Functional Adaptation of LPS-affected Dentoalveolar Fibrous Joints in Rats.

INTRODUCTION: The functional interplay between cementum of the root and alveolar bone of the socket is tuned by a uniquely positioned 70-80 µm wide fibrous and lubricious ligament in a dentoalveolar joint (DAJ). In this study, structural and biomechanical properties of the DAJ, periodontal ligament space (PDL-space also known as the joint space), alveolar bone of the socket, and cementum of the tooth root that govern the biomechanics of a lipopolysaccharide (LPS)-affected DAJ were mapped both in space and time. METHODS: The hemi-maxillae from 20 rats (4 control at 6 weeks of age, 4 control and 4 LPS-affected at 12 weeks of age, 4 control and 4 LPS-affected at 16 weeks of age) were investigated using a hybrid technique; micro-X-ray computed tomography (5 µm resolution) in combination with biomechanical testing in situ. Temporal variations in bone and cementum volume fractions were evaluated. Trends in mineral apposition rates (MAR) in additional six Sprague Dawley rats (3 controls, 3 LPS-affected) were revealed by transforming spatial fluorochrome signals to functional growth rates (linearity factor - RW) of bone, dentin, and cementum using a fast Fourier transform on fluorochrome signals from 100-µm hemi-maxillae sections. RESULTS: An overall change in LPS-affected DAJ biomechanics (a 2.5-4.5X increase in tooth displacement and 2X tooth rotation at 6 weeks, no increase in displacement and a 7X increase in rotation at 12 weeks; 27% increase in bone effective strain at 6 weeks and 11% at 12 weeks relative to control) was associated with structural changes in the coronal regions of the DAJ (15% increase in PDL-space from 0 to 6 weeks but only 5% from 6 to 12 weeks compared to control). A significant increase (p < 0.05) in PDL-space between ligated and age-matched control was observed. The bone fraction of ligated at 12 weeks was significantly lower than its age-matched control, and no significant differences (p > 0.05) between groups were observed at 6 weeks. Cementum in the apical regions grew faster but nonlinearly (11% and 20% increase in cementum fraction (CF) at 6 and 12 weeks) compared to control. Alveolar bone revealed site-specific nonlinear growth with an overall increase in MAR (108.5 µm/week to 126.7 µm/week after LPS treatment) compared to dentin (28.3 µm/week in control vs. 26.1 µm/week in LPS-affected) and cementum (126.5 µm/week in control vs. 119.9 µm/week in LPS-affected). A significant increase in CF (p < 0.05) in ligated specimens was observed at 6 weeks of age. CONCLUSIONS: Anatomy-specific responses of cementum and bone to the mechano-chemo stimuli, and their collective temporal contribution to observed changes in PDL-space were perpetuated by altered tooth movement. Data highlight the "resilience" of DAJ function through the predominance of nonlinear growth response of cementum, changes in PDL-space, and bone architecture. Despite the significant differences in bone and cementum architectures, data provided insights into the reactionary effects of cementum as a built-in compensatory mechanism to reestablish functional competence of the DAJ. The spatial shifts in architectures of alveolar bone and cementum, and consequently ligament space, highlight adaptations farther away from the site of insult, which also is another novel insight from this study. These adaptations when correlated within the context of joint function (biomechanics) illustrate that they are indeed necessary to sustain DAJ function albeit being pathological.

Mineralized Peyronie's plaque has a phenotypic resemblance to bone.

