Differentiation of human mesenchymal stem cells on niobium-doped fluorapatite glass-ceramics

Abstract

Objective

Our goal was to characterize the response of human mesenchymal stem cells (hMSCs) to a niobium-doped fluorapatite-based glass-ceramic (FAp).

Methods

The glass was prepared by twice melting at 1525 °C for 3 h, and cast into cylindrical ingots later sectioned into discs and heat-treated to promote crystallization of fluorapatite submicrometer crystals. Tissue culture polystyrene (TCP) was used as control. The surface of the FAp discs was either left as-heat treated, ground or etched. Initial cell attachment was assessed at 3 h. Proliferation and alkaline phosphatase (ALP) expression data were collected at days 1, 4, and 8. Cell morphology was examined using SEM, at days 2 and 4. Mineralization was evaluated by Alizarin Red staining and SEM.

Results

Initial cell attachment on as heat-treated, etched, or ground surfaces was similar to that of the positive control group ( p > 0.05). The percentage of area covered by living cells increased significantly on as heat-treated, etched, or ground surfaces between days 1 and 8 ( p < 0.05). There was no significant difference among groups in cell coverage at day 8, compared to TCP control. SEM revealed well spread polygonal cells with numerous filopodia, either attached to the ceramic surface or connected to neighboring cells. ALP expression at day 8 was significantly higher in osteogenic media compared to growth media on both FAp and control. FAp discs stained positively with Alizarin Red and calcium-rich mineralized granules associated with fibrils were observed by SEM at day 35.

Significance

hMSCs displayed excellent attachment, proliferation, and differentiation on niobium-doped FAp glass-ceramic.

Introduction

Currently available materials for bone replacement include autologous bone, allografts, xenografts, and various synthetic materials. Autologous bone, is considered a gold standard but presents some drawbacks, such as donor site morbidity and, in some cases, limited availability . Both allografts and xenografts possess shortcomings of potential disease transmission, unpredictable resorption rate, and graft rejection . To overcome these challenges, synthetic materials are being developed, among which bioactive glass-ceramics have raised considerable interest. A biomaterial is described as bioactive when “an interfacial bond forms between the tissue and the implant” . Bioactive glass-ceramics encompass a wide variety of crystalline phases, ranging from micas to calcium silicates and various apatites . Fluorapatite glass-ceramics containing mica have been commercially available for use as inter-vertebral spacers and ossicles of the middle ear since the early 1990s .

When selecting materials for bone replacement, surface topography is considered to be critical in determining the fate of early peri-implant healing . It is well established that topographical features provide cues through focal adhesions that can interact with cell proliferation and differentiation. This is true at various scales from macrotopographical features to nanotopographical features. The placement of an implant at a surgical site triggers a cascade of molecular and cellular activities that leads to new tissue formation on the biomaterial surface. Briefly, fibrin from the blood is adsorbed on the implant surface, to which the platelets adhere and get activated. These activated platelets then release cytokines to recruit undifferentiated cells on implant site and trigger their osteogenic differentiation (osteoinduction). The combination of recruitment and migration of osteogenic cells (osteoconduction) and bone formation by these cells on the implant surface is known as contact osteogenesis . This phenomenon can be favorably assisted by the presence of a microtopographically complex implant surface by increasing the available surface area for fibrin attachment, leading to enhanced platelet activation followed by the recruitment of osteogenic cells. Additionally, efficient bone binding may occur through inter-digitation of newly formed mineralized matrix . Titanium implants modified by nano-crystalline deposition have been shown to improve osteoconduction in vitro . Acid-etching of titanium implants was also shown to lead to enhanced endosseous integration in a rabbit model . Moreover, a gradually increasing body of preclinical and clinical findings associates topographic features at the nanometer and submicrometer levels and improved implant integration .

Fluorapatite (FAp) is structurally and chemically similar to hydroxyapatite, and is known to be more chemically stable and easier to synthesize as a stoichiometric compound . Additionally, FAp can provide fluoride release at a controlled rate and several studies have demonstrated the stimulating effect of fluoride on bone formation . FAp-based glass-ceramics have long attracted considerable interest . Depending on the composition of the base glass, the crystallization temperature has been shown to vary with the Ca:P ratio and particle size of the glasses, reaching a minimum value for the glasses with the apatite stoichiometry of 1.67 . In vitro studies showed that addition of up to 40 wt.% fluorapatite to thermally sprayed hydroxyapatite coatings led to elevated expression of several proteins involved in bone metabolism regulation in osteoblast cultures . Similarly, fluorapatite glass-ceramic coatings on alumina elicited greater proliferation response of osteoblast-like cells compared to uncoated alumina . In vivo studies have shown that fluorapatite coatings on implants in non-weight-bearing models were more chemically stable than hydroxyapatite coatings . Previous work in our laboratory revealed that doping fluorapatite glass-ceramic compositions with small amounts of niobium oxide promoted the formation of spherical submicrometer fluorapatite crystals (200–300 nm in diameter), while the undoped composition exhibited long needle-shaped crystals (5 μm in length) . The introduction of niobium oxide is thought to decrease the diffusivity of the glassy matrix, thereby limiting the growth of fluorapatite crystals to a sub-micrometer level. Subsequent cytotoxicity experiments with human gingival fibroblasts on these niobium-doped fluorapatite glass-ceramics demonstrated their good cytocompatibility , prompting the need for a more complete assessment of cell attachment, proliferation and differentiation on this material.

Mesenchymal stem cells in the bone marrow stroma are a well-established source of osteoblasts in vivo . Moreover, these newly formed osteoblasts are majorly responsible for de novo bone formation at the time of injury or on implant surface . The purpose of this study was to investigate the behavior of human mesenchymal stem cells (hMSCs) on a niobium-doped fluorapatite glass-ceramic. This experimental glass-ceramic has many potential uses; we anticipate that it could be used as a coating, as bulk part in non-load bearing implants or as a macroporous ceramic scaffold for bone grafts. Our experimental design therefore includes several groups with different surface topographies (as heat-treated, etched or ground) in the initial attachment, cell morphology and cell proliferation studies, with a polished group as negative control in the initial cell attachment evaluation. These surface finishes are directly applicable to bulk implants and coatings. Meanwhile, our research plan focuses on tridimensional macroporous scaffolds, these sponge-like cellular structures will be either left as heat-treated or chemically etched to reveal the unique microstructural features of this material. The cell differentiation studies are thus targeted at chemically etched substrates. The hypotheses tested were that the expression of submicrometer topographical features via various surface treatment techniques could affect initial cell attachment and that these features would provide an enhanced environment for cell proliferation, protein expression, and differentiation. Our long-term goal is to evaluate this glass-ceramic as a bone substitute in order to support osteo-specific differentiation through nanoscale disorder of fluorapatite isometric crystals.

Materials and methods

Specimen preparation

A fluorapatite-based glass composition doped with 1 wt.% of niobium oxide was prepared by mixing reagent grade oxides and carbonates as previously described . The batch ingredients were melted at 1525 °C for 3 h in platinum crucibles. After quenching, the glass frit was powdered and melted again at 1525 °C for 3 h. The glass was cast into pre-heated cylindrical stainless steel molds to form 12 mm × 60 mm rods. Rods were furnace-cooled to room temperature and cut into discs (12 mm in diameter, 1.5 mm thick) using a low speed diamond saw. The glass discs were then heat treated at 950 °C for 1 h to promote nucleation and crystallization of spherical submicrometer fluorapatite crystals, as previously established . Three types of experiments were conducted: (1) initial cell attachment and cell morphology, (2) cell proliferation and (3) alkaline phosphatase (ALP) activity and cell differentiation. Glass-ceramic discs were randomly divided into three groups ( n = 8 per group): specimens were either left as-heat treated, etched with a diluted hydrofluoric solution for 60 s or ground with a 600-grit diamond disc under water irrigation. Additional specimens for initial cell attachment experiments were polished to a 1-μm finish and served as negative controls. As mentioned earlier, ALP activity and cell differentiation experiments were conducted on the etched group only, as most revealing of the submicrometer FAp crystallites. Glass-ceramic specimens were sterilized in a steam autoclave at 134 °C for 30 min. Tissue culture polystyrene (TCP) was used as a positive control for all cell culture experiments.

Cell culture

Human mesenchymal stem cells (hMSCs, Lonza, Walkersville, MD) were cultured in growth medium (Mesenchymal Stem Cell Growth Medium Bullet Kit, Lonza, Walkersville, MD), passaged by trypsinizing (0.05% trypsin/EDTA) and sub-cultured at a density of 2500–5000 cells/cm 2 . Cells between the third and fifth passage were used for all experiments. The cell cultures were maintained under standard culture conditions (37 °C, 95% relative humidity, and 5% CO 2 ). hMSC Osteogenic Bullet Kit (Lonza, Walkersville, MD) was selected for osteogenic induction during experiments. Normal Human Osteoblasts (NhOsts, Lonza, Walkersville, MD) served as positive controls for analysis of extracellular matrix secretion. NhOsts were seeded in growth medium (Osteoblast Growth Medium Bullet Kit) and mineralization was induced by osteogenic medium (Osteogenic bullet Kit, Lonza, Walkersville, MD).

Initial attachment

Discs from each experimental group ( n = 4 per group) were seeded with 3000 cells. Tissue culture polystyrene (TCP) surface was used as positive control. At 3 h, the discs were stained with green calcein dye (Invitrogen, Carlsbad, CA). In order to count the number of live cells on each substrate, digital micrographs were taken at three random locations on each disc. NIH Image J (version 1.440) software was used for digital image analyses . The experiment was performed in duplicate.

Cell proliferation

A live/dead assay was used to assess cell proliferation at days 1, 4 and 8. This well-established assay uses a membrane-permeant calcein-AM dye, which is cleaved by esterases in a live cell, yielding green fluorescence, and a membrane impermeant ethidium homodimer-1, which binds to nucleic acids in membrane, leading to a red fluorescence in compromised cells. For each time point, three steam-sterilized discs from each group were seeded with 3000 cells. TCP was used as positive control. At days 1, 4 and 8, the cell culture medium was replaced with Dulbecco’s phosphate-buffered saline (pH 7.2) containing 0.002 mmol/L calcein-AM and 0.002 mmol/L ethidium homodimer-1 (Invitrogen, Carlsbad, CA). After 30 min of incubation at room temperature, the cells were observed using epifluorescence microscopy (Olympus, BX 51, Tokyo, Japan). Digital micrographs were taken at five random locations on each disc. Images were analyzed using Image J software. The amount of cell attachment was measured on each micrograph as the percentage of specimen surface area covered by live cells.

Early cell morphology

In order to examine the cell–glass ceramic interaction and early cell morphology, hMSCs were cultured on steam-sterilized glass-ceramic discs for 2–4 days and prepared for examination by scanning electron microscopy (SEM). Specimens were rinsed with phosphate buffered saline and fixed in formaldehyde, followed by graded ethanol dehydrations up to final dehydration in absolute ethanol. Specimens were then dried in hexamethyldisilizane (HMDS)/ethanol solution series and gold-coated prior to SEM examination in secondary electron imaging and ultra high-resolution mode (Sirion, FEG scanning electron microscope, FEI Company).

Alkaline phosphatase (ALP) activity

ALP is an enzyme expressed by cells during osteogenesis and is well established as a differentiation marker. A colorimetric p -nitrophenol phosphate assay (Stanbio, Boerne, TX) was used to measure ALP expression. hMSCs were seeded on etched glass-ceramic and TCP discs in either osteogenic or control growth medium. At days 1, 4, and 8 cells were lyzed using a mammalian protein extraction reagent (Thermo Scientific, Rockford, IL). Cell lysates were assayed for ALP activity using the manufacturer’s protocol. ALP activity was normalized to total protein content using a Bradford assay (Thermo Scientific, Rockford, IL).

Cell differentiation

Mineral formation was evaluated by seeding both hMSCs and NhOsts (as control) on etched glass-ceramic discs. hMSCs and NhOsts were allowed to proliferate for a week. Osteogenesis was then induced by replacing growth medium with osteogenic and mineralization medium, respectively. At 21 days some hMSCs specimens were stained with Alizarin Red to visualize calcium-rich nodules. Briefly, discs were washed with PBS and fixed with ice-cold ethanol at 4 °C for 1 h. The fixed cells were soaked in 1% Alizarin Red (Sigma) solution for 5 min at room temperature (pH 4.0) and washed with water to remove the residual dye, prior to examination by optical microscopy. Specimens were fixed for SEM at 21 and 35 days as described earlier, and gold-coated prior to examination. The cell morphology, as well as nodules and fibril formation was investigated in ultra-high resolution mode.

Statistical analysis

All experiments were performed twice with at least three specimens per treatment condition for a total of 6–8 specimens per group. Graphical results are presented as mean ± standard deviation (SD). A p -value of less than 0.05 was considered statistically significant. ANOVA was used to detect differences between group means. Tukey’s simultaneous tests were used for comparison between groups.

Materials and methods

Specimen preparation

A fluorapatite-based glass composition doped with 1 wt.% of niobium oxide was prepared by mixing reagent grade oxides and carbonates as previously described . The batch ingredients were melted at 1525 °C for 3 h in platinum crucibles. After quenching, the glass frit was powdered and melted again at 1525 °C for 3 h. The glass was cast into pre-heated cylindrical stainless steel molds to form 12 mm × 60 mm rods. Rods were furnace-cooled to room temperature and cut into discs (12 mm in diameter, 1.5 mm thick) using a low speed diamond saw. The glass discs were then heat treated at 950 °C for 1 h to promote nucleation and crystallization of spherical submicrometer fluorapatite crystals, as previously established . Three types of experiments were conducted: (1) initial cell attachment and cell morphology, (2) cell proliferation and (3) alkaline phosphatase (ALP) activity and cell differentiation. Glass-ceramic discs were randomly divided into three groups ( n = 8 per group): specimens were either left as-heat treated, etched with a diluted hydrofluoric solution for 60 s or ground with a 600-grit diamond disc under water irrigation. Additional specimens for initial cell attachment experiments were polished to a 1-μm finish and served as negative controls. As mentioned earlier, ALP activity and cell differentiation experiments were conducted on the etched group only, as most revealing of the submicrometer FAp crystallites. Glass-ceramic specimens were sterilized in a steam autoclave at 134 °C for 30 min. Tissue culture polystyrene (TCP) was used as a positive control for all cell culture experiments.

Cell culture

Human mesenchymal stem cells (hMSCs, Lonza, Walkersville, MD) were cultured in growth medium (Mesenchymal Stem Cell Growth Medium Bullet Kit, Lonza, Walkersville, MD), passaged by trypsinizing (0.05% trypsin/EDTA) and sub-cultured at a density of 2500–5000 cells/cm 2 . Cells between the third and fifth passage were used for all experiments. The cell cultures were maintained under standard culture conditions (37 °C, 95% relative humidity, and 5% CO 2 ). hMSC Osteogenic Bullet Kit (Lonza, Walkersville, MD) was selected for osteogenic induction during experiments. Normal Human Osteoblasts (NhOsts, Lonza, Walkersville, MD) served as positive controls for analysis of extracellular matrix secretion. NhOsts were seeded in growth medium (Osteoblast Growth Medium Bullet Kit) and mineralization was induced by osteogenic medium (Osteogenic bullet Kit, Lonza, Walkersville, MD).

