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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
gene expression; osteogenesis; angiogenesis
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 -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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Blum JS, Barry MA, and Mikos AG. Bone regeneration through transplantation of genetically modified cells. Clin Plast Surg 30: 611620, 2003.[CrossRef][ISI][Medline]
3. Blum JS, Barry MA, Mikos AG, and Jansen JA. In vivo evaluation of gene therapy vectors in ex vivo-derived marrow stromal cells for bone regeneration in a rat critical-size calvarial defect model. Hum Gene Ther 14: 16891701, 2003.[CrossRef][ISI][Medline]
4. Chang SC, Chuang HL, Chen YR, Chen JK, Chung HY, Lu YL, Lin HY, Tai CL, and Lou J. Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration. Gene Ther 10: 20132019, 2003.[CrossRef][ISI][Medline]
5. Chang SC, Wei FC, Chuang H, Chen YR, Chen JK, Lee KC, Chen PK, Tai CL, and Lou J. Ex vivo gene therapy in autologous critical-size craniofacial bone regeneration. Plast Reconstr Surg 112: 18411850, 2003.[ISI][Medline]
6. Chen G, Ushida T, and Tateishi T. A biodegradable hybrid sponge nested with collagen microsponges. J Biomed Mater Res 51: 273279, 2000.[CrossRef][ISI][Medline]
7. Chen Y, Cheung KM, Kung HF, Leong JC, Lu WW, and Luk KD. In vivo new bone formation by direct transfer of adenoviral-mediated bone morphogenetic protein-4 gene. Biochem Biophys Res Commun 298: 121127, 2002.[CrossRef][ISI][Medline]
8. Chen Y, Luk KD, Cheung KM, Xu R, Lin MC, Lu WW, Leong JC, and Kung HF. Gene therapy for new bone formation using adeno-associated viral bone morphogenetic protein-2 vectors. Gene Ther 10: 13451353, 2003.[CrossRef][ISI][Medline]
9. Cukierman E, Pankov R, Stevens DR, and Yamada KM. Taking cell-matrix adhesions to the third dimension. Science 294: 17081712, 2001.
10. Damasio EE, Cerri R, Masoudi B, and Risso M. Bone marrow aspiration from the posterior iliac crest and spine using a sterile and pyrogen free disposable spinal needle (Yale spinal 18 Gx31/2) without local anesthetic. Haematologica 85: 871, 2000.
11. Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, and Semenza GL. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16: 46044613, 1996.[Abstract]
12. Franceschi RT, Yang S, Rutherford RB, Krebsbach PH, Zhao M, and Wang D. Gene therapy approaches for bone regeneration. Cells Tissues Organs 176: 95108, 2004.[CrossRef][ISI][Medline]
13. Gerber HP and Ferrara N. Angiogenesis and bone growth. Trends Cardiovasc Med 10: 223228, 2000.[CrossRef][ISI][Medline]
14. Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, and Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med 5: 623628, 1999.[CrossRef][ISI][Medline]
15. Goidin D, Mamessier A, Staquet MJ, Schmitt D, and Berthier-Vergnes O. Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem 295: 1721, 2001.[CrossRef][ISI][Medline]
16. Greenwald AS, Boden SD, Goldberg VM, Khan Y, Laurencin CT, and Rosier RN. Bone-graft substitutes: facts, fictions, and applications. J Bone Joint Surg Am 83: 98103, 2001.[Medline]
17. Harper J and Kalgsbrun M. Cartilage to boneangiogenesis leads the way. Nat Med 5: 617618, 1999.[CrossRef][ISI][Medline]
18. Huang W, Carlsen B, Wulur I, Rudkin G, Ishida K, Wu B, Yamaguchi DT, and Miller TA. BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold. Exp Cell Res 299: 325334, 2004.[CrossRef][ISI][Medline]
19. Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ, and Mikos AG. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J Biomed Mater Res 36: 1728, 1997.[CrossRef][ISI][Medline]
20. Ishaug SL, Yaszemski MJ, Bizios R, and Mikos AG. Osteoblast function on synthetic biodegradable polymers. J Biomed Mater Res 28: 14451453, 1994.[CrossRef][ISI][Medline]
21. Ishaug-Riley SL, Crane-Kruger GM, Yaszemski MJ, and Mikos AG. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers. Biomaterials 19: 14051412, 1998.[CrossRef][ISI][Medline]