Mineralized Peyronie's plaque (MPP) impairs penile function. The association, colocalization, and dynamic interplay between organic and inorganic constituents can provide insights into biomineralization of Peyronie's plaque. Human MPPs (n = 11) were surgically excised, and the organic and inorganic constituents were spatially mapped using multiple high-resolution imaging techniques. Multiscale image analyses resulted in spatial colocalization of elements within a highly porous material with heterogenous composition, lamellae, and osteocytic lacuna-like features with a morphological resemblance to bone. The lower (520 ±â€¯179 mg/cc) and higher (1024 ±â€¯155 mg/cc) mineral density regions were associated with higher (11%) and lower (7%) porosities in MPP. Energy dispersive X-ray and micro-X-ray fluorescent spectroscopic maps in the higher mineral density regions of MPP revealed higher counts of calcium (Ca) and phosphorus (P), and a Ca/P ratio of 1.48 ±â€¯0.06 similar to bone. More importantly, higher counts of zinc (Zn) were localized at the interface between softer (more organic to inorganic ratio) and harder (less organic to inorganic ratio) tissue regions of MPP and adjacent softer matrix, indicating the involvement of Zn-related proteins and/or pathways in the formation of MPP. In particular, dentin matrix protein-1 (DMP-1) was colocalized in a matrix rich in proteoglycans and collagen that contained osteocytic lacuna-like features. This combined materials science and biochemical with correlative microspectroscopic approach provided insights into the plausible cellular and biochemical pathways that incite mineralization of an existing fibrous Peyronie's plaque. STATEMENT OF SIGNIFICANCE: Aberrant human penile mineralization is known as mineralized Peyronie's plaque (MPP) and often results in a loss of form and function. This study focuses on investigating the spatial association of matrix proteins and elemental composition of MPP by colocalizing calcium, phosphorus, and trace metal zinc with dentin matrix protein 1 (DMP-1), acidic proteoglycans, and fibrillar collagen along with the cellular components using high resolution correlative microspectroscopy techniques. Spatial maps provided insights into cellular and biochemical pathways that incite mineralization of fibrous Peyronie's plaque in humans.

Food hardness can regulate orthodontic tooth movement in mice.

BACKGROUND AND OBJECTIVES: Orthodontic treatment is often accompanied with prescription of softer foods to patients. The question to ask is, is this prescribed load regimen congruent with Wolff's law, and does it provide an adequate mechanical stimulus to maintain the functional health of periodontal complex? This question was answered by studying the effects of mice chewing on soft food (SF) and hard food (HF) while undergoing experimental tooth movement (ETM). METHODS: Three-week-old C57BL/6 mice (n = 18) were fed either hard pellet (HF; n = 9) or soft-chow food (SF; n = 9). ETM was performed on mice at 8 weeks of age, and mice were euthanized at 1 min, 2 weeks, and 4 weeks (8, 10, and 12 weeks old, respectively). A logistic regression model was applied to the experimental data to extrapolate the prolonged effects of ETM on the physical features of the dentoalveolar joint (DAJ). RESULTS: By 12 weeks, mice that chewed on SF expressed wider periodontal ligament space than those that chewed on HF. Mice that chewed on SF demonstrated increased alveolar socket roughness with larger alveoli and decreased bone volume fraction but with significantly lower bone mineral density and reduced overall tooth movement. CONCLUSIONS: These altered physical features when contextualized within the DAJ illustrated that (a) the regions farther away from the "site of insult" also undergo significant adaptation, and (b) these adaptations vary between mesial and distal sides of the periodontal complex and topographically differentiate in the direction of the ETM. These insights underpin the main conclusion, in that there is a need to "regulate chewing loads" as a therapeutic dose following ETM to encourage regeneration of periodontal complex as an effective clinical outcome. The discussed multiscale image analyses also can be used on patient cone beam computed tomography data to identify the effectiveness of orthodontic treatment within the realm of masticatory function.

Spatial survey of non-collagenous proteins in mineralizing and non-mineralizing vertebrate tissues ex vivo.

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca10(PO4)6(OH)2) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.

Functional adaptation of interradicular alveolar bone to reduced chewing loads on dentoalveolar joints in rats.

OBJECTIVES: The effects of reduced chewing loads on load bearing integrity of interradicular bone (IB) within dentoalveolar joints (DAJ) in rats were investigated. METHODS: Four-week-old Sprague Dawley rats (N = 60) were divided into two groups; rats were either fed normal food, which is hard-pellet food (HF) (N = 30), or soft-powdered chow (SF) (N = 30). Biomechanical testing of intact DAJs and mapping of the resulting mechanical strains within IBs from 8- through 24-week-old rats fed HF or SF were performed. Tension- and compression-based mechanical strain profiles were mapped by correlating digital volumes of IBs at no load with the same IBs under load. Heterogeneity within IB was identified by mapping cement lines and TRAP-positive multinucleated cells using histology, and mechanical properties using nanoindentation technique. RESULTS: Significantly decreased interradicular functional space, IB volume fraction, and elastic modulus of IB in the SF group compared with the HF group were observed, and these trends varied with an increase in age. The elastic modulus values illustrated significant heterogeneity within IB from HF or SF groups. Both compression- and tension-based strains were localized at the coronal portion of the IB and the variation in strain profiles complemented the observed material heterogeneity using histology and nanoindentation. SIGNIFICANCE: Interradicular space and IB material-related mechanoadaptations in a DAJ are optimized to meet soft food related chewing demands. Results provided insights into age-specific regulation of chewing loads as a plausible "therapeutic dose" to reverse adaptations within the periodontal complex as an attempt to regain functional competence of a dynamic DAJ.

