Mesoscale structural gradients in human tooth enamel.

The outstanding mechanical and chemical properties of dental enamel emerge from its complex hierarchical architecture. An accurate, detailed multiscale model of the structure and composition of enamel is important for understanding lesion formation in tooth decay (dental caries), enamel development (amelogenesis) and associated pathologies (e.g., amelogenesis imperfecta or molar hypomineralization), and minimally invasive dentistry. Although features at length scales smaller than 100 nm (individual crystallites) and greater than 50 µm (multiple rods) are well understood, competing field of view and sampling considerations have hindered exploration of mesoscale features, i.e., at the level of single enamel rods and the interrod enamel (1 to 10 µm). Here, we combine synchrotron X-ray diffraction at submicrometer resolution, analysis of crystallite orientation distribution, and unsupervised machine learning to show that crystallographic parameters differ between rod head and rod tail/interrod enamel. This variation strongly suggests that crystallites in different microarchitectural domains also differ in their composition. Thus, we use a dilute linear model to predict the concentrations of minority ions in hydroxylapatite (Mg2+ and CO32-/Na+) that plausibly explain the observed lattice parameter variations. While differences within samples are highly significant and of similar magnitude, absolute values and the sign of the effect for some crystallographic parameters show interindividual variation that warrants further investigation. By revealing additional complexity at the rod/interrod level of human enamel and leaving open the possibility of modulation across larger length scales, these results inform future investigations into mechanisms governing amelogenesis and introduce another feature to consider when modeling the mechanical and chemical performance of enamel.

Sea urchins have teeth? A review of their microstructure, biomineralization, development and mechanical properties.

Sea urchins possess a set of five teeth which are self-sharpening and which continuously replace material lost through abrasion. The continuous replacement dictates that each tooth consists of the range of developmental states from discrete plates in the plumula, the least mineralized and least mature portion, to plates and needle-prisms separated by cellular syncytia at the beginning of the tooth shaft to a highly dense structure at the incisal end. The microstructures and their development are reviewed prior to a discussion of current understanding of the biomineralization processes operating during tooth formation. For example, the mature portions of each tooth consist of single crystal calcite but the early stages of mineral formation (e.g. solid amorphous calcium carbonate, ions in solution) continue to be investigated. The second stage mineral that cements the disparate plates and prisms together has a much higher Mg content than the first stage prisms and needles and allows the tooth to be self-sharpening. Mechanically, the urchin tooth's calcite performs better than inorganic calcite, and aspects of tooth functionality that are reviewed include the materials properties themselves and the role of the orientations of the plates and prisms relative to the axes of the applied loads. Although the properties and microarchitecture of sea urchin teeth or other mineralized tissues are often described as optimized, this view is inaccurate because these superb solutions to the problem of constructing functional structures are intermediaries not endpoints of evolution.

Calcite orientations and composition ranges within teeth across Echinoidea.

Sea urchin's teeth from four families of order Echinoida and from orders Temnopleuroida, Arbacioida and Cidaroida were studied with synchrotron X-ray diffraction. The high and very high Mg calcite phases of the teeth, i.e. the first and second stage mineral constituents, respectively, have the same crystallographic orientations. The co-orientation of first and second stage mineral, which the authors attribute to epitaxy, extends across the phylogenic width of the extant regular sea urchins and demonstrates that this is a primitive character of this group. The range of compositions Δx for the two phases of Ca1-xMgxCO3 is about 0.20 or greater and is consistent with a common biomineralization process.

A Mechanistic and Preclinical Assessment of BioRestore Bioactive Glass as a Synthetic Bone Graft Extender and Substitute for Osteoinduction and Spine Fusion.

