- Open Access
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.
- Osteon architecture
- Extracellular Matrix
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.
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 18.104.22.168).
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.
- Murugan R, Ramakrishna S: Development of nanocomposites for bone grafting. Compos Sci Technol 2005, 65: 2385-2406. 10.1016/j.compscitech.2005.07.022View ArticleGoogle Scholar
- Teo WE, Liao S, Chan C, Ramakrishna S: Fabrication and characterization of hierarchically organized nanoparticle-reinforced nanofibrous composite scaffolds. Acta Biomater 2011, 7: 193-202. 10.1016/j.actbio.2010.07.041View ArticleGoogle Scholar
- Martin BR, Burr DB, Sharkey NA: Skeletal Tissue Mechanics. New York: Springer-Verlag; 1998.View ArticleGoogle Scholar
- Carter CB, Norton MG: Ceramic Materials: Science and Engineering. New York: Springer; 2007.Google Scholar
- Chim H, Hutmacher DW, Chou AM, Oliveira AL, Reis RL, Lim TC, Schantz JT: A comparative analysis of scaffold material modifications for load-bearing applications in bone tissue engineering. Int J Oral Maxillofac Surg 2006, 35: 928-934. 10.1016/j.ijom.2006.03.024View ArticleGoogle Scholar
- Gibson I, Savalani MM, Lam CXF, Olkowski R, Ekaputra AK, Tan KC, Hutmacher DW: Towards a medium/high load-bearing scaffold fabrication system. Tsinghua Sci Technol 2009,14(1):13–-19.View ArticleGoogle Scholar
- Brandoff JF, Silber JS, Vaccaro AR: Contemporary alternatives to synthetic bone grafts for spine surgery. Am J Orthop (Belle Mead NJ) 2008, 37: 410-414.Google Scholar
- Buma P, Schreurs W, Verdonschot N: Skeletal tissue engineering-from in vitro studies to large animal models. Biomaterials 2004, 25: 1487-1495. 10.1016/S0142-9612(03)00492-7View ArticleGoogle Scholar
- Cowan CM, Soo C, Ting K, Wu B: Evolving concepts in bone tissue engineering. Curr Top Dev Biol 2005, 66: 239-285.View ArticleGoogle Scholar
- Drosse I, Volkmer E, Capanna R, De Biase P, Mutschler W, Schieker M: Tissue engineering for bone defect healing: an update on a multi-component approach. Injury 2008,39(Suppl 2):S9-S20.View ArticleGoogle Scholar
- LeGeros RZ: Properties of osteoconductive biomaterials: calcium phosphates. Clin Orthop Relat Res 2002, 395: 81-98.View ArticleGoogle Scholar
- Mistry AS, Mikos AG: Tissue engineering strategies for bone regeneration. Adv Biochem Eng Biot 2005, 94: 1-22.Google Scholar
- Bauer TW, Togawa D: Bone graft substitutes: towards a more perfect union. Orthopedics 2003, 26: 925-926.Google Scholar
- Roohani-Esfahani S-I: The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites. Biogeosciences 2010, 31: 5498-5509.Google Scholar
- Delloye C: Bone substitutes in 2003: an overview. Acta Orthop Belg 2003, 69: 1-8.Google Scholar
- Gleeson JP, Plunkett NA, O’Brien FJ: Addition of hydroxyapatite improves stiffness, interconnectivity and osteogenic potential of a highly porous collagen-based scaffold for bone tissue regeneration. Eur Cell Mater 2010, 20: 218-230.Google Scholar
- Roshan-Ghias A, Terrier A, Bourban PE, Pioletti DP: In vivo cyclic loading as a potent stimulatory signal for bone formation inside tissue engineering scaffold. Eur Cell Mater 2010, 19: 41-49.Google Scholar
- Aguirre A, Gonzalez A, Navarro M, Castano O, Planell JA, Engel E: Control of microenvironmental cues with a smart biomaterial composite promotes endothelial progenitor cell angiogenesis. Eur Cell Mater 2012, 24: 90-106. discussion 106Google Scholar
- Seefried L, Mueller-Deubert S, Schwarz T, Lind T, Mentrup B, Kober M, Docheva D, Liedert A, Kassem M, Ignatius A, et al.: A small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes. Eur Cell Mater 2010, 20: 344-355.Google Scholar
- Torcasio A, van Lenthe GH, Van Oosterwyck H: The importance of loading frequency, rate and vibration for enhancing bone adaptation and implant osseointegration. Eur Cell Mater 2008, 16: 56-68.Google Scholar
- Wagoner Johnson AJ, Herschler BA: A review of the mechanical behavior of CaP and CaP/polymer composites for applications in bone replacement and repair. Acta Biomater 2011, 7: 16-30. 10.1016/j.actbio.2010.07.012View ArticleGoogle Scholar
- Babis GC, Soucacos PN: Bone scaffolds: the role of mechanical stability and instrumentation. Injury 2005, 36: S38-S44.View ArticleGoogle Scholar
- Nandi SK, Roy S, Mukherjee P, Kundu B, De DK, Basu D: Orthopaedic applications of bone graft & graft substitutes: a review. Indian J Med Res 2010, 132: 15-30.Google Scholar
- Gerich TG, Wilmes P, Nackenhorst U, Gosling T, Ziefle M, Krettek C: A clinical, radiological and computational analysis of the thrust plate prosthesis in young patients. Bull Soc Sci Med Grand Duche Luxemb 2011, 2: 57-70.Google Scholar
- Hutmacher DW: Scaffolds in tissue engineering bone and cartilage. Biogeosciences 2000, 21: 2529-2543.Google Scholar
- Bhumiratana S, Grayson WL, Castaneda A, Rockwood DN, Gil ES, Kaplan DL, Vunjak-Novakovic G: Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds. Biogeosciences 2011, 32: 2812-2820.Google Scholar
- Wu C, Zhang Y, Zhu Y, Friis T, Xiao Y: Structure–property relationships of silk-modified mesoporous bioglass scaffolds. Biogeosciences 2010, 31: 3429-3438.Google Scholar
- Zhou H: Nanoscale hydroxyapatite particles for bone tissue engineering. Acta Biomater 2011, 7: 2769-2781. 10.1016/j.actbio.2011.03.019View ArticleGoogle Scholar
- Tanase CE, Popa MI, Verestiuc L: Biomimetic bone scaffolds based on chitosan and calcium phosphates. Mater Lett 2011, 65: 1681-1683. 10.1016/j.matlet.2011.02.077View ArticleGoogle Scholar
- Black J, Mattson R, Korostoff E: Haversian osteons: size, distribution, internatl structure, and orientation. J Biomed Mater Res 1973, 8: 299-319.View ArticleGoogle Scholar
- Wheeless CR Book Wheeless Textbook of Orthopaedics. In Wheeless Textbook of Orthopaedics. City: Editor ed.^eds; 1996.Google Scholar
- Balaban NQ, Schwarz US, Riveline D, Goichberg P, Tzur G, Sabanay I, Mahalu D, Safran S, Bershadsky A, Addadi L, Geiger B: Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol 2001, 3: 466-472. 10.1038/35074532View ArticleGoogle Scholar
- Lam MT, Sim S, Zhu X, Takayama S: The effect of continuous wavy micropatterns on silicone substrates on the alignment of skeletal muscle myoblasts and myotubes. Biogeosciences 2006, 27: 4340-4347.Google Scholar
- Lehnert D, Wehrle-Haller B, David C, Weiland U, Ballestrem C, Imhof BA, Bastmeyer M: Cell behaviour on micropatterned substrata: limits of extracellular matrix geometry for spreading and adhesion. J Cell Sci 2004, 117: 41-52. 10.1242/jcs.00836View ArticleGoogle Scholar
- Nikkhah M, Strobl JS, Agah M: Geometry-dependent behavior of fibroblast cells in three-dimensional silicon microstructures. Conf Proc IEEE Eng Med Biol Soc 2007, 2007: 6078-6081.Google Scholar
- Noireaux V: Growing an actin gel on spherical surfaces. Biophys J 2000, 78: 1643-1654. 10.1016/S0006-3495(00)76716-6View ArticleGoogle Scholar
- Pathak A, Deshpande VS, McMeeking RM, Evans AG: The simulation of stress fibre and focal adhesion development in cells on patterned substrates. J R Soc Interface 2008, 5: 507-524. 10.1098/rsif.2007.1182View ArticleGoogle Scholar
- Schwartz IM, Ehrenberg M, Bindschadler M, McGrath JL: The role of substrate curvature in actin-based pushing forces. Curr Biol 2004, 14: 1094-1098. 10.1016/j.cub.2004.06.023View ArticleGoogle Scholar
- Appleford MR, Oh S, Cole JA, Carnes DL, Lee M, Bumgardner JD, Haggard WO, Ong JL: Effects of trabecular calcium phosphate scaffolds on stress signaling in osteoblast precursor cells. Biogeosciences 2007, 28: 2747-2753.Google Scholar
- Yim EKF, Reano RM, Pang SW, Yee AF, Chen CS, Leong KW: Nanopattern-induced changes in morphology and motility of smooth muscle cells. Biogeosciences 2005, 26: 5405-5413.Google Scholar
- Kazakia GJ, Nauman EA, Ebenstein DM, Halloran BP, Keaveny TM: Effects of in vitro bone formation on the mechanical properties of a trabeculated hydroxyapatite bone substitute. J Biomed Mater Res A 2006, 77A: 688-699. 10.1002/jbm.a.30644View ArticleGoogle Scholar
- Oliver WCaP GM: An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res 1992, 7: 1564-1583. 10.1557/JMR.1992.1564View ArticleGoogle Scholar
- Rho JY, Tsui TY, Pharr GM: Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biogeosciences 1997, 18: 1325-1330.Google Scholar
- Reilly DT, Burstein AH, Frankel VH: The elastic modulus for bone. J Biomech 1974, 7: 271-275. 10.1016/0021-9290(74)90018-9View ArticleGoogle Scholar
- Zhao Z, Zhao M, Xiao G, Franceschi RT: Gene transfer of the Runx2 transcription factor enhances osteogenic activity of bone marrow stromal cells in vitro and in vivo. Mol Ther 2005, 12: 247-253. 10.1016/j.ymthe.2005.03.009View ArticleGoogle Scholar
- Oste L, Bervoets AR, Behets GJ, Dams G, Marijnissen RL, Geryl H, Lamberts LV, Verberckmoes SC, Van Hoof VO, De Broe ME, D’Haese PC: Time-evolution and reversibility of strontium-induced osteomalacia in chronic renal failure rats. Kidney Int 2005, 67: 920-930. 10.1111/j.1523-1755.2005.00156.xView ArticleGoogle Scholar
- Brunette DM: Spreading and orientation of epithelial cells on grooved substrata. Exp Cell Res 1986, 167: 203-217. 10.1016/0014-4827(86)90217-XView ArticleGoogle Scholar
- Brunette DM: Fibroblasts on micromachined substrata orient hierarchically to grooves of different dimensions. Exp Cell Res 1986, 164: 11-26. 10.1016/0014-4827(86)90450-7View ArticleGoogle Scholar
- Brunette DM, Kenner GS, Gould TRL: Grooved titanium surfaces orient growth and migration of cells from human gingival explants. J Dent Res 1983, 62: 1045-1048. 10.1177/00220345830620100701View ArticleGoogle Scholar
- Kapoor A, Caporali EH, Kenis PJ, Stewart MC: Microtopographically patterned surfaces promote the alignment of tenocytes and extracellular collagen. Acta Biomater 2010, 6: 2580-2589. 10.1016/j.actbio.2009.12.047View ArticleGoogle Scholar
- Clark P, Connolly P, Curtis AS, Dow JA, Wilkinson CD: Topographical control of cell behaviour: II. Multiple grooved substrata. Dev 1990, 108: 635-644.Google Scholar
- Recknor JB, Recknor JC, Sakaguchi DS, Mallapragada SK: Oriented astroglial cell growth on micropatterned polystyrene substrates. Biogeosciences 2004, 25: 2753-2767.Google Scholar
- Clarke JC, Tuft BW, Clinger JD, Levine R, Figueroa LS, Allan Guymon C, Hansen MR: Micropatterned methacrylate polymers direct spiral ganglion neurite and Schwann cell growth. Hear Res 2011, 278: 96-105. 10.1016/j.heares.2011.05.004View ArticleGoogle Scholar
- Bowden N, Huck WTS, Paul KE, Whitesides GM: The controlled formation of ordered, sinusoidal structures by plasma oxidation of an elastomeric polymer. Appl Phys Lett 1999, 75: 2557-2559. 10.1063/1.125076View ArticleGoogle Scholar
- Charest JL, García AJ, King WP: Myoblast alignment and differentiation on cell culture substrates with microscale topography and model chemistries. Biogeosciences 2007, 28: 2202-2210.Google Scholar
- Théry M, Pépin A, Dressaire E, Chen Y, Bornens M: Cell distribution of stress fibres in response to the geometry of the adhesive environment. Cell Motil Cytoskeleton 2006, 63: 341-355. 10.1002/cm.20126View ArticleGoogle Scholar
- Aubin JE: Bone stem cells. J Cell Biochem Suppl 1998, 30–31: 73-82.View ArticleGoogle Scholar
- Chen J, Shi ZD, Ji X, Morales J, Zhang J, Kaur N, Wang S: Enhanced osteogenesis of human mesenchymal stem cells by periodic heat shock in self-assembling peptide hydrogel. Tissue Eng Part A 2013, 19: 716-728. 10.1089/ten.tea.2012.0070View ArticleGoogle Scholar
- Chyun YS, Kream BE, Raisz LG: Cortisol decreases bone formation by inhibiting periosteal cell proliferation. Endocrinol 1984, 114: 477-480. 10.1210/endo-114-2-477View ArticleGoogle Scholar
- Alcantara EH, Shin M-Y, Sohn H-Y, Park Y-M, Kim T, Lim J-H, Jeong H-J, Kwon S-T, Kwun I-S: Diosgenin stimulates osteogenic activity by increasing bone matrix protein synthesis and bone-specific transcription factor Runx2 in osteoblastic MC3T3-E1 cells. J Nutr Biochem 2011, 22: 1055-1063. 10.1016/j.jnutbio.2010.09.003View ArticleGoogle Scholar
- Gadeau AP, Chaulet H, Daret D, Kockx M, Daniel-Lamazière JM, Desgranges C: Time course of osteopontin, osteocalcin, and osteonectin accumulation and calcification after acute vessel wall injury. J Histochem Cytochem 2001, 49: 79-86. 10.1177/002215540104900108View ArticleGoogle Scholar
- Li S, Kong H, Yao N, Yu Q, Wang P, Lin Y, Wang J, Kuang R, Zhao X, Xu J, et al.: The role of runt-related transcription factor 2 (Runx2) in the late stage of odontoblast differentiation and dentin formation. Biochem Biophys Res Commun 2011, 410: 698-704. 10.1016/j.bbrc.2011.06.065View ArticleGoogle Scholar
- Mikami Y, Takahashi T, Kato S, Takagi M: Dexamethasone promotes DMP1 mRNA expression by inhibiting negative regulation of Runx2 in multipotential mesenchymal progenitor, ROB-C26. Cell Biol Int 2008, 32: 239-246. 10.1016/j.cellbi.2007.08.033View ArticleGoogle Scholar
- Narayanan K, Ramachandran A, Hao J, He G, Park KW, Cho M, George A: Dual functional roles of dentin matrix protein 1. Implications in biomineralization and gene transcription by activation of intracellular Ca2+ store. J Biol Chem 2003, 278: 17500-17508. 10.1074/jbc.M212700200View ArticleGoogle Scholar
- Luan Y, Praul CA, Gay CV: Confocal imaging and timing of secretion of matrix proteins by osteoblasts derived from avian long bone. Comparat Biochem Physiol - Part A: Mol; Integ Physiol 2000, 126: 213-221. 10.1016/S1095-6433(00)00200-2View ArticleGoogle Scholar
- Reffitt DM, Ogston N, Jugdaohsingh R, Cheung HFJ, Evans BAJ, Thompson RPH, Powell JJ, Hampson GN: Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro. Bone 2003, 32: 127-135. 10.1016/S8756-3282(02)00950-XView ArticleGoogle Scholar
- Kacena MA, Todd P, Gerstenfeld LC, Landis WJ: Experiments with osteoblasts cultured under varying orientations with respect to the gravity vector. Cytotechnology 2002, 39: 147-154. 10.1023/A:1023936503105View ArticleGoogle Scholar
- Rho J-Y, Roy ME, Tsui TY, Pharr GM: Elastic properties of microstructural components of human bone tissue as measured by nanoindentation. J Biomed Mater Res 1999, 45: 48-54. 10.1002/(SICI)1097-4636(199904)45:1<48::AID-JBM7>3.0.CO;2-5View ArticleGoogle Scholar
- Roy ME, Rho J-Y, Tsui TY, Evans ND, Pharr GM: Mechanical and morphological variation of the human lumbar vertebral cortical and trabecular bone. J Biomed Mater Res 1999, 44: 191-197. 10.1002/(SICI)1097-4636(199902)44:2<191::AID-JBM9>3.0.CO;2-GView ArticleGoogle Scholar
- Turner CH, Rho J, Takano Y, Tsui TY, Pharr GM: The elastic properties of trabecular and cortical bone tissues are similar: results from two microscopic measurement techniques. J Biomech 1999, 32: 437-441. 10.1016/S0021-9290(98)00177-8View ArticleGoogle Scholar
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