Publications

2006
Leach KJ, Kaigler D, Wang Z, Krebsbach PH, Mooney DJ. Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration. Biomaterials. 2006;27 (17) :3249-55.Abstract
Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth factor (VEGF) has demonstrated benefit in bone regeneration as well. We hypothesized that the specific combination of prolonged localized VEGF presentation from a matrix coated with a bioactive glass may enhance bone regeneration. To test this hypothesis, the capacity of VEGF-releasing polymeric scaffolds with a bioactive glass coating was examined in vitro and in vivo using a rat critical-sized defect model. In the presence of a bioactive glass coating, we did not detect pronounced differences in the differentiation of human mesenchymal stem cells in vitro. However, we observed significantly enhanced mitogenic stimulation of endothelial cells in the presence of the bioactive glass coating, with an additive effect with VEGF release. This trend was maintained in vivo, where coated VEGF-releasing scaffolds demonstrated significant improvements in blood vessel density at 2 weeks versus coated control scaffolds. At 12 weeks, bone mineral density was significantly increased in coated VEGF-releasing scaffolds versus coated controls, while only a slight increase in bone volume fraction was observed. The results of this study suggest that a bioactive glass coating on a polymeric substrate participates in bone healing through indirect processes which enhance angiogenesis and bone maturation and not directly on osteoprogenitor differentiation and bone formation. The mass of bioactive glass used in this study provides a comparable and potentially additive, response to localized VEGF delivery over early time points. These studies demonstrate a materials approach to achieve an angiogenic response formerly limited to the delivery of inductive growth factors.
Smith MK, Riddle KW, Mooney DJ. Delivery of hepatotrophic factors fails to enhance longer-term survival of subcutaneously transplanted hepatocytes. Tissue Eng. 2006;12 (2) :235-44.Abstract
Tissue engineering approaches have been investigated as a strategy for hepatocyte transplantation; however the death of a majority of transplanted cells critically limits success of these approaches. In a previous study, a transient increase in hepatocyte survival was achieved through delivery of vascular endothelial growth factor (VEGF) from the porous polymer scaffold utilized for cell delivery. To enhance longer-term survival of the hepatocytes, this delivery system was modified to additionally deliver epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in a sustained manner. Hepatocytes were subcutaneously implanted in SCID mice on scaffolds containing EGF and/or HGF, in addition to VEGF, and survival was monitored for two weeks. A short-term enhancement of hepatocyte survival was observed after one week and is attributed to VEGF-enhanced vascularization, which was not altered by EGF or HGF. Surprisingly, long-term hepatocyte engraftment was not improved, as survival declined to the level of control conditions for all growth factor combinations after two weeks. This investigation indicates that the survival of hepatocytes transplanted into heterotopic locations is dependent on multiple signals. The delivery system developed for the current study may be useful in elucidating the specific factors controlling this process, and bring therapeutic transplantation of hepatocytes closer to implementation.
Hill E, Boontheekul T, Mooney DJ. Designing scaffolds to enhance transplanted myoblast survival and migration. Tissue Eng. 2006;12 (5) :1295-304.Abstract
Myoblast transplantation is currently limited by poor survival and integration of these cells into host musculature. Transplantation systems that enhance the viability of the cells and induce their outward migration to populate injured muscle may enhance the success of this approach to muscle regeneration. In this study, enriched populations of primary myoblasts were seeded onto delivery vehicles formed from alginate, and the role of vehicle design and local growth factor delivery in cell survival and migration were examined. Only 5 +/- 2.5% of cells seeded into nanoporous alginate gels survived for 24 h and only 4 +/- 0.5% migrated out of the gels. Coupling cell adhesion peptides (G4RGDSP) to the alginate prior to gelling slightly increased the viability of cells within the scaffold to 16 +/- 1.4% and outward migration to 6 +/- 1%. However, processing peptide-modified alginate gels to yield macroporous scaffolds, in combination with sustained delivery of HGF and FGF2 from the material, dramatically increased the viability of seeded cells over a 5-day time course and increased outward migration to 110 +/- 12%. This data indicate long-term survival and migration of myoblasts placed within polymeric delivery vehicles can be greatly increased by appropriate scaffold composition, architecture, and growth factor delivery. This system may be particularly useful in the regeneration of muscle tissue and be broadly useful in the regeneration of other tissues as well.