Strong Enantiomeric Preference on the Macroion-Counterion Interaction Induced by Weakly Associated Chiral Counterions.

The role of chiral counterions on the attraction and self-assembly of chiral Pd12L24 metal organic cages (MOCs) with NO3- being the original counterion is studied by laser light scattering and isothermal titration calorimetry. Nitrates can trigger the self-assembly of macrocationic Pd12L24 into hollow spherical blackberry-type supramolecular structures via counterion-mediated attraction. Although chiral counteranions, such as N-(tert-butoxycarbonyl)-alanine (Boc-Ala), have weaker interaction with the MOCs compared to NO3-, they can induce different assembly behaviors between two enantiomeric MOCs by inhibiting the MOC-nitrate binding and weakening the interaction between them. The d-counterions are capable of selectively suppressing and slowing down the assembly of l-MOCs and also considerably decreasing their assembly size due to the much weaker MOC-nitrate interaction. The same scenario is observed for l-counterions when interacting with the d-MOCs. This study unveils the role of weakly associated chiral counterions on the central chiral macroions, especially their supramolecular structure formation, and provides additional evidence on the mechanism of the homochirality phenomenon.

Oligo(l-glutamic acids) in Calcium Phosphate Precipitation: Chain Length Effect.

The understanding of calcium phosphate precipitation is of major interest in different fields of science, including medicine, biomaterials, and physical chemistry. The presence of additive biomacromolecules has been known to influence various stages of the precipitation process from nucleation to crystal growth. In the current work, well-defined sequences of short, negatively charged peptides, oligo(l-glutamic acids), were utilized as a model, inspired by contiguous sequences of acidic amino acids in natural biomineralization proteins. The precipitate morphology and phases, the element time profile in solution and in the precipitates, as well as the kinetics during the precipitation process were analyzed to explain the effect of these short peptides on calcium phosphate precipitation. The results show that peptides can delay the phase transformation of an amorphous precursor phase to hydroxyapatite and that there is an optimal chain length for this effect at a given concentration of peptide. This study is the first part of a two-part series and is followed by a subsequent work to reveal the mechanism by which these short peptides influence the calcium phosphate precipitation.

Oligo(l-glutamic acids) in Calcium Phosphate Precipitation: Mechanism of Delayed Phase Transformation.

Proteins and their mimics that contain negatively charged sequences are important in natural and biomimetic mineralization. The mechanism by which these sequences affect calcium phosphate mineralization is not well understood. Here, peptides containing different numbers of repeat units of contiguous glutamic acid residues, oligo(l-glutamic acid)n (n = 3, 7, 8, 10), were investigated with regards to the mechanism in delaying the crystallization of amorphous calcium phosphate (ACP) while holding the amount of carboxylic acid groups in solution constant. Increasing peptide chain length increases the stability of ACP at a certain total amount of carboxylic acid groups in solution. This effect is shown to be due to stronger binding as well as binding to more calcium ions per peptide by the longer oligopeptides compared to the shorter ones. It is proposed that these associations delay the structural rearrangement of calcium ions and the dehydration of ACP, which are required for the crystallization of hydroxyapatite. The initial nucleation and the local structure of ACP, however, do not vary with chain length. This second part of a two-part series provides an improved mechanistic understanding of how organic additives, especially those with contiguous acidic amino acid sequences, modulate the kinetics of calcium phosphate precipitation and phase transformation.

Unraveling Chiral Selection in the Self-assembly of Chiral Fullerene Macroions: Effects of Small Chiral Components Including Counterions, Co-ions, or Neutral Molecules.