STUDY DESIGN: Preclinical animal study. OBJECTIVE: Evaluate the osteoinductivity and bone regenerative capacity of BioRestore bioactive glass. SUMMARY OF BACKGROUND DATA: BioRestore is a Food and Drug Administration (FDA)-approved bone void filler that has not yet been evaluated as a bone graft extender or substitute for spine fusion. METHODS: In vitro and in vivo methods were used to compare BioRestore with other biomaterials for the capacity to promote osteodifferentiation and spinal fusion. The materials evaluated (1) absorbable collagen sponge (ACS), (2) allograft, (3) BioRestore, (4) Human Demineralized Bone Matrix (DBM), and (5) MasterGraft. For in vitro studies, rat bone marrow-derived stem cells (BMSC) were cultured on the materials in either standard or osteogenic media (SM, OM), followed by quantification of osteogenic marker genes ( Runx2, Osx, Alpl, Bglap, Spp1 ) and alkaline phosphatase (ALP) activity. Sixty female Fischer rats underwent L4-5 posterolateral fusion (PLF) with placement of 1 of 5 implants: (1) ICBG from syngeneic rats; (2) ICBG+BioRestore; (3) BioRestore alone; (4) ICBG+Allograft; or (5) ICBG+MasterGraft. Spines were harvested 8 weeks postoperatively and evaluated for bone formation and fusion via radiography, blinded manual palpation, microCT, and histology. RESULTS: After culture for 1 week, BioRestore promoted similar expression levels of Runx2 and Osx to cells grown on DBM. At the 2-week timepoint, the relative ALP activity for BioRestore-OM was significantly higher ( P <0.001) than that of ACS-OM and DBM-OM ( P <0.01) and statistically equivalent to cells grown on allograft-OM. In vivo, radiographic and microCT evaluation showed some degree of bridging bone formation in all groups tested, with the exception of BioRestore alone, which did not produce successful fusions. CONCLUSIONS: This study demonstrates the capacity of BioRestore to promote osteoinductivity in vitro. In vivo, BioRestore performed similarly to commercially available bone graft extender materials but was incapable of producing fusion as a bone graft substitute. LEVEL OF EVIDENCE: Level V.

A supramolecular polymer-collagen microparticle slurry for bone regeneration with minimal growth factor.

Recombinant bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive growth factor that can promote bone regeneration for challenging skeletal repair and even for ectopic bone formation in spinal fusion procedures. However, serious clinical side effects related to supraphysiological dosing highlight the need for advances in novel biomaterials that can significantly reduce the amount of this biologic. Novel biomaterials could not only reduce clinical side effects but also expand the indications for use of BMP-2, while at the same time lowering the cost of such procedures. To achieve this objective, we have developed a slurry containing a known supramolecular polymer that potentiates BMP-2 signaling and porous collagen microparticles. This slurry exhibits a paste-like consistency that stiffens into an elastic gel upon implantation making it ideal for minimally invasive procedures. We carried out in vivo evaluation of the novel biomaterial in the rabbit posterolateral spine fusion model, and discovered efficacy at unprecedented ultra-low BMP-2 doses (5 µg/implant). This dose reduces the growth factor requirement by more than 100-fold relative to current clinical products. This observation is significant given that spinal fusion involves ectopic bone formation and the rabbit model is known to be predictive of human efficacy. We expect the novel biomaterial can expand BMP-2 indications for difficult cases requiring large volumes of bone formation or involving patients with underlying conditions that compromise bone regeneration.

Sea urchin tooth mineralization: calcite present early in the aboral plumula.

In both vertebrate bone, containing carbonated hydroxyapatite as the mineral phase, and in invertebrate hard tissue comprised of calcium carbonate, a popular view is that the mineral phase develops from a long-lived amorphous precursor which later transforms into crystal form. Important questions linked to this popular view are: when and where is the crystallized material formed, and is amorphous solid added subsequently to the crystalline substrate? Sea urchin teeth, in which the earliest mineral forms within isolated compartments, in a time and position dependent manner, allow direct investigation of the timing of crystallization of the calcite primary plates. Living teeth of the sea urchin Lytechinus variegatus, in their native coelomic fluid, were examined by high-energy synchrotron X-ray diffraction. The diffraction data show that calcite is present in the most aboral portions of the plumula, representing the very earliest stages of mineralization, and that this calcite has the same crystal orientation as in the more mature adoral portions of the same tooth. Raman spectroscopy of the aboral plumula confirms the initial primary plate mineral material is calcite and does not detect amorphous calcium carbonate; in the more mature adoral incisal flange, it does detect a broader calcite peak, consistent with two or more magnesium compositions. We hypothesize that some portion of each syncytial membrane in the plumula provides the information for nucleation of identically oriented calcite crystals that subsequently develop to form the complex geometry of the single crystal sea urchin tooth.

