marginal gaps created between a bioactive cement (calcium aluminate/glass ionomer (D)) and a dentin tooth substrate.

FIG. 9 is a graph illustrating marginal gap closure data for baseline artificial gaps created between calcium aluminate/glass ionomer (D) cement and dentin ranging from 50 to 100 μιη (top four plots), up to 300 μιη (bottom single plot) for up to 35 days in simulated body fluid (phosphate buffered saline).

FIG. 10 are micro-CT transverse images through artificial marginal gaps demonstrating the presence of radiodense deposits (after 4 months of incubation in phosphate buffered saline (PBS)) within marginal gap area of bioactive cements (top four images) and absence of deposits for nonbioactive cements (lower three images).

FIG. 11 are digital photomicrographs of artificial margin gaps at 8 months of incubation in phosphate buffered saline (PBS).

Described herein is a test method and method of analysis which provides a more rapid, definitive, clinically relevant and easier determination of whether a material is bioactive compared to prior art methods described herein. The clinical relevance of this method of analysis is the ability to: 1) provide an efficient method to differentiate bioactive and non-bioactive materials; 2) develop and establish a test method for bioactivity that relates directly to improved efficacy for a dental or medical biomaterial; 3) determine materials which are bioactive, determine their relative "degree" of bioactivity; and 4) provide an efficient test method to establish the presence and degree of bioactivity in newly formulated biomaterials intended to possess and utilize this property of bioactivity for improved clinical performance. Dental or medical materials that can reseal marginal defects or gaps could potentially reduce recurrent decay around existing restorations (fillings) or permit more stable and durable orthopedic implants and appliances which are implanted in bone. The subject test method may be utilized to assess the bioactivity of dental restorative material or cement, or any biomaterial used in medicine, podiatry, or dentistry. In one embodiment, the test may relate to bioactivity in relationship to the hard tissue elements of teeth. In other embodiments, the test may relate to a cement or material of any type used in other hard tissue applications, such as bone reconstruction and rehabilitation.

The subject test method and assembly described herein permits the identification of bioactive materials in a straightforward and relatively non-complex fashion. This method has serendipitously demonstrated the ability to differentiate a bioactive material from a material lacking bioactivity.

As used herein, the term "bioactivity" refers to the ability of restorative materials or cements, or any material of interest, to produce an external layer of a calcium phosphate or hydroxyapatite material on the surface of the test cement or material, when the test cement or material in question is immersed or incubated in a solution containing physiologic concentrations of inorganic phosphate.

Recently, several cement-like materials which contain calcium as an elemental component have been demonstrated a bioactive response. Current methods of analysis have focused strictly on the use of light-based or electron microscopy to determine calcium phosphate or hydroxyapatite material formation on the surface of the set cement. Other current methods require sophisticated chemical or X-ray methods to analyze the surface of a material. The actual significance of this "bioactivity" phenomenon from a clinical perspective in either dentistry or medicine has been lacking. Little is understood concerning the potential clinical significance of this bioactivity behavior, especially in the area of restorative dentistry.

The discovery that certain types of bioactive materials demonstrate closure of artificially created gaps, yet materials lacking bioactivity do not, has led to this straightforward, efficient and economical analytical test method to determine whether a given material or composition is truly bioactive, and whether this bioactivity is at a level of clinical significance.

In one embodiment, the subject test method utilizes the orientation and positioning of at least one first material placed in "close approximation", that is, at a pre-determined distance (discussed below) from at least one second material. The first and second materials may comprise substantially similar or different materials. The at least one first material may be in a parallel alignment with the at least one second material. In another embodiment, the at least one first material may be in a contiguous alignment with the at least one second material. The at least one first material and/or the at least one second material may be substantially flat, semi-flat, concave, convex, or circular or semi-circular. The at least one first material and/or the at least one second material may comprise segments having different degrees of flatness.

The alignment of the opposing surfaces of the materials should be such that the space created between them should be reasonably uniform in dimensions (thickness or width of the space bounded by the first and second material). As such, the most straightforward configuration of the first and second materials are relatively flat, parallel surfaces, such that the width of the space between is relatively uniform. However, the test method is viable when the gap width or thickness deviates as much as 50% or more over the length of the gap being monitored. As a result, the gap created may not have parallel boundaries above and below the gap (although this is the optimal configuration), but such a situation may still permit assessment of the gap-closing property of bioactivity.

In one embodiment, the at least one first material and/or the at least one second material may comprise a biologic material such as biological tissue. The first material (i.e., the biomaterial/test material) could be a formulated biomaterial being tested for its bioactivity. Examples of this substrate include but are not limited to dental restorative materials, dental cements, dental adhesives, dental cements, orthopedic materials, bone graft substitute materials, bone cements, bone graft materials, and devices for the controlled delivery of therapeutic substances. Examples of biologic material (i.e., the substrate), without limitation, may include a flat or semi-flat segment of tooth, a flat or semi-flat segment of bone, a flat or semi-flat segment of connective tissue such as a ligament, tendon, or cartilage.

Although the "test" material and "substrate" have been thus far differentiated by way of illustration, and the subject method and assembly are intended to assess the bioactive qualities of the test material; it should be noted that both materials' interfaces are elements of the analysis and should be considered as a distinct test assembly. In certain instances, both materials may be considered "test" materials.

In another embodiment, the at least one first material or the at least one second material may comprise a non-biologic (i.e., not a natural tissue) such as a synthetic or semi-synthetic material for which one may wish to assess possible interactions between the at least one first material and/or the at least one second material. In this particular application, crack healing behavior or possible adhesive interactions between biologic and non-biologic materials may be analyzed by the subject method.

In yet another embodiment, the at least one first material and/or the at least one second material may comprise biologic and/or non-biologic segments. For example, without limitation, the at least one first material and/or the at least one second material may comprise a biologic segment and a non-biologic segment, or the at least one first material and/or the at least one second material may comprise more than one segment of different biologic materials, different non-biologic materials, or combinations thereof.

One element of the subject test method is the maintenance of a defined gap or space between the at least one first material and the at least one second material, such as a test material and a reference substrate. The dimensions of this gap or space can vary considerably, ranging from gaps on the order of millimeters to the order of nanometers. The gap space dimensions may range from about 5 to about 10,000 microns (μηι), i.e. 10 millimeters; in certain embodiments from about 20 to about 1000 microns (μιη); and in particular embodiments from about 20 to about 800 microns (μιη). Alternatively, the gap space may be so small that the at least one first material makes physical contact with the at least one second material resulting in a contiguous alignment, or represents a "virtual" contact between the materials.

In one embodiment, the subject test method assesses bioactivity of a material comprising: forming an assembly comprising at least one first material and at least one second material, wherein a gap space is defined between the least one first material and the at least one second material, recording the dimensions of the gap space, incubating the assembly in a body fluid or simulated body fluid for a time period, recording the dimensions of the gap space following incubation, and comparing the change in gap space dimensions.

In one embodiment, the least one first material and the at least one second material are substantially similar. In another embodiment, the least one first material and the at least one second material are substantially different.

In one embodiment, the at least one first material is at least partially a biologic material and the at least one second material is at least partially a non-biologic material. In another embodiment, the at least one first material is at least partially a non-biologic material and the at least one second material is at least partially a biologic material. In one embodiment, the at least one first material and/or the at least one second material is substantially flat, semi-flat, concave, convex, or circular.

In one embodiment, the at least one first material and/or the at least one second material comprise i) a segment of biologic material and a segment of non-biologic material, ii) a segment of biologic material and a segment of a substantially different biologic material, iii) a segment of non-biologic material and a segment of a substantially different non-biologic material, or iv) combinations thereof.

In one embodiment, the at least one first material and/or the at least one second material comprise at least one of a dental restorative material, a polymeric cement, an inorganic cement, tooth enamel, tooth dentin, tooth cementum, bone cartilage, or soft tissue elements.

In one embodiment, the least one first material and the at least one second material are positioned in substantially parallel planes. In another embodiment, the least one first material and the at least one second material are positioned in a substantially contiguous alignment.

In one embodiment, the gap space is formed by at least one spacer element. In some embodiments, the spacer element includes any synthetic or natural polymer which can form a flexible, semi-rigid or rigid film, sheet or spacer. In some embodiments, the spacer elements is a polyester film made from polyethylene terephthalate such as biaxially-oriented polyethylene terephthalate (BoPET) (trademark MYLAR®). In some embodiments, the spacing element comprises at least one of: acetyl sheet polymers, polymers comprising at least one of polyethylene, polypropylene, polyester, polymethylmethacrylate, polycarbonate, or elastomers such as silicone or rubber elastomers.

In one embodiment, the at least one adhesive is applied to or proximate at least one end of the at least one first material and the at least one second material. In certain embodiments, the at least one adhesive comprises a light-cure adhesive or dental cement. In other embodiments, the at least one adhesive comprises a light-curable or dual-curable (by light and chemical) adhesive and/or a light-curable or dual-curable (by light and chemical) dental cement.

In one embodiment, a material is applied over the adhesive to form at least one pillar to maintain the pre-determined gap space. In certain embodiments, the at least one pillar comprises a composite resin. In some embodiments, the at least one pillar comprises a polymer, a polymeric material, a composite resin, a cement or a polymer cement, that is applied and subsequently cured into a rigid form.

In one embodiment, the simulated body fluid is a phosphate-containing medium. In some embodiments, the phosphate-containing medium is a sterile phosphate-buffered saline solution.

In one embodiment, the gap space dimensions are recorded by micro computer tomography (Micro CT), scanning electron microscopy (SEM) or transmission electron microscopy (TEM).

In one embodiment, after incubating the assembly in the body fluid or simulated body fluid for the specified time period, the at least one first material is separated from the at least one second material to expose at least one interfacial surface.

In one embodiment, the exposed interfacial surface of the at least one first material and/or the at least one second material is analyzed by scanning or transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray spectroscopy, infra-red spectroscopy, X-ray photoelectron spectroscopy or atomic force microscopy.

In one embodiment, the assembly comprises a fixture having at least one side member connecting opposing platforms, to each of which platforms one of the at least one first material or the at least one second material are affixed, defining the gap space there between. In certain embodiments, the fixture comprises plastic, metal, ceramic, or combinations thereof.

As is noted above, the subject test method and method of analysis facilitates a more rapid, definitive, clinically relevant and easier determination of whether a biomaterial is bioactive, as compared to prior art methods. The subject test method also permits materials to be differentiated in terms of their bioactivity based on the degree of new mineral deposits observed to be deposited on the material interfaces, that is, on the surfaces opposite one another.

In one embodiment, an intended use of the subject test method is to discriminate between bioactive materials and non-bioactive materials based on their ability to form hydroxyapatite or some form of calcium phosphate mineral, when incubated in a physiologic phosphate buffered saline medium. Furthermore, the findings resulting from the subject test method provides far more clinically relevant information regarding the behavior of materials of interest for use as potential dental and medical biomaterials.

The orientation and stabilization of the at least one first material and/or the at least one second material according to the subject method permits the virtual elimination of material creep that may alter the gap dimension, resulting in more accurate and consistent measurements of the gap dimensions pre- and post-incubation.

The subject test method emphasizes any changes in the gap dimensions or morphology due to deposition of new material in the gap interface area. The subject test method provides a wide range of potential information and data not previously explored, both of a qualitative and quantitative nature. The dimensions and changes in dimensions of the marginal gap can be assessed by digital light microscopy via calibrated measurement software. The qualitative changes in the gap micro-morphology can also be assessed by light microscopy, but also by other methods such as scanning and transmission electron microscopy. An additional quantitative or semi-quantitative measure in this analysis is the kinetics (time-dependent change) of gap closure. Measurement of the initial rate of gap closure and curve-fitting methods to derive "best-fit" kinetic equations can aid in differentiating the type and intensity of bioactivity by various biomaterials and tissue -base substrates.

The subject test method is also highly versatile, permitting analysis of microscopy by reflected or transmitted light, surface analysis by scanning or transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray spectroscopy (EDXS), infra-red spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy, and non-contact (laser-based) or contact-based profilometry.

Using these techniques permits analysis of changes in the gap dimensions and/or changes in morphology due to the formation of new mineral deposits, such as some form of calcium phosphate mineral. Qualitative, semi-quantitative, and quantitative changes in gap dimensions can be assessed using digital microscopy techniques. Qualitative micromorphology of the material occluding the marginal gap area can be determined with high resolution light microscopy, polarized light microscopy, atomic force microscopy (AFM), non-contact (laser-based) or contact-based profilometry, and scanning electron microscopy (SEM). Using these techniques, one can observe and even categorize the "relative" fill of the gap space using a predetermined scoring rubric. Alternatively, using calibrated digital measurement software, the change in gap dimensions can be measured in units (such as microns) and recorded. Scanning electron microscopy with energy- dispersive X-ray spectroscopy (SEM-EDXS), infra-red spectroscopy, and X-ray photoelectron spectroscopy (XPS) comprise some of the analytical techniques to assess the chemical composition of the deposits found in the marginal gap areas. Micro computerized tomography (Micro-CT) can also provide qualitative and quantitative data concerning the changes in gap dimensions and fill, as well as providing three-dimensional analysis to analyze the internal morphology of the deposition of radio-dense mineral within the depths of the artificial gap. Other test methods such as various gas or liquid-based diffusion test methods to determine the nature of microleakage or the diffusion of molecules or microorganisms through the gap closure may be employed.

The subject test method will now be demonstrated by the following non- limiting illustrative examples of its use and application. EXAMPLE 1

The configuration used in the subject test method includes the positioning and fixation of a segment of at least one first material positioned in a substantially parallel plane to the at least one second material. While it is optimal to achieve a uniform (constant) gap dimension between the test material and substrate, deviations in gap dimensions of even 40 to 60% over the length of the test assembly still provide adequate artificial gaps which can differentiate the presence or absence of bioactivity in a test material. Thus deviations of approximately 10-40° (degrees) from parallel are acceptable, if not optimal.

The subject test method assembly involves an initial step of preparing the at least one first material, such as a test material. In one embodiment, the at least one first material is formed into a disc of various dimensions, such as from less than 1 mm in thickness to several millimeters in thickness, with a radius varying from approximately 1 mm to several centimeters. In various embodiments the dimensions of the test material can vary with respect to geometry and dimensions. Various shapes, including but not limited to, circular discs, cylinders, blocks, square or rectangular plates, and the like, may be utilized both as test materials or substrates for this test method. The thickness of these test or specimen components can vary from a thin layer of 0.1 mm thickness to as large as 10 to 100 mm in cross-section thickness in the same plane as the artificial marginal gap. The length and width dimensions of the specimen in the plane perpendicular to the plane facing the artificial marginal gap can vary considerably depending on the ability to orient the specimen in a configuration to create a stable and uniform assembly, thereby creating a stable and relatively uniform gap space.

The length and width dimensions of the test material and underlying substrate or additional test material or article can be from approximately 2 to 4 mm in width and length and/or diameter, to several centimeters (such as 1 to 5 cm). In certain embodiments, the thickness of the "test" specimen is about 0.5 to about 30 mm and the rectangular length and width dimensions from about 4 to about 60 mm in width and about 4 to about 60 mm in length. Any dimensions are appropriate assuming that the elements of the assembly can be oriented and stabilized to create stable and relatively uniform artificial marginal gaps. If the test method is being applied in- vivo or in-situ, the dimensions of the test configuration described here can be on the order of the dimensions of natural teeth or bones, which can also approach in some cases tens of centimeters in dimensions of width and length, and gap widths on the order of several thousand microns (several millimeters) in thickness.

