Translating Dental, Oral, and Craniofacial Regenerative Medicine Innovations to the Clinic through Interdisciplinary Commercial Translation Architecture.

Few university-based regenerative medicine innovations in the dental, oral, and craniofacial (DOC) space have been commercialized and affected clinical practice in the United States. An analysis of the commercial translation literature and National Institute for Dental and Craniofacial Research's (NIDCR's) portfolio identified barriers to commercial translation of university-based DOC innovations. To overcome these barriers, the NIDCR established the Dental Oral Craniofacial Tissue Regeneration Consortium. We provide generalized strategies to inform readers how to bridge the "valley of death" and more effectively translate DOC technologies from the research laboratory or early stage company environment to clinical trials and bring needed innovations to the clinic. Three valleys of death are covered: 1) from basic science to translational development, 2) from translational technology validation to new company formation (or licensing to an existing company), and 3) from new company formation to scaling toward commercialization. An adapted phase-gate model is presented to inform DOC regenerative medicine teams how to involve regulatory, manufacturability, intellectual property, competitive assessments, business models, and commercially oriented funding mechanisms earlier in the translational development process. An Industrial Partners Program describes how to conduct market assessments, industry maps, business development processes, and industry relationship management methods to sustain commercial translation through the later-stage valley of death. Paramount to successfully implementing these methods is the coordination and collaboration of interdisciplinary teams around specific commercial translation goals and objectives. We also provide several case studies for translational projects with an emphasis on how they addressed DOC biomaterials for tissue regeneration within a rigorous commercial translation development environment. These generalized strategies and methods support innovations within a university-based and early stage company-based translational development process, traversing the many funding gaps in dental, oral, and craniofacial regenerative medicine innovations. Although the focus is on shepherding technologies through the US Food and Drug Administration, the approaches are applicable worldwide.

Biological and Mechanical Evaluation of Novel Prototype Dental Composites.

The breakdown of the polymeric component of contemporary composite dental restorative materials compromises their longevity, while leachable compounds from these materials have cellular consequences. Thus, a new generation of composite materials needed to be designed to have a longer service life and ensure that any leachable compounds are not harmful to appropriate cell lines. To accomplish this, we have developed concurrent thiol-ene-based polymerization and allyl sulfide-based addition-fragmentation chain transfer chemistries to afford cross-linked polymeric resins that demonstrate low shrinkage and low shrinkage stress. In the past, the filler used in dental composites mainly consisted of glass, which is biologically inert. In several of our prototype composites, we introduced fluorapatite (FA) crystals, which resemble enamel crystals and are bioactive. These novel prototype composites were benchmarked against similarly filled methacrylate-based bisphenol A diglycidyl ether dimethacrylate / triethylene glycol dimethacrylate (bisGMA/TEGDMA) composite for their cytotoxicity, mechanical properties, biofilm formation, and fluoride release. The leachables at pH 7 from all the composites were nontoxic to dental pulp stem cells. There was a trend toward an increase in total toughness of the glass-only-filled prototype composites as compared with the similarly filled bisGMA/TEGDMA composite. Other mechanical properties of the glass-only-filled prototype composites were comparable to the similarly filled bisGMA/TEGDMA composite. Incorporation of the FA reduced the mechanical properties of the prototype and bisGMA/TEGDMA composite. Biofilm mass and colony-forming units per milliliter were reduced on the glass-only-filled prototype composites as compared with the glass-only-filled bisGMA/TEGDMA composite and were significantly reduced by the addition of FA to all composites. Fluoride release at pH 7 was greatest after 24 h for the bisGMA/TEGDMA glass + FA composite as compared with the similarly filled prototypes, but overall the F- release was marginal and not at a concentration to affect bacterial metabolism.

Tissue engineering: state of the art in oral rehabilitation.