22. Kale S, Biermann S, Edwards C, Tarnowski C, Morris M, Long MW. Three-dimensional cellular development is essential for ex vivo formation of human bone. Nat Biotechnol 18: 954958, 2000.[CrossRef][ISI][Medline]
23. Kim BS, Nikolovski J, Bonadio J, Smiley E, and Mooney DJ. Engineered smooth muscle tissues: regulating cell phenotype with the scaffold. Exp Cell Res 251: 318328, 1999.[CrossRef][ISI][Medline]
24. Kim HD and Valentini RF. Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro. J Biomed Mater Res 59: 573584, 2002.[CrossRef][ISI][Medline]
25. Kim HH, Lee SE, Chung WJ, Choi Y, Kwack K, Kim SW, Kim MS, Park H, and Lee ZH. Stabilization of hypoxia-inducible factor-1 is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells. Cytokine 17: 1427, 2002.[CrossRef][ISI][Medline]
26. Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, Laurie GW, and Martin GR. Basement membrane complexes with biological activity. Biochemistry 25: 312318, 1986.[CrossRef][ISI][Medline]
27. Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, and Martin GR. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21: 61886193, 1982.[CrossRef][ISI][Medline]
28. Laurencin CT, Attawia MA, Lu LQ, Borden MD, Lu HH, Gorum WJ, and Lieberman JR. Poly(lactide-co-glycolide)/hydroxyapatite delivery of BMP-2-producing cells: a regional gene therapy approach to bone regeneration. Biomaterials 22: 12711277, 2001.[CrossRef][ISI][Medline]
29. Lewinson D, Maor G, Rozen N, Rabinovich I, Stahl S, and Rachmiel A. Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system. Histochem Cell Biol 116: 381388, 2001.[CrossRef][ISI][Medline]
30. Lu HH, El-Amin SF, Scott KD, and Laurencin CT. Three-dimensional, bioactive, biodegradable, polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro. J Biomed Mater Res 64: 465474, 2003.[CrossRef]
31. Mauney JR, Blumberg J, Pirun M, Volloch V, Vunjak-Novakovic G, and Kaplan DL. Osteogenic differentiation of human bone marrow stromal cells on partially demineralized bone scaffolds in vitro. Tissue Eng 10: 8192, 2004.[CrossRef][ISI][Medline]
32. Mikos AG, Lyman MD, Freed LE, and Langer R. Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture. Biomaterials 15: 5558, 1994.[CrossRef][ISI][Medline]
33. Murphy WL, Kohn DH, and Mooney DJ. Growth of continuous bonelike mineral within porous poly(lactide-co-glycolide) scaffolds in vitro. J Biomed Mater Res 50: 5058, 2000.[CrossRef][ISI][Medline]
34. Murphy WL, Peters MC, Kohn DH, and Mooney DJ. Sustained release of vascular endothelial growth factor from mineralized poly(lactide-co-glycolide) scaffolds for tissue engineering. Biomaterials 21: 25212527, 2000.[CrossRef][ISI][Medline]
35. Murphy WL, Simmons CA, Kaigler D, and Mooney DJ. Bone regeneration via a mineral substrate and induced angiogenesis. J Dent Res 83: 204210, 2004.
36. Nussenbaum B, Rutherford RB, Teknos TN, Dornfeld KJ, and Krebsbach PH. Ex vivo gene therapy for skeletal regeneration in cranial defects compromised by postoperative radiotherapy. Hum Gene Ther 14: 11071115, 2003.[CrossRef][ISI][Medline]
37. Nuttelman CR, Tripodi MC, and Anseth KS. In vitro osteogenic differentiation of human mesenchymal stem cells photoencapsulated in PEG hydrogels. J Biomed Mater Res 68: 773782, 2004.[CrossRef]
38. Ochi K, Chen G, Ushida T, Gojo S, Segawa K, Tai H, Ueno K, Ohkawa H, Mori T, Yamaguchi A, Toyama Y, Hata J, and Umezawa A. Use of isolated mature osteoblasts in abundance acts as desired-shaped bone regeneration in combination with a modified poly-DL-lactic-co-glycolic acid (PLGA)-collagen sponge. J Cell Physiol 194: 4553, 2003.[CrossRef][ISI][Medline]
39. Park J, Ries J, Gelse K, Kloss F, von der Mark K., Wiltfang J., Neukam FW, and Schneider H. Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer: a comparison of adenoviral vectors and liposomes. Gene Ther 10: 10891098, 2003.[CrossRef][ISI][Medline]
40. Paulman PM. Bone marrow sampling. Am Fam Physician 40: 8589, 1989.[ISI][Medline]