Initial attachment

Discs from each experimental group ( n = 4 per group) were seeded with 3000 cells. Tissue culture polystyrene (TCP) surface was used as positive control. At 3 h, the discs were stained with green calcein dye (Invitrogen, Carlsbad, CA). In order to count the number of live cells on each substrate, digital micrographs were taken at three random locations on each disc. NIH Image J (version 1.440) software was used for digital image analyses . The experiment was performed in duplicate.

Cell proliferation

A live/dead assay was used to assess cell proliferation at days 1, 4 and 8. This well-established assay uses a membrane-permeant calcein-AM dye, which is cleaved by esterases in a live cell, yielding green fluorescence, and a membrane impermeant ethidium homodimer-1, which binds to nucleic acids in membrane, leading to a red fluorescence in compromised cells. For each time point, three steam-sterilized discs from each group were seeded with 3000 cells. TCP was used as positive control. At days 1, 4 and 8, the cell culture medium was replaced with Dulbecco’s phosphate-buffered saline (pH 7.2) containing 0.002 mmol/L calcein-AM and 0.002 mmol/L ethidium homodimer-1 (Invitrogen, Carlsbad, CA). After 30 min of incubation at room temperature, the cells were observed using epifluorescence microscopy (Olympus, BX 51, Tokyo, Japan). Digital micrographs were taken at five random locations on each disc. Images were analyzed using Image J software. The amount of cell attachment was measured on each micrograph as the percentage of specimen surface area covered by live cells.

Early cell morphology

In order to examine the cell–glass ceramic interaction and early cell morphology, hMSCs were cultured on steam-sterilized glass-ceramic discs for 2–4 days and prepared for examination by scanning electron microscopy (SEM). Specimens were rinsed with phosphate buffered saline and fixed in formaldehyde, followed by graded ethanol dehydrations up to final dehydration in absolute ethanol. Specimens were then dried in hexamethyldisilizane (HMDS)/ethanol solution series and gold-coated prior to SEM examination in secondary electron imaging and ultra high-resolution mode (Sirion, FEG scanning electron microscope, FEI Company).

Alkaline phosphatase (ALP) activity

ALP is an enzyme expressed by cells during osteogenesis and is well established as a differentiation marker. A colorimetric p -nitrophenol phosphate assay (Stanbio, Boerne, TX) was used to measure ALP expression. hMSCs were seeded on etched glass-ceramic and TCP discs in either osteogenic or control growth medium. At days 1, 4, and 8 cells were lyzed using a mammalian protein extraction reagent (Thermo Scientific, Rockford, IL). Cell lysates were assayed for ALP activity using the manufacturer’s protocol. ALP activity was normalized to total protein content using a Bradford assay (Thermo Scientific, Rockford, IL).

Cell differentiation

Mineral formation was evaluated by seeding both hMSCs and NhOsts (as control) on etched glass-ceramic discs. hMSCs and NhOsts were allowed to proliferate for a week. Osteogenesis was then induced by replacing growth medium with osteogenic and mineralization medium, respectively. At 21 days some hMSCs specimens were stained with Alizarin Red to visualize calcium-rich nodules. Briefly, discs were washed with PBS and fixed with ice-cold ethanol at 4 °C for 1 h. The fixed cells were soaked in 1% Alizarin Red (Sigma) solution for 5 min at room temperature (pH 4.0) and washed with water to remove the residual dye, prior to examination by optical microscopy. Specimens were fixed for SEM at 21 and 35 days as described earlier, and gold-coated prior to examination. The cell morphology, as well as nodules and fibril formation was investigated in ultra-high resolution mode.

Statistical analysis

All experiments were performed twice with at least three specimens per treatment condition for a total of 6–8 specimens per group. Graphical results are presented as mean ± standard deviation (SD). A p -value of less than 0.05 was considered statistically significant. ANOVA was used to detect differences between group means. Tukey’s simultaneous tests were used for comparison between groups.

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Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Differentiation of human mesenchymal stem cells on niobium-doped fluorapatite glass-ceramics
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