The at least one first material may be contoured on one side of its radius to create a flat edge. The length of this flat edge can vary from less than 1 mm to several millimeters, such as ten or twenty millimeters, or even centimeters or tens of centimeters. The flat edge side may form a rectangular plane. The dimensions of this rectangular plane area can vary as necessary for the subsequent analyses of the area of interest in this test method.

The opposing at least one second material may be a biologic material such as a slice of segment of human dentin sectioned from an extracted tooth. The at least one second material may have at least one of its edge surfaces contoured on one side of its radius to create a flat edge. The length of this flat edge, which contains the length of gap "window", can vary from less than 1 mm to several millimeters or centimeters. A practical range of the length of flat edge for assemblies created for in-vitro analysis may range approximately from about 2 mm to about 15 mm, thus creating a size of the assembly that would be of a reasonable size for storage and incubation in the simulated body fluid (SBF) and positioning in various analytical devices. The flat edge side may form a rectangular planar area which may be positioned in alignment with the flat planed surface of the adjacent at least one first (test) material, such that both flat edges sit on top of the other in approximately the same vertical plane. Any disparities in the planar alignment of the flat edges can be adjusted by holding the materials fixed and gently grinding (for example using a table top horizon grinding device with silicon carbide discs) the flat edges to be approximately flush in a similar plane which is approximately perpendicular (forming an approximate 90 degree angle) to the top and bottom surface of the test material and underlying substrate.

A spacing element such as a Mylar spacer (or a spacer of similar material) is then positioned between the at least one first material and the opposing at least one second material (in this example a slice of human dentin) to create a gap space of a specific dimensional range. This gap space creates a window between the upper and lower segments of first and second materials. The spacer element may include any synthetic or natural polymer which can form a flexible, semi-rigid, or rigid film, sheet or spacer. Non-limiting examples of commercially available synthetic polymers in addition to Mylar include acetyl sheet polymers such as DELRIN®, polymers comprising at least one of polyethylene, polypropylene, polyester, polymethylmethacrylate, polycarbonate, or elastomers such as silicone or rubber elastomers.

With the spacer element in place and the segments of material stabilized, an adhesive may be added to the ends of each segment of material and allowed to cure. In this illustrative example, a dental, light-cure adhesive was applied to the end of the segments of material and light cured. Care was taken to prevent flow of the uncured adhesive into the open space being maintained by the polyester film spacer between the segments of material.

After curing the adhesive, a composite resin was applied over the adhesive layer to provide a structural support, in order to maintain the open gap space, or window, between the segments of material. The composite resin may be contoured to a shape and thickness to form sufficient strength to maintain the segments of material in a fixed position. Composite and/or composite resin materials utilized for dental and medical purposes are composed of the basic elements of a filler material, such as silica/inorganic glass or ceramic particles/prepolymerized polymer particles; and a polymerizable or formable matrix material in which the filler is uniformly dispersed. An illustrative example of such basic elements include a resin-based oligomer matrix such as a Bisphenol A-glycidyl methacrylate (BISGMA) or urethane dimethacrylate (UDMA). Unfilled polymeric materials, such as polymethyl and polyethyl methacrylate-based materials may also be utilized.

The resin composite may approximate the form of two pillars covering the external walls of segments of both materials. Some composite may be positioned in the gap space at the external aspects of the segments, leaving the central portion of the gap space, or window open. The result of this construction is an assembly comprising a complex of at least one first (test) material, a gap space or window of varying dimensions, and at least one second (reference substrate) material on the opposite side of the gap space; all in a stable assembly. If the opposing surfaces of the test and adjacent substrate materials are not substantially parallel, the width of the gap will vary in one test assembly (as described above) enough to be measurable. Dimensions can also vary between test assemblies. However, by utilizing a spacer of a given thickness, the variation between test assemblies in a given experiment can be kept to a minimum. Nevertheless, there may still be some variability between different test assemblies in a similar group.

A basic depiction of the test assembly 10 as described in this example is illustrated in FIG. 1, wherein area 20 is a disc of the at least one first material 20 being measured for its potential bioactivity. Area 30 is a segment of the at least one second material 30 with a flat rectangular exposed area immediately below the white or gap space area 50. The assembly of FIG. 2 utilizes a dentin substrate 35 as the at least one second material 30. The vertical bars 40 represent adhesive and resin composite pillars 40 which support and stabilize the assembly to maintain the approximate dimensions of the gap space area.

Using a light microscope interfaced to a computer using digital measurement software, digital images of the gap space 50 between the at least one first material 20 and the at least one second material 30 are recorded to examine the nature of the gap space area 50 and the dimensions of the gap space 50 itself.

Qualitative, semi-quantitative and quantitative measurements of the gap space area 50 and the immediate edge surfaces of the first material (e.g., the test material) and the second material (reference substrate material, e.g., dentin) segments may be taken at baseline or the beginning (time zero) of the experiment, using the techniques discussed above.

Baseline measurements may include digital photographs and the digital measurement of the gap space area between the segments in microns using digital measurement software. As described herein, additional analytical measurements of the test assembly and gap space area may be employed.

After obtaining baseline measurements, the assembly may be immersed in a calcification-initiating solution of sterile phosphate-buffered saline (PBS) for varying periods of time. Timeframes for observation beyond the initial or baseline observation may generally range from less than 1 day (hours, less than 24 hours) to several days, up to several weeks or months. In some instances where the presence of gap closure is evident but very slow (such as large marginal gaps), the time period for observation could be one year or longer. The assembly in this solution may be maintained at about 37°C, or at other desired temperatures and test conditions. Test temperatures may vary, however, from just above freezing (0°C), for example 3-7°C; to temperatures in excess of 40-50°C to observe the effect of elevated temperature on the kinetics of gap closure.

The assembly is periodically removed from the sterile phosphate-buffered saline solution, excess fluid is removed from the assembly, and the assembly is placed under the digital light microscope to examine the gap space interface and the related magnified edges of the at least one first (test) material and/or the adjacent at least one second (reference substrate) material (dentin, in this example). Any residual moisture in the gap space area which could obscure visualization of the gap space is allowed to dissipate prior to examining the gap space area, measuring the open gap space area present, and taking any digital photographs.

Examples of suitable incubation solutions include Phosphate Buffered Saline (PBS) or Hanks Balanced Salt Solution (HBSS). Dulbecco's Phosphate Buffered Saline was used in the examples listed below. The composition for this Dulbecco's PBS is: Inorganic salts grams/liter

CaCl₂ ' 2H₂0 0.133

MgCl₂ ' 6H₂0 0.1

KC1 0.2

KH₂PO 0.2

NaCl 8.0

Na₂HP0₄ (anhydrous) 1.15

Other simulated body fluids (SBF) that could be utilized are various balanced, pH adjusted salt solutions such as Hanks Balanced Salt Solution (HBSS), a custom prepared solution as described in Kokubo, et al, "Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W", J. Biomed. Mater. Res., 24, 721-734 (1990) and Kokubo, et al., "Ca,P-rich layer formed on high-strength bioactive glass-ceramic A-W", J. Biomed. Mater. Res., 24, 331-343 (1990), or commercially available artificial saliva. Actual body fluids such as simulated and non-simulated human or animal saliva may be utilized.

The gap space area 50 can also be compared to an established scoring rubric to give a numerical score concerning gap space change or closure. Evidence of bioactivity is demonstrated by the presence of mineral deposits on one or more edges or interfacial surfaces of either or both the at least one first (test) material 20 and the at least one second (reference or substrate) material 30.

High bioactivity materials may demonstrate not only clearly visible evidence of new mineral deposit formation on the test material 25 and adjacent slice of dentin 35, but also actual filling and even occlusion of the gap space 50 itself.

Using the subject method, materials which had previously been demonstrated not to be bioactive, and therefore were not classified as bioactive; did not demonstrate deposits or even any evidence of gap occlusion.

The microphotographs in Fig. 3 illustrate the results generated by the subject test assembly 10 with a material lacking bioactivity in terms of changes in gap morphology. The microphotograph 3 A on the left-hand side 60 shows a test material 25, a dentin substrate 35, and an open gap space 50 between the test material 25 and the dentin substrate 35 at a baseline reading The microphotograph 3B on the right-hand side 70 shows a test material 25, a dentin substrate 35, and an open gap space 50 between the test material 25 and the dentin substrate 35 after 22 days immersed in sterile phosphate buffered saline. As evident from the post-incubation microphotograph 70, there is a complete absence of any formation of mineral deposits in the gap space 50 between the opposed materials 25, 35.

The microphotographs at baseline and a later time period in Fig. 4 illustrate the results generated by the subject test assembly and method with a bioactive material in terms of changes in gap morphology. The microphotograph 4A on the left-hand side 80 shows a test material 25, a dentin substrate 35, and an open gap space 50 between the test material 25 and the dentin substrate 35 at the baseline reading. The microphotograph 4B on the right-hand side 90 shows the test material 25, the dentin substrate 35, and the gap space area 50 between the test material 25 and the dentin substrate 35 after 23 days immersed in sterile phosphate buffered saline. As evident from the post-incubation microphotograph 90, the gap space area 50 between the materials was completely occluded by the formation of mineral deposits 55.

The degree of bioactivity appears to relate to how quickly deposits and gap occlusion occur, but also the degree and depth of mineral deposits and gap occlusion. The subject test method provides a straightforward, cost and time-effective method for a clear differentiation between bioactive and non-bioactive materials. The subject test method also provides a basis to determine the degree of bioactivity of a particular material of interest, that is, a basis to differentiate strong bioactive materials from weak bioactive materials. A high bioactive material ultimately, over the time-course of the test method, completely or virtually completely fills the entire aspect of the gap window. The subject test method therefore provides a new, clinically relevant approach to establishing whether a material is bioactive and the degree to which that bioactivity is manifested.

EXAMPLE 2

The test method utilized in Example 1 may be modified in a number of ways which are not limited to the following illustrative examples.

The dimensions and thickness of the at least one first material and/or the adjacent, opposing at least one second material may be varied over a wide range of dimensions.

The dimensions and locations of the open window areas entering into the gap space areas may be varied. The gap space on one side of the assembly of the at least one first material and the at least one second material, i.e., the test specimen/substrate assembly, may be occluded with a variety of materials. One could use, as examples (but not being limited to), the following materials to occlude one of the two open gap areas or windows: an adhesive, a dental material of various chemistries that could be placed and cured, or allowed to cure, a preformed material such as a polyester film spacer that may or may not be stabilized with an adhesive. One may wish to occlude one of the gap spaces or "windows" so as to see the effect of altering, reducing, or limiting the actual diffusion of the fluid medium (i.e., simulated body fluid) through the open gap space and volume. This approach, in some instances, may more accurately simulate the actual clinical or in- situ situation. Alternatively, occluding "pillars" or "struts" can be placed within the length of the gap space window, especially if additional strength or stability of the assembly or maintenance of the gap space dimensions is desired.

The type and arrangement of the materials in the test assembly can be varied over a wide range of options. Numerous types and configurations of test materials and substrates may be utilized in the subject test method, for determining whether the material displays bioactivity in terms of the production of insoluble calcium phosphate and/or hydroxyapatite at the interfaces of the assembly materials and within the gap space.

The at least one first material and/or the at least one second material of the assembly utilized in the subject test method may be substantially similar or different. For example, without limitation, a wide variety of dental materials, including polymer and inorganic cement compositions may be fabricated and utilized in (examined with) the subject test method. Alternatively, biomaterials used for medical applications may also be utilized in or examined with the subject test method.

Also, the configurations of the at least one first material and/or the at least one second material may be substantially parallel, staggered, or contiguous with each other. The at least one first (test) material may be positioned against a tissue-based material, for example tooth enamel, tooth dentin, tooth cementum, bone, cartilage, or even various soft tissue elements (as the reference substrate material). In terms of the substrate materials placed in a parallel configuration opposite the gap space adjacent to the test material, a wide range of options are available.

The gap space may be developed using a polyester film spacer or a spacer elemetn of other material, or the at least one first (test) material may be positioned directly on the surface of the at least one second material such as a tissue substrate, resulting in a gap of extremely small dimensions or no gap at all. The presence of no gap at all is especially useful if the "test" material and the adjacent "substrate" were actually the same material or different materials. This approach would be a simulated situation to evaluate the ability of the material or materials to undergo "self-repair" of internal defects in the material itself, by depositing apatite-like material in such a defect, and/or even promoting an adhesive bond of some type between the contiguous materials.

Alternatively, at least one first material may be positioned against at least one second material comprising a biopolymer derived from a native biological tissue. For example, at least one first material may be positioned against at least one second material comprising a membrane or sponge-like configuration of Type 1 collagen, a segment of deproteinized bone, or some other extracted material or modified native tissue material.

Alternatively, at least one first material and/or at least one second material (such as a biological substrate) may be positioned against a carrier containing compounds, biopolymers or another substrate material, or a polymer impregnated with a material or materials of interest.

In another approach, specimens of an at least one first material and an at least one second material of similar or identical composition may be positioned against one another. This approach not only assesses bioactivity but also can indicate whether the materials demonstrate any degree of self-adhesive or self-repair properties.

Thus it can be seen that a wide variety of materials can be used in the subject test method as a substrate opposing the test material.

Once the assembly is constructed and baseline measurements have been taken, the assembly may be immersed and/or incubated in a wide variety of solutions and/or environments. For example, in Example 1, the incubation solution used was phosphate-buffered saline solution. This solution is the basic liquid composition used to establish the formation of surface apatite on bioactive materials, which has been previously documented in scientific literature. There can be a wide range of modifications to the phosphate-buffered saline solution composition. For example, additional salts, chemical compounds, substrates, and/or other substances, may be added to these solutions.

For purposes of illustration but not limitation, additives may include salts such as zinc salts, fluoride and fluoride salts, nano-hydroxyapatite, and amorphous calcium phosphate (ACP); polyelectrolytes such as polyacids, phosphate and polyphosphate compounds; bioactive glasses, such as in a powdered form; and various peptides, oligopeptides, and phosphopeptide. These additives may act as mineralization adjuvants. See Cao et al, Int. J. Mol. Sci. 2015, 16, pp. 4615-4627; van Loveren C (ed), "Toothpastes", Monogr. Oral Sci. Basel, Karger, 2013, 23, pp. 15-26; and International Dentistry SA, vol. 11, No. 6, Dec. 2009, pp. 6-16.

The surfaces of the interfacial area, and in some embodiments, the gap space area of at least one first material and/or at least one second material may be modified by energy, chemical or other means. For example, the slice or segment of dentin may be treated with an acid to reduce the surface mineral component immediately adjacent and opposite of the gap space in the test assembly.

EXAMPLE 3

In one embodiment, a basic design feature used in the subject test method is the positioning and fixation of a flat slice, slab, or segment of a biomaterial positioned in a relatively parallel plane in the test assembly configuration.

