Influence of substrate curvature on osteoblast orientation and extracellular matrix deposition
© Pilia et al.; licensee BioMed Central Ltd. 2013
Received: 2 July 2013
Accepted: 27 September 2013
Published: 3 October 2013
The effects of microchannel diameter in hydroxyapatite (HAp) substrates on osteoblast behavior were investigated in this study. Microchannels of 100, 250 and 500 μm diameter were created on hydroxyapatite disks. The changes in osteoblast precursor growth, differentiation, extra cellular matrix (ECM) secretion and cell attachment/orientation were investigated as a function of microchannel diameter.
Curvature did not impact cellular differentiation, however organized cellular orientation was achieved within the 100 and 250 μm microchannels (mc) after 6 days compared to the 12 days it took for the 500mc group, while the flat substrate remained disorganized. Moreover, the 100, 250 and 500mc groups expressed a specific shift in orientation of 17.45°, 9.05°, and 22.86° respectively in 24 days. The secreted/mineralized ECM showed the 100 and 250mc groups to have higher modulus (E) and hardness (h) (E = 42.6GPa; h = 1.6GPa) than human bone (E = 13.4-25.7GPa; h = 0.47-0.74GPa), which was significantly greater than the 500mc and control groups (p < 0.05). It was determined that substrate curvature affects the cell orientation, the time required for initial response, and the shift in orientation with time.
These findings demonstrate the ability of osteoblasts to organize and mineralize differentially in microchannels similar to those found in the osteons of compact bone. These investigations could lead to the development of osteon-like scaffolds to support the regeneration of organized bone.
Natural bone achieves much of its mechanical strength through cortical bone, specifically through the organization of its osteons. The structural organization of native bone directly contributes to the mechanical strength of bone tissue, which is critical since load bearing and providing mechanical support are the primary functions of the skeleton. The lamellar rings that surround the central microchannel-like structure of the osteon are formed by the secretion of Type-I Collagen (Col-I) by osteoblasts during osteonal development. While Col-I and other organic molecules make up 70% of total bone composition, the remaining 30% is inorganic, composed of bone minerals, specifically nano-size crystals of hydroxyapatite (HAp) . The bone minerals are responsible for the hardness of bone whereas the organic portion gives skeletal tissue its elasticity . In addition, the secretion of Col-I along different orientations, followed by the deposition of bone minerals, gives cortical bone its high compressive strength and toughness . The compressive strength of cortical bone ranges between 100–230 MPa, whereas trabecular bone ranges between 2–12 MPa .
The goal of this study is to determine the effect of curved substrates on osteoblast growth, differentiation and organization within the microchannels, as well as ECM secretion, mineralization and hardness as a function of the substrate curvature. To accomplish this, HAp substrates with microchannels of various diameters were built to match the microchannel diameter range of natural osteons. On these patterned substrates, an osteoblast precursor cell was cultured in vitro to investigate cell responses to the various curvatures.
Materials and methods
Fabrication of the HAp disks
The HAp disks were made by solution casting as shown in Figure 2D. A HAp slurry was created by using a previously described method  using synthetic nano-size HAp (OssGen, South Korea). Briefly, the binders used to stabilize the slurry structure included 3% high molecular weight polyvinyl alcohol, 1% v/v carboxymethylcellulose, 1% v/v ammonium polyacrylate dispersant, and 3% v/v N,N-dimethylformamide drying agent. The solution was then cast into the constructed molds and sintered in a high temperature furnace (Thermolyne, Dubuque, IA). Before sintering, each disk measured 10 mm diameter and 5 mm height. The sintering process profile contained a ramp increase in temperature of 5°C/min up to 300°C with a hold time of 1 hour, then up at the same rate to 600°C for 1 hour hold, and finally to 1230°C for a 5 hour hold. The sintered disks were then cooled at a rate of 5°C/min until room temperature is achieved.
