Sunita P. Ho

Enhanced osteocalcin expression by osteoblast-like cells (MC3T3-E1) exposed to bioactive coating glass (SiO2-CaO-P2O5-MgO-K2O-Na2O system) ions.

This study tested the hypothesis that bioactive coating glass (SiO(2)-CaO-P(2)O(5)-MgO-K(2)O-Na(2)O system), used for implant coatings, enhanced the induction of collagen type 1 synthesis and in turn enhanced the expression of downstream markers alkaline phosphatase, Runx2 and osteocalcin during osteoblast differentiation. The ions from experimental bioactive glass (6P53-b) and commercial Bioglass(TM) (45S5) were added to osteoblast-like MC3T3-E1 subclone 4 cultures as a supplemented ion extract (glass conditioned medium (GCM)). Ion extracts contained significantly higher concentrations of Si and Ca (Si, 47.9+/-10.4 ppm; Ca, 69.8+/-14.0 for 45S5; Si, 33.4+/-3.8 ppm; Ca, 57.1+/-2.8 ppm for 6P53-b) compared with the control extract (Si<0.1 ppm, Ca 49.0 ppm in alpha-MEM) (ANOVA, p<0.05). Cell proliferation rate was enhanced (1.5x control) within the first 3 days after adding 45S5 and 6P53-b GCM. MC3T3-E1 subclone 4 cultures were then studied for their response to the addition of test media (GCM and control medium along with ascorbic acid (AA; 50 ppm)). Each GCM+AA treatment enhanced collagen type 1 synthesis as observed in both gene expression results (day 1, Col1alpha1, 45S5 GCM+AA: 3x control+AA; 6P53-b GCM+AA: 4x control+AA; day 5, Col1alpha2, 45S5 GCM+AA: 3.15x control+AA; 6P53-b GCM+AA: 2.35x control+AA) and in histological studies (Picrosirius stain) throughout the time course of early differentiation. Continued addition of each GCM and AA treatment led to enhanced expression of alkaline phosphatase (1.4x control+AA after 5 days, 2x control+AA after 10 days), Runx2 (2x control+AA after 7 days) and osteocalcin gene (day 3, 45S5 GCM+AA: 14x control+AA; day 5, 6P53-b GCM+AA: 19x control+AA) and protein expression (40x-70x control+AA after 6 days). These results indicated the enhanced effect of bioactive glass ions on key osteogenic markers important for the bone healing process.

Matrix metalloproteinase–13 is required for osteocytic perilacunar remodeling and maintains bone fracture resistance

Like bone mass, bone quality is specified in development, actively maintained postnatally, and disrupted by disease. The roles of osteoblasts, osteoclasts, and osteocytes in the regulation of bone mass are increasingly well defined. However, the cellular and molecular mechanisms by which bone quality is regulated remain unclear. Proteins that remodel bone extracellular matrix, such as the collagen‐degrading matrix metalloproteinase (MMP)‐13, are likely candidates to regulate bone quality. Using MMP‐13–deficient mice, we examined the role of MMP‐13 in the remodeling and maintenance of bone matrix and subsequent fracture resistance. Throughout the diaphysis of MMP‐13–deficient tibiae, we observed elevated nonenzymatic cross‐linking and concentric regions of hypermineralization, collagen disorganization, and canalicular malformation. These defects localize to the same mid‐cortical bone regions where osteocyte lacunae and canaliculi exhibit MMP‐13 and tartrate‐resistant acid phosphatase (TRAP) expression, as well as the osteocyte marker sclerostin. Despite otherwise normal measures of osteoclast and osteoblast function, dynamic histomorphometry revealed that remodeling of osteocyte lacunae is impaired in MMP‐13−/− bone. Analysis of MMP‐13−/− mice and their wild‐type littermates in normal and lactating conditions showed that MMP‐13 is not only required for lactation‐induced osteocyte perilacunar remodeling, but also for the maintenance of bone quality. The loss of MMP‐13, and the resulting defects in perilacunar remodeling and matrix organization, compromise MMP‐13−/− bone fracture toughness and postyield behavior. Taken together, these findings demonstrate that osteocyte perilacunar remodeling of mid‐cortical bone matrix requires MMP‐13 and is essential for the maintenance of bone quality.

Frictional properties of poly(MPC-co-BMA) phospholipid polymer for catheter applications.

A fundamental understanding of surface properties of the biomaterials at a nanometer scale should be generated in order to understand cellular responses of the tissue to biomaterials thereby minimizing or eliminating tissue trauma at a macrometer scale. In this study poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) ([poly(MPC-co-BMA]) was evaluated as a potential coating material for vascular applications to provide smooth catheterization using atomic force microscopy (AFM) techniques.A uniform coating of [poly(MPC-co-BMA] equivalent to a thickness of 2.5 microm on a polyurethane (PU) catheter material was provided using dip casting technique. Using a contact mode AFM, no significant difference in surface roughness (R(a)) and frictional force (f) between uncoated (R(a)=10.2+/-1.9 nm, f=0.907+/-0.02) and coated (R(a)=11.7+/-1.8 nm, f=0.930+/-0.06) surfaces was observed under dry conditions. However, under wet conditions the R(a) of the coated surface (3.4+/-1.0 nm) was significantly lower than uncoated PU surface (9.0+/-1.8 nm). The coating on PU substrate offered the least frictional resistance (f=0.004+/-0.001) illustrating enhanced boundary lubrication capability due to hydration of phosphorylcholine polymer as compared to a significantly higher f for uncoated PU (0.017+/-0.007) surfaces. These tribological and chemical characteristics of the [poly(MPC-co-BMA)] coating could increase the overall efficacy of PU for clinical applications.

Functional Remineralization of Dentin Lesions Using Polymer-Induced Liquid-Precursor Process

It was hypothesized that applying the polymer-induced liquid-precursor (PILP) system to artificial lesions would result in time-dependent functional remineralization of carious dentin lesions that restores the mechanical properties of demineralized dentin matrix. 140 µm deep artificial caries lesions were remineralized via the PILP process for 7–28 days at 37°C to determine temporal remineralization characteristics. Poly-L-aspartic acid (27 KDa) was used as the polymeric process-directing agent and was added to the remineralization solution at a calcium-to-phosphate ratio of 2.14 (mol/mol). Nanomechanical properties of hydrated artificial lesions had a low reduced elastic modulus (ER = 0.2 GPa) region extending about 70 μm into the lesion, with a sloped region to about 140 μm where values reached normal dentin (18–20 GPa). After 7 days specimens recovered mechanical properties in the sloped region by 51% compared to the artificial lesion. Between 7–14 days, recovery of the outer portion of the lesion continued to a level of about 10 GPa with 74% improvement. 28 days of PILP mineralization resulted in 91% improvement of ER compared to the artificial lesion. These differences were statistically significant as determined from change-point diagrams. Mineral profiles determined by micro x-ray computed tomography were shallower than those determined by nanoindentation, and showed similar changes over time, but full mineral recovery occurred after 14 days in both the outer and sloped portions of the lesion. Scanning electron microscopy and energy dispersive x-ray analysis showed similar morphologies that were distinct from normal dentin with a clear line of demarcation between the outer and sloped portions of the lesion. Transmission electron microscopy and selected area electron diffraction showed that the starting lesions contained some residual mineral in the outer portions, which exhibited poor crystallinity. During remineralization, intrafibrillar mineral increased and crystallinity improved with intrafibrillar mineral exhibiting the orientation found in normal dentin or bone.

The biomechanical characteristics of the bone-periodontal ligament-cementum complex

The relative motion between the tooth and alveolar bone is facilitated by the soft-hard tissue interfaces which include periodontal ligament-bone (PDL-bone) and periodontal ligament-cementum (PDL-cementum). The soft-hard tissue interfaces are responsible for attachment and are critical to the overall biomechanical efficiency of the bone-tooth complex. In this study, the PDL-bone and PDL-cementum attachment sites in human molars were investigated to identify the structural orientation and integration of the PDL with bone and cementum. These attachment sites were characterized from a combined materials and mechanics perspective and were related to macro-scale function. High resolution complimentary imaging techniques including atomic force microscopy, scanning electron microscopy and micro-scale X-ray computed tomography (Micro XCT) illustrated two distinct orientations of PDL; circumferential-PDL (cir-PDL) and radial-PDL (rad-PDL). Within the PDL-space, the primary orientation of the ligament was radial (rad-PDL) as is well known. Interestingly, circumferential orientation of PDL continuous with rad-PDL was observed adjacent to alveolar bone and cementum. The integration of the cir-PDL was identified by 1-2 microm diameter PDL-inserts or Sharpeys fibers in alveolar bone and cementum. Chemically and biochemically the cir-PDL adjacent to bone and cementum was identified by relatively higher carbon and lower calcium including the localization of small leucine rich proteins responsible for maintaining soft-hard tissue cohesion, stiffness and hygroscopic nature of PDL-bone and PDL-cementum attachment sites. The combined structural and chemical properties provided graded stiffness characteristics of PDL-bone (E(r) range for PDL: 10-50 MPa; bone: 0.2-9.6 GPa) and PDL-cementum (E(r) range for cementum: 1.1-8.3 GPa), which was related to the macro-scale function of the bone-tooth complex.