On the formation and functions of high and very high magnesium calcites in the continuously growing teeth of the echinoderm Lytechinus variegatus: development of crystallinity and protein involvement.

Sea urchin teeth grow continuously and develop a complex mineralized structure consisting of spatially separate but crystallographically aligned first stage calcitic elements of high Mg content (5-15 mol% mineral). These become cemented together by epitaxially oriented second stage very high Mg calcite (30-40 mol% mineral). In the tooth plumula, ingressing preodontoblasts create layered cellular syncytia. Mineral deposits develop within membrane-bound compartments between cellular syncytial layers. We seek to understand how this complex tooth architecture is developed, how individual crystalline calcitic elements become crystallographically aligned, and how their Mg composition is regulated. Synchrotron microbeam X-ray scattering was performed on live, freshly dissected teeth. We observed that the initial diffracting crystals lie within independent syncytial spaces in the plumula. These diffraction patterns match those of mature tooth calcite. Thus, the spatially separate crystallites grow with the same crystallographic orientation seen in the mature tooth. Mineral-related proteins from regions with differing Mg contents were isolated, sequenced, and characterized. A tooth cDNA library was constructed, and selected matrix-related proteins were cloned. Antibodies were prepared and used for immunolocaliztion. Matrix-related proteins are acidic, phosphorylated, and associated with the syncytial membranes. Time-of-flight secondary ion mass spectroscopy of various crystal elements shows unique amino acid, Mg, and Ca ion distributions. High and very high Mg calcites differ in Asp content. Matrix-related proteins are phosphorylated. Very high Mg calcite is associated with Asp-rich protein, and it is restricted to the second stage mineral. Thus, the composition at each part of the tooth is related to architecture and function.

3D-Printed Ceramic-Demineralized Bone Matrix Hyperelastic Bone Composite Scaffolds for Spinal Fusion.

Although numerous spinal biologics are commercially available, a cost-effective and safe bone graft substitute material for spine fusion has yet to be proven. In this study, "3D-Paints" containing varying volumetric ratios of hydroxyapatite (HA) and human demineralized bone matrix (DBM) in a poly(lactide-co-glycolide) elastomer were three-dimensional (3D) printed into scaffolds to promote osteointegration in rats, with an end goal of spine fusion without the need for recombinant growth factor. Spine fusion was evaluated by manual palpation, and osteointegration and de novo bone formation within scaffold struts were evaluated by laboratory and synchrotron microcomputed tomography and histology. The 3:1 HA:DBM composite achieved the highest mean fusion score and fusion rate (92%), which was significantly greater than the 3D printed DBM-only scaffold (42%). New bone was identified extending from the host transverse processes into the scaffold macropores, and osteointegration scores correlated with successful fusion. Strikingly, the combination of HA and DBM resulted in the growth of bone-like spicules within the DBM particles inside scaffold struts. These spicules were not observed in DBM-only scaffolds, suggesting that de novo spicule formation requires both HA and DBM. Collectively, our work suggests that this recombinant growth factor-free composite shows promise to overcome the limitations of currently used bone graft substitutes for spine fusion. Impact Statement Currently, there exists a no safe, yet highly effective, bone graft substitute that is well accepted for use in spine fusion procedures. With this work, we show that a three-dimensional printed scaffold containing osteoconductive hydroxyapatite and osteoinductive demineralized bone matrix that promotes new bone spicule formation, osteointegration, and successful fusion (stabilization) when implemented in a preclinical model of spine fusion. Our study suggests that this material shows promise as a recombinant growth factor-free bone graft substitute that could safely promote high rates of successful fusion and improve patient care.

Plasticity of mandibular biomineralization in myostatin-deficient mice.

Compared with the normal or wild-type condition, knockout mice lacking myostatin (Mstn), a negative regulator of skeletal muscle growth, develop significant increases in relative masticatory muscle mass as well as the ability to generate higher maximal muscle forces. Wild-type and myostatin-deficient mice were compared to assess the postweaning influence of elevated masticatory loads because of increased jaw-adductor muscle and bite forces on the biomineralization of mandibular cortical bone and dental tissues. Microcomputed tomography (microCT) was used to quantify bone density at a series of equidistant external and internal sites in coronal sections for two symphysis and two corpus locations. Discriminant function analyses and nonparametric ANOVAs were used to characterize variation in biomineralization within and between loading cohorts. Multivariate analyses indicated that 95% of the myostatin-deficient mice and 95% of the normal mice could be distinguished based on biomineralization values at both symphysis and corpus sections. At the corpus, ANOVAs suggest that between-group differences are due to the tendency for cortical bone mineralization to be higher in myostatin-deficient mice, coupled with higher levels of dental biomineralization in normal mice. At the symphysis, ANOVAs indicate that between-group differences are related to significantly elevated bone-density levels along the articular surface and external cortical bone in the knockout mice. Both patterns, especially those for the symphysis, appear because of the postweaning effects of increased masticatory stresses in the knockout mice versus normal mice. The greater number of symphyseal differences suggest that bone along this jaw joint may be characterized by elevated plasticity. Significant differences in bone-density levels between normal and myostatin-deficient mice, coupled with the multivariate differences in patterns of plasticity between the corpus and symphysis, underscore the need for a comprehensive analysis of the plasticity of masticatory tissues vis-à-vis altered mechanical loads.

In vitro effect of amorphous calcium phosphate paste applied for extended periods of time on enamel remineralization.

OBJECTIVES: Dental applications based on the unique characteristics of amorphous calcium phosphate stabilized by casein phosphopeptides (CPP-ACP) have been proposed, as well as the improvement of its properties. The objective of this study was to determine the ability of topically applied CPP-ACP from a commercial product to remineralize subsurface lesions when applied for extended periods of time (3 h and 8 h). MATERIAL AND METHODS: Artificially induced carious lesions were produced in 50 bovine enamel blocks previously selected by surface hardness. After treatments with gel without F and CPP-ACP applied for 1 minute (Placebo); 2% NaF neutral gel applied for 1 minute (Fluoride 1 min); CPP-ACP applied for 3 min (ACP 3 min); and CPP-ACP applied for 3 h (ACP 3 h) and for 8 h (ACP 8 h), the enamel blocks were submitted to the remineralization pH-cycling. Surface hardness and synchrotron micro-tomography were used to determine the percentage of surface hardness recovery (%SHR) and to calculate mineral concentration (gHAp.cm-3), respectively. The data were submitted to ANOVA followed by the Student-Newman-Keuls test (p<0.05). RESULTS: Fluoride gel presented higher %SHR followed by ACP 3 min (p<0.001). No difference (p = 0.148) was found for Placebo, ACP 3 h and ACP 8 h groups for %SHR. Fluoride gel showed greater mineral concentration (p<0.001) when compared with the other groups. ACP 3 min demonstrated a significant difference (p<0.001) from ACP 3 h and ACP 8 h. The ACP 3 h and 8 h presented a subsurface lesion with development of laminations in all blocks. CONCLUSION: In this in vitro study the use of CPP-ACP for extended periods of time did not produce an additive effect in the remineralization process.

Biomineralization and adaptive plasticity of the temporomandibular joint in myostatin knockout mice.

Mice lacking myostatin (GDF-8), a negative regulator of skeletal muscle growth, show a significant increase in muscle mass versus normal mice. We compared wild-type and myostatin deficient mice to assess the postnatal effect of elevated masticatory loads due to increased jaw-adductor muscle activity and greater bite forces on mandibular condyle morphology. Microcomputed tomography (microCT) was used to provide details of internal condylar morphology and quantify bone density in three condylar regions. Biomineralization levels, as well as external mandibular dimensions, were used to characterize within-slice, within-joint, within-group and between-group variation. Dimensions of the mandible and mandibular condyle were similar between the myostatin knockout and normal mice. Knockout mice exhibited significantly more biomineralization on the outer surface of the condylar subchondral bone and along the condylar neck, most notably on the buccal side of the condylar neck. The buccal side of the inner aspect of the condyle was significantly less biomineralized in knockout mice, both for the pooled data and for the posterior and anterior condylar slices. Whilst normal mice had symmetric subchondral bone surfaces, those of knockout mice were asymmetric, with a lower, less convex surface on the buccal side versus the lingual side. This appears related to the ontogenetic effects of increased masticatory stress in the mandibles of knockout mice as compared to normal mice. Significant differences in biomineralization between normal and myostatin knockout mice, coupled with the lack of significant differences in certain external dimensions, underscores a need for information on the external and internal morphology of mineralized tissues vis-à-vis altered or excessive mechanical loads.

Hyperelastic "bone": A highly versatile, growth factor-free, osteoregenerative, scalable, and surgically friendly biomaterial.

Despite substantial attention given to the development of osteoregenerative biomaterials, severe deficiencies remain in current products. These limitations include an inability to adequately, rapidly, and reproducibly regenerate new bone; high costs and limited manufacturing capacity; and lack of surgical ease of handling. To address these shortcomings, we generated a new, synthetic osteoregenerative biomaterial, hyperelastic "bone" (HB). HB, which is composed of 90 weight % (wt %) hydroxyapatite and 10 wt % polycaprolactone or poly(lactic-co-glycolic acid), could be rapidly three-dimensionally (3D) printed (up to 275 cm(3)/hour) from room temperature extruded liquid inks. The resulting 3D-printed HB exhibited elastic mechanical properties (~32 to 67% strain to failure, ~4 to 11 MPa elastic modulus), was highly absorbent (50% material porosity), supported cell viability and proliferation, and induced osteogenic differentiation of bone marrow-derived human mesenchymal stem cells cultured in vitro over 4 weeks without any osteo-inducing factors in the medium. We evaluated HB in vivo in a mouse subcutaneous implant model for material biocompatibility (7 and 35 days), in a rat posterolateral spinal fusion model for new bone formation (8 weeks), and in a large, non-human primate calvarial defect case study (4 weeks). HB did not elicit a negative immune response, became vascularized, quickly integrated with surrounding tissues, and rapidly ossified and supported new bone growth without the need for added biological factors.

Piecewise-constant-model-based interior tomography applied to dentin tubules.

Dentin is a hierarchically structured biomineralized composite material, and dentin's tubules are difficult to study in situ. Nano-CT provides the requisite resolution, but the field of view typically contains only a few tubules. Using a plate-like specimen allows reconstruction of a volume containing specific tubules from a number of truncated projections typically collected over an angular range of about 140°, which is practically accessible. Classical computed tomography (CT) theory cannot exactly reconstruct an object only from truncated projections, needless to say a limited angular range. Recently, interior tomography was developed to reconstruct a region-of-interest (ROI) from truncated data in a theoretically exact fashion via the total variation (TV) minimization under the condition that the ROI is piecewise constant. In this paper, we employ a TV minimization interior tomography algorithm to reconstruct interior microstructures in dentin from truncated projections over a limited angular range. Compared to the filtered backprojection (FBP) reconstruction, our reconstruction method reduces noise and suppresses artifacts. Volume rendering confirms the merits of our method in terms of preserving the interior microstructure of the dentin specimen.

In vitro effect of amorphous calcium phosphate paste applied for extended periods of time on enamel remineralization

Abstract Dental applications based on the unique characteristics of amorphous calcium phosphate stabilized by casein phosphopeptides (CPP-ACP) have been proposed, as well as the improvement of its properties. Objectives: The objective of this study was to determine the ability of topically applied CPP-ACP from a commercial product to remineralize subsurface lesions when applied for extended periods of time (3 h and 8 h). Material and Methods: Artificially induced carious lesions were produced in 50 bovine enamel blocks previously selected by surface hardness. After treatments with gel without F and CPP-ACP applied for 1 minute (Placebo); 2% NaF neutral gel applied for 1 minute (Fluoride 1 min); CPP-ACP applied for 3 min (ACP 3 min); and CPP-ACP applied for 3 h (ACP 3 h) and for 8 h (ACP 8 h), the enamel blocks were submitted to the remineralization pH-cycling. Surface hardness and synchrotron micro-tomography were used to determine the percentage of surface hardness recovery (%SHR) and to calculate mineral concentration (gHAp.cm−3), respectively. The data were submitted to ANOVA followed by the Student-Newman-Keuls test (p<0.05). Results: Fluoride gel presented higher %SHR followed by ACP 3 min (p<0.001). No difference (p = 0.148) was found for Placebo, ACP 3 h and ACP 8 h groups for %SHR. Fluoride gel showed greater mineral concentration (p<0.001) when compared with the other groups. ACP 3 min demonstrated a significant difference (p<0.001) from ACP 3 h and ACP 8 h. The ACP 3 h and 8 h presented a subsurface lesion with development of laminations in all blocks. Conclusion: In this in vitro study the use of CPP-ACP for extended periods of time did not produce an additive effect in the remineralization process.

Nanocomposite therapy as a more efficacious and less inflammatory alternative to bone morphogenetic protein-2 in a rodent arthrodesis model.

The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in spine fusion has led to concerns regarding a potential accompanying inflammatory response. This study evaluates a combination therapy (TrioMatrix®; Pioneer Surgical, Inc., Marquette, MI) comprised of a demineralized bone matrix (DBM), hydroxyapatite, and a nanofiber-based collagen scaffold in a rodent spine fusion model. Thirty-six athymic rats that underwent a posterolateral intertransverse spinal fusion were randomly assigned to 1 of 5 treatment groups: absorbable collagen sponge alone (ACS, negative control), 10 µg rhBMP-2 on ACS (positive control), TrioMatrix®, Grafton® (Osteotech, Inc., Eatontown, NJ), and DBX® (Synthes, Inc., West Chester, PA). Both TrioMatrix® and rhBMP-2-treated animals demonstrated 100% fusion rates as graded by manual palpation scores 8 weeks after implantation. This rate was significantly greater than those of the ACS, Grafton®, and DBX® groups. Notably, the use of TrioMatrix® as evaluated by microCT quantification led to a greater fusion mass volume when compared to all other groups, including the rhBMP-2 group. T2-weighted axial MRI images of the fusion bed demonstrated a significant host response associated with a large fluid collection with the use of rhBMP-2; this response was significantly reduced with the use of TrioMatrix®. Our results therefore demonstrate that a nanocomposite therapy represents a promising, cost-effective bone graft substitute that could be useful in spine fusions where BMP-2 is contraindicated.

Layered PLG scaffolds for in vivo plasmid delivery.

Gene delivery from tissue engineering scaffolds can induce localized expression of tissue inductive factors to direct the function of progenitor cells, either endogenous or transplanted. In this report, we developed a layering approach for fabricating scaffolds with encapsulated plasmid, and investigated in vivo gene transfer following implantation into intraperitoneal fat, a widely used site for cell transplantation. Porous poly(lactide-co-glycolide) (PLG) scaffolds were fabricated using a gas foaming method, in which a non-porous layer containing plasmid was inserted between two porous polymer layers. The layered scaffold design decouples the scaffold structural requirements from its function as a drug delivery vehicle, and significantly increased the plasmid incorporation efficiency relative to scaffolds formed without layers. For multiple plasmid doses (200, 400, and 800mug), transgene expression levels peaked during the first few days and then declined over a period of 1-2 weeks. Transfected cells were observed both in the surrounding adipose tissue and within the scaffold interior. Macrophages were identified as an abundantly transfected cell type. Scaffolds delivering plasmid encoding fibroblast growth factor-2 (FGF-2) stimulated a 40% increase in the total vascular volume fraction relative to controls at 2 weeks. Scaffold-based gene delivery systems capable of localized transgene expression provide a platform for inductive and cell transplantation approaches in regenerative medicine.