41. Perets A, Baruch Y, Weisbuch F, Shoshany G, Neufeld G, and Cohen S. Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres. J Biomed Mater Res 65: 489497, 2003.[CrossRef]
42. Peter SJ, Lu L, Kim DJ, and Mikos AG. Marrow stromal osteoblast function on a poly(propylene fumarate)/beta-tricalcium phosphate biodegradable orthopaedic composite. Biomaterials 21: 12071213, 2000.[CrossRef][ISI][Medline]
43. Porter BD, Oldham JB, He SL, Zobitz ME, Payne RG, An KN, Currier BL, Mikos AG, and Yaszemski MJ. Mechanical properties of a biodegradable bone regeneration scaffold. J Biomech Eng 122: 286288, 2000.[CrossRef][ISI][Medline]
44. Rago R, Mitchen J, and Wilding G. DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water. Anal Biochem 191: 3134, 1990.[CrossRef][ISI][Medline]
45. Ribatti D, Conconi MT, Nico B, Baiguera S, Corsi P, Parnigotto PP, and Nussdorfer GG. Angiogenic response induced by acellular brain scaffolds grafted onto the chick embryo chorioallantoic membrane. Brain Res 989: 915, 2003.[CrossRef][ISI][Medline]
46. Rowe NM, Mehrara BJ, Luchs JS, Dudziak ME, Steinbrech DS, Illei PB, Fernandez GJ, Gittes GK, and Longaker MT. Angiogenesis during mandibular distraction osteogenesis. Ann Plast Surg 42: 470475, 1999.[ISI][Medline]
47. Rutherford RB, Nussenbaum B, and Krebsbach PH. Bone morphogenetic protein 7 ex vivo gene therapy. Drug News Perspect 16: 510, 2003.[CrossRef][ISI][Medline]
48. Saito N, Okada T, Horiuchi H, Ota H, Takahashi J, Murakami N, Nawata M, Kojima S, Nozaki K, and Takaoka K. Local bone formation by injection of recombinant human bone morphogenetic protein-2 contained in polymer carriers. Bone 32: 381386, 2003.[CrossRef][ISI][Medline]
49. Sato M, Ishikawa O, and Miyachi Y. Distinct patterns of collagen gene expression are seen in normal and keloid fibroblasts grown in three-dimensional culture. Br J Dermatol 138: 938943, 1998.[CrossRef][ISI][Medline]
50. Shea LD, Wang D, Franceschi RT, and Mooney DJ. Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds. Tissue Eng 6: 605617, 2000.[CrossRef][ISI][Medline]
51. Steinbrech DS, Mehrara BJ, Saadeh PB, Greenwald JA, Spector JA, Gittes GK, and Longaker MT. VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism. Am J Physiol Cell Physiol 278: C853C860, 2000.
52. Steinbrech DS, Mehrara BJ, Saadeh PB, Chin G, Dudziak ME, Gerrets RP, Gittes GK, and Longaker MT. Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro. Plast Reconstr Surg 104: 738747, 1999.[ISI][Medline]
53. Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, van Bruggen N, Redmond HP, Carano RA, and Filvaroff EH. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA 99: 96569661, 2002.
54. Tsuda H, Wada T, Ito Y, Uchida H, Dehari H, Nakamura K, Sasaki K, Kobune M, Yamashita T, and Hamada H. Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector. Mol Ther 7: 354365, 2003.[CrossRef][ISI][Medline]
55. Turgeman G, Aslan H, Gazit Z, and Gazit D. Cell-mediated gene therapy for bone formation and regeneration. Curr Opin Mol Ther 4: 390394, 2002.[ISI][Medline]
56. Wang F, Weaver VM, Petersen OW, Larabell CA, Dedhar S, Briand P, Lupu R, and Bissell MJ. Reciprocal interactions between 1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology. Proc Natl Acad Sci USA 95: 1482114826, 1998.
57. Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C, and Bissell MJ. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol 137: 231245, 1997.
58. Webb K, Li W, Hitchcock RW, Smeal RM, Gray SD, and Tresco PA. Comparison of human fibroblast ECM-related gene expression on elastic three-dimensional substrates relative to two-dimensional films of the same material. Biomaterials 24: 46814690, 2003.[CrossRef][ISI][Medline]
59. Yaszemski MJ, Payne RG, Hayes WC, Langer R, and Mikos AG. Evolution of bone transplantation: molecular, cellular and tissue strategies to engineer human bone. Biomaterials 17: 175185, 1996.[CrossRef][ISI][Medline]
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |