80 research outputs found
Discerning the Subfibrillar Structure of Mineralized Collagen Fibrils: A Model for the Ultrastructure of Bone
<div><p>Biomineralization templated by organic molecules to produce inorganic-organic nanocomposites is a fascinating example of nature using bottom-up strategies at nanoscale to accomplish highly ordered multifunctional materials. One such nanocomposite is bone, composed primarily of hydroxyapatite (HA) nanocrystals that are embedded within collagen fibrils with their <i>c</i>-axes arranged roughly parallel to the long axis of the fibrils. Here we discern the ultra-structure of biomimetic mineralized collagen fibrils (MCFs) as consisting of bundles of subfibrils with approximately 10 nm diameter; each one with an organic-inorganic core-shell structure. Through an amorphous calcium phosphate precursor phase the HA nanocrystals were specifically grown along the longitudinal direction of the collagen microfibrils and encapsulated them within the crystal lattice. They intercalated throughout the collagen fibrils such that the mineral phase surrounded the surface of collagen microfibrils forming an interdigitated network. It appears that this arrangement of collagen microfibrils in collagen fibrils is responsible for the observed ultrastructure. Such a subfibrillar nanostructure in MCFs was identified in both synthetic and natural bone, suggesting this is the basic building block of collagen-based hard tissues. Insights into the ultrastructure of mineralized collagen fibrils have the potential to advance our understanding on the biomineralization principles and the relationship between bone’s structure and mechanical properties, including fracture toughness mechanisms. We anticipate that these principles from biological systems can be applied to the rational design of new nanocomposites with improved performance.</p> </div
Pure and mineralized collagen fibrils.
<p>(A) SEM and TEM image of non-crosslinked collagen fibrils showing their native banding patterns. (B) SEM image of non-crosslinked collagen fibrils after mineralization appearing a filamentous substructure. D-banding can be only observed on areas without subfibrillar structure. SEM (C) and AFM (D) image of crosslinked collagen fibrils with native banding patterns. Dotted line in (C) marks the width of a single collagen fibril. The microfibrillar structure is visible with careful observation on the AFM image in (D). (E) (F) and (G) SEM images of crosslinked collagen fibrils after mineralization composed of bundles of subfibrils. (H) A cross-sectional view of crosslinked collagen fibrils after mineralization. Dashed circles in (F) and (H) mark the outer edges of the individual MCFs. Dotted line in G marks the width of a MCF. Some of the multiple subfibrils are pointed by arrows in (E) and (H).</p
XRD of MCFs and native hard tissues.
<p>XRD spectra of the crosslinked collagen fibrils after biomimetic mineralization, bovine cortical bone and dentin. Diffraction in all types of samples corresponded to hydroxyapatite nanocrystals. The reconstituted collagen fibrils were compressed as a sheet, resulting in oriented crystals with higher peak intensity for (300) and (210) planes in MCFs than those of native hard tissues.</p
TEM micrographs of bovine cortical bone.
<p>(A) and (B) Longitudinally-sectioned bovine bone showing distinct individual hydroxyapatite crystals as dark strands. Multiple long (20-190 nm) crystal strands are paired. The thickness of the crystal strands is 2-4 nm and the separation distance between two strands is approximately 2.5 nm. (C) and (D) Crushed bovine bone showing crystal fragments with widths from 5.7 to 10 nm. Dimensions of the widths of the crystals in (C) and (D) were consistent with those shown in (B).</p
TEM images of the crosslinked collagen fibrils after mineralization.
<p>(A) A representative TEM micrograph of transversely-sectioned mineralized fibrils that shows hundreds of irregular mineral fragments. (B) Magnified view of subfibrils in the area marked by the dotted square in (A). (C) Lattice image of subfibrils from a crushed MCF showing low mass contrast in the center of the subfibrils. (D) Transversely-sectioned individual subfibril showing low mass contrast in the center. (C) and (D) suggest the formation of a core-shell collagen-HA subfibrillar structure where HA crystals encapsulate collagen microfibrils.</p
SEM image of the calcium phosphate mineral.
<p>The mineral was precipitated from a PILP solution after 14 days of incubation without collagen fibrils. Some of the thin crystals with curved shapes are pointed by arrows.</p
Comparison of the subfibrillar structure produced <i>in vitro</i> to native bone.
<p>(A) Transverse view of the fractured collagen sheet produced by the PILP process showing hundreds of subfibrils. (B) Fractured surface of bovine cortical bone exhibits similar subfibrils (arrow) with similar size scale to that of the biomimetic mineralized collagen fibrils in (A).</p
TEM micrographs of unstained mineralized collagen fibrils.
<p>(A) and (B) typical TEM images of non-crosslinked collagen fibrils after mineralization showing the bundles of subfibrils. (C) Typical TEM image of crosslinked collagen fibrils after mineralization. (D) Dark-Field TEM image of crosslinked collagen fibril after mineralization constructed by selecting one of the (002) arcs with the objective aperture. It illuminates some of the [001] aligned hydroxyapatite single crystals, which appear as short bright strands. (E) Selected area electron diffraction pattern of one mineralized collagen fibril in (C) with labeled lattice planes of hydroxyapatite crystals. (D) A 9.7-nm wide isolated subfibril from crosslinked collagen fibrils after mineralization.</p
Antimicrobial GL13K Peptide Coatings Killed and Ruptured the Wall of Streptococcus gordonii and Prevented Formation and Growth of Biofilms
<div><p>Infection is one of the most prevalent causes for dental implant failure. We have developed a novel antimicrobial peptide coating on titanium by immobilizing the antimicrobial peptide GL13K. GL13K was developed from the human salivary protein BPIFA2. The peptide exhibited MIC of 8 µg/ml against planktonic Pseudonomas aeruginosa and their biofilms were reduced by three orders of magnitude with 100 µg/ml GL13K. This peptide concentration also killed 100% of Streptococcus gordonii. At 1 mg/ml, GL13K caused less than 10% lysis of human red blood cells, suggesting low toxicity to mammalian cells. Our GL13K coating has also previously showed bactericidal effect and inhibition of biofilm growth against peri-implantitis related pathogens, such as Porphyromonas gingivalis. The GL13K coating was cytocompatible with human fibroblasts and osteoblasts. However, the bioactivity of antimicrobial coatings has been commonly tested under (quasi)static culture conditions that are far from simulating conditions for biofilm formation and growth in the oral cavity. Oral salivary flow over a coating is persistent, applies continuous shear forces, and supplies sustained nutrition to bacteria. This accelerates bacteria metabolism and biofilm growth. In this work, the antimicrobial effect of the coating was tested against Streptococcus gordonii, a primary colonizer that provides attachment for the biofilm accretion by P. gingivalis, using a drip-flow biofilm bioreactor with media flow rates simulating salivary flow. The GL13K peptide coatings killed bacteria and prevented formation and growth of S. gordonii biofilms in the drip-flow bioreactor and under regular mild-agitation conditions. Surprisingly the interaction of the bacteria with the GL13K peptide coatings ruptured the cell wall at their septum or polar areas leaving empty shell-like structures or exposed protoplasts. The cell wall rupture was not detected under regular culture conditions, suggesting that cell wall rupture induced by GL13K peptides also requires media flow and possible attendant biological sequelae of the conditions in the bioreactor.</p></div
Antimicrobial effects of GL13K-peptide coatings on <i>S. gordonii</i> cultured for 3d in the drip flow bioreactor.
<p>A) Live/dead cell fluorescence staining; and B) CFU and ATP activity demonstrated the significant (*, p<0.05) and strong antimicrobial effect of the GL13K peptide coatings in comparison to all control surfaces. <i>S. gordonii bacteria</i> were killed on the GL13K coated surfaces. Error bars are the standard deviation of at least three samples in each group.</p
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