Using "Mighty Mouse" to understand masticatory plasticity: myostatin-deficient mice and musculoskeletal function.

Knockout mice lacking myostatin (Mstn), a negative regulator of the growth of skeletal muscle, develop significant increases in the relative mass of masticatory muscles as well as the ability to generate higher maximal muscle forces. Wild-type and Mstn-deficient mice were compared to investigate the postnatal influence of elevated masticatory loads due to increased jaw-adductor and bite forces on the biomineralization of mandibular articular and cortical bone, the internal structure of the jaw joints, and the composition of temporomandibular joint (TMJ) articular cartilage. To provide an interspecific perspective on the long-term responses of mammalian jaw joints to altered loading conditions, the findings on mice were compared to similar data for growing rabbits subjected to long-term dietary manipulation. Statistically significant differences in joint proportions and bone mineral density between normal and Mstn-deficient mice, which are similar to those observed between rabbit loading cohorts, underscore the need for a comprehensive analysis of masticatory tissue plasticity vis-à-vis altered mechanical loads, one in which variation in external and internal structure are considered. Differences in the expression of proteoglycans and type-II collagen in TMJ articular cartilage between the mouse and rabbit comparisons suggest that the duration and magnitude of the loading stimulus will significantly affect patterns of adaptive and degradative responses. These data on mammals subjected to long-term loading conditions offer novel insights regarding variation in ontogeny, life history, and the ecomorphology of the feeding apparatus.

Pushing the limit: masticatory stress and adaptive plasticity in mammalian craniomandibular joints.

Excessive, repetitive and altered loading have been implicated in the initiation of a series of soft- and hard-tissue responses or ;functional adaptations' of masticatory and locomotor elements. Such adaptive plasticity in tissue types appears designed to maintain a sufficient safety factor, and thus the integrity of given element or system, for a predominant loading environment(s). Employing a mammalian species for which considerable in vivo data on masticatory behaviors are available, genetically similar domestic white rabbits were raised on diets of different mechanical properties so as to develop an experimental model of joint function in a normal range of physiological loads. These integrative experiments are used to unravel the dynamic inter-relationships among mechanical loading, tissue adaptive plasticity, norms of reaction and performance in two cranial joint systems: the mandibular symphysis and temporomandibular joint (TMJ). Here, we argue that a critical component of current and future research on adaptive plasticity in the skull, and especially cranial joints, should employ a multifaceted characterization of a functional system, one that incorporates data on myriad tissues so as to evaluate the role of altered load versus differential tissue response on the anatomical, cellular and molecular processes that contribute to the strength of such composite structures. Our study also suggests that the short-term duration of earlier analyses of cranial joint tissues may offer a limited notion of the complex process of developmental plasticity, especially as it relates to the effects of long-term variation in mechanical loads, when a joint is increasingly characterized by adaptive and degradative changes in tissue structure and composition. Indeed, it is likely that a component of the adaptive increases in rabbit TMJ and symphyseal proportions and biomineralization represent a compensatory mechanism to cartilage degradation that serves to maintain the overall functional integrity of each joint system. Therefore, while variation in cranial joint anatomy and performance among sister taxa is, in part, an epiphenomenon of interspecific differences in diet-induced masticatory stresses characterizing the individual ontogenies of the members of a species, this behavioral signal may be increasingly mitigated in over-loaded and perhaps older organisms by the interplay between adaptive and degradative tissue responses.