Proteomic Analyses Discern the Developmental Inclusion of Albumin in Pig Enamel: A New Model for Human Enamel Hypomineralization.

Excess albumin in enamel is a characteristic of the prevalent developmental dental defect known as chalky teeth or molar hypomineralization (MH). This study uses proteomic analyses of pig teeth to discern between developmental origin and post-eruptive contamination and to assess the similarity to hypomineralized human enamel. Here, the objective is to address the urgent need for an animal model to uncover the etiology of MH and to improve treatment. Porcine enamel is chalky and soft at eruption; yet, it hardens quickly to form a hard surface and then resembles human teeth with demarcated enamel opacities. Proteomic analyses of enamel from erupted teeth, serum, and saliva from pigs aged 4 (n = 3) and 8 weeks (n = 2) and human (n = 4) molars with demarcated enamel opacities show alpha-fetoprotein (AFP). AFP expression is limited to pre- and perinatal development and its presence in enamel indicates pre- or perinatal inclusion. In contrast, albumin is expressed after birth, indicating postnatal inclusion into enamel. Peptides were extracted from enamel and analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) after tryptic digestion. The mean total protein number was 337 in the enamel of all teeth with 13 different unique tryptic peptides of porcine AFP in all enamel samples but none in saliva samples. Similarities in the composition, micro-hardness, and microstructure underscore the usefulness of the porcine model to uncover the MH etiology, cellular mechanisms of albumin inclusion, and treatment for demarcated opacities.

Rapid post-eruptive maturation of porcine enamel.

The teeth of humans and pigs are similar in size, shape, and enamel thickness. While the formation of human primary incisor crowns takes about 8 months, domestic pigs form their teeth within a much shorter time. Piglets are born after 115 days of gestation with some of their teeth erupted that must after weaning meet the mechanical demands of their omnivorous diet without failure. We asked whether this short mineralization time before tooth eruption is combined with a post-eruptive mineralization process, how fast this process occurs, and how much the enamel hardens after eruption. To address this question, we investigated the properties of porcine teeth at two, four, and sixteen weeks after birth (N = 3 animals per time point) through analyses of composition, microstructure, and microhardness. We collected data at three standardized horizontal planes across the tooth crown to determine the change of properties throughout the enamel thickness and in relation to soft tissue eruption. Our findings indicate that porcine teeth erupt hypomineralized compared to healthy human enamel and reach a hardness that is similar to healthy human enamel within less than 4 weeks.

Osteopontin regulates type I collagen fibril formation in bone tissue.

Osteopontin (OPN) is a non-collagenous protein involved in biomineralization of bone tissue. Beyond its role in biomineralization, we show that osteopontin is essential to the quality of collagen fibrils in bone. Transmission electron microscopy revealed that, in Opn-/- tissue, the organization of the collagen fibrils was highly heterogeneous, more disorganized than WT bone and comprised of regions of both organized and disorganized matrix with a reduced density. The Opn-/- bone tissue also exhibited regions in which the collagen had lost its characteristic fibrillar structure, and the crystals were disorganized. Using nanobeam electron diffraction, we show that damage to structural integrity of collagen fibrils in Opn-/- bone tissue and their organization causes mineral disorganization, which could ultimately affect its mechanical integrity. STATEMENT OF SIGNIFICANCE: This study presents new evidence about the role of osteopontin (OPN) - a non-collagenous protein - on the structure and organization of the organic and mineral matrix in bone. In previous work, osteopontin has been suggested to regulate the nucleation and growth of bone mineral crystals and to form sacrificial bonds between mineralized collagen fibrils to enhance bone's toughness. Our findings show that OPN plays a crucial role before mineralization, during the formation of the collagen fibrils. OPN-deficient bones present a lower collagen content compared to wild type bone and, at the tissue level, collagen fibrils organization can be significantly altered in the absence of OPN. Our results suggest that OPN is critical for the formation and/or remodeling of bone collagen matrix. Our findings could lead to the development of new therapeutic strategies of bone diseases affecting collagen formation and remodeling.

The different distribution of enzymatic collagen cross-links found in adult and children bone result in different mechanical behavior of collagen.

Enzymatic collagen cross-linking has been shown to play an important role in the macroscopic elastic and plastic deformation of bone across ages. However, its direct contribution to collagen fibril deformation is unknown. The aim of this study is to determine how covalent intermolecular connections from enzymatic collagen cross-links contribute to collagen fibril elastic and plastic deformation of adults and children's bone matrix. We used ex vivo data previously obtained from biochemical analysis of children and adults bone samples (n = 14; n = 8, respectively) to create 22 sample-specific computational models of cross-linked collagen fibrils. By simulating a tensile test for each fibril, we computed the modulus of elasticity (E), ultimate tensile and yield stress (σu and σy), and elastic, plastic and total work (We, Wp and Wtot) for each collagen fibril. We present a novel difference between children and adult bone in the deformation of the collagen phase and suggest a link between collagen fibril scale and macroscale for elastic behavior in children bone under the influence of immature enzymatic cross-links. We show a parametric linear correlation between We and immature enzymatic collagen cross-links at the collagen fibril scale in the children population that is similar to the one we found at the macroscale in our previous study. Finally, we suggest the key role of covalent intermolecular connections to stiffness parameters (e.g. elastic modulus and We) in children's collagen fibril and to toughness parameters in adult's collagen fibril, respectively.

Molecular level detection and localization of mechanical damage in collagen enabled by collagen hybridizing peptides.

Mechanical injury to connective tissue causes changes in collagen structure and material behaviour, but the role and mechanisms of molecular damage have not been established. In the case of mechanical subfailure damage, no apparent macroscale damage can be detected, yet this damage initiates and potentiates in pathological processes. Here, we utilize collagen hybridizing peptide (CHP), which binds unfolded collagen by triple helix formation, to detect molecular level subfailure damage to collagen in mechanically stretched rat tail tendon fascicle. Our results directly reveal that collagen triple helix unfolding occurs during tensile loading of collagenous tissues and thus is an important damage mechanism. Steered molecular dynamics simulations suggest that a likely mechanism for triple helix unfolding is intermolecular shearing of collagen α-chains. Our results elucidate a probable molecular failure mechanism associated with subfailure injuries, and demonstrate the potential of CHP targeting for diagnosis, treatment and monitoring of tissue disease and injury.

Protein-free formation of bone-like apatite: New insights into the key role of carbonation.

The nanometer-sized plate-like morphology of bone mineral is necessary for proper bone mechanics and physiology. However, mechanisms regulating the morphology of these mineral nanocrystals remain unclear. The dominant hypothesis attributes the size and shape regulation to organic-mineral interactions. Here, we present data supporting the hypothesis that physicochemical effects of carbonate integration within the apatite lattice control the morphology, size, and mechanics of bioapatite mineral crystals. Carbonated apatites synthesized in the absence of organic molecules presented plate-like morphologies and nanoscale crystallite dimensions. Experimentally-determined crystallite size, lattice spacing, solubility and atomic order were modified by carbonate concentration. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations predicted changes in surface energy and elastic moduli with carbonate concentration. Combining these results with a scaling law predicted the experimentally observed scaling of size and energetics with carbonate concentration. The experiments and models describe a clear mechanism by which crystal dimensions are controlled by carbonate substitution. Furthermore, the results demonstrate that carbonate substitution is sufficient to drive the formation of bone-like crystallites. This new understanding points to pathways for biomimetic synthesis of novel, nanostructured biomaterials.

Large Deformation Mechanisms, Plasticity, and Failure of an Individual Collagen Fibril With Different Mineral Content.

Mineralized collagen fibrils are composed of tropocollagen molecules and mineral crystals derived from hydroxyapatite to form a composite material that combines optimal properties of both constituents and exhibits incredible strength and toughness. Their complex hierarchical structure allows collagen fibrils to sustain large deformation without breaking. In this study, we report a mesoscale model of a single mineralized collagen fibril using a bottom-up approach. By conserving the three-dimensional structure and the entanglement of the molecules, we were able to construct finite-size fibril models that allowed us to explore the deformation mechanisms which govern their mechanical behavior under large deformation. We investigated the tensile behavior of a single collagen fibril with various intrafibrillar mineral content and found that a mineralized collagen fibril can present up to five different deformation mechanisms to dissipate energy. These mechanisms include molecular uncoiling, molecular stretching, mineral/collagen sliding, molecular slippage, and crystal dissociation. By multiplying its sources of energy dissipation and deformation mechanisms, a collagen fibril can reach impressive strength and toughness. Adding mineral into the collagen fibril can increase its strength up to 10 times and its toughness up to 35 times. Combining crosslinks with mineral makes the fibril stiffer but more brittle. We also found that a mineralized fibril reaches its maximum toughness to density and strength to density ratios for a mineral density of around 30%. This result, in good agreement with experimental observations, attests that bone tissue is optimized mechanically to remain lightweight but maintain strength and toughness.

Influence of cross-link structure, density and mechanical properties in the mesoscale deformation mechanisms of collagen fibrils.

Collagen is a ubiquitous protein with remarkable mechanical properties. It is highly elastic, shows large fracture strength and enables substantial energy dissipation during deformation. Most of the connective tissue in humans consists of collagen fibrils composed of a staggered array of tropocollagen molecules, which are connected by intermolecular cross-links. In this study, we report a three-dimensional coarse-grained model of collagen and analyze the influence of enzymatic cross-links on the mechanics of collagen fibrils. Two representatives immature and mature cross-links are implemented in the mesoscale model using a bottom-up approach. By varying the number, type and mechanical properties of cross-links in the fibrils and performing tensile test on the models, we systematically investigate the deformation mechanisms of cross-linked collagen fibrils. We find that cross-linked fibrils exhibit a three phase behavior, which agrees closer with experimental results than what was obtained using previous models. The fibril mechanical response is characterized by: (i) an initial elastic deformation corresponding to the collagen molecule uncoiling, (ii) a linear regime dominated by molecule sliding and (iii) the second stiffer elastic regime related to the stretching of the backbone of the tropocollagen molecules until the fibril ruptures. Our results suggest that both cross-link density and type dictate the stiffness of large deformation regime by increasing the number of interconnected molecules while cross-links mechanical properties determine the failure strain and strength of the fibril. These findings reveal that cross-links play an essential role in creating an interconnected fibrillar material of tunable toughness and strength.

A new murine model of osteoblastic/osteolytic lesions from human androgen-resistant prostate cancer.

BACKGROUND: Up to 80% of patients dying from prostate carcinoma have developed bone metastases that are incurable. Castration is commonly used to treat prostate cancer. Although the disease initially responds to androgen blockade strategies, it often becomes castration-resistant (CRPC for Castration Resistant Prostate Cancer). Most of the murine models of mixed lesions derived from prostate cancer cells are androgen sensitive. Thus, we established a new model of CRPC (androgen receptor (AR) negative) that causes mixed lesions in bone. METHODS: PC3 and its derived new cell clone PC3c cells were directly injected into the tibiae of SCID male mice. Tumor growth was analyzed by radiography and histology. Direct effects of conditioned medium of both cell lines were tested on osteoclasts, osteoblasts and osteocytes. RESULTS: We found that PC3c cells induced mixed lesions 10 weeks after intratibial injection. In vitro, PC3c conditioned medium was able to stimulate tartrate resistant acid phosphatase (TRAP)-positive osteoclasts. Osteoprotegerin (OPG) and endothelin-1 (ET1) were highly expressed by PC3c while dikkopf-1 (DKK1) expression was decreased. Finally, PC3c highly expressed bone associated markers osteopontin (OPN), Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP) and produced mineralized matrix in vitro in osteogenic conditions. CONCLUSIONS: We have established a new CRPC cell line as a useful system for modeling human metastatic prostate cancer which presents the mixed phenotype of bone metastases that is commonly observed in prostate cancer patients with advanced disease. This model will help to understand androgen-independent mechanisms involved in the progression of prostate cancer in bone and provides a preclinical model for testing the effects of new treatments for bone metastases.