More than 85% of the global population requires repair or replacement of a craniofacial structure. These defects range from simple tooth decay to radical oncologic craniofacial resection. Regeneration of oral and craniofacial tissues presents a formidable challenge that requires synthesis of basic science, clinical science and engineering technology. Identification of appropriate scaffolds, cell sources and spatial and temporal signals (the tissue engineering triad) is necessary to optimize development of a single tissue, hybrid organ or interface. Furthermore, combining the understanding of the interactions between molecules of the extracellular matrix and attached cells with an understanding of the gene expression needed to induce differentiation and tissue growth will provide the design basis for translating basic science into rationally developed components of this tissue engineering triad. Dental tissue engineers are interested in regeneration of teeth, oral mucosa, salivary glands, bone and periodontium. Many of these oral structures are hybrid tissues. For example, engineering the periodontium requires growth of alveolar bone, cementum and the periodontal ligament. Recapitulation of biological development of hybrid tissues and interfaces presents a challenge that exceeds that of engineering just a single tissue. Advances made in dental interface engineering will allow these tissues to serve as model systems for engineering other tissues or organs of the body. This review will begin by covering basic tissue engineering principles and strategic design of functional biomaterials. We will then explore the impact of biomaterials design on the status of craniofacial tissue engineering and current challenges and opportunities in dental tissue engineering.

Ultrastructural observation of electron irradiation damage of lamellar bone.

The ultrastructure of murine femoral lamellar bone and the effect of electron irradiation (200 kV) on collagen and mineral features were investigated using in situ high resolution transmission electron microscopy (HRTEM). Bands of collagen fibrils were mostly aligned parallel to the long axis of the bones, with some bands of fibrils inclined in longitudinal sections. The similarity of the ultrastructure between the longitudinal and transverse sections supports the rotated plywood structure of the lamellar bone. The collagen fibrils appeared damaged and the mineral crystals were coarsened after electron irradiation. Continuous diffraction rings became spotty and the contrast between rings and the background became sharper, further suggesting coarsening of apatite crystals and increased crystallinity after irradiation. No new phases were observed after irradiation. Both the damage to collagen and coarsening of apatite crystals can deteriorate the strength and integrity of bone, and may provide insight into fracture in patients who have undergone radiation therapy.

Ultrastructural analyses of nanoscale apatite biomimetically grown on organic template.

The ultrastructure of nanoscale apatite biomimetically formed on an organic template from a supersaturated mineralizing solution was studied to examine the morphological and crystalline arrangement of mineral apatites. Needle-shaped apatite crystal plates with a size distribution of ~100 to ~1000 nm and the long axis parallel to the c axis ([002]) were randomly distributed in the mineral films. Between these randomly distributed needle-shaped apatite crystals, amorphous phases and apatite crystals (~20-40 nm) with the normal of the grains quasi-perpendicular to the c axis were observed. These observations suggest that the apatite film is an interwoven structure of amorphous phases and apatite crystals with various orientations. The mechanisms underlying the shape of the crystalline apatite plate and aggregated apatite nodules are discussed from an energy-barrier point of view. The plate or needle-shaped apatite is favored in single-crystalline form, whereas the granular nodules are favored in the polycrystalline apatite aggregate. The similarity in shape in both single-crystalline needle-shaped apatite and polycrystalline granular apatite over a wide range of sizes is explained by the principle of similitude, in which the growth and shape are determined by the forces acting upon the surface area and the volume.

Ultrastructural changes accompanying the mechanical deformation of bone tissue: a Raman imaging study.

Raman spectroscopy and imaging are known to be valuable tools for the analysis of bone, the determination of protein secondary structure, and the study of the composition of crystalline materials. We have utilized all of these attributes to examine how mechanical loading and the resulting deformation affects bone ultrastructure, addressing the hypothesis that bone spectra are altered, in both the organic and inorganic regions, in response to mechanical loading/deformation. Using a cylindrical indenter, we have permanently deformed bovine cortical bone specimens and investigated the ultrastructure in and around the deformed areas using hyperspectral Raman imaging coupled with multivariate analysis techniques. Indent morphology was further examined using scanning electron microscopy. Raman images taken at the edge of the indents show increases in the low-frequency component of the amide III band and high-frequency component of the amide I band. These changes are indicative of the rupture of collagen crosslinks due to shear forces exerted by the indenter passing through the bone. However, within the indent itself no evidence was seen of crosslink rupture, indicating that only compression of the organic matrix takes place in this region. We also present evidence of what is possibly a pressure-induced structural transformation occurring in the bone mineral within the indents, as indicated by the appearance of additional mineral factors in Raman image data from indented areas. These results give new insight into the mechanisms and causes of bone failure at the ultrastructural level.

Sustained release of vascular endothelial growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering.

Strategies to engineer bone tissue have focused on either: (1) the use of scaffolds for osteogenic cell transplantation or as conductive substrates for guided bone regeneration; or (2) release of inductive bioactive factors from these scaffold materials. This study describes an approach to add an inductive component to an osteoconductive scaffold for bone tissue engineering. We report the release of bioactive vascular endothelial growth factor (VEGF) from a mineralized, porous, degradable polymer scaffold. Three dimensional, porous scaffolds of the copolymer 85 : 15 poly(lactide-co-glycolide) were fabricated by including the growth factor into a gas foaming/particulate leaching process. The scaffold was then mineralized via incubation in a simulated body fluid. Growth of a bone-like mineral film on the inner pore surfaces of the porous scaffold is confirmed by mass increase measurements and quantification of phosphate content within scaffolds. Release of 125I-labeled VEGF was tracked over a 15 day period to determine release kinetics from the mineralized scaffolds. Sustained release from the mineralized scaffolds was achieved, and growth of the mineral film had only a minor effect on the release kinetics from the scaffolds. The VEGF released from the mineralized and non-mineralized scaffolds was over 70% active for up to 12 days following mineralization treatment, and the growth of mineral had little effect on total scaffold porosity.

Raman spectroscopic imaging markers for fatigue-related microdamage in bovine bone.

Raman spectroscopic markers have been determined for fatigue-related microdamage in bovine bone. Microdamage was induced using a cyclic fatigue loading regime. After loading, the specimens were stained en-bloc with basic fuchsin to facilitate damage visualization and differentiate fatigue-induced damage from cracks generated during subsequent histological sectioning. Bone tissue specimens were examined by light microscopy and hyperspectral near-infrared Raman imaging microscopy. Three regions were defined-tissue with no visible damage, tissue with microcracks, and tissue with diffuse damage. Raman transects, lines of 150-200 Raman spectra, were used for initial tissue surveys. Exploratory factor analysis of the transect Raman spectra has identified spectroscopically distinct chemical microstructures of the bone specimens that correlate with damage. In selected regions of damage, full hyperspectral Raman images were obtained with 1.4-microm spatial resolution. In regions of undamaged tissue, the phosphate nu1 band is found at 957 cm(-1), as expected for the carbonated hydroxyapatic bone mineral. However, in regions of visible microdamage, an additional phosphate nu1 band is observed at 963 cm(-1) and interpreted as a more stoichiometric, less carbonated mineral species. Raman imaging confirms the qualitative relationship between the Raman spectral signature of bone mineral and the type of microdamage in bovine bone. Two tentative explanations for the presence of less carbonated phosphate in damaged regions are proposed.

Growth of continuous bonelike mineral within porous poly(lactide-co-glycolide) scaffolds in vitro.

Strategies to engineer bone have focused on the use of natural or synthetic degradable materials as scaffolds for cell transplantation or as substrates to guide bone regeneration. The basic requirements of the scaffold material are biocompatibility, degradability, mechanical integrity, and osteoconductivity. A major design problem is satisfying each of these requirements with a single scaffold material. This study addresses this problem by describing an approach to combine the biocompatibility and degradability of a polymer scaffold with the osteoconductivity and mechanical reinforcement of a bonelike mineral film. We report the nucleation and growth of a continuous carbonated apatite mineral on the interior pore surfaces of a porous, degradable polymer scaffold via a one step, room temperature incubation process. A 3-dimensional, porous scaffold of the copolymer 85:15 poly(lactide-co-glycolide) was fabricated by a solvent casting, particulate leaching process. Fourier transform IR spectroscopy and scanning electron microscopy (SEM) analysis after different incubation times in a simulated body fluid (SBF) demonstrate the growth of a continuous bonelike apatite layer within the pores of the polymer scaffold. Quantification of phosphate on the scaffold displays the growth and development of the mineral film over time with an incorporation of 0.43 mg of phosphate (equivalent to 0.76 mg of hydroxyapatite) per scaffold after 14 days in SBF. The compressive moduli of polymer scaffolds increased fivefold with formation of a mineral film after a 16-day incubation time as compared to control scaffolds. In summary, this biomimetic treatment provides a simple, one step, room temperature method for surface functionalization and subsequent mineral nucleation and growth on biodegradable polymer scaffolds for tissue engineering.

Fluoride release and re-uptake in direct tooth colored restorative materials.

OBJECTIVES: Glass ionomers may be "recharged" through topical fluoride (F-) treatments; however, this reported "recharging," may be attributed to surface changes after F- treatment. This study examined differences in F- release and re-uptake among dual-cured and chemically-cured glass ionomers, and a photo-cured F- releasing composite. A secondary goal was to determine if tensile strength or surface roughness changed due to F- release, or F- re-uptake and re-release. METHODS: In Phase 1, initial surface roughness and diametral tensile strength were measured. F- release was measured for 30 days. Strength and roughness were then remeasured. In Phase 2, surface roughness was measured, then materials were treated with a 5000 ppm neutral F- gel, the same gel without F-, or phosphoric acid. F- release was measured for 30 days, then final surface roughness and strength were determined. RESULTS: Significant differences were found in amount and rate of F- release, and F- re-uptake and re-release among study materials and enamel controls (p < 0.001). The amount and rate of F- re-release after NaF treatment differed significantly from F- release after acid treatment in glass ionomers, although both groups showed increased F- release after surface treatment (p < 0.001). There were no significant changes in tensile strength or surface roughness after F- release or F- re-uptake and re-release as determined by ANOVA. SIGNIFICANCE: The results of this in vitro study indicate that applications of neutral 5000 ppm F- gel to aged glass ionomer restorations results in a significant fluoride uptake and subsequent release. The data suggest that the application of neutral fluoride gel to glass ionomer restorations in situ may result in increases in oral fluoride concentrations, without affecting the restoration's surface roughness or tensile strength.

Stress-related molar responses to the transpalatal arch: a finite element analysis.

The finite element method of analysis (FEM) was used to analyze theoretically the effects of a transpalatal arch (TPA) on periodontal stresses of molars that were subjected to typical retraction forces. The purposes of this investigation were (1) to construct an appropriate finite element model, (2) to subject the model to orthodontic forces and determine resultant stress patterns and displacements with and without the presence of a TPA, and (3) to note any differences in stress patterns and displacements between models with and without a TPA. Because anchorage is stress-dependent, a TPA must be able to modify periodontal stresses as a prerequisite for increasing orthodontic anchorage. A finite element model, consisting of two maxillary first molars, their associated periodontal ligaments and alveolar bone segments, and a TPA, was constructed. The model was subjected to simulated orthodontic forces (2 N per molar) with and without the presence of the TPA. Resultant stress patterns at the root surface, periodontal ligament, and alveolar bone, as well as displacements with and without a TPA, were calculated. Analysis of the results revealed minute differences of less than 1% of the stress range in stress values with respect to the presence of a TPA. Modification of bone properties to allow for increased displacement levels confirmed the ability of the TPA to control molar rotations; however, no effect on tipping was noted. Results suggested that the presence of a TPA has no effect on molar tipping, decreases molar rotations, and affects periodontal stress magnitudes by less than 1%. The final results suggest an inability of the TPA to modify orthodontic anchorage through modification of periodontal stresses.

Development and characterization of a biodegradable polyphosphate.

A biodegradable polyphosphate polymer (Mn = 18,000, Mw/Mn = 3.2) matrix system was developed as a potential delivery vehicle for growth factors. As a model system, release of recombinant human osteogenic protein-1 (OP-1) from this polymer was evaluated. The polyphosphate was synthesized using a triethylamine catalyst in an argon environment, and characterized using elemental analysis, gel permeation chromatography (GPC), and Fourier transform infrared spectroscopy (FTIR). Degradation kinetics of the polyphosphate polymer in phosphate-buffered saline (PBS) were represented by a second-order polynomial while degradation in bovine serum was linear with time. The polymer degraded faster in PBS than in bovine serum. In vitro release of OP-1 was also faster in PBS than in serum. Release kinetics of OP-1 in PBS and serum were represented by second-order polynomials. The OP-1 release from this physically dispersed polymeric matrix may be described by several possible mechanisms: diffusion, bulk polymer degradation, ion complexation, and interactions among the protein (OP-1), polymer, proteins, and enzymes in the media. This polyphosphate may be an effective carrier for morphogens, growth factors, or other classes of bioactive molecules.

