Selective cell affinity of biomimetic micro-nano-hybrid structured TiO 2overcomes the biological dilemma of osteoblasts

Abstract

Objective

There is a great demand for dental implant surfaces to accelerate the process of peri-implant bone generation to reduce its healing time and enable early loading. To this end, an inverse correlation between the proliferation and functional maturation (differentiation) in osteoblasts presents a challenge for the rapid generation of greater amounts of bone. For instance, osteoblasts exhibit faster differentiation but slower proliferation on micro-roughened titanium surfaces. Using a unique micro-nano-hierarchical topography of TiO 2 that mimics biomineralized matrices, this study demonstrates that this challenge can be overcome without the use of biological agents.

Methods

Titanium disks of grade 2 commercially pure titanium were prepared by machining (smooth surface). To create a microtexture with peaks and valleys (micropit surface), titanium disks were acid-etched. To create 200-nm TiO 2 nanonodules within the micropits (nanonodule-in-micropit surface), TiO 2 was sputter-deposited onto the acid-etched surface. Rat bone marrow-derived osteoblasts and NIH3T3 fibroblasts were cultured on machined smooth, micropit, and nanonodule-in-micropit surfaces.

Results

Despite the substantially increased surface roughness, the addition of 200-nm nanonodules to micropits increased osteoblast proliferation while enhancing their functional differentiation. In contrast, this nanonodule-in-micropit surface decreased proliferation and function in fibroblasts.

Significance

The data suggest the establishment of cell-selectively functionalized nano-in-micro smart titanium surfaces that involve a regulatory effect on osteoblast proliferation, abrogating the inhibitory mechanism on the micropitted surface, while enhancing their functional differentiation. Biomimetic and controllable nature of this nanonodules-in-micropits surface may offer a novel micro-to-nanoscale hierarchical platform to biologically optimize nanofeatures of biomaterials. Particularly, this micro-nano-hybrid surface may be an effective approach to improve current dental implant surfaces for accelerated bone integration.

Introduction

A major goal in developing successful implant materials in bone and bone engineering scaffolds is the rapid formation of greater amounts of bone tissue. A considerable effort has been made to find microfeatures of material surfaces that create better bioactivity . Generally, microfeatures promote the rate of differentiation of differentiating cells, e.g., stem cell/osteoblastic differentiation . Several microtopographical textures have been implemented in various commercial implant devices in dental and orthopedic fields .

There is one barrier that has to be overcome for faster generation of greater amounts of bone. A biological dilemma exists in osteoblasts, due to an inverted correlation between proliferation and differentiation rates . Proliferation and differentiation are regulated by growth factors that counteract each other in osteoblasts . This applies to the bone formation around biomaterials. For instance, micro-roughened titanium surfaces have advantages over machined, smooth surfaces in that they promote osteoblastic differentiation , resulting in faster onset of bone formation . The bone mass, however, is smaller than that around the machined surface , due to the diminished osteoblastic proliferation . To compensate for the reduced rate of proliferation in the differentiating osteoblasts, the treatment of the cells with growth factors, e.g., transforming growth factors (TGF), that specifically stimulate their proliferation, may be a biological strategy . We hypothesized that addition of another phase of structures on the existing microstructures would offer an optimized osteogenic environment, improving the undesirable biological properties of the micro-featured surface while preserving their established desirable properties of bioactivity.

Biological tissues are structurally heterogeneous, and created in biologically complex, yet sophisticated and functionally adapted hierarchy in living systems . Mimicking more fundamental scales of the constituent components of biological tissue may provide additional functionality to biomaterials (concept of biomimetics) . In light of the recent advancement in surface technology from micronscale to nanoscale, a hierarchical organization of heterogeneous surface structures, in particular, with the coexistence of micro- and nanofeatures should be explored for optimized biological interfaces. Here, we show a micro-nano-hybrid structured TiO 2 surface that resembles biomineralized matrices, and is selectively functionalized for allowing bone-forming cells to modify their undesirable biological principles.

Technically, the creation of controllable hybrid of nano- and micro-structures has been challenging. Recently, controllable self-assembly of nanonodules that occurs during chemical depositioning of materials onto specifically conditioned microtopographical surfaces was reported . The emergence of the nanonodules was associated with substrate surface topography that was >200 nm in the root mean square roughness and >400 nm in the peak-to-valley roughness . Also, the inter-irregularities space ranging 1000 nm to 1.5 μm seems to develop well-isolated and defined nanonodules. The nanonodular self-assembly does not take place on relatively smooth surfaces without micron-level configuration, such as machined metal surfaces . This self-assembly was proven to be feasible in various types of target and substrate materials, even in a heterogeneous combination. The substrate can also be non-metallic materials, such as the biodegradable polymers used for tissue engineering scaffolds, under a condition that they have the required microarchitecture. In this report, we explore the creation of biocompatible surfaces with micro-nano-hybrid features utilizing this technique and further investigate its potential as a biological interface. We used TiO 2 as a target for depositioning and bulk titanium as a substrate material because they are proven biocompatible materials and are extensively used as implantable devices in dental and orthopedic therapies as well as tissue engineering materials. Despite the well-established record of implant treatment as a standard treatment modality in modern dental reconstructive works, there is still a desire to accelerate the process of osseointegration to shorten the healing time and to ensure early loading.

Methods

Creation of TiO 2 micro-nano-hybrid topography

Titanium disks (20 mm in diameter and 1 mm in thickness) of grade 2 commercially pure titanium were prepared by machining (machine-smooth surface). To create a microtexture with peaks and valleys (micropit surface), titanium samples were acid-etched with 3% HF and 66% H 2 SO 4 . We employed the previously established method of controllable nanonodule self-assembly to create 200-nm TiO 2 nanonodular structures within the micropits . TiO 2 was deposited onto the acid-etched surface using a sputter deposition system (Denton Discovery 550, Moorestown, NJ) for 108 min with a deposition rate of 18.5 Å/min according to the established equation for the diameter of nanonodules. The surface morphology of these surfaces was examined by scanning electron microscopy (SEM) (XL30, Philips, Eindhoven, Netherlands) and a laser profile microscope (VK-8500, KEYENCE, Osaka, Japan) was used to measure the average roughness, peak-to-valley length, inter-irregularity space (Sm), and surface area.

Protein adsorption assay

Bovine serum albumin, fraction V (Pierce Biotechnology, Inc., Rockford, IL) was used as model proteins. Following the established protocol , 300 μl of protein solution (1 mg/ml protein/saline) was pipetted onto titanium disks with either micropits alone or micropits with 125 nm nodules. After incubation for 2 and 24 h at 37 °C, nonadherent proteins were removed and mixed with microbicinchoninic acid (Pierce Biotechnology) at 37 °C for 60 min. The amount of removed albumin, as well as the total amount of albumin inoculated, was quantified using a microplate reader at 562 nm. The rate of albumin adsorption was calculated as the percentage of albumin adsorbed to titanium disks relative to the total amount.

Cell culture

Osteoblasts were isolated from bone marrow cells following the established protocol . Bone marrow cells were harvested from the femur of 8-week-old male Sprague–Dawley rats and placed into alpha-modified Eagle’s medium supplemented with 15% FBS 50 mg/ml ascorbic acid, 10 mM Na-β-glycerophosphate, 10 −8 M dexamethasone and antibiotic-antimycotic solution containing 10,000 units/ml penicillin G sodium, 10,000 mg/ml streptomycin sulfate and 25 mg/ml amphotericin B. Cells were incubated in a humidified atmosphere of 95% air, 5% CO 2 at 37 °C. At 80% confluency, the cells were detached using 0.25% Trypsin-1 mM EDTA-4Na and seeded onto titanium disks with machine-smooth, micropitted, or micro-nano-hybrid surfaces at a density of 5 × 10 4 cells/cm 2 . NIH3T3 fibroblasts, cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Grand Island, NY), supplemented with 10% Fetal Bovine Serum and antibiotic–antimycotic solution, were also inoculated onto titanium disks. To compare the morphology of the micro-nano-hybrid titanium surface and biomineralized matrices, osteoblasts were cultured on culture-grade polystyrene dishes for 14 days for SEM examination. The culture medium was renewed every 3 days for both cell types. The University of California at Los Angeles Chancellor’s Animal Research Committee approved this protocol and all experimentation was performed in accordance with the United States Department of Agriculture guidelines for animal research.

Cell attachment, density, and proliferation assays

Initial attachment of osteoblasts and fibroblasts to titanium surfaces was evaluated by measuring the amount of cells attached to titanium substrates after 6 and 24 h of incubation. The propagated cells were quantified in terms of cell density at 2 and 5 culture days. Both quantifications were performed using WST-1 based colorimetry (WST-1, Roche Applied Science, Mannheim, Germany). The culture well was incubated at 37 °C for 4 h with 100 μl tetrazolium salt (WST-1) reagent. The amount of formazan product was measured using an ELISA reader at 420 nm. The proliferative activity of cells was measured by BrdU incorporation during DNA synthesis. At day 2 of culture, 100 μl of 100 mM BrdU solution (Roche Applied Science) was added to the culture wells and incubated for 10 h. After trypsinizing cells and denaturing DNA, cultures were incubated with anti-BrdU conjugated with peroxidase for 90 min and reacted with tetramethylbenzidine for color development. Absorbance at 370 nm was measured using an ELISA reader (Synergy HT, BioTek Instruments, Winooski, VT).

Morphology and morphometry of cells

Confocal laser scanning microscopy was used to examine cell morphology and cytoskeletal arrangement in osteoblasts and fibroblasts seeded onto various titanium surfaces. After 6 h of culture, cells were fixed in 10% formalin and stained using the fluorescent dye rhodamine phalloidin (actin filament, red color; Molecular Probes, OR). Cultures were also immunochemically stained with mouse anti-vinculin monoclonal antibody (Abcam, Cambridge, MA), followed by a FITC-conjugated anti-mouse secondary antibody (Abcam). The area, perimeter and Feret’s diameter of cells were quantified using an image analyzer (ImageJ, NIH, Bethesda, ML).

Alkaline phosphatase (ALP) activity

ALP activity of osteoblasts was examined at day 7 using a colorimetry-based assay. Cultures were rinsed with double-distilled water (ddH 2 O), followed by the addition of 250 μl p-nitrophenylphosphate (LabAssay ATP, Wako Pure Chemicals, Richmond, VA) and then incubated at 37 °C for 15 min. ALP activity was evaluated as the amount of nitrophenol released through the enzymatic reaction and measured at 405 nm using an ELISA reader, and then standardized relative to cell number.

Gene expression analysis

Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA in osteoblasts cultured for 7 days was extracted using TRIzol (Invitrogen, Carlsbad, CA) on a purification column (RNeasy, Qiagen, Valencia, CA). Following DNAse I treatment, reverse transcription of 0.5 μg of total RNA was performed using MMLV reverse transcriptase (Clontech, Carlsbad, CA) in the presence of oligo(dT) primers (Clontech). PCR reaction was performed using Taq DNA polymerase (EX Taq, Takara Bio, Madison, WN) to detect osteopontin and osteocalcin mRNA using the primer designs and PCR conditions optimized previously . PCR products were visualized on 1.5% agarose gel by ethidium bromide staining. Band intensity was detected and quantified under UV light and normalized to GAPDH mRNA.

Mineralization assay

The mineralization capability of cultured osteoblasts was examined by o-cresolphthalein complexone method at day 21. Cultures were washed with PBS and incubated overnight in 1 ml of 0.5 M HCl solution with gentle shaking. The solution was mixed with o-cresolphthalein complexone in an alkaline medium (calcium-binding and buffer reagent, Sigma, St Louis, MO) to produce a red calcium–cresolphthalein complexone complex. Color intensity was measured by an ELISA reader at 575 nm absorbance.

Collagen production

Sirius red staining-based colorimetric assay was employed to quantify collagen production in fibroblasts as reported previously . Cultures on titanium disks were washed with 1× PBS and fixed with Bouin’s fluid for 1 h at room temperature. Following the treatment with 0.2% aqueous phosphomolybdic acid, the day 7 cultures were stained with Sirius red dye (C.I. No. 35780, Pfaltz and Bauer, Stamford, CT, USA) for 90 min. After non-bound dye was removed with 0.01 N hydrochloric acid, the culture was incubated in 600 μl of 0.1 N sodium hydroxide for 30 min at room temperature. Then, the optical density of the solution was measured using a spectrophotometer at 550 nm. To visualize collagen deposition, cultures were examined using a confocal laser scanning microscope following dual staining with Sirius red and a green fluorescent nucleic-acid stain (SYTO 13, Molecular Probe, Eugene, OR).

Statistical analyses

The number of samples was three for all studies, except for roughness analyses for titanium substrates ( n = 9) and cell morphometry ( n = 10). Two-way ANOVA was used to examine the effects of culture/healing time and titanium surface features. If necessary, the post hoc Bonferroni test was used as a multiple comparison test; p < 0.05 was considered significant. If data were available at only one time point, one-way ANOVA was used to determine the differences among different surface groups.

Methods

Creation of TiO 2 micro-nano-hybrid topography

Titanium disks (20 mm in diameter and 1 mm in thickness) of grade 2 commercially pure titanium were prepared by machining (machine-smooth surface). To create a microtexture with peaks and valleys (micropit surface), titanium samples were acid-etched with 3% HF and 66% H 2 SO 4 . We employed the previously established method of controllable nanonodule self-assembly to create 200-nm TiO 2 nanonodular structures within the micropits . TiO 2 was deposited onto the acid-etched surface using a sputter deposition system (Denton Discovery 550, Moorestown, NJ) for 108 min with a deposition rate of 18.5 Å/min according to the established equation for the diameter of nanonodules. The surface morphology of these surfaces was examined by scanning electron microscopy (SEM) (XL30, Philips, Eindhoven, Netherlands) and a laser profile microscope (VK-8500, KEYENCE, Osaka, Japan) was used to measure the average roughness, peak-to-valley length, inter-irregularity space (Sm), and surface area.

Protein adsorption assay

Bovine serum albumin, fraction V (Pierce Biotechnology, Inc., Rockford, IL) was used as model proteins. Following the established protocol , 300 μl of protein solution (1 mg/ml protein/saline) was pipetted onto titanium disks with either micropits alone or micropits with 125 nm nodules. After incubation for 2 and 24 h at 37 °C, nonadherent proteins were removed and mixed with microbicinchoninic acid (Pierce Biotechnology) at 37 °C for 60 min. The amount of removed albumin, as well as the total amount of albumin inoculated, was quantified using a microplate reader at 562 nm. The rate of albumin adsorption was calculated as the percentage of albumin adsorbed to titanium disks relative to the total amount.

Cell culture

Osteoblasts were isolated from bone marrow cells following the established protocol . Bone marrow cells were harvested from the femur of 8-week-old male Sprague–Dawley rats and placed into alpha-modified Eagle’s medium supplemented with 15% FBS 50 mg/ml ascorbic acid, 10 mM Na-β-glycerophosphate, 10 −8 M dexamethasone and antibiotic-antimycotic solution containing 10,000 units/ml penicillin G sodium, 10,000 mg/ml streptomycin sulfate and 25 mg/ml amphotericin B. Cells were incubated in a humidified atmosphere of 95% air, 5% CO 2 at 37 °C. At 80% confluency, the cells were detached using 0.25% Trypsin-1 mM EDTA-4Na and seeded onto titanium disks with machine-smooth, micropitted, or micro-nano-hybrid surfaces at a density of 5 × 10 4 cells/cm 2 . NIH3T3 fibroblasts, cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Grand Island, NY), supplemented with 10% Fetal Bovine Serum and antibiotic–antimycotic solution, were also inoculated onto titanium disks. To compare the morphology of the micro-nano-hybrid titanium surface and biomineralized matrices, osteoblasts were cultured on culture-grade polystyrene dishes for 14 days for SEM examination. The culture medium was renewed every 3 days for both cell types. The University of California at Los Angeles Chancellor’s Animal Research Committee approved this protocol and all experimentation was performed in accordance with the United States Department of Agriculture guidelines for animal research.

Cell attachment, density, and proliferation assays

Initial attachment of osteoblasts and fibroblasts to titanium surfaces was evaluated by measuring the amount of cells attached to titanium substrates after 6 and 24 h of incubation. The propagated cells were quantified in terms of cell density at 2 and 5 culture days. Both quantifications were performed using WST-1 based colorimetry (WST-1, Roche Applied Science, Mannheim, Germany). The culture well was incubated at 37 °C for 4 h with 100 μl tetrazolium salt (WST-1) reagent. The amount of formazan product was measured using an ELISA reader at 420 nm. The proliferative activity of cells was measured by BrdU incorporation during DNA synthesis. At day 2 of culture, 100 μl of 100 mM BrdU solution (Roche Applied Science) was added to the culture wells and incubated for 10 h. After trypsinizing cells and denaturing DNA, cultures were incubated with anti-BrdU conjugated with peroxidase for 90 min and reacted with tetramethylbenzidine for color development. Absorbance at 370 nm was measured using an ELISA reader (Synergy HT, BioTek Instruments, Winooski, VT).

Morphology and morphometry of cells

Confocal laser scanning microscopy was used to examine cell morphology and cytoskeletal arrangement in osteoblasts and fibroblasts seeded onto various titanium surfaces. After 6 h of culture, cells were fixed in 10% formalin and stained using the fluorescent dye rhodamine phalloidin (actin filament, red color; Molecular Probes, OR). Cultures were also immunochemically stained with mouse anti-vinculin monoclonal antibody (Abcam, Cambridge, MA), followed by a FITC-conjugated anti-mouse secondary antibody (Abcam). The area, perimeter and Feret’s diameter of cells were quantified using an image analyzer (ImageJ, NIH, Bethesda, ML).

Alkaline phosphatase (ALP) activity

ALP activity of osteoblasts was examined at day 7 using a colorimetry-based assay. Cultures were rinsed with double-distilled water (ddH 2 O), followed by the addition of 250 μl p-nitrophenylphosphate (LabAssay ATP, Wako Pure Chemicals, Richmond, VA) and then incubated at 37 °C for 15 min. ALP activity was evaluated as the amount of nitrophenol released through the enzymatic reaction and measured at 405 nm using an ELISA reader, and then standardized relative to cell number.

Gene expression analysis

Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA in osteoblasts cultured for 7 days was extracted using TRIzol (Invitrogen, Carlsbad, CA) on a purification column (RNeasy, Qiagen, Valencia, CA). Following DNAse I treatment, reverse transcription of 0.5 μg of total RNA was performed using MMLV reverse transcriptase (Clontech, Carlsbad, CA) in the presence of oligo(dT) primers (Clontech). PCR reaction was performed using Taq DNA polymerase (EX Taq, Takara Bio, Madison, WN) to detect osteopontin and osteocalcin mRNA using the primer designs and PCR conditions optimized previously . PCR products were visualized on 1.5% agarose gel by ethidium bromide staining. Band intensity was detected and quantified under UV light and normalized to GAPDH mRNA.

Mineralization assay

The mineralization capability of cultured osteoblasts was examined by o-cresolphthalein complexone method at day 21. Cultures were washed with PBS and incubated overnight in 1 ml of 0.5 M HCl solution with gentle shaking. The solution was mixed with o-cresolphthalein complexone in an alkaline medium (calcium-binding and buffer reagent, Sigma, St Louis, MO) to produce a red calcium–cresolphthalein complexone complex. Color intensity was measured by an ELISA reader at 575 nm absorbance.

Collagen production

Sirius red staining-based colorimetric assay was employed to quantify collagen production in fibroblasts as reported previously . Cultures on titanium disks were washed with 1× PBS and fixed with Bouin’s fluid for 1 h at room temperature. Following the treatment with 0.2% aqueous phosphomolybdic acid, the day 7 cultures were stained with Sirius red dye (C.I. No. 35780, Pfaltz and Bauer, Stamford, CT, USA) for 90 min. After non-bound dye was removed with 0.01 N hydrochloric acid, the culture was incubated in 600 μl of 0.1 N sodium hydroxide for 30 min at room temperature. Then, the optical density of the solution was measured using a spectrophotometer at 550 nm. To visualize collagen deposition, cultures were examined using a confocal laser scanning microscope following dual staining with Sirius red and a green fluorescent nucleic-acid stain (SYTO 13, Molecular Probe, Eugene, OR).

Statistical analyses

The number of samples was three for all studies, except for roughness analyses for titanium substrates ( n = 9) and cell morphometry ( n = 10). Two-way ANOVA was used to examine the effects of culture/healing time and titanium surface features. If necessary, the post hoc Bonferroni test was used as a multiple comparison test; p < 0.05 was considered significant. If data were available at only one time point, one-way ANOVA was used to determine the differences among different surface groups.

Results

Creation of TiO 2 micro-nano-hybrid structured surface

We aimed to create a micro-nano-hybrid feature of titanium consisting of nodules with an appropriate nanoscale size within micropits, as schematically illustrated in Fig. 1 a . We took advantage of the micropitted titanium surface created by acid-etching that is well characterized topographically and biologically, and extensively used in dental and orthopedic implants. The micropits render: (1) increased roughness, (2) sharp ridges, (3) pits, and (4) increased surface area as compared to smoother surfaces, such as machined titanium surface ( Fig. 1 a). After producing nanonodular structures on the micropit surface, we aimed to enhance the surface morphology with (1) further increased roughness, (2) rounded ridges, (3) even deeper pits, (4) even larger surface areas, and (5) extensive geographical undercuts. First, the smooth Ti surface was prepared by machining commercially pure Ti disks. The micropits, with compartmental architectures ranging 0.5–1.5 μm in peak-to-peak distance (average, approximately 1 μm), were created by acid-etching the disks using a combination of hydrofluoric acid and sulfuric acid. This micropit feature is one of the microtextures that allow to induce the nanonodular self-assembly as we previously demonstrated . To create 200 nm nanonodules, TiO 2 sputter deposition was performed onto the micropitted titanium surfaces was performed with a deposition rate of 18.5 Å/min. According to the equation established previously, the deposition time was adjusted to 108 min, which resulted in an actual measured nodule diameter of 198.5 ± 22.3 nm (coefficient variation = 11.2%).

Nov 30, 2017 | Posted by in Dental Materials | Comments Off on Selective cell affinity of biomimetic micro-nano-hybrid structured TiO 2overcomes the biological dilemma of osteoblasts
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