Micron-scale Topography Research Articles

Membrane Remodeling and Matrix Dispersal Intermediates During Mammalian Acrosomal Exocytosis.

To become fertilization-competent, mammalian sperm must undergo a complex series of biochemical and morphological changes in the female reproductive tract. These changes, collectively called capacitation, culminate in the exocytosis of the acrosome, a large vesicle overlying the nucleus. Acrosomal exocytosis is not an all-or-nothing event but rather a regulated process in which vesicle cargo disperses gradually. However, the structural mechanisms underlying this controlled release remain undefined. In addition, unlike other exocytotic events, fusing membranes are shed as vesicles; the cell thus loses the entire anterior two-thirds of its plasma membrane and yet remains intact, while the remaining nonvesiculated plasma membrane becomes fusogenic. Precisely how cell integrity is maintained throughout this drastic vesiculation process is unclear, as is how it ultimately leads to the acquisition of fusion competence. Here, we use cryoelectron tomography to visualize these processes in unfixed, unstained, fully hydrated sperm. We show that paracrystalline structures within the acrosome disassemble during capacitation and acrosomal exocytosis, representing a plausible mechanism for gradual dispersal of the acrosomal matrix. We find that the architecture of the sperm head supports an atypical membrane fission–fusion pathway that maintains cell integrity. Finally, we detail how the acrosome reaction transforms both the micron-scale topography and the nanoscale protein landscape of the sperm surface, thus priming the sperm for fertilization.

Coupling between macrophage phenotype, angiogenesis and bone formation by calcium phosphates

The ability of calcium phosphate (CaP) materials to induce bone formation varies with their physicochemical properties, with surface topography as one of the most crucial triggers. In view of the natural wound healing processes (e.g., inflammation, angiogenesis, tissue formation and remodeling) initiated after surgical implantation, we here comparatively investigated the biological cascades occurring upon ectopic implantation of a tricalcium phosphate with submicron surface topography (TCP-S, osteoinductive) and a tricalcium phosphate with micron-scale topography (TCP-B, non-osteoinductive). In vitro, TCP-S facilitated M2 polarization of macrophages derived from a human leukemic cell line (THP-1) as shown by the enhanced secretion of TGF-β and CCL18. Interestingly, the conditioned media of polarized M2 macrophages on TCP-S enhanced tube formation by human umbilical vein endothelial cells (HUVECs), while had no influence on the osteogenic differentiation of human bone marrow stromal cells (HBMSCs). Following an intramuscular implantation in canines, TCP-S locally increased typical M2 macrophage markers (e.g., IL-10) at week 1 to 3 and enhanced blood vessel formation after week 3 as compared to TCP-B. Bone formation was observed histologically in TCP-S 6 weeks after implantation, and bone formation was inhibited when an angiogenesis inhibitor (KRN633) was loaded onto TCP-S. No bone formation was observed for TCP-B. The data presented herein suggest strong links between macrophage polarization, angiogenesis and CaP-induced bone formation. Statement of significanceThe ability of calcium phosphate (CaP) materials to induce bone formation varies with their physicochemical properties, and the key physicochemical properties relevant to CaP-induced bone formation have been outlined in the last two decades. However, the biological mechanism underlying this material-driven osteoinduction remains largely unknown. This manuscript presented demonstrates strong links between surface topography, macrophage polarization, angiogenesis and bone formation in CaP materials implanted in non-osseous sites. The finding may provide new clues for further exploring the possible mechanism underlying osteoinduction by CaP materials.

Coupling between Macrophage Phenotype, Angiogenesis and Bone Formation by Calcium Phosphates

The ability of calcium phosphate (CaP) materials to induce bone formation varies with their physicochemical properties, with surface topography as one of the most crucial triggers. In view of the natural wound healing processes initiated after surgical implantation, involving inflammation, angiogenesis, tissue formation and remodeling, we here comparatively investigated the biological cascades occurring upon ectopic implantation of a tricalcium phosphate with submicron surface topography (TCP-S, osteoinductive) and a tricalcium phosphate with micron-scale topography (TCP-B, non-osteoinductive). In vitro, TCP-S facilitated M2 polarization of macrophages derived from a human leukemic cell line (THP-1) as shown by the enhanced secretion of TGF-β and CCL18. Interestingly, the conditioned media of polarized M2 macrophages on TCP-S enhanced tube formation by human umbilical vein endothelial cells (HUVECs), while had no influence on the osteogenic differentiation of human bone marrow stromal cells (HBMSCs). Following implantation in a canine intramuscular defect, TCP-S locally increased typical M2 macrophage markers (e.g. IL-10) at week 1 and enhanced blood vessel formation after week 3 compared to TCP-B. Bone formation was observed histologically for TCP-S 6 weeks after implantation, and bone formation was reduced when an angiogenesis inhibitor (KRN633) was added to TCP-S. No bone formation was observed for TCP-B. The data here presented demonstrates strong links between surface topography, macrophage polarization, angiogenesis and bone formation in CaP materials implanted in non-osseous sites.

Regulation of Osteoblast Differentiation by Acid-Etched and/or Grit-Blasted Titanium Substrate Topography Is Enhanced by 1,25(OH)2D3 in a Sex-Dependent Manner.

This study assessed contributions of micron-scale topography on clinically relevant titanium (Ti) to differentiation of osteoprogenitor cells and osteoblasts; the interaction of this effect with 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3); and if the effects are sex-dependent. Male and female rat bone marrow cells (BMCs) were cultured on acid-etched (A, R a = 0.87 μm), grit-blasted (GB, R a = 3.90 μm), or grit-blasted/acid-etched (SLA, R a = 3.22 μm) Ti. BMCs were sensitive to surface topography and underwent osteoblast differentiation. This was greatest on SLA; acid etching and grit blasting contributed additively. Primary osteoblasts were also sensitive to SLA, with less effect from individual structural components, demonstrated by enhanced local factor production. Sex-dependent responses of BMCs to topography varied with parameter whereas male and female osteoblasts responded similarly to surface treatment. 1α,25(OH)2D3 enhanced cell responses on all surfaces similarly. Effects were sex-dependent and male cells grown on a complex microstructured surface were much more sensitive than female cells. These results indicate that effects of the complex SLA topography are greater than acid etching or grit blasting alone on multipotent BMCs and committed osteoblasts and that individual parameters are sex-specific. The effect of 1α,25(OH)2D3 was sex dependent. The results also suggest that levels of 1α,25(OH)2D3 in the patient may be important in osseointegration.

Characterization of Nb SRF cavity materials by white light interferometry and replica techniques

Much work has shown that the topography of the interior surface is an important contributor to the performance of Nb superconducting radiofrequency (SRF) accelerator cavities. Micron-scale topography is implicated in non-linear loss mechanisms that limit the useful accelerating gradient range and impact cryogenic cost. Aggressive final chemical treatments in cavity production seek to reliably obtain “smoothest” surfaces with superior performance. Process development suffers because the cavity interior surface cannot be viewed directly without cutting out pieces, rendering the cavities unavailable for further study. Here we explore replica techniques as an alternative, providing imprints of cavity internal surface that can be readily examined. A second matter is the topography measurement technique used. Atomic force microscopy (AFM) has proven successful, but too time intensive for routine use in this application. We therefore introduce white light interferometry (WLI) as an alternative approach. We examined real surfaces and their replicas, using AFM and WLI. We find that the replica/WLI is promising to provide the large majority of the desired information, recognizing that a trade-off is being made between best lateral resolution (AFM) and the opportunity to examine much more surface area (WLI).

Osteoblastic and cytokine gene expression of implant‐adherent cells in humans

Implant surface topography is a key determinant affecting osteoblastic differentiation and cell-cell signaling of implant-adherent cells. To assess the early osteoinductive and cell-cell signaling events in adherent cells, commercially pure titanium implants (2.2×5mm) with nanotopography (HF-treated TiO2 grit-blasted) were compared with micron-scale topography TiO2 grit-blasted (micron-scale, control) implants in vivo. Six implants (n=3/surface) were placed in 10 systemically healthy subjects and removed by reverse threading at 1, 3, and 7days. Gene expression profiles of adherent cells were interrogated using low-density RT-PCR arrays. Osteoinduction was not observed at day 1 on either surface. At 3days, elevated levels of BMP6, osteopontin, and osterix (OSX) were observed in RNA of cells adherent to both micron-scale and nanotopography surfaces. Both surfaces supported osteoinductive gene expression at 7days; however, modest elevations of most mRNAs and significantly higher OSX mRNA levels were measured for cells adhered to nanotopography implants. Further, chemokine and cytokine profiles including CXCL10, CXCL14, IL-9, IL-22, and TOLLIP were upregulated on nanotopographic surfaces as compared with microtopographic surfaces. Implants with superimposed nanoscale topography generate a greater induction of genes linked to osteogenesis and cell-cell signaling during the early phases of osseointegration.

The effect of different surface morphology and roughness on osteoblast‐like cells

Increased magnitude of biomaterial surface roughness and micromachined-grooved surfaces has both been shown to stimulate osteoblast activity, but have not been compared in the same study quantitatively. A series of titanium alloy (Ti6Al4V) samples were prepared using simple machining techniques to undertake such a comparison. Samples were either grit blasted (Gb) or shot peened (Sp) to give random discontinuities, or silicon carbide ground (SiC) to produce ordered grooves. These were compared with micropolished samples (Mp). The samples were coated with a 1 mum continuous coating of hydroxyapatite to remove differences in surface chemistry. Human osteoblast-like cells were seeded onto the materials and metabolic activity, proliferation, alkaline phosphatase activity, and osteocalcin production assessed. Cell responses were highly dependent on the substrate that they were cultured on. Cells cultured on the smooth and ordered (Mp and SiC, respectively) samples had higher metabolic activity and a more elongated morphology than those cultured on the randomly structured Gb or Sp samples. Over 21 days, cell metabolic activity peaked relative to the control between 7 and 14 days on the Mp sample, and between 14 and 21 days on the Gb, Sp, and SiC samples. In common with other researchers, we note that micron scale topography may have potential for influencing osseointegration. More importantly, as the magnitude of the discontinuities on SiC, Gb, and Sp were similar, the differences in cell responses does not appear to lie with the size of the features, but whether the features showed an ordered or disordered structure.

Integrin α5 controls osteoblastic proliferation and differentiation responses to titanium substrates presenting different roughness characteristics in a roughness independent manner

Integrin alpha(5)beta(1) regulates osteoblast proliferation and differentiation on smooth synthetic surfaces presenting different chemistries, but it is not known whether this integrin controls osteoblast behavior on surfaces that have micron-scale rough topographies. We cultured MG63 human osteoblast-like cells on titanium substrates with three different roughness characteristics: chemically polished (PT), grit blasted and acid etched with a complex topography consisting of 20-100 mum craters and 0.5-2 mum micropits (SLA), and plasma-sprayed Ti with irregular projections (TPS). Cells spread well on PT but displayed a smaller footprint on SLA or TPS. Nuclei were larger on PT as well. alpha(5)beta(1) binding and FAK phosphorylation were greater on the rougher surfaces (TPS > SLA > PT). Antibodies against the alpha(5)beta(1) binding site on fibronectin had no effect on cell number at 3 days, but [(3)H]-thymidine incorporation was increased, suggesting that binding to fibronectin was necessary for cell cycle regulation. Antibodies to the alpha(5) subunit reduced cell number at 3 days on PT and TPS and reduced DNA synthesis on all substrates in a surface microstructure-independent manner. At 7 days, cell numbers were reduced on PT, and DNA synthesis was reduced by 50% on all surfaces. At 7 days, anti-alpha(5) antibodies caused a partial reduction in alkaline phosphatase enzyme activity on all surfaces, but this effect was independent of surface microstructure. These results indicate that surface micron-scale topography modulates alpha(5)beta(1) integrin binding and FAK activation. Signaling via alpha(5)-dependent mechanisms is required for DNA synthesis and regulation of alkaline phosphatase, but this effect is independent of surface microstructure.

Microstructural and tensile properties of elastin‐based polypeptides crosslinked with Genipin and pyrroloquinoline quinone

Elastin is an elastomeric, self-assembling extracellular matrix protein with potential for use in biomaterials applications. Here, we compare the microstructural and tensile properties of the elastin-based recombinant polypeptide (EP) EP20-244 crosslinked with either genipin (GP) or pyrroloquinoline quinone (PQQ). Recombinant EP-based sheets were produced via coacervation and subsequent crosslinking. The micron-scale topography of the GP-crosslinked sheets examined with atomic force microscopy revealed the presence of extensive mottling compared with that of the PQQ-crosslinked sheets, which were comparatively smoother. Confocal microscopy showed that the subsurface porosity in the GP-crosslinked sheets was much more open. GP-crosslinked EP-based sheets exhibited significantly greater tensile strength (P < or = 0.05). Mechanistically, GP appears to yield a higher crosslink density than PQQ, likely due to its capacity to form short-range and long-range crosslinks. In conclusion, GP is able to strongly modulate the microstructural and mechanical properties of elastin-based polypeptide biomaterials forming membranes with mechanical properties similar to native insoluble elastin.

Osteoblast‐like cells are sensitive to submicron‐scale surface structure

Studies showing that osteoblasts exhibit a more differentiated phenotype on rough titanium (Ti) surfaces and osteoclast-resorbed bone surfaces used materials characterized by average peak to valley distance (Ra). Other surface features impacting the cells include distance between peaks, curvature of the valleys, and relative distribution of flat and smooth regions. We used novel Ti surfaces prepared by electrochemical micromachining as models to examine specific contributions of individual design features to osteoblast response. Results show that micron-scale topography modulates cell number, cell morphology and prostaglandin E2 (PGE2). In the presence of the appropriate microtopography, submicron-scale rugosity modulates differentiation and transforming growth factor-beta1 (TGF-beta1) levels. In this study, we examined the role of different types of submicron-scale structures. Thirty micrometer diameter craters on Ti disks were produced by photolithography resulting in an electropolished smooth surface, and arranged so that inside crater area vs. outside flat area was 6 (30/6). Submicron-scale structures were superposed by acid etching and porous anodization. Ra's were 700, 400, 60 nm on acid-etched, porous anodized and smooth 30/6 surfaces, respectively. MG63 osteoblast-like cells were sensitive to submicron-scale architecture. Cell morphology on anodized surfaces was similar to morphology on smooth surfaces, whereas on etched surfaces, cells had a more elongated differentiated shape. Cell number was greatest on smooth surfaces > anodized > etched. Osteocalcin and PGE2 were affected in a reverse manner. Active TGF-beta1 was greatest on etched 30/6 surfaces > anodized > smooth; latent TGF-beta1 was elevated on all rough surfaces. These results support our previous observations that submicron-scale structures modulate osteoblastic phenotype and show that the physical properties of the submicron-scale structures are important variables in determining osteoblast response to substrate topography.