In vitro comparison of parameters affecting the fixation strength of sagittal split osteotomies.

PURPOSE: The goal of this study was to determine how different parameters affect the bending strength of human cadaver mandibles that have undergone a sagittal split osteotomy. MATERIALS AND METHODS: The effects of screw material (titanium [Ti] vs polylactic acid/polyglycolic acid [PLA/PGA]), screw configuration (linear vs inverted L-shape), screw diameter (2.0 mm vs 2.7 mm), material into which screws were inserted (human mandible, bovine rib, synthetic polymer), and loading rate (1.0 mm/min vs 10.0 mm/min) were quantified. Also, biomechanical principles were used to model shear stress and displacement. Variable lever arms, screw material, screw diameter, screw configuration, distance between screws, and bone properties were all evaluated in this model. RESULTS: Accounting for variable mandible geometries and differentiating between deflections (and shear stresses) due to bending and due to torsion, in vitro mechanical testing revealed that there was a statistically significant difference in total shear stress at 3 mm of deflection depending on screw material (Ti > PLA/PGA), screw diameter, and material into which screws are inserted (mandibles > ribs = synthetic polymer). There was no significant difference in total shear stress depending on screw configuration or strain rate. CONCLUSION: Total shear stress and deflections are important and more viable parameters than load to assess parameters of clinical importance in osteotomy or fracture fixation.

Acoustic emission and nondestructive evaluation of biomaterials and tissues.

Acoustic emission (AE) is an acoustic wave generated by the release of energy from localized sources in a material subjected to an externally applied stimulus. This technique may be used nondestructively to analyze tissues, materials, and biomaterial/tissue interfaces. Applications of AE include use as an early warning tool for detecting tissue and material defects and incipient failure, monitoring damage progression, predicting failure, characterizing failure mechanisms, and serving as a tool to aid in understanding material properties and structure-function relations. All these applications may be performed in real time. This review discusses general principles of AE monitoring and the use of the technique in 3 areas of importance to biomedical engineering: (1) analysis of biomaterials, (2) analysis of tissues, and (3) analysis of tissue/biomaterial interfaces. Focus in these areas is on detection sensitivity, methods of signal analysis in both the time and frequency domains, the relationship between acoustic signals and microstructural phenomena, and the uses of the technique in establishing a relationship between signals and failure mechanisms.

Fractographic analysis of failed porous and surface-coated cobalt-chromium alloy total joint replacements.

Fractographic analyses were performed on retrieved porous and surface-coated cobalt-chromium alloy prostheses which were revised because of metallurgical fracture. Two femoral neck fractures and one fractured post of a femoral component of a total knee replacements were retrieved and analyzed via light, stereo and scanning electron microscopy (SEM). In all cases, fatigue was the mechanism of failure. The life time of these prostheses was 3-5 years. The porous coating; microstructural features, including large grains, carbides, porosity, inclusions and defects; design and manufacturing defects were all likely causative factors in these fatigue failures. In light of these and other reported fractures, further study of fatigue mechanisms and improvement of design and manufacturing processes are warranted.

Acoustic emission during fatigue of porous-coated Ti-6Al-4V implant alloy.

Acoustic emission (AE) events and event intensities (e.g., event amplitude, counts, duration, and energy counts) were recorded and analyzed during fatigue loading of uncoated and porous-coated Ti-6Al-4V. AE source location, spatial filtering, event, and event intensity distributions were used to detect, monitor, analyze, and predict failures. AE provides the ability to spatially and temporally locate multiple fatigue cracks, in real time. Fatigue of porous-coated Ti-6Al-4V is governed by a sequential, multimode fracture process of: transverse fracture in the porous coating; sphere/sphere and sphere/substrate debonding; substrate fatigue crack initiation; slow and rapid substrate fatigue crack propagation. Because of the porosity of the coating, the different stages of fracture within the coating occur in a discontinuous fashion. Therefore, the AE events generated are intermittent and the onset of each mode of fracture in the porous coating can be detected by increases in AE event rate. Changes in AE event rate also correspond to changes in crack extension rate, and may therefore be used to predict failure. AE offers two distinct advantages over conventional optical and microscopic methods of analyzing fatigue cracks--it is more sensitive and it can determine the time history of damage progression. The magnitude of the AE event intensities increased with increasing stress. Failure mechanisms are best differentiated by analyzing AE event amplitudes. Intergranular fracture and microvoid coalescence generated the highest AE event amplitudes (100 dB), whereas, plastic flow and friction generated the lowest AE event amplitudes (55-65 dB). Fractures in the porous coating were characterized by AE event amplitudes of less than 80 dB.