Bone micromechanical properties are compromised during long-term alendronate therapy independently of mineralization.

In the treatment of postmenopausal osteoporosis (PMOP), the use of alendronate (ALN) leads to a decrease in the risk of vertebral and nonvertebral fractures. To explore the possible adverse effects of prolonged ALN therapy, we studied the effects of 8 ± 2 years (6-10 years) of ALN treatment on the iliac cortical bone mineral and collagen quality and micromechanical properties; by design, our study examined these parameters, independent of the degree of mineralization. From six ALN-treated and five age-matched untreated PMOP women, 153 bone structural units have been chosen according their degree of mineralization to obtain the same distribution in each group. In those bone structural units, Fourier transform infrared spectroscopy, quantitative microradiography, and nanoindentation were used to assess bone quality. Irrespective of the degree of mineralization, ALN treatment was associated with higher collagen maturity (+7%, p < 0.001, c.v. = 13% and 16% in treated and untreated women, respectively) and lower mineral crystallinity than that observed in the untreated PMOP group (-2%, p < 0.0001, c.v. = 3% in both groups). Bone matrix from ALN-treated women also had lower elastic modulus (-12%, p < 0.0001, c.v. = 14% in both groups) and, contact hardness (-6%, p < 0.05, c.v. = 14% in both groups) than that of untreated women. Crystallinity (which reflects the size and perfection of crystals) was associated with both elastic modulus and contact hardness in treated women exclusively (r = 0.43 and r = 0.54, p < 0.0001, respectively), even after adjustment for the amount of mineral. We infer that long-term ALN treatment compromises micromechanical properties of the bone matrix as assessed ex vivo. The strength deficits are in part related to difference in crystallinity, irrespective of the mineral amount and mineral maturity. These novel findings at local levels of bone structure will have to be taken into account in the study of the pathophysiology of bone fragilities associated with prolonged ALN treatment.

Dual function of ERRα in breast cancer and bone metastasis formation: implication of VEGF and osteoprotegerin.

Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERRα) has been implicated in breast cancer and bone development, prompting us to examine whether ERRα may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERRα reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERRα upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERRα reduced breast cancer cell growth in bone. In contrast, ERRα overexpression increased breast cancer cell growth in the mammary gland. ERRα-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERRα mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERRα plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone.

Effects of preexisting microdamage, collagen cross-links, degree of mineralization, age, and architecture on compressive mechanical properties of elderly human vertebral trabecular bone.

Previous studies have shown that the mechanical properties of trabecular bone are determined by bone volume fraction (BV/TV) and microarchitecture. The purpose of this study was to explore other possible determinants of the mechanical properties of vertebral trabecular bone, namely collagen cross-link content, microdamage, and mineralization. Trabecular bone cores were collected from human L2 vertebrae (n = 49) from recently deceased donors 54-95 years of age (21 men and 27 women). Two trabecular cores were obtained from each vertebra, one for preexisting microdamage and mineralization measurements, and one for BV/TV and quasi-static compression tests. Collagen cross-link content (PYD, DPD, and PEN) was measured on surrounding trabecular bone. Advancing age was associated with impaired mechanical properties, and with increased microdamage, even after adjustment by BV/TV. BV/TV was the strongest determinant of elastic modulus and ultimate strength (r² = 0.44 and 0.55, respectively). Microdamage, mineralization parameters, and collagen cross-link content were not associated with mechanical properties. These data indicate that the compressive strength of human vertebral trabecular bone is primarily determined by the amount of trabecular bone, and notably unaffected by normal variation in other factors, such as cross-link profile, microdamage and mineralization.