Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds

Reza Jarrahy,1,2 Weibiao Huang,1,2 George H. Rudkin,1,2 Jane M. Lee,1 Kenji Ishida,1 Micah D. Berry,1 Modar Sukkarieh,1 Benjamin M. Wu,3 Dean T. Yamaguchi,1 and Timothy A. Miller1,2

1Plastic Surgery Section, Veterans Administration Greater Los Angeles Healthcare System, Los Angeles; 2Division of Plastic Surgery, Department of Surgery, University of California Los Angeles School of Medicine, Los Angeles, and 3Department of Bioengineering, University of California Los Angeles, Los Angeles, California

Submitted 21 April 2004 ; accepted in final form 23 March 2005


    ABSTRACT
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Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(L-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.

gene expression; osteogenesis; angiogenesis


THE IDEAL BONE GRAFT SUBSTITUTE combines the osteogenic potential of autologous osteoprogenitor cells with a biocompatible three-dimensional (3D) delivery vehicle to generate replacement tissue that is structurally and functionally equivalent to surrounding bone (33, 50). These bone grafts must allow for cell attachment, migration, proliferation, differentiation, and ultimately deposition of a mineralized osteoid matrix (16, 59). Because of its regenerative properties, bone marrow, from which many native bone precursor cells can be obtained with relative ease, provides a self-renewing source of potential graft material (10, 31, 40).

To date, numerous in vitro and in vivo models of bone graft substitutes combining various cell types and delivery vehicles capable of depositing mineralized tissue have been described (19, 27, 38, 43, 50). Some authors have attempted to improve the efficiency of osteogenesis by altering carrier design; different angiogenic, osteoinductive, and osteoconductive proteins have been directly incorporated into scaffold frameworks (6, 27, 34, 41, 42). Others have focused on cell manipulation to genetically engineer osteoprogenitor cells with improved ability to direct local osseous tissue regeneration (25, 7, 8, 12, 31, 36, 39, 47, 54, 55). Because the metabolic needs of developing bone depend on successful neovascularization, still others have examined the role played by angiogenesis in effective bone regeneration (13, 14, 17, 32, 46, 53).

Despite the attention given to tissue engineering in the pursuit of a clinically relevant bone graft substitute, the effect of a 3D culture environment on differentiation of osteoprogenitor cells has not yet been determined. In particular, there is no available data that specifically and quantitatively compares the expression of genes associated with angiogenesis and osteogenesis in osteoprogenitor cells cultured on scaffolds compared with cells cultured on conventional two-dimensional (2D) culture surfaces. Such information is valuable to the design of a 3D bone graft substitute: elucidation of the molecular events that drive angiogenesis and osteogenesis at the cell-scaffold interface will help determine the optimal approach to using autologous osteoprogenitor cells in scaffold-based reconstruction of bone defects.

In this study, we hypothesized that gene expression of angiogenic and osteogenic markers would be significantly altered in MC3T3-E1 murine preosteoblastic cells cultured on poly-L-lactide-co-glycolide (PLGA) scaffolds compared with cells cultured on conventional 2D surfaces. We further predicted that any observed differences in transcriptional events related to angiogenesis would be reflected in differences in the secretion and function of angiogenic factors between the two culture systems. To test these hypotheses, we compared the expression of alkaline phosphatase (ALP) and osteocalcin (OCN) (early and late markers of osteogenic induction, respectively), as well as the angiogenic factor vascular endothelial growth factor (VEGF) in MC3T3-E1 cells cultured in 2D and 3D environments. We also examined the amount of VEGF protein secretion by cells cultured in 2D and 3D systems, and studied the efficacy with which conditioned medium from these environments was able to induce in vitro angiogenesis activity.


    METHODS
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Fabrication of PLGA films and scaffolds. Polymer-based scaffolds were prepared from a 15% wt/wt solution of PLGA pellets (Birmingham Polymers, Birmingham, AL; inherent viscosity = 0.72 dl/g) dissolved in chloroform. A solvent-casting/particulate-leaching technique to was used to create scaffolds measuring 35 mm in diameter and 3 mm in thickness with 95% porosity and 200- to 300-µm pore size range. To achieve the desired porosity, 1 g of 15% PLGA/chloroform solution and 0.46 g of methanol were magnetically stirred until no phase separation was observed. Next, 3.6 g of 200- to 300-µm diameter sucrose particles (C & H, Crockett, CA) were added, and the resulting paste was mixed with a spatula. The paste mixture was packed into ultrasonically cleaned stainless steel molds of the desired dimensions. Scaffolds were stored under vacuum and immersed in ddH20 for 24 h before use to dissolve the porogen. Ethanol was used to prewet and sterilize the scaffolds (32), which were rinsed and cut into pie-shaped wedges measuring one-eighth of the original scaffold area before cell seeding.