While FIGS. 1-4 show the least one first material 20, 25 and/or the at least one second material 30, 35 configured in a substantially parallel plane with respect to each other, in other embodiments, the assembly and gap space area 50 may assume different types, sizes and/or configurations. For example, a concentric circular or semi circular configuration of the at least one first material (such as a test biomaterial) and/or the at least one second material (such as a natural tissue specimen), or vice versa, with a concentric gap or space between the materials may also be used in this test method.

Alternatively, a circular disc may be removed from the at least one first material 20. The circular disc of the at least one first material is then inserted or fitted within a circular space of the at least one second material 30, wherein a uniform or non-uniform gap space 50 is created between the inner circumference of the at least one second material and the outer circumference of the at least one first material.

FIG. 5 illustrates such a modified test assembly using an annular geometry to simulate an actual dental filling material marginal behavior and potential marginal defects. The inner or outer annular material can be an actual biological hard tissue, such as bone or dentin.

In a further modification, a segment of the at least one second material

(e.g., dentin) 35 may have a circular opening 100 created through the dentin slice 35. A spacer element (not shown) may be applied or adapted to a portion of the circumferential aspect of the opening. An uncured material 25 such as unset dental cement can be injected into the circular disc opening 100 in the dentin 35, and allowed to set. When the spacer element is removed, a semi-circular gap space 50 exists on the outer circumference of the previously open disc space. This circular gap space 50 then becomes the gap space area or window of interest for examination of bioactivity of the at least one first material (e.g., dental cement filling) in the rest of the internal portion of the circular area in the dentin slice 35. As an illustrative example of this approach, a segment or disc of a biological substrate, for example human dentin, can have a circular opening created through the dentin slice. A short flexible spacer element or some other plastic material may be applied to a portion of the circumferential aspect of the opening. An uncured material, for example an unset dental cement, can be injected into the circular disc opening in the dentin, and allowed to set. When the spacer element or plastic spacer is removed, a semi-circular gap space will then exist on the outer circumference of the previously open disc space for examination of bioactivity.

FIG. 6 illustrates such a modified test assembly using a natural extracted tooth 110 to simulate an actual filling material 120 marginal behavior and potential marginal gap defects 50.

EXAMPLE 4

In one embodiment, the assembly used in the subject method is an actual metal, plastic or ceramic fixture with at least one side member connected to opposing platforms, to which platforms are affixed the at least one first material segment and/or the at least one second material segment, and that can hold the first and second materials in the assembly in close approximation for testing and analysis. This design also permits the materials utilized in the test method (as discussed in Example 1 , a test material placed in close approximation to a human dentin substrate) to be separated at various time points for examination of the underlying surfaces. By holding the materials involved in a precise fixed position, the materials can also be repositioned in their original location, with or without the use of a gap spacer element, such as a plastic gap spacer.

EXAMPLE 5

In addition to optical microscopy for assessing reduction/filling of the gap space, other analytical test methods may be utilized to assess the reduction in the marginal gap space and the nature and microstructure of the material which fills the gap space area.

For example, the degree of fill, that is, the formation of mineral deposits, in the gap space area may be assessed in a non-destructive way with micro computer tomography (Micro CT). Additionally, the microstructure of the gap space and both interface areas may be assessed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Any precise or semi-precise analytical method for analyzing surfaces or interfaces at the micro- or nano- level of accuracy and precision may be utilized in conjunction with the subject test method.

EXAMPLE 6

In addition to assessing the ability of a material to display bioactivity in relationship to a biologic tissue, the degree of bioactivity or interaction with other non-biologic materials may be analyzed using the basic features or assembly designs of the subject test method described herein.

For example, an artificial gap space in a concentric annular configuration may be created to assess the ability of bioactive materials to fill artificial gap spaces in non-linear geometries. Additionally, in extracted teeth or bone specimens, mechanical preparations can be made with a gap space of varying dimensions created along one of the marginal interfaces, then filled with the test material and subsequently incubated in a simulated body fluid to assess marginal gap fill. This model provides a close approximation to an actual clinical situation in dentistry.

"Mechanical preparations" refers to creating or "cutting" a restorative cavity preparation in an extracted or actual in-vivo tooth (or in bone if simulating an orthopedic testing situation) using well-known mechanical instrumentation such as a high- or low- speed hand piece, and/or hand instruments, to create a cavity preparation. Prior to filling the cavity preparation, removable spacers (such as Mylar films or "shims") are stabilized within the cavity preparation to create article gaps in one or more areas or portions of the cavity preparation. After the remaining portion of the cavity is filled with the "test" material and that material is permitted to cure or set sufficiently, the spacers or shims are removed, thus creating the artificial marginal spaces or gaps.

EXAMPLE 7

After utilizing any one of the above described embodiments of the subject test assembly to assess bioactivity via incubation in a simulated body fluid, such as phosphate buffered saline (PBS), the assembly may be taken apart by separating the at least one first material (e.g., test material) from the at least one second material (e.g., biological or alloplastic substrate material).

The segments may be separated mechanically by a variety of means, exposing the interfacial surfaces of the test material and the biological/alloplastic substrates. These exposed surfaces may then be analyzed by a variety of surface microanalytical methods, including without limitation, scanning or transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray spectroscopy (EDXS), infra-red spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy, or other advanced surface analytical methods.

EXAMPLE 8

The use of the test assembly containing an artificial marginal gap can also applied to a novel modification of an in-situ model (See Zero DT, "In situ caries models", Adv. Dent. Res., 1995 Nov., 9(3), pp. 214-30; discussion 231-4.) to assess the ability of a test dental biomaterial to reseal or close a marginal gap in which the marginal gap assemblies are subjected to actual in-oral conditions. For example, the assemblies created with a test material and an adjacent dentin or enamel slab, separated by a defined marginal gap, are inserted into a removable palatal appliance so that it can be worn for pre-defined periods intra-orally. The clinical subjects wear removable palatal appliances mounted with the test assemblies with the artificial marginal gaps. The clinical subjects can wear the appliances up to 24 hours daily for any time period from days to several months, removing just during meals and oral hygiene. The appliances may also be removed during the test period to be analyzed non-destructively with various methods to assess any changes in the dimensions of the artificial marginal gap. The clinical subjects can then receive the appliances back to wear them for additional time periods. The research subjects can also rinse and clean the appliances as they would normally. The appliances can also be removed and subjected to various additional external treatments that may alter or modify the ability of the test materials to close or reseal the marginal gaps in the test assembly. After the experimental period, the dental blocks are removed from the appliance and examined by visual, microscopic, and other analytical methods previously described. The degree of gap closure or resealing by the test materials is then assessed by these analytical methods. This modification of the zero in-situ test method permits the assessment of gap resealing and closure in artificial margin gaps directly under the actual conditions of the oral environment. Thus, in one embodiment, the assembly may be configured for in-vivo (in-oral) incubation. EXAMPLE 9

MATERIALS AND METHODS

Freshly extracted, unrestored human permanent posterior teeth free of carious lesions or obvious defects were collected under an institutional review board (IRB) exempt protocol. Teeth were stored in sterile distilled water at 4°C. for up to two weeks prior to processing. The outer surface of each specimen was cleaned with a soft-bristle toothbrush and sterile distilled water, sectioned perpendicular to the longitudinal axis using a diamond saw (Leco Corporation, VC-50, St. Joseph, MI, USA) under water, to create a tooth disk from the mid-coronal dentin. The surfaces were ground with 600-grit silicon carbide paper in a water lubricated surface polisher (Model 900, Electron Microscopy Sciences, Hatfield, PA) under running water for 30 seconds. The edges of the discs were also planed to create exposed dentin running lengthwise (approximately 5 to 6 mm in the longest dimension) and to create an approximate rectangular-shaped dentin slice. Approximately 1.0 mm thick dentin/enamel discs were positioned adjacent to the second disc of one of the five possible dental cements, thereby defining or creating a controlled gap between the two discs, as described below. The commercial cements utilized in the study were: (A) Self-Adhesive Resin Cement (Rely X Unicem Cement. 3M-ESPE, Minneapolis, MN, USA); (B) Resin-Modified Glass Ionomer (Rely X Luting Cement. 3M-ESPE, Minneapolis, MN, USA); (C) Conventional Glass Ionomer Luting Cement (Fuji I Luting Cement, GC,Tokyo, Japan); (D) Calcium Aluminate-Glass Ionomer (Ceramir Crown & Bridge, Doxa Dental AB, Uppsala, Sweden); and (E) a calcium silicate -based, "Portland-type" cement (White ProRoot MTA/Dentsply International, York, PA, USA).

The cement materials were mixed (triturated in the case of the Unicem and

Ceramir capsules) and then dispensed into polyethylene annular molds to form discs (N=5) approximately 1.2 cm in diameter and approximately 0.8 mm thick. The remaining materials were hand mixed from powder-liquid components as prescribed in the manufacturers' directions and similarly dispensed into the polyethylene molds. The top and bottom surface was covered with a Mylar film and a glass cover slip, forming a flat surface, and the materials were allowed to cure under ambient conditions as per their indicated setting times. For the Mineral Trioxide Aggregate (MTA) material, due to its prolonged setting behavior, the material was retained in its mold but placed in 100% humidity in a sealed container for 6 hours to facilitate complete setting prior to separation from the mold. For all cements tested, after 24 hours incubation at 37°C. in distilled water, the cured cement disc was placed on the human tooth disc surface separated by a 50 micron thick Mylar strip spacer. The Mylar strip was not interposed between the discs at one edge, thereby creating standard gaps (ranging from approximately 50 to 110 microns wide). The edges of these disc assemblies (adjacent to the artificial gap) were planed flat (lightly abraded with 1200 grit silicon carbide) establishing the edges of the cement and dentin segments in a common vertical plane with the artificial marginal gap. With a Mylar spacer positioned between the test material and the dentin segment, a "window" or artificial marginal gap was created between the upper and lower segments or slices. With the Mylar spacer in place and the segments stabilized, a dentin adhesive and composite resin were applied at the opposite ends of the separated segments to maintain and stabilize the gap space between the tooth and cement segments according to the assembly configuration shown in FIG. 2.

The result of this construction was an assembly comprising a complex of test material, an artificial marginal gap, and the dentin slice or segment on the opposite side of the gap space; all in a stable assembly. Prior to microscopic examination of the artificial marginal gap, the tooth-gap-cement assembly was thoroughly rinsed with distilled water and oil-free compressed air to remove any surface debris. The tooth-gap-cement assembly was then dried with oil-free compressed air. A light microscope (Nikon, Japan), using fixed incident light for illumination, was interfaced to a computer using digital imaging and measurement software (uEye, IDS Imaging Development Systems GmbH ). The tooth-gap-cement assembly was again examined visually with the light microscope to insure that the assembly was stable, and that the artificial marginal gap was open and absent of any significant debris in the gap space which could produce an artifact in visual interpretation. Digital images of the interfacial gap between the test material and substrate at 50X magnification were recorded at baseline (time zero), and at intermittent time periods from 1 day to 8 months after incubation in phosphate buffered saline (Dulbecco's Phosphate Buffered Saline (PBS), Sigma-Aldrich). The nature of the gap space and its approximate dimensions were assessed. Qualitative and quantitative measurements of changes in the artificial gap area (i.e. mineral deposition within the gap or actual occlusion/closure of the gap dimension) were monitored over time.

The assembly was maintained at 37°C. in the phosphate buffered saline (PBS) solution and the PBS was replaced weekly for the first two months and then every two weeks thereafter. The assembly was periodically removed from the PBS solution, excess fluid was removed from the assembly, and the assembly was placed under the digital light microscope to examine the gap interface and the related magnified edges of the test material and the adjacent dentin segment. The degree of gap closure or occlusion at 30 + 3 days and 8 months + 10 days was also scored categorically using the following rubric to give a numerical score concerning gap change or closure: No apparent change in gap = 1; greater than zero up to 1/2 the gap dimension filled with deposits = 2; greater than 1/2 the gap dimension but less than complete gap occlusion filled with deposits = 3; and complete visual closure of the gap dimension = 4. Non-parametric ANOVA utilizing the Kruskal-Wallis Test and pair- wise comparisons using the Mann- Whitney U test were conducted to assess significant differences between the ranking scores for the cement groups (p<0.05). Additional measurements at intervening time points were conducted over the course of the experiment to confirm the categorical scoring method of assessing gap closure using selected digital photographs and calibrated digital measurement software (uEyeTools, IDS Imaging Development Systems GmbH) to measure changes in the narrowing of the artificial gap dimensions.

Additional analysis of the gap area of various randomly-selected cement specimens, representative of each experimental group at approximately five months incubation time in PBS, was conducted using Micro CT imaging. The objective of this analysis was to identify and to confirm the actual deposition of "mineralized" /X-Ray radiographically dense material both at the outer boundaries of and within the artificial gap space. Each specimen was positioned in a sample holder using parafilm at 100% humidity with a moist cotton pellet and scanned by micro-CT (SkyScan 1172; Skyscan, Aartselaar, Belgium) with 11 megapixel resolution. The scanning parameters were: accelerating voltage of 70 kV, current of 142 μΑ, exposure time of 1050 ms per frame, Al + Cu filter, and rotation step at 0.52° (180° rotation). The X-ray beam was irradiated perpendicularly to the preparation long axis, and the image pixel size was 5 μιη. The X-ray projections were reconstructed using Sky Scan's volumetric reconstruction software (Nrecon) that uses the set of acquired angular projections to create a set of cross section slices through the object. This program uses a modified Feldkamp algorithm with automatic adaptation to the scan geometry in each micro-CT scanner. Reconstructed slices were saved as a stack of bmp-type files. Beam hardening correction of 20% and ring artifact correction of 12 were used for the reconstruction. The CTAn software (Skyscan, Aartselaar, Belgium) was used to obtain cross-section images through the center of the specimen's original gap area in the transverse plane to assess the presence of mineralized, radio-dense material deposited in the gap. The resultant images, representing a view of the transverse plane (bisecting the top/bottom portion) of the marginal gap space, were examined and evaluated to confirm the assessment of gap closure by light microscopy.

ANALYSIS The analysis of gap closure for the various materials indicated a complete lack of any changes in the artificial margin gaps created for the glass ionomer (C), resin-modified glass ionomer (B), or self-adhesive resin (A) cements at any time point in the evaluation. In contrast, both the calcium silicate-Portland cement type cement (E) and the calcium aluminate-glass ionomer cement (D) clearly demonstrated mineral deposits and partial/total occlusion of the artificial marginal gaps created in their specimens. Furthermore, all bioactive cement specimens (both the calcium silicate/Portland Cement Type (E) and the calcium aluminate/glass ionomer (D)) clearly demonstrated complete gap closure in gaps ranging from about 50 to about 120 microns in initial dimensions. One exception was the single calcium aluminate/glass ionomer cement specimen with an initial marginal gap of approximately 250 - 300 microns. In this case, the kinetics of gap closure was much slower and virtually complete closure required a longer time period of approximately 11-13 weeks. FIG. 7 depicts in a progressive time sequence, microscopic photos of artificial gap changes over approximately 58 days during incubation in phosphate buffered saline of five dental cement test materials 25 and dentin tooth substrates 35 configured according to the test assembly of FIG. 2. The significant changes within the artificial gaps 55 for both bioactive cements evaluated, the calcium aluminate/glass ionomer (D) and the calcium silicate-based/Portland cement-type (E) cements, can clearly be seen. In contrast, the artificial gaps 50 for the glass ionomer (C), resin-modified glass ionomer (B), and the self-adhesive resin (A) cements appear completely unchanged and open without any evidence of visible mineral deposition within the gap space 50. FIG. 8 are microscopic photos illustrating progressive closure over time of artificial marginal gaps created between a bioactive cement (D) (calcium aluminate/glass ionomer) 25 and a dentin tooth 35 substrate, with a baseline gap dimension of approximately 75 microns. The calcium silicate (Mineral Trioxide Aggregate) specimens, with initial artificial marginal gaps of approximately 50 to 120 microns in width, demonstrated a very rapid rate of marginal gap closure, with virtually complete gap closure in all specimens within 24 to 48 hours.