Characterization of the disks
Human fetal osteoblast cell culture
Human Fetal Osteoblasts (HFObs) (Cell Applications, Inc.- San Diego, CA) were used to evaluate bone cell response to the varying curvatures. Even though primary Mesenchymal cells are pluripotent compared to HFObs which are committed to the osteogenic lineage, they were not used because the objective of this study was to investigate the effect of local micro-architecture on the matrix production, organization and maturation of osteoblast-like cells, rather than to investigate the commitment of progenitor cells to an osteoblast-like phenotype. The cells were cultured in growth media containing Dubecco Modifed Eagle Medium (DMEM), 10% Fetal Bovine Serum (FBS), and 1% Penicillin Streptomycin Amphotericin B Solution (PSA) (all purchased from Invitrogen, USA). When cells reached confluence on the cell culture-flask, the HFObs were washed with phosphate buffered saline (PBS) and then 0.25% Trypsin/EDTA was added in osteogenic media (DMEM, 3% FBS, 1% PSA, 10 mM Glycerolphosphate, 50 μg/mL Ascorbic acid and 10nM Dexamethasone). The cells in solution were counted (Z2 Coulter® Particle Count and Size Analyzer; Beckman Coulter™ - Brea, CA) and seeded on the disks at confluence (55,000 cells/cm2). Four time points were tested: 6, 12, 18 and 24 days (n = 12). For n = 8 disks, media was collected. Each disk was then washed with PBS, followed by cell permeabilization using 0.1% Triton X-100 in PBS (PBS-T), and after a freeze/thaw cycle the supernatant was collected. The remaining n = 4 disks were stored in 4% formaldehyde for imaging.
In vitro osteoblast tracking on the HAp disks
Cell numbers were measured directly from the cell lysate solution. Specifically, 25 μL of lysate was added to the Quant-iT™ PicoGreen® dsDNA kit (Invitrogen, USA). This assay was performed in black opaque 96 well plates and the fluorescence was assessed using a microplate reader (Biotek Synergy 2 – Winooski, VT). The plate was excited at 485/20 nm, and the emitted light was measured at 528/20 nm. This reading was used to assess the change in cell numbers over time.
In vitro differentiation assays of the osteoblasts on the HAp disks
HFOb differentiation was determined by testing the cell lysates for the protein product of the bone-specific transcription factor runt-related transcription factor 2 (RUNX2), Alkaline Phosphatase (ALP), Dental Matrix Protein 1 (DMP1), and Osteopontin (OPN) activity. RUNX2 activity was measured from the lysate using a phosphospecific antibody cell-based enzyme linked immunoabsorbent assay (PACE). Specifically, 50 μL of lysate were pipetted into a protein-attachment ready microplate and diluted in PBS-T solution at a ratio of 1:1. After 24 hours the wells were endogenous peroxide quenched in 0.6% H2O2, and blocked in 10% fetal bovine serum (FBS). Anti-RUNX2 primary antibody (Cat # 41–1400, Invitrogen) was then added overnight using a concentration of 1 μg/mL, followed by PBS-T washes and a secondary antibody (Cat # 81–6720, Invitrogen) for one hour using a concentration of 0.214 μg/mL. Following 3 PBS washes, Pierce 1-step ultra TMB was added to each well and the reaction was stopped using 2 M H2SO4. The plate absorbance was measured operating the same microplate reader used in the DNA analysis. The absorbance was read at 450 nm with reference at 655 nm. Primary to secondary antibody ratio was optimized by identifying highest signal-to-noise ratio (highest signal to noise ratio found using 1ug/mL primary, 0.214 μg/mL secondary with these antibodies). ALP activity was assessed from the cell lysate using an ALP Fluorescence Detection Kit (APF, Sigma-Aldrich). 10 μL of lysate were added in black opaque 96 well plates to fluorescence assay buffer following kit manufacturer instructions. Fluorescence was read after exactly 45 minutes with an excitation of 360 nm, and an emission of 460 nm. DMP1 was detected and quantified using the same PACE technique used for the RUNX2 assay. All of the steps remained the same with the difference that the primary antibody used was anti-DMP1 (code ab76632, Abcam) at a concentration of 2.5 μg/mL, and the secondary antibody used was the same used in the RUNX2 and was used at a concentration of 0.30 μg/mL. As in RUNX2, primary to secondary antibody ratio was optimized for maximum signal-to-noise ratio (strongest signal to noise ratio found using 2.5 μg/mL primary, 0.30 μg/mL secondary with these antibodies). OPN detection from the cell lysate solution was performed using the bone panel Milliplex kit (Millipore, USA). This kit also tested for osteocalcin and osteoprotegerin. Specifically, 10 μL of lysate were used for this test and combined with assay buffer following kit manufacturer instructions.