Lactic acid-functionalized chiral fullerene (C60) molecules are used as models to understand chiral selection in macroionic solutions involving chiral macroions, chiral counterions, and/or chiral co-ions. With the addition of Zn2+ cations, the C60 macroions exhibit slow self-assembly behavior into hollow, spherical, blackberry-type structures, as confirmed by laser light scattering (LLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques. Chiral counterions with high charge density show no selection to the chirality of AC60 macroions (LAC60 and DAC60) during their self-assembly process, while obvious chiral discrimination between the assemblies of LAC60 and DAC60 is observed when chiral counterions with low charge density are present. Compared with chiral counterions, chiral co-ions show weaker effects on chiral selection with larger amounts needed to trigger the chiral discrimination between LAC60 and DAC60. However, they can induce a higher degree of discrimination when abundant chiral co-ions are present in solution. Furthermore, the self-assembly of chiral AC60 macroions is fully suppressed by adding significant amounts of neutral molecules with opposite chirality. Thermodynamic parameters from isothermal titration calorimetry (ITC) reveal that chiral selection is controlled by the ion pairing and the destruction of solvent shells between ions, and meanwhile originates from the delicate balance between electrostatic interaction and molecular chirality.

Structure-Activity Relationships of Hydroxyapatite-Binding Peptides.

Elucidating the structure-activity relationships between biomolecules and hydroxyapatite (HAP) is essential to understand bone mineralization mechanisms, develop HAP-based implants, and design drug delivery vectors. Here, four peptides identified by phage display were selected as model HAP-binding peptides (HBPs) to examine the effects of primary amino acid sequence, phosphorylation of serine, presence of charged amino acid residues, and net charge of the peptide on (1) HAP-binding affinity, (2) secondary conformation, and (3) HAP nucleation and crystal growth. Binding affinities were determined by obtaining adsorption isotherms by mass depletion, and the conformations of the peptides in solution and bound states were observed by circular dichroism. Results showed that the magnitude of the net charge primarily controlled binding affinity, with little dependence on the other HBP features. The binding affinity and conformation results were in good agreement with our previous molecular dynamics simulation results, thus providing an excellent benchmark for the simulations. Transmission electron microscopy was used to explore the effect of these HBPs on calcium phosphate (Ca-PO4) nucleation and growth. Results indicated that HBPs may inhibit nucleation of Ca-PO4 nanoparticles and their phase transition to crystalline HAP, as well as control crystal growth rates in specific crystallographic directions, thus changing the classical needle-like morphology of inorganically grown HAP crystals to a biomimetic plate-like morphology.

Toward the Understanding of Small Protein-Mediated Collagen Intrafibrillar Mineralization.

The design of improved materials for orthopedic implants and bone tissue engineering scaffolds relies on materials mimicking the properties of bone. Calcium phosphate (Ca-PO4)-mineralized collagen fibrils arranged in a characteristic hierarchical structure constitute the building blocks of mineralized vertebrate tissues and control their biomechanical and biochemical properties. Large, flexible, acidic noncollagenous proteins (ANCPs) have been shown to influence collagen mineralization but little is known about mineralization mechanisms with the aid of small proteins. Osteocalcin (OCN) is a small, highly structured biomolecule known as a multifunctional hormone in its undercarboxylated form. Here, we examined the potential mechanism of collagen intrafibrillar mineralization in vitro mediated by OCN as a model protein. Rapid and random extrafibrillar mineralization of flakey Ca-PO4 particles was observed by transmission electron microscopy mainly on the outer surfaces of collagen fibrils of a preformed collagen scaffold in the absence of the protein. In contrast, the protein stabilized hydrated, spherical nanoclusters of Ca-PO4 on the outer surface of the fibrils, thereby retarding extrafibrillar mineralization. The nanoclusters then infiltrated the fibrils resulting in intrafibrillar mineralization with HAP crystals aligned with the fibrils. This mechanism is similar to that observed for unstructured ANCPs. Results of fibrillogenesis and immunogold labeling studies showed that OCN was associated primarily with the fibrils, consistent with ex vivo studies on mineralizing turkey tendon. The present findings contribute to expanding our understanding of collagen intrafibrillar mineralization and provide insight into design synthetic macromolecular matrices for orthopedic implants and bone regeneration.

Biological Response of and Blood Plasma Protein Adsorption on Silver-Doped Hydroxyapatite.

Hydroxyapatite (HAP) is an extensively used orthopedic biomaterial because of its high biocompatibility and osteoconductivity. Implant-related infection is a major cause of orthopedic device failure. Previous research showed that silver-doped hydroxyapatite nanoparticles (Ag-HAP NPs) have prominent antimicrobial activity, but their biocompatibility and plasma protein response remained unexplored. Here we investigated the effects of synthesis conditions on Ag-HAP NP antimicrobial (E. coli and S. epidermidis) activity, biocompatibility, and the adsorption of two blood plasma proteins, human serum albumin (HSA) and fibrinogen (Fib). It was found that synthesis pH affected the Ag content of Ag-HAP NPs and subsequent Ag+ release from the NPs in solution. This, in turn, affected antimicrobial efficiency and cytotoxicity to murine preosteoblast cells (MC3T3-E1). More HSA than Fib was adsorbed on a molar basis. The conformation of HSA changed drastically from predominantly α-helix and minor ß-sheet content in solution to greater ß-sheet than α-helix content when adsorbed. Correspondingly, the melting temperature Tm of HSA changed significantly from 76 °C in solution to ∼65-66 °C when adsorbed. Fib exhibited a modest decrease in α-helix content while the ß-sheet content increased modestly upon adsorption and its Tm remained unchanged at ∼60 °C. These differences in behavior of HSA and Fib are ascribed to the much smaller size of HSA, which allows a greater molecular packing density on the surface, which induces greater conformational changes. The protein adsorption behavior on Ag-HAP was similar to that on pure HAP. Thus, we show that Ag-HAP NPs have antimicrobial activity without deleterious effects on biocompatibility and blood plasma protein adsorption.

Orthosilicic acid, Si(OH)4, stimulates osteoblast differentiation in vitro by upregulating miR-146a to antagonize NF-κB activation.

UNLABELLED: Accumulating evidence over the last 40years suggests that silicate from dietary as well as silicate-containing biomaterials is beneficial to bone formation. However, the exact biological role(s) of silicate on bone cells are still unclear and controversial. Here, we report that orthosilicic acid (Si(OH)4) stimulated human mesenchymal stem cells (hMSCs) osteoblastic differentiation in vitro. To elucidate the possible molecular mechanisms, differential microRNA microarray analysis was used to show that Si(OH)4 significantly up-regulated microRNA-146a (miR-146a) expression during hMSC osteogenic differentiation. Si(OH)4 induced miR-146a expression profiling was further validated by quantitative RT-PCR (qRT-PCR), which indicated miR-146a was up-regulated during the late stages of hMSC osteogenic differentiation. Inhibition of miR-146a function by anti-miR-146a suppressed osteogenic differentiation of MC3T3 pre-osteoblasts, whereas Si(OH)4 treatment promoted osteoblast-specific genes transcription, alkaline phosphatase (ALP) production, and mineralization. Furthermore, luciferase reporter assay, Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence showed that Si(OH)4 decreased TNFα-induced activation of NF-κB, a signal transduction pathway that inhibits osteoblastic bone formation, through the known miR-146a negative feedback loop. Our studies established a mechanism for Si(OH)4 to promote osteogenesis by antagonizing NF-κB activation via miR-146a, which might be interesting to guide the design of osteo-inductive biomaterials for treatments of bone defects in humans. STATEMENT OF SIGNIFICANCE: Accumulating evidence over 40years suggests that silicate is beneficial to bone formation. However, the biological role(s) of silicate on bone cells are still unclear and controversial. Here, we report that Si(OH)4, the simplest form of silicate, can stimulate human mesenchymal stem cells osteoblastic differentiation. We identified that miR-146a is the expression signature in bone cells treated with Si(OH)4. Further analysis of miR-146a in bone cells reveals that Si(OH)4 upregulates miR-146a to antagonize the activation of NF-κB. Si(OH)4 was also shown to deactivate the same NF-κB pathway to suppress osteoclast formation. Our findings are important to the development of third-generation cell-and gene affecting biomaterials, and suggest silicate and miR-146a can be used as pharmaceuticals for bone fracture prevention and therapy.