PLGA films were created by evenly pouring 2 ml of 5% wt/wt solution of PLGA dissolved in chloroform into 50-mm glass culture dishes. The films were allowed to air dry, and ethanol was used to sterilize the PLGA films before cell culture.

Seeding and culture of PLGA scafflolds and films. MC3T3-E1 cells were a generous gift from Dr. G. Rodan (Merck, Sharpe, and Dohme, West Point, PA). Cells were plated on polystyrene surfaces and cultured in {alpha}-modification minimal essential medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma) in humidified 5% CO2-95% air at 37°C. At 85% confluence, monolayers were lifted using a 0.05% trypsin/1 mM EDTA solution (Sigma). Cells were concentrated via centrifuge, resuspended in a measured amount of medium, and counted with a hemocytometer.

Harvested cells were seeded onto prepared PLGA scaffolds (3D culture) and onto PLGA films (2D control) in 200-µl aliquots containing 2 x 106 cells each. Scaffolds and films were cultured in differentiation medium consisting of standard culture medium supplemented with 10 mM {beta}-glycerol phosphate (Sigma) and 50 µg/ml L-ascorbic acid (Sigma). Cells were cultured for 1, 2, 4, 8, 12, 16, 24, and 48 h or 1, 2, 3, 5, 7, 10, and 14 days. Culture medium was changed every 48 h for long-term cultures. The experiment was repeated three times, using distinct cell populations for each repeated experiment.

Design of oligonucleotide primers. Gene sequences for murine ALP, OCN, VEGF, and 18S rRNA were obtained from the National Center for Biotechnology Information gene database. Based on these sequences, oligonucleotide sense and antisense primers were designed with attention to AT-GC balance and likelihood of dimer or hairpin loop formation (Table 1). Stock solutions of primers were run on 1% agarose gels to verify the purity of the oligonucleotides before their use in amplification.


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Table 1. Sense and antisense primer sequences used in quantitative real-time RT-PCR

 
Quantitative real-time RT-PCR. Cells grown on PLGA films were harvested using trypsin, and cells grown on PLGA scaffolds were processed without being separated from the scaffolds. Total RNA was prepared using the RNeasy kit (Qiagen, Valencia, CA). The 260-nm optical density of the prepared total RNA was measured in a spectrophotometer and used to determine RNA concentration. One hundred nanograms of total RNA from each sample were applied to 1% agarose gels to determine the purity of RNA by ethidium bromide staining.

To perform quantitative real-time RT-PCR, each 25-µl RT-PCR mix contained 12.5 µl of 2x QuantiTect SYBR Green RT-PCR Master Mix (Qiagen), 0.5 µl of QuantiTect RT Mix (Qiagen), 500 nM forward and reverse primers, 10 ng of total RNA template, and RNase-free water. Cycle conditions for RT-PCR were as follows: reverse transcription at 50°C for 30 min; activation of HotStarTaq DNA polymerase (Qiagen) and inactivation of reverse transcriptases at 95°C for 15 min; 45 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for 45 s. 18S rRNA was used to control the amount of template for each sample (15). Standard curves for each amplification product were generated from 10-fold dilutions of pooled total RNA to determine primer efficiency. Raw PCR values were determined by plotting signals generated from individual wells against standard curves. Each reaction was run in triplicate with the results averaged. The data are representative of the means ± SD of three independent experiments. Annealing curves were performed to ensure the absence of primer dimmers. Amplification products were also run on 1% agarose gels to ensure one single band was generated for each of the tested genes.

Results were analyzed with Microsoft Excel to generate raw expression values. Differences between levels of gene expression in 2D and 3D cultures were analyzed using Student's t-test.