FIG. 9 is a graph illustrating marginal gap closure data, percent marginal reduction over time, for baseline artificial gaps created between calcium aluminate/glass ionomer cement (D) and dentin ranging from about 50 to about 100 μιη (top four plots), up to 300 μιη (bottom single plot) for up to 35 days in simulated body fluid (phosphate buffered saline). One specimen out of all those prepared for both bioactive cements (a calcium aluminate/glass ionomer cement specimen) was fabricated with a significantly larger average gap dimension of 250-300 microns, in contrast in the approximately 50 to 100 μιη range for all other specimens. The data for the calcium aluminate/glass ionomer cement (D) seen in Fig. 9 indicates that the artificial marginal gaps with dimensions of about 50 to about 100 μιη appeared to close very rapidly, with apparent first order behavior in gap closure kinetics; yet the single specimen with a significantly larger artificial gap in the range of about 250-300 microns demonstrated a slower rate of gap closure and with an apparently different kinetic profile of gap closure. The calcium silicate/Portland cement-type bioactive cement (E) also demonstrated a rapid rate of closure of artificial marginal gaps with dimensions in the range of about 50-120 microns.

Despite the variation in gap sizes, the ability of both bioactive materials to ultimately close all tested artificial marginal gaps, regardless of gap size, was demonstrated.

FIG. 10 are micro-CT transverse images through artificial marginal gaps demonstrating the presence of radiodense deposits (after 4 months of incubation in phosphate buffered saline (PBS)) within marginal gap area of bioactive cements (top four images) and absence of deposits for nonbioactive cements (lower three images). The upper three images from left to right are transverse images of calcium aluminate/glass ionomer cement (D) specimens; far upper right image is transverse image of an Mineral Trioxide Aggregate (MTA) (E) specimen. The lower transverse micro-CT images from left to right are of specimens of glass ionomer (C), resin-modified glass ionomer (B), and self-adhesive resin (A) cement, respectively. Apparent absence of radiopaque deposits in artificial gaps of these nonbioactive cements (lower three images) is evident. Arrows 56 indicate radiodense deposits at marginal areas of bioactive cement specimens. Arrows 57 indicate areas deeper within the marginal gap that demonstrate radiodense deposits. Arrows 58 indicate an artifact created by edge-overlap created by self-adhesive cement and not a radiodense deposit within the artificial marginal gap. These images in the transverse plane of the marginal gap demonstrated radio-dense material at the outer marginal interfaces of both classes of bioactive cements (D), (E), as well as deposits in the internal gap space areas of certain bioactive cement specimens. In contrast, there was no evidence of any radio-dense material in any of the transverse sections through the gaps of the other, non-bioactive cements (A), (B), (C).

The ability of the micro-computerized tomography (micro CT) to visualize internal structures of 3 -dimensional objects was utilized to confirm the findings of the digital microscopy regarding the presence of radio-dense material within the artificial marginal gap space. This micro CT analysis was limited to planar images passing through the transverse plane through the entire extent of the marginal gap. The micro-CT image permitted assessment of the presence of material deposited not only at the external margins of the gap, but also in the internal portions of the gap between the restorative material and tooth slice. The resultant images derived from this analysis not only confirmed the presence of radio-dense deposits along the outer, exposed areas marginal areas of the artificial gap, but also material irregularly deposited within the inner portions of the gap for certain specimens. While the control materials failed to demonstrated deposited material either at the borders or even the central portion of gap, the bioactive cements demonstrated greater radiolucency in the center of the gap and were more uniformly radiopaque at the edges. This phenomenon may be related to diffusional and mass-transfer phenomenon related to mineral formation with the gap space itself.

FIG. 11 are digital photomicrographs of marginal areas of cement-gap-tooth slice assemblies at 8 months of incubation in phosphate buffered saline (PBS). Upper left photomicrograph is Fuji I (glass ionomer material (C)), approximately a 110 μιη gap; upper right is Rely X Luting Cement (resin-modified glass ionomer (B)), approximately an 80-100 μιη gap; lower left ProRoot Mineral Trioxide Aggregate (MTA) (calcium silicate/Portland cement- like hydraulic cement (E)), approximately a 100 μιη gap; and lower right Ceramir Crown & Bridge (calcium aluminate/glass ionomer cement (D)), approximately a 300 μιη gap. Cement material 25 is positioned above the gap space, whereas dentin segment 35 is below the artificial gap 50 as in FIG. 2.

Complete closure of both types of bioactive cements (D), (E) is evident in artificial marginal gaps of varying dimensions, whereas none of the three non-bioactive cement types failed to demonstrate any evidence of gap closure. In FIG. 11, neither the glass ionomer (C) or resin-modified glass ionomer (B) specimens, representative of all specimens of the non-bioactive cements tested, demonstrated any evidence of gap closure or formation of interfacial deposits; even long-term at 8 months contact with PBS. In contrast, both the calcium aluminate/glass ionomer (D) and calcium silicate (E) cements, based on microscopic visual analysis, continued to demonstrate virtually complete closure 55 of the artificial marginal gaps.

Analysis of the categorical ranking for each of the cement groups at approximately 1 month and 8 months indicated values of 1 (no change in marginal gap/no evidence of gap closure) at both time periods for the glass ionomer, resin-modified glass ionomer, and the self-adhesive resin cement. Categorical ranking for the calcium aluminate/glass ionomer (D) gave ranks of complete marginal closure for three specimens (ranking of 4), and between half and full marginal closure for two specimens (ranking of 3) at 1 month evaluation. Categorical ranking for the calcium silicate/Portland cement-type (E) cement give ranks of complete marginal closure for all specimens (ranking of 4) at approximately 1 month observation time. Non-parametric ANOVA utilizing the Kruskal- Wallis Test and pair-wise comparisons using the Mann- Whitney U test were conducted indicating significant differences between the ranking scores for the two bioactive cement groups as compared to the three other cement systems (A), (B), (C) at approximate 1 month observation (p<0.002, and p<0.008, respectively). Categorical ranking for both bioactive cements, the calcium aluminate/glass ionomer (D) and the calcium silicate/Portland cement-type (E) cement, gave ranks of complete marginal closure for all specimens (ranking of 4) at 8 months evaluation. Similarly, non- parametric ANOVA utilizing the Kruskal- Wallis Test and pair-wise comparisons using the Mann- Whitney U test were conducted indicated significant differences between the ranking scores for the two bioactive cement groups as compared to the three other cement systems at approximate 8 month observation (p<0.002, and p<0.008, respectively).

The use of scanning electron microscopy (SEM)/energy dispersive X-ray analysis (EDX) is a technique that has been utilized to establish the formation of calcium- and phosphate-containing apatite on the surface of bioactive materials. This methodology could potentially be utilized for analysis of the composition of the material deposited within the artificial marginal gap space. Likewise, physical properties, such as micro- or nano-hardness of the gap-deposited material, may be able to be assessed by micro- or nano-indentation instruments and methods. These techniques could provide a clearer picture of the chemical and physical characteristics of this material deposited within the gap space by these bioactive materials.

The subject test method has demonstrated that a bioactive biomaterial, as described herein, can result in partial or total occlusion and/or filling of marginal gap areas between the biomaterial and a substrate, such as a tooth or dentin substrate, in a stable assembly.

The subject test method may be utilized in the development of various types of bioactive materials that can interact with an adjacent substrate. The subject test method assists in analyzing clinically meaningful materials currently available, but more importantly, may assist in the modification of existing materials and development of new bioactive materials. Utilizing the subject test method may assist in developing a new class of filling and restorative materials that may be applied to a cavity preparation as a classical restorative material, or as a sealant to the marginal area of an existing restoration. If a bioactive sealant is formulated, such a material could be brushed or applied to the marginal area to improve or better assure marginal sealing. This material and technique may be applied immediately after placement of the restorative material, or alternatively, to an existing marginal area which may have developed a marginal gap of some type.

Various embodiments of the subject test method and assembly for use therein include, among others, the following. It is important to note that while the following embodiments refer to a "disc", the physical configuration of the test material and/or the substrate does not need to be a "disc" in terms of a circular element. In fact, the "disc" would have a flat surface on one or more sides as described above. The term "disc" should be interpreted as a sample of a material of a suitable geometry, such as a flat slice, slab, or segment of the material, as described above.

1) A method to assess the degree of bioactivity of a material or of a

substrate modification comprising:

a) Creating a sample, or disc of the test material or substrate;

b) Creating a sample, or disc of a similar or different material or biologically-derived substrate;

c) Placing one disc adjacent to the second disc thereby defining or creating a controlled gap between the two discs; d) Creating or Fixing the discs so that they remain relatively stable in relation to the gap created;

e) Taking a photomicrograph or using some other imaging technique to record the morphology and dimensions of the gap;

f) Immersing the assembly in a simulated body fluid containing some concentration of phosphate for a pre-determined time period;

g) Taking one or more follow-up photomicrograph(s) or using said other imaging technique to record the morphology and dimensions of any changes in the morphology of deposits within the gap in order to assess bioactive behavior (i.e., deposition of a calcium-containing deposit within the gap) .Assessing and characterizing the presence of bioactivity and the degree of bioactive response by the test material using the method described in embodiment 1.

) The method of embodiment 1 where a suitable, non-bioactive material is included in the described test methodology to permit comparison.

) The method of embodiment 1 where the gap area is created in annular area between an inner circumference of the substrate and an outer circumference of the test material (or vice versa).

) The method of embodiment 1 where a modified test assembly to measure bioactivity, using a natural, extracted tooth and/or tissue specimen of some type, to simulate an actual filling material marginal behavior and potential marginal defects.

) The method of embodiment 1 where the test material and the substrate which was placed adjacent to it during the incubation period are separated and are examined by various analytical methods.

) A biomaterial (a currently available biomaterial, a modification thereof, or a newly formulated biomaterial), identified and thereby selected by the test method described in embodiment 1 or any of the subsequent embodiments, that possesses the ability to partially or substantially fully occlude and/or seal a marginal gap or opening between the biomaterial and the biological/non- biological substrate. 7) A bioactive filling or sealing material that is applied to a marginal area described in any of the above embodiments to secure sealing or occlusion of the marginal area or space.

8) A method of sealing or resealing a marginal gap area, either partially or substantially completely, by applying a bioactive restorative material adjacent to the marginal area, or by applying the bioactive material directly on the marginal gap area as a sealant, in order to affect a sealing or resealing of the marginal gap area, either partially or substantially completely.

While the test method and method of analyzing bioactivity have been described in connection with various embodiments, it is to be understood that other similar embodiments may be used, or modifications and additions may be made to the described embodiments for performing the same function. It will be understood that the embodiments described herein are merely exemplary, and that one skilled in the art may make variations and modifications without departing from the spirit and scope of the invention. Further, all embodiments disclosed are not necessarily in the alternative, as various embodiments may be combined to provide the desired result.

Claims

What is claimed is:

1. A method for assessing bioactivity of a material comprising: forming an assembly comprising at least one first material and at least one second material, wherein a gap space is defined between the least one first material and the at least one second material, recording the dimensions of the gap space, incubating the assembly in a body fluid or simulated body fluid for a time period, recording the dimensions of the gap space following incubation, and comparing the change in gap space dimensions.

2. The method of claim 1, wherein the least one first material and the at least one second material are substantially similar.

3. The method of claim 1, wherein the least one first material and the at least one second material are substantially different.

4. The method of claim 1, wherein the at least one first material is at least partially a biologic material and wherein the at least one second material is at least partially a non- biologic material.

5. The method of claim 1, wherein the at least one first material is at least partially a non-biologic material and wherein the at least one second material is at least partially a biologic material.

6. The method of claim 1, wherein the at least one first material and/or the at least one second material is substantially flat, semi-flat, concave, convex, or circular.

7. The method of claim 1, wherein the at least one first material and/or the at least one second material comprise i) a segment of biologic material and a segment of non-biologic material, ii) a segment of biologic material and a segment of a substantially different biologic material, iii) a segment of non-biologic material and a segment of a substantially different non-biologic material, or iv) combinations thereof.

8. The method of claim 1, wherein the least one first material and/or the at least one second material comprise at least one of dental restorative material, a polymeric cement, an inorganic cement, tooth enamel, tooth dentin, tooth cementum, bone cartilage, or soft tissue elements.

9. The method of claim 1, wherein the least one first material and the at least one second material are positioned in substantially parallel planes.

10. The method of claim 1, wherein the least one first material and the at least one second material are positioned in a substantially contiguous alignment.

11. The method of claim 1, wherein the gap space is formed by at least one spacer element.

12. The method of claim 11, wherein the at least one spacer element comprises a synthetic or natural polymer which can form a flexible, semi-rigid or rigid film, sheet or spacer.

13. The method of claim 12, wherein the at least one spacer element comprises at least one of: acetyl sheet polymers, polymers comprising at least one of polyethylene, polyethylene terephthalate, biaxially-oriented polyethylene terephthalate, polypropylene, polyester, polymethylmethacrylate, polycarbonate, or silicone or rubber elastomers.

14. The method of claim 1, wherein at least one adhesive is applied to or proximate at least one end of the at least one first material and the at least one second material.

15. The method of claim 14, wherein the at least one adhesive comprises a light-curable or dual-curable adhesive and/or a light-curable or dual-curable dental cement.

16. The method of claim 14, wherein a material is applied over the adhesive to form at least one pillar to maintain the pre-determined gap space.

17. The method of claim 16, wherein the at least one pillar comprises a polymer, a polymeric material, a composite resin, a cement or a polymer cement, that is applied and subsequently cured into a rigid form.

18. The method of claim 1, wherein the simulated body fluid is a phosphate-containing medium.

19. The method of claim 18, wherein the phosphate-containing medium is a sterile phosphate-buffered saline solution.

20. The method of claim 1, wherein the gap space dimensions are recorded by micro computer tomography, scanning electron microscopy or transmission electron microscopy.

21. The method of claim 1, wherein after incubating the assembly in the body fluid or simulated body fluid for the time period, the at least one first material is separated from the at least one second material to expose at least one interfacial surface.

22. The method of claim 21, where in the exposed interfacial surface of the at least one first material and/or the at least one second material is analyzed by scanning or transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray spectroscopy, infra-red spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy, non-contact (laser-based) or contact-based profilometry.

23. The method of claim 1 wherein the assembly comprises a fixture having at least one side member connecting opposing platforms, to each of which platforms one of the at least one first material or the at least one second material are affixed, defining the gap space therebetween.

24. The method of claim 21, wherein the fixture comprises plastic, metal, ceramic, or combinations thereof.

25. The method of claim 1, wherein the assembly is configured for in- vivo incubation.