Overview of factors important in implant design.

Forty-two percent of the population over the age of 65 is totally edentulous. The use of dental implants as a means of treating these patients has accelerated in the last decade, and there are now 300,000 dental implants used in the United States. It is therefore imperative that a greater understanding of the parameters which govern the long-term success of implants be developed. In order for the effectiveness of implants to be better quantified, a fundamental, quantitative understanding of the physical parameters governing the complex synthetic material/tissue aggregate is needed. The design of an "optimal" implant requires the integration of material, physical, chemical, mechanical, biological, and economic factors. The approach taken for a specific property objective to be met should be based on a materials science approach, in which the synergistic relationships among processing, composition, structure, and properties are characterized. Implant success is a function of biomaterials and biomechanical factors, including: materials and material processing; mechanisms of implant/tissue attachment; mechanical properties; implant design; loading type; tissue properties; stress and strain distributions; initial stability and mechanisms of enhancing osseointegration; biocompatibility; and surface chemistry, mechanics, and bone-binding ability of the implant. This paper presents an overview of physical parameters important to implantology. Following a general presentation of implantology concepts, the physical parameters listed above are discussed in greater detail.

Micromechanics of implant/tissue interfaces.

A series of finite element models was developed for evaluation of the micromechanics of implant/tissue interfaces. Conventional finite element global models of a dental implant, assuming a continuum implant/bone interface, were developed so that general stress patterns in the implant and surrounding tissue could be obtained. Stresses in bone were concentrated on the alveolar crest and apex region for all global models having a direct bone/implant contact. The addition of a 100-microns-thick layer of fibrous tissue into the bone/implant interface concentrated the stresses in the middle third of the bone adjacent to the implant surface. Stresses in the middle third were ten times higher than in the cases without fibrous tissue. Interfaces modeled under the assumption of a volume-weighted average material stiffness of bone tissue and metal confirmed these general stress patterns, but provided no stress details of the interfacial zone. Finally, the equivalent material constants of the interfacial zone with and without fibrous tissue were calculated by homogenization theory. From these equivalent constants, local strains around single threads were calculated. These equivalent material properties are sensitive to the microstructure. Therefore, it is now possible for stress patterns within the interfacial zone to be quantified and the local micromechanical behavior around individual surface structures for whole implants accounted for.

A parametric study of the factors affecting the fatigue strength of porous coated Ti-6A1-4V implant alloy.

The high cycle fatigue strength of porous coated Ti-6A1-4V is approximately 75% less than the fatigue strength of uncoated Ti-6A1-4V. This study separates the effects of three parameters thought to be responsible for this reduction: interfacial geometry, microstructure, and surface alterations brought about by sintering. To achieve the goal of one parameter variations, hydrogen-alloying treatments, which refined the lamellar microstructure of beta-annealed and porous coated Ti-6A1-4V, were formulated. The fatigue strength of smooth-surfaced Ti-6A1-4V subjected to hydrogen-alloying treatments is 643-669 MPa, significantly greater than the fatigue strength of beta-annealed Ti-6A1-4V (497 MPa) and also greater than the fatigue strength of pre-annealed, equiaxed Ti-6A1-4V (590 MPa). The fatigue strength of porous coated Ti-6A1-4V, however, is independent of microstructure. This leads to the conclusion that the notch effect of the surface porosity does not allow the material to take advantage of the superior fatigue crack initiation resistance of a refined alpha-grain size. Thus, sinternecks acts as initiated microcracks and fatigue of porous coated Ti-6A1-4V is propagation controlled.