In vitro Collagen I semi-quantification assays of the HAp disks
A total of 10 fluorescence immunohistochemistry readings for each group were analyzed after testing semi-quantitatively for the presence of Col-I. The disks, previously fixed in 4% formaldehyde, were quenched hydrogen peroxide in 0.6% H2O2, blocked in 10% FBS, and a polyclonal anti-Col-I primary antibody (code ab34710, Abcam) was added overnight at a concentration of 10 μg/mL. This step was followed by PBS-T washes and the addition of a FITC linked secondary antibody (Code ab96895, Abcam) also using a concentration of 5 μg/mL. As in RUNX2 quantification, the strongest signal to noise ratio was found using 10 μg/mL primary, 5 μg/mL secondary with these antibodies. The disks were washed 2x in PBS-T and ProLong® Gold Antifade with DAPI was added to the bottom of the plate where the disks were inverted for fluorescent microscopy. A total of 10 intensity readings were obtained from different channels of the disks. Readings were averaged and Col-I was quantified using intensity measurements with reference to control surfaces.
In vitro cell orientation assays of the HAp disks
In vitro mechanical ECM characterization of the HAp disks
300 random readings were taken from each disk.
In which Pmax is the peak load and A is the projected area of contact at peak load evaluated by a function which relates the cross-sectional area of the indenter to the vertical distance from its tip (h c ) .
The modulus and hardness readings that fell into the control HAp readings ± standard deviation were discarded because it was assumed that the indent was measuring the actual HAp layer and not the cell ECM.
All data is reported as average ± standard error. The statistical test used to analyze the data was a two-way analysis of variance, and Tukey’s comparison test was used to determine statistical significance between individual groups. All statistical tests were performed using SigmaPlot® (version 11.0, Systat Software, Inc.). Differences were considered significant at P < 0.05. The difference between variances of distribution was evaluated using an F-Test (MedCalc® V 220.127.116.11).
Characterization of the Hap disks
Results of morphology characterization of the HAp disks
92.32 ± 0.57
42.83 ± 0.92
169.55 ± 2.12
79.78 ± 0.87
229.92 ± 1.07
111.84 ± 2.24
430.27 ± 5.41
81.25 ± 0.92
525.07 ± 2.32
203.18 ± 3.40
880.27 ± 9.00
72.78 ± 0.51
HFOb growth and differentiation in the microchannels
In vitro extracellular matrix (ECM) assays of the HAp disks
In vitro cell organization and orientation assays of the HAp disks
In vitro mechanical ECM characterization of the HAp disks
Multiple materials, surface morphologies, and cell types have been previously investigated to identify the role of surface architecture on tissue regeneration. In fact, micropatterned surfaces have been previously created on different material substrates using a variety of techniques. Primarily these methods were micromachining [47–49], low voltage electron beam lithography , standard photolithographic [50–52] and photopolymerization process , plasma oxidation [33, 54], and single mask fabrication technique . All these techniques can achieve a different array of substrates, but none of them was useful to create the microchannel template on HAp. Other materials used to identify the effects of substrate on cell behavior include silicon wafers [33–35, 48], polystyrene [36, 52], methacrylate , Perspex , SU-8 5 photoresist , latex , and ligand patches . HAp posed as a more challenging material to machine and/or pattern due to the strength and brittleness of its nature. Multiple cell types including tenocytes , fibroblasts [32, 35, 37, 48], myoblasts [33, 55], neurons , spiral ganglion cells , retinal endothelial cells , epithelial cells , astroglial cells , HeLa S3 cancerous cell line , and Mouse B16F1 melanoma cells  have been previously investigated regarding their response to surface architectural cues. However, there have been very few (or no known reports to the authors knowledge) reports on the response of fetal osteoblasts on such curved or micro-patterned substrates. In this study, a mold template/casting technique for HAp was employed. This allowed for precise, repeatable (<5% variation), and consistent (< 2.5% variation) patterned morphologies resembling the natural range of osteon curvature (Table 1). Using this system, we systematically studied the effect of a changing substrate curvature within the range of native osteon diameter on osteoblast response while simulating lamellar organization.
Osteoblast precursor cells such as the ones used in this study are not fully differentiated bone cells, however they are already committed to the osteogenic lineage. These cells undergo three key phases of cell activity: proliferation, differentiation and mineralization . These steps are well defined and can overlap one another . The osteoblast precursor cells seeded on the disks were induced to differentiate into mature osteoblasts using the glucocorticoid dexamethasone. A common drawback of this steroid is a reduction in the rate of replication of the cells . Thus, as osteoblast precursor cells start to differentiate, proliferation ceases. This is demonstrated with the findings on DNA analysis in the current study which showed no significant change in cell number but rather only differentiation. The assay used to determine cell numbers relies on testing from cell lysate solution. Given both the geometry of the substrate and the time for which the cells were cultured, it is not surprising that the DNA yield was not optimal, translating into high standard error. Another drawback related to use of Dexamethasone is the inhibition of bone formation in vivo due to decreased collagen synthesis . Overall in this experiment the dexamethasone ρρdid not inhibit collagen secretion altogether, since actual Col-I deposition was seen (Figure 8). Our results show a strong differentiation response consistent with the effect of dexamethasone, glucocorticoid receptor induced activation of osteoblast gene expression such as osteocalcin, collagen Iα1 and transforming growth factor-β1. Osteoblasts differentiation can be detected by analyzing specific early and late markers which have been thoroughly studied and characterized . Perhaps the earliest activation factor is RUNX2, followed by ALP (an early-mid marker), OPN, OC, ON . The four week experiment resulted in no changes in osteoblast differentiation/mineralization rates between the four experimental groups, concluding that the curvature associated with different microchannel diameters had little or no effect on HFOb rate of differentiation. The rate of differentiation for all groups in this study was consistent with the literature. RUNX2 peaked early at week 1, followed by ALP, which was activated first at day 12 and then at day 24. OPN, which is activated by RUNX2 , did not show activation until day 18, with the highest levels seen at day 24, showing continuous differentiation throughout the study. The expression of DMP1 was limited after 24 days, which does not agree with the study by Mikami et al. which showed early expression of DMP1 from mRNA using RT-PCR when using dexamethasone as stimulant . However, the current study did not analyze DMP1 expression from mRNA but rather quantified the actual Human DMP1 protein present in the cell lysate at the different time points. It is difficult to determine whether natural DMP1 expression follows the same in vitro trend. Narayanan et al. gave an insight into the role DMP1 . According to their study in the very early stages of differentiation, DMP1 serves as a transcription factor that stimulates further differentiation and expression of such markers as RUNX2. Only at the last stage of Ob maturity (near collagen mineralization) is DMP1 secreted by the differentiating osteoblast and triggers mineralization . This contradicts the findings that showed different modulus and hardness of the deposited ECM, suggesting that mineralization is occurring in the curved substrates, whereas there were no differences in the flat substrate. According to the findings in the current study, a noticeable increase in DMP1 should have been expected at four weeks. These contradicting discoveries could be due to the deterioration of the DMP1 after freeze/thaw cycles, or due to the low sensitivity of the DMP1 ELISA. Thus, it is reasonable to conclude from the present study that negligible increases in DMP1 expression observed from day 6 to 24 in all tested groups indicated the absence of phenotypic transition from early osteoblast to late mineralization behavior. DMP1 is also a marker for osteoblast differentiation into osteocyte. The lack of DMP1 could be explained by the cells not reaching mature osteoblast status at 24 days, or by the inhibited proliferation of the cells in culture, preventing layering effects in the microchannels.