Comisar WA, Hsiong SX, Kong H-J, Mooney DJ, Linderman JJ. Multi-scale modeling to predict ligand presentation within RGD nanopatterned hydrogels. Biomaterials. 2006;27 (10) :2322-9.Abstract
The adhesion ligand RGD has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters cell adhesion, proliferation, and differentiation. However, elucidating the impact of nanopattern parameters on cellular responses has been stymied by a lack of understanding of the actual ligand presentation within these systems. We have developed a multi-scale predictive modeling approach to characterize the adhesion ligand nanopatterns within an alginate hydrogel matrix. The models predict the distribution of ligand islands, the spacing between ligands within an island and the fraction of ligands accessible for cell binding. These model predictions can be used to select pattern parameter ranges for experiments on the effects of individual parameters on cellular responses. Additionally, our technique could also be applied to other polymer systems presenting peptides or other signaling molecules.
Hsiong SX, Mooney DJ. Regeneration of vascularized bone. Periodontol 2000. 2006;41 :109-22.
Hill E, Boontheekul T, Mooney DJ. Regulating activation of transplanted cells controls tissue regeneration. Proc Natl Acad Sci U S A. 2006;103 (8) :2494-9.Abstract
Current approaches to tissue regeneration are limited by the death of most transplanted cells and/or resultant poor integration of transplanted cells with host tissue. We hypothesized that transplanting progenitor cells within synthetic microenvironments that maintain viability, prevent terminal differentiation, and promote outward migration would significantly enhance their repopulation and regeneration of damaged host tissue. This hypothesis was addressed in the context of muscle regeneration by transplanting satellite cells to muscle laceration sites on a delivery vehicle releasing factors that induce cell activation and migration (hepatocyte growth factor and fibroblast growth factor 2) or transplantation on materials lacking factor release. Controls included direct cell injection into muscle, the implantation of blank scaffolds, and scaffolds releasing factors without cells. Injected cells demonstrated a limited repopulation of damaged muscle and led to a slight improvement in muscle regeneration, as expected. Delivery of cells on scaffolds that did not promote migration resulted in no improvement in muscle regeneration. Strikingly, delivery of cells on scaffolds that promoted myoblast activation and migration led to extensive repopulation of host muscle tissue and increased the regeneration of muscle fibers at the wound and the mass of the injured muscle. This previously undescribed strategy for cell transplantation significantly enhances muscle regeneration from transplanted cells and may be broadly applicable to the various tissues and organ systems in which provision and instruction of a cell population competent to participate in regeneration may be clinically useful.
Storrie H, Mooney DJ. Sustained delivery of plasmid DNA from polymeric scaffolds for tissue engineering. Adv Drug Deliv Rev. 2006;58 (4) :500-14.Abstract
The encapsulation of DNA into polymeric depot systems can be used to spatially and temporally control DNA release, leading to a sustained, local delivery of therapeutic factors for tissue regeneration. Prior to encapsulation, DNA may be condensed with cationic polymers to decrease particle size, protect DNA from degradation, promote interaction with cell membranes, and facilitate endosomal release via the proton sponge effect. DNA has been encapsulated with either natural or synthetic polymers to form micro- and nanospheres, porous scaffolds and hydrogels for sustained DNA release and the polymer physical and chemical properties have been shown to influence transfection efficiency. Polymeric depot systems have been applied for bone, skin, and nerve regeneration as well as therapeutic angiogenesis, indicating the broad applicability of these systems for tissue engineering.
Ennett AB, Kaigler D, Mooney DJ. Temporally regulated delivery of VEGF in vitro and in vivo. J Biomed Mater Res A. 2006;79 (1) :176-84.Abstract
The exposure duration and tissue distribution will likely dictate the success of vascular endothelial growth factor (VEGF) in therapeutic angiogenesis. We hypothesized that these variables can be regulated via the manner in which the VEGF is incorporated into polymer constructs (formed with a gas foaming technique) used for its delivery. VEGF was incorporated directly into poly(lactide-co-glycolide) (PLG) scaffolds or pre-encapsulated in PLG microspheres used to fabricate scaffolds. Protein release kinetics and tissue distribution were determined using iodinated VEGF. VEGF was positioned predominantly adjacent to scaffold pores when incorporated directly and was released rapidly (40-60% in 5 days). Pre-encapsulation led to the VEGF being more deeply embedded and resulted in a delayed release. Alterations in polymer composition, scaffold size, and matrix composition generated minor variations in release kinetics. In vivo, the released VEGF generated local protein concentrations above 10 ng/mL at distances up to 2 cm from the implant site for the 21 days of the experiment, with negligible release into the systemic circulation, and significantly enhanced local angiogenesis. These data indicate that VEGF can be administered in a sustained and localized fashion in vivo, and the timing of VEGF delivery can be altered with the mechanism of incorporation into polymer scaffolds used for its delivery.