Western blot analysis. Supernatant samples collected at 48 h from 2D and 3D cultures were concentrated by centrifuging at 14,000 rpm across a 3,000 molecular weight filter (Millipore, Bedford, MA) at 4°C for 90 min. Samples were then electrophoresed alongside VEGF protein standards (R&D Systems, Minneapolis, MN) on a 14% polyacrylamide gel and blotted onto a Hybond C Extra nitrocellulose membrane (Whatman, Newton, MA). The membrane was incubated in blocking buffer (5% nonfat milk in PBS) and probed with goat anti-mouse VEGF primary antibody (R&D Systems). The blots were then incubated with peroxidase-conjugated donkey anti-goat secondary antibody (Sigma). Bound antibody was detected using chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ) and analyzed using BioQuant Image Analysis Software (R&M Biometrics, Nashville, TN).

DNA quantification assay. For Western blot assays, cell numbers were normalized using a technique based on the DNA-binding fluorochrome Hoechst 33258 (44). Briefly, 200 µl of working dye solution was added to PLGA films and scaffolds collected after 48 h in culture. Samples were then subjected to a triplicate freeze-thaw cycle by immersion in dry ice followed by placement in a 37°C bath. Sample aliquots of 2 µl were added to 2 ml of working dye solution, and bound fluorescence was measured on the Perkin-Elmer LS-3B fluorometer (PerkinElmer Life Sciences, Boston, MA) with excitation and emission wavelengths set at 350 and 450 nm, respectively. Serial dilutions of salmon testis DNA (Sigma) were used in a range of concentrations from 0.38 to 50 µg/ml to generate a standard curve. Cell standards were generated by measuring the bound fluorescence of known concentrations of MC3T3-E1 cell dilutions also subjected to triplicate freeze-thaw cycles. Fluorometer measurements from 2D and 3D culture samples were compared with DNA and cell standard curves to determine the amounts of DNA and cells contained in the samples.

In vitro angiogenesis assays. Growth factor-reduced Matrigel (BD Biosciences, Bedford, MA), a mixture of extracellular and basement membrane proteins derived from the mouse Engelbreth-Holm-Swarm sarcoma line (26, 27), was used to compare the ability of conditioned medium from dish and scaffold cultures to induce angiogenesis. The Matrigel was diluted with supernatant collected from 48-h 2D and 3D MC3T3-E1 cultures. Triplicate 30-µl aliquots of the Matrigel dilu-tions were added to 96-well plates and allowed to polymerize at 37°C for 60 min. Human umbilical vein endothelial cells (Cambrex, East Rutherford, NJ) were then added to each well at a concentration of 3 x 104 cells/200 µl and incubated at 37°C for 4 h, after which wells were examined under light microscopy. Human umbilical vein endothelial cells migration patterns were qualitatively evaluated, and the average number of tubule formations in randomly selected high-power field (x40) views of 2D- and 3D-conditioned assays were counted and compared. Differences between the numbers of tubule formations in 2D- and 3D-conditioned Matrigel assays were subject to Student's t-test analysis.


    RESULTS
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Expression of osteogenic markers is downregulated in 3D culture. To identify short- and long-term differences in differentiation between MC3T3-E1 cells cultured in 2D and 3D, we used quantitative real-time RT-PCR analysis to compare levels of ALP and OCN expression in the two different culture systems. The 2D cultures were carried out on PLGA films to control for any effects of the copolymer on gene expression. There were no statistically significant differences observed in the expression of the 18S rRNA reference gene in 2D and 3D cultures at any time point (Fig. 1, A and B).



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Fig. 1. Quantitative RT-PCR (QPCR) analysis of 18S rRNA (A and B), alkaline phosphatase (ALP; C and D), and and osteocalcin (OCN; E and F) expression in 2- (2D) and 3-dimensional (3D) cultures. MC3T3-E1 cells were cultured in {alpha}-minimal essential medium ({alpha}-MEM) supplemented with {beta}-GP/L-ascorbic acid (L-AA) for varying time points up to 48 h (A, C, E) and 14 days (B, D, F). Quantitative RT-PCR analysis of 18S rRNA, ALP, and OCN expression by cells cultured in 2D (black bars) and 3D (gray bars) environments is shown. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.