Structure, chemical composition and mechanical properties of human and rat cementum and its interface with root dentin

This work seeks to establish comparisons of the physical properties of rat and human cementum, root dentin and their interface, including the cementum-dentin junction (CDJ), as a basis for future studies of the entire periodontal complex using rats as animal models. In this study the structure, site-specific chemical composition and mechanical properties of cementum and its interface with root dentin taken from 9- to 12-month-old rats were compared to the physiologically equivalent 40- to 55-year-old human age group using qualitative and quantitative characterization techniques, including histology, atomic force microscopy (AFM), micro-X-ray computed tomography, Raman microspectroscopy and AFM-based nanoindentation. Based on results from this study, cementum taken from the apical third of the respective species can be represented as a woven fabric with radially and circumferentially oriented collagen fibers. In both species the attachment of cementum to root dentin is defined by a stiffness-graded interface (CDJ/cementum-dentin interface). However, it was concluded that cementum and the cementum-dentin interface from a 9- to 12-month-old rat could be more mineralized, resulting in noticeably decreased collagen fiber hydration and significantly higher modulus values under wet conditions for cementum and CDJ (E(rat-cementum)=12.7+/-2.6 GPa; E(rat-CDJ)=11.6+/-3.2 GPa) compared to a 40- to 55-year-old human (E(human-cementum)=3.73+/-1.8 GPa; E(human-CDJ)=1.5+/-0.7 GPa). The resulting data illustrated that the extensions of observations made from animal models to humans should be justified with substantial and equivalent comparison of data across age ranges (life spans) of mammalian species.

Age-related adaptation of bone-PDL-tooth complex: Rattus-Norvegicus as a model system.

Functional loads on an organ induce tissue adaptations by converting mechanical energy into chemical energy at a cell-level. The transducing capacity of cells alters physico-chemical properties of tissues, developing a positive feedback commonly recognized as the form-function relationship. In this study, organ and tissue adaptations were mapped in the bone-tooth complex by identifying and correlating biomolecular expressions to physico-chemical properties in rats from 1.5 to 15 months. However, future research using hard and soft chow over relevant age groups would decouple the function related effects from aging affects. Progressive curvature in the distal root with increased root resorption was observed using micro X-ray computed tomography. Resorption was correlated to the increased activity of multinucleated osteoclasts on the distal side of the molars until 6 months using tartrate resistant acid phosphatase (TRAP). Interestingly, mononucleated TRAP positive cells within PDL vasculature were observed in older rats. Higher levels of glycosaminoglycans were identified at PDL-bone and PDL-cementum entheses using alcian blue stain. Decreasing biochemical gradients from coronal to apical zones, specifically biomolecules that can induce osteogenic (biglycan) and fibrogenic (fibromodulin, decorin) phenotypes, and PDL-specific negative regulator of mineralization (asporin) were observed using immunohistochemistry. Heterogeneous distribution of Ca and P in alveolar bone, and relatively lower contents at the entheses, were observed using energy dispersive X-ray analysis. No correlation between age and microhardness of alveolar bone (0.7 ± 0.1 to 0.9 ± 0.2 GPa) and cementum (0.6 ± 0.1 to 0.8 ± 0.3 GPa) was observed using a microindenter. However, hardness of cementum and alveolar bone at any given age were significantly different (P<0.05). These observations should be taken into account as baseline parameters, during development (1.5 to 4 months), growth (4 to 10 months), followed by a senescent phase (10 to 15 months), from which deviations due to experimentally induced perturbations can be effectively investigated.

Structure, chemical composition and mechanical properties of coronal cementum in human deciduous molars

OBJECTIVES It was hypothesized that the coronal cementum containing collagen forms a weak junction with enamel unlike the well integrated DEJ and CDJ. METHODS The hypothesis was investigated in two parts: (1) evaluate the structure, chemical composition and mechanical properties of coronal cementum and its junction with enamel using scanning electron microscopy, micro-X-ray computed tomography, and atomic force microscopy. The chemical composition and mechanical properties were determined by evaluating the spatial variations of inorganic (PO(4)(3-)nu(1) mode at 960 cm(-1)) and organic (C-H deformation at 1452 cm(-1); C-H stretch at 2940 cm(-1)) contents using Raman microspectroscopy and elastic modulus and hardness values using nanoindentation. (2) Estimate the strength and evaluate the microstructure of coronal cementum interface with enamel using SEM and MicroXCT. RESULTS AND CONCLUSIONS Coronal cementum is heterogeneous because it is a combination of laminar acellular afibrillar cementum and acellular extrinsic fiber cementum with relatively higher organic content. It integrates micromechanically via a scallop-like weak interface with enamel unlike the biomechanically efficient DEJ and CDJ and is continuous with primary root cementum. A single tooth could exhibit all three types of cementum enamel junctions; an overlap, butt and a gap depending on the sectioning plane. The elastic modulus of coronal cementum (11.0+/-5.8 GPa) is significantly lower (p<0.05; Students t-test with 95% confidence interval) than primary cementum (15.8+/-5.3 GPa).

Removal of SOST or blocking its product sclerostin rescues defects in the periodontitis mouse model

Understanding periodontal ligament (PDL) biology and developing an effective treatment for bone and PDL damage due to periodontitis have been longstanding aims in dental medicine. Here, we first demonstrated by cell lineage tracing and mineral double‐labeling approaches that murine PDL progenitor cells display a 2‐ and 3‐fold higher mineral deposition rate than the periosteum and endosteum at the age of 4 weeks, respectively. We next proved that the pathologic changes in osteocytes (Ocys; changes from a spindle shape to round shape with a >50% reduction in the dendrite number/length, and an increase in SOST) are the key pathologic factors responsible for bone and PDL damage in periostin‐null mice (a periodontitis animal model) using a newly developed 3‐dimensional FITC‐Imaris technique. Importantly, we proved that deleting the Sost gene (a potent inhibitor of WNT signaling) or blocking sclerostin function by using the mAb in this periodontitis model significantly restores bone and PDL defects (n = 4‐5; P < 0.05). Together, identification of the key contribution of the PDL in normal alveolar bone formation, the pathologic changes of the Ocys in periodontitis bone loss, and the novel link between sclerostin and Wnt signaling in the PDL will aid future drug development in the treatment of patients with periodontitis.—Ren, Y., Han, X., Ho, S. P., Harris, S. E., Cao, Z., Economides, A. N., Qin, C., Ke, H., Liu, M., Feng, J. Q. Removal of SOST or blocking its product sclerostin rescues defects in the periodontitis mouse model. FASEB J. 29, 2702‐2711 (2015). www.fasebj.org

Essential role of osterix for tooth root but not crown dentin formation.

Tooth is made of crown and root. It is widely believed that dentin formation in crown and root uses the same regulatory mechanism. However, identification of nuclear factor 1 C (NFIC)s unique function in determining root but not crown dentin formation challenges the old thinking. In searching for the target molecules downstream of NFIC, we unexpectedly found a sharp reduction of osterix (OSX), the key transcription factor in skeleton formation, in the Nfic knockout (Nfic‐KO) tooth root. We then demonstrated a dose‐dependent increase of Osx in the odontoblast cell line due to a transient transfection of Nfic expression plasmid. Studies of global and conditional Osx‐KO mice revealed no apparent changes in the crown dentin tubules and dentin matrix. However, the OSX conditional KO (cKO) mice (crossed to the 2.3‐kb collagen type 1 [Col1]‐Cre) displayed an increase in cell proliferation but great decreases in expressions of root dentin matrix proteins (dentin matrix protein 1 [DMP1] and dentin sialophosphoprotein [DSPP]), leading to an inhibition in odontoblast differentiation, and short, thin root dentin with few dentin tubules. Compared to the Nfic‐KO tooth, which contains essentially no dentin tubules and remains in a “root‐less” status at adult stages, the Osx‐cKO root phenotype had partially improved at the late stage, indicating that other factors can compensate for OSX function. Thus, we conclude that OSX, one of the key downstream molecules of NFIC, plays a critical role in root, but not crown, formation.