When Luan et al. tested osteoblast cultures for Col-I secretion at 4, 8, and 12 days they were unable to see any significant differences at each time point . Refitt et al. instead investigated the function of silicone in Col-I secretion. The Col-I measurements they described were done indirectly on the amount of carboxy-terminal propeptide of type 1 procollagen liberated into the culture medium . In this study, although a definite trend can be seen in collagen secretion up to day 18 in all tested groups, no significant differences in collagen deposition over time or between groups were seen. However, a change of trend in the Col-I fluorescence was observed at day 24 and was characterized by a decrease in fluorescence intensity throughout all four experimental groups. This change, although not significant, was attributed to early mineralization that contributed to the masking of the Col-I antibody labeling. The control group did not show any changes in Col-I secretion within the study, which is consistent with data reported by Luan et al..
Previous studies on cell orientation within micropatterned substrata all agreed that the narrower the channel, the higher the degree of alignment the cell presents, and the deeper the groove, also the higher the degree of alignment [49–53, 55]. There are however a few basic differences between the cited research and those performed in the current study. The micropatterning described in the literature is in the range of few micrometers and does not necessarily reflect the concave shape that this study created. In fact Recknor et al. and Kapoor et al. had squared grooves [50, 52], Brunette et al. had both vertical and sloped walls , and Charest et al. had 10 μm holes and 10 μm grooves . Another important difference is that most studies only lasted up to 3 days and the cells were seeded at minimal confluence with the purpose of analyzing individual cell’s behavior. The present study, however, lasted 24 days, and the disks were seeded at full confluence with the goal of analyzing osteoblast behavior as a tissue, observing its mineralization and tissue orientation in relation to the substrate. The behavior of the individual cells was not as imperative. This experiment was able to show overall organization in the 100 μm, 250 μm and 500 μm, as well as the change in orientation with time which may be part of the signaling mechanism for the change in lamellar alignment observed in natural alternating osteons. Although initial cell layering was observed in the channel valleys from the beginning, it was not shown to change density over 24 days. A previous study by Kacena et al. showed that gravity plays no effect on fully confluent osteoblast that are not in proliferation mode . No effect of gravity or cell slippage within the microchannels was observed in the current study either.
The nanomechanical analysis demonstrated that by day 24 the ECM modulus and hardness values were much higher in the 100mc than the flat substrate and other microchannel groups. It is hypothesized that the high modulus and hardness values seen were due to the process of mineralization of the ECM by day 24, a process that perhaps was not seen in the flat substrates. The cell line investigated lacked phenotypic transition from early osteoblast to one that has late mineralization behavior. This can be explained by the cells not reaching mature osteoblast status at 24 days, or by the inhibited proliferation of the cells in culture, preventing layering effects in the microchannels.
It can be speculated that the curvature of the substrate indirectly increased the rate of mineralization of the HFObs on the ECM. This mineralization trend was also supported by the ALP activity and the OPN levels, which were significantly higher in the 100mc group than the flat control group. When visually comparing the organization of the cells and their change in orientation with the modulus and the hardness, it was observed that the higher the organization of the cells in the substrate, the higher the modulus and the hardness. As observed above, at day 24 the 100mc displayed the highest cell organization, followed by the 500mc and the 250mc. On the other hand, the flat substrate never demonstrated orientation. As the cells aligned onto the substrate, they were able to organize faster and secrete organized ECM with higher modulus and hardness. After 24 days in cell culture, cells produced their own extracellular matrix that could modify the topography of the substrates. In fact, the rate of change on each of the microchannels groups is probably different since the cells are probably laying down ECM to vary their local micro-environment. This could explain the different temporal response elicited on each microchannel. During nano-indentation testing we measured E and h of the plain HAp disks to determine baseline values (E = 125.30 ± 9.52 and h = 8.98 ± 1.25). This value was considered the internal control to verify that all the indents on the in vitro cultured substrates were measuring actual secreted ECM properties and not the underlying HAp surfaces. Also, HAp’s E was 2 fold higher than the 100mc group and 6 fold higher than the control flat groups. The hardness was 4 fold higher than the 100mc group and 9 fold higher than the Flat disk control. The elastic modulus value of the flat substrate (7.8GPa) was slightly lower than what has been previously reported in the literature for dehydrated human bone (12.4GPa) [41, 43, 68–70]. However, the three experimental groups with different diameter microchannels resulted in a much higher modulus. Specifically, the 100 μm group showed the highest modulus amongst the groups (42.6GPa) and tested above human bone (13.4-25.7GPa) . The hardness values of the flat control disk (0.2GPa) averaged below previously reported values for human bone (0.47-0.74GPa) [41, 43, 68–70]. The experimental groups with microchannels have much higher hardness than what was previously reported, and once again, the hardness in the 100mc (1.6GPa) is above natural bone values. It is hypothesized that the reason this group tested above cortical bone is because the experimental setup in this study allowed us to measure modulus and hardness directly on the surface of the osteon. When testing natural bone, the values come from an area outside the osteon, and this could contribute to the differences seen here. Potentially due to a largely two dimensional environment, in this study there were no significant changes in differentiation markers, although a significant difference in matrix modulus and hardness was observed, indicating different collagen expression and mineralization between groups occurred. This is something that should be further investigated in future studies, especially in a 3D environment.
The goal of this study was to determine osteoblast response to being cultured on a curved substrate mimicking the native curvature of an osteon. This was investigated through the alignment of cells in the microchannels (organization), the change in orientation with time, the secretion of collagen, and the production of a mineralized extracellular matrix. In this study we showed that the substrate affects physical properties of cellular organization and orientation, as well as the composition and quality of the ECM secreted. Regardless of what substrate the cells were cultured in, they all maintained equal number of cells over time while differentiating at statistical equal rates. Although this model is only the first step in depicting osteoblast behavior within a three dimensional osteon, it is critical since it has allowed us to demonstrate that when cultured in the appropriate substrate curvature and appropriate conditions, osteoblasts can organize as a tissue and change orientation with time, producing a mineralized ECM that is hard and tough. Based on these findings, future studies will focus on recreating three-dimensional scaffolds with longitudinal microchannels that resemble naturally occurring osteons. The scaffolds will then be characterized for their ability to recreate an in vitro osteon.
Overall, in this study microchannels of 100, 250 and 500 μm diameter were successfully created on the surface of HAp disk. A 24 day in vitro study was performed on these different substrates and demonstrated an early osteogenic phenotype and extensive collagen deposition Moreover, cell alignment within the microchannels were assessed to find that the 100mc and the 250mc groups induced fast orientation and a higher degree of organization, while the 500mc only started showing organization after 12 days and the control flat group did not show alignment towards a specific direction. This study was also able to demonstrate a significant increase in elastic modulus and hardness in the microchannel substrates compared to a flat control, suggesting that curvature plays an important role in the quality of the ECM secreted and mineralized. Understanding how bone cells grow and secrete ECM in these curved substrates is the first step in understanding the mechanism involved in creating artificial osteons and in the long run regenerating organized cortical bone.
Thanks to Dr. Daniel Oh for his help and expertise in the casting of HAp materials, Beth Pollot for her assistance with scanning electron microscopy, and Cameron Taylor for his support analyzing cell orientation. Also a special thanks to the dental research staff at the United States Army Institute of Surgical research at Fort Sam Houston, San Antonio TX for allowing us to use their laser profilometer. This study was supported in part by the UT System South Texas Technology Management (STTM) POC Sparc grant program.
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