 
Cells cultured on PLGA scaffolds demonstrated significant decreases in the expression of ALP and OCN compared with cells grown on PLGA films in short- and long-term cultures. Expression of ALP in 3D culture remained comparatively low throughout the 48-h culture period (Fig. 1C). At 12 h, fivefold differences between expression in 2D and 3D cultures were observed. ALP expression in 3D culture increased steadily over 14 days but remained significantly lower than expression in 2D culture at 5, 7, 10, and 14 days (Fig. 1D). Expectedly, levels of OCN expression remained low in both systems early in short-term culture. However, anticipated late increases in OCN expression were significantly more pronounced in 2D culture. (Fig. 1, E and F).

VEGF expression is enhanced in 3D culture. We used quantitative real-time RT-PCR analysis of VEGF expression to identify differences in the elaboration of this angiogenic factor between MC3T3-E1 cells cultured in 2D and 3D. MC3T3-E1 cells cultured on PLGA scaffolds demonstrated increased levels of VEGF expression during 48-h and 14-day cultures relative to cells cultured on PLGA films (Fig. 2, A and B). In short-term cultures, levels of VEGF expression in 2D initially increased, then dropped, and remained persistently low throughout the remainder of the culture period. VEGF expression in 3D culture demonstrated a similar pattern but rebounded to levels that were four times greater than those observed in 2D culture at 48 h. Levels of VEGF expression in 2D increased during long-term culture but remained significantly lower than levels of 3D expression at all time points during the culture period. Maximal differences were observed at 3 days, when 3D expression was increased nearly fourfold over 2D.



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Fig. 2. Vascular endothelial growth factor (VEGF) expression in 2D and 3D cultures. MC3T3-E1 cells were cultured in {alpha}-MEM supplemented with {beta}-GP/L-AA for varying time points up to 48 h (A) and 14 days (B). Quantitative RT-PCR analysis of VEGF expression by cells cultured in 2D (black bars) and 3D (gray bars) environments is shown. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.

 
VEGF secretion is increased in 3D culture. To determine whether differences between VEGF mRNA expression in 2D and 3D cultures also contributed to differences in protein translation, we used Western blots to compare the amount of VEGF protein secreted by cells cultured in 2D and 3D. Western blot analysis of conditioned medium collected from 2D and 3D culture systems was performed following confirmation of equivalent numbers of cells in 2D and 3D samples via DNA quantification assay (Fig. 3A). Comparative densitometry of the blots demonstrated that MC3T3-E1 cells cultured on PLGA scaffolds secreted triple the amount of VEGF protein secreted by cells cultured on 2D PLGA films (Fig. 3B).



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Fig. 3. VEGF protein secretion in 2D and 3D cultures. A: DNA quantification assay based on the DNA-binding fluorochrome Hoechst-33258 was performed on 2D (black bar) and 3D (gray bar) cell cultures used for Western blots. Cultures contained equal amounts of DNA. B: samples of conditioned medium from 2D and 3D cultures of MC3T3-E1 cells were concentrated and used for Western blot analysis. Bands represent VEGF standard (lane 1), duplicate samples from 2D cultures (lanes 2 and 3), and duplicate samples from 3D cultures (lanes 4 and 5). Comparative densitometry revealed a 3-fold increase in the intensity of bands derived from 3D culture medium compared with 2D controls.

 
Conditioned medium from 3D culture induces greater in vitro angiogenesis activity. To assess the functional implications of the above-described differences in VEGF mRNA expression and protein secretion, we examined the migration patterns of human umbilical vein endothelial cells placed on polymerized Matrigel diluted with supernatant collected from 2D and 3D cultures. Differences were used to estimate the relative efficiency with which cells cultured in these different environments could induce in vitro angiogenic activity. Tubules on Matrigel diluted with supernatant from 2D cultures were more primordial in their morphology. In contrast, tubules on Matrigel mixed with 3D culture supernatant were generally organized into a more uniform and extensive network (Fig. 4, A and B). Endothelial cells seeded onto Matrigel prepared with medium from 3D cultures formed a significantly greater number of branches than 2D controls (Fig. 4C).



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Fig. 4. In vitro angiogenesis assays derived from 2D and 3D cultures. Matrigel was diluted with conditioned medium from 2D (A) and 3D (B) MC3T3-E1 cell cultures and allowed to polymerize. Aliquots of human umbilical vein endothelial cells were placed on the Matrigel surfaces, and migration patterns were observed after 4 h of incubation. C: average numbers of tubule branch points in randomly selected high-power field (x40) views of assays derived from 2D (black bar) and 3D (gray bar) cultures were quantified. **P ≤ 0.005.

 

    DISCUSSION
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Although most studies on osteoblast differentiation have been performed in conventional 2D culture systems, the importance of identifying key differences in phenotype between osteoblasts cultured in 2D and 3D environments should not be ignored. In fact, in vitro studies of osteoblasts cultured in 3D should have greater relevance to understanding the process of in vivo bone formation, since these cells develop within a 3D framework in the body. There is evidence that 3D cellular development is essential for in vitro bone formation (22); greater use of 3D culture models can lead to more accurate assessment of the responses of osteoblasts to various osteoinductive agents or mechanical stimuli. We have previously identified differential responses to bone morphogenetic protein-2 exposure by rabbit bone marrow stromal cells in 2D and 3D culture systems (18). In this study, we have identified fundamental differences in the patterns of expression of genes related to osteoblastic differentiation and neovascularization due strictly to the addition of a structural component to tissue culture.

Our results show that, in this model, the expression of genes related to bone induction is downregulated in 3D culture. Levels of expression of both ALP and OCN in cells introduced to a structural culture environment were significantly lower during short- and long-term cultures compared with 2D controls. These observations are consistent with the results of Nuttleman et al. (37), who in a recent report described a decrease in ALP expression in human marrow stromal cells encapsulated and cultured in 3D poly(ethylene glycol) hydrogels compared with cells cultured in monolayer. By comparison, VEGF expression in 3D culture was substantially enhanced relative to 2D controls. Moreover, greater levels of VEGF transcription were directly related to increases in secretion of VEGF protein and resulted in increased induction of angiogenic by cell cultured in 3D. Local VEGF release from macroporous PLGA scaffolds has previously been shown to increase deposition and vascularization of mineralized tissue (35). Our results suggest that autografted scaffold-associated osteoprogenitor cells have the potential to effect strong paracrine influences over new blood vessel recruitment to the site of bone regeneration. This notion is supported by reports that describe patterns of increased vascularization in the chick chorioallantoic membrane following exposure to known angiogenic cytokines (1, 45). To our knowledge, ours is the first report of quantitative differences in the expression of VEGF specifically due to the incorporation of a 3D component into the culture environment.

The precise mechanisms driving these observed differences remain unclear. We are currently exploring the possibility that the elaboration of a factor that is sensitive to a structural culture environment, a "dimension response element," may be involved in the regulation of osteogenic and angiogenic gene expression at the transcriptional level. Hypoxia may also play a role in the upregulation of VEGF expression observed in 3D culture. It is known that hypoxia is a characteristic of the fracture environment and likely a potent stimulus for angiogenesis at the site of fracture healing (51, 52). It has also been shown that rat calvarial osteoblasts cultured on PLGA scaffolds form a superficial layer around the scaffold periphery, with a relative paucity of cells migrating to the scaffold interior (30). This organization may be indicative of the formation of an effective hypoxic or nutritional gradient within the scaffold. Osteoblasts may respond to rates of relative hypoxia or malnutrition by releasing signals that stimulate the proliferation and differentiation of endothelial cells, which in turn contribute to the process of neovascularization (11, 31).

The 2D studies, which provide the basis of our knowledge of intracellular events governing the differentiation of osteoblasts and on which ongoing research endeavors in osseous tissue engineering are founded, do not account for potentially different patterns of gene expression when 3D structures are incorporated into the culture environment. In recent years, greater attention has been given to developing 3D culture models in various areas of biomedical research (9, 26, 49, 5658). PLGA has been identified as a particularly suitable vehicle due to its ability to support attachment, migration, proliferation, and differentiation of osteoblasts while being degraded and ultimately replaced by bone (1820). We believe that an appreciation of the impact of a structural culture component on the molecular mechanisms driving angiogenesis and osteogenesis in osteoprogenitor cells, as identified in this study, is directly relevant to the future designs of bone graft substitutes and that the specific effects of 3D culture on gene expression warrant continued study.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. A. Miller, Plastic Surgery Laboratory, VA GLAHS, 11301 Wilshire Blvd., Rm. 221, Bldg. 114, Los Angeles, CA 90073 (e-mail: millerlab{at}hotmail.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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