Kaigler D, Krebsbach PH, Wang Z, West ER, Horger K, Mooney DJ. Transplanted endothelial cells enhance orthotopic bone regeneration. J Dent Res. 2006;85 (7) :633-7.Abstract
The aim of this study was to determine if endothelial cells could enhance bone marrow stromal-cell-mediated bone regeneration in an osseous defect. Using poly-lactide-co-glycolide scaffolds as cell carriers, we transplanted bone marrow stromal cells alone or with endothelial cells into 8.5-mm calvarial defects created in nude rats. Histological analyses of blood vessel and bone formation were performed, and microcomputed tomography (muCT) was used to assess mineralized bone matrix. Though the magnitude of the angiogenic response between groups was the same, muCT analysis revealed earlier mineralization of bone in the co-transplantation condition. Ultimately, there was a significant increase (40%) in bone formation in the co-transplantation group (33 +/- 2%), compared with the transplantation of bone marrow stromal cells alone (23 +/- 3%). Analysis of these data demonstrates that, in an orthotopic site, transplanted endothelial cells can influence the bone-regenerative capacity of bone marrow stromal cells.
Kaigler D, Wang Z, Horger K, Mooney DJ, Krebsbach PH. VEGF scaffolds enhance angiogenesis and bone regeneration in irradiated osseous defects. J Bone Miner Res. 2006;21 (5) :735-44.Abstract
UNLABELLED: Bone regeneration is challenging in sites where the blood supply has been compromised by radiation. We examined the potential of a growth factor (VEGF) delivery system to enhance angiogenesis and bone formation in irradiated calvarial defects. VEGF-releasing polymers significantly increased blood vessel density and vascular perfusion in irradiated defects and increased bone formation relative to control conditions. INTRODUCTION: Radiation therapy causes damage to tissues and inhibits its regenerative capacity. Tissue injury from radiation is in large part caused by a compromised vascular supply and reduced perfusion of tissues. The aim of this study was to determine if delivery of vascular endothelial growth factor (VEGF) from a biodegradable PLGA (copolymer of D,L-lactide and glycolide) scaffold could enhance neovascularization and bone regeneration in irradiated osseous defects. MATERIALS AND METHODS: An isolated area of the calvarium of Fisher rats was irradiated (12 Gy) 2 weeks preoperatively, and two 3.5-mm osseous defects were created in this area, followed by the placement of PLGA scaffolds or VEGF scaffolds (PLGA scaffolds with incorporated VEGF) into the defects. Laser Doppler perfusion imaging was performed to measure perfusion of these areas at 1, 2, and 6 weeks. Implants were retrieved at 2, 6, and 12 weeks, and histologic and muCT analyses were performed to determine neovascularization and bone regeneration. RESULTS: Histological analyses revealed statistically significant increases in blood vessel formation (>2-fold) and function (30%) within the VEGF scaffolds compared with PLGA scaffolds. Additionally, evaluation of bone regeneration through bone histomorphometric and muCT analyses revealed significantly greater bone coverage (26.36 +/- 6.91% versus 7.05 +/- 2.09% [SD]) and increased BMD (130.80 +/- 58.05 versus 71.28 +/- 42.94 mg/cm(3)) in VEGF scaffolds compared with PLGA scaffolds. CONCLUSIONS: Our findings show that VEGF scaffolds have the ability to enhance neovascularization and bone regeneration in irradiated osseous defects, outlining a novel approach for engineering tissues in hypovascular environments.
Augst AD, Kong H, Mooney DJ. Alginate hydrogels as biomaterials. Macromol Biosci. 2006;6 (8) :623-33.Abstract
[Image: see text] Alginate hydrogels are proving to have a wide applicability as biomaterials. They have been used as scaffolds for tissue engineering, as delivery vehicles for drugs, and as model extracellular matrices for basic biological studies. These applications require tight control of a number of material properties including mechanical stiffness, swelling, degradation, cell attachment, and binding or release of bioactive molecules. Control over these properties can be achieved by chemical or physical modifications of the polysaccharide itself or the gels formed from alginate. The utility of these modified alginate gels as biomaterials has been demonstrated in a number of in vitro and in vivo studies.Micro-CT images of bone-like constructs that result from transplantation of osteoblasts on gels that degrade over a time frame of several months leading to improved bone formation.
Matsumoto T, Mooney DJ. Cell instructive polymers. Adv Biochem Eng Biotechnol. 2006;102 :113-37.Abstract
Polymeric materials used in tissue engineering were initially used solely as delivery vehicles for transplanting cells. However, these materials are currently designed to actively regulate the resultant tissue structure and function. This control is achieved through spatial and temporal regulation of various cues (e.g., adhesion ligands, growth factors) provided to interacting cells from the material. These polymeric materials that control cell function and tissue formation are termed cell instructive polymers, and recent trends in their design are outlined in this chapter.
Kong H, Boontheekul T, Mooney DJ. Quantifying the relation between adhesion ligand-receptor bond formation and cell phenotype. Proc Natl Acad Sci U S A. 2006;103 (49) :18534-9.Abstract
One of the fundamental interactions in cell biology is the binding of cell receptors to adhesion ligands, and many aspects of cell behavior are believed to be regulated by the number of these bonds that form. Unfortunately, a lack of methods to quantify bond formation, especially for cells in 3D cultures or tissues, has precluded direct probing of this assumption. We now demonstrate that a FRET technique can be used to quantify the number of bonds formed between cellular receptors and synthetic adhesion oligopeptides coupled to an artificial extracellular matrix. Similar quantitative relations were found between bond number and the proliferation and differentiation of MC3T3-E1 preosteoblasts and C2C12 myoblasts, although the relation was distinct for each cell type. This approach to understanding 3D cell-extracellular matrix interactions will allow one to both predict cell behavior and to use bond number as a fundamental design criteria for synthetic extracellular matrices.
2005
D'Souza KA, Mooney DJ, Russell AE, MacIsaac AI, Aylward PE, Prior DL. Abnormal septal motion affects early diastolic velocities at the septal and lateral mitral annulus, and impacts on estimation of the pulmonary capillary wedge pressure. J Am Soc Echocardiogr. 2005;18 (5) :445-53.Abstract
Abnormal motion of the interventricular septum (ASM), seen post cardiac operation, with left bundle branch block or right ventricular pacing, may affect septal mitral annular motion and correlation of the ratio between the velocity of early diastolic mitral inflow and the early diastolic mitral annular velocity (E/Ea) with pulmonary capillary wedge pressure (PCWP). We examined the effect of ASM on the relationship between E/Ea and E/Vp (propagation velocity of mitral inflow) ratios and PCWP in adult patients in the intensive care unit (14 with normal septal motion [NSM], 36 with ASM) undergoing echocardiography and pulmonary artery catheterization. E/Ea correlated well with PCWP during NSM ( r = 0.86 lateral annulus, r = 0.75 septal annulus), but poorly during ASM ( r = 0.36 lateral annulus, r = 0.39 septal annulus). E/Vp correlated poorly with PCWP ( r = 0.05 NSM, r = 0.17 ASM). For patients who are critically ill, E/Vp ratios poorly estimate PCWP. During NSM, E/Ea ratios measured at the lateral or septal annulus correlate well with PCWP. ASM affects E/Ea ratios at both the septal and lateral annulus, making E/Ea ratios unreliable for estimating PCWP in this group.
Boontheekul T, Kong H-J, Mooney DJ. Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. Biomaterials. 2005;26 (15) :2455-65.Abstract
Degradability is often a critical property of materials utilized in tissue engineering. Although alginate, a naturally derived polysaccharide, is an attractive material due to its biocompatibility and ability to form hydrogels, its slow and uncontrollable degradation can be an undesirable feature. In this study, we characterized gels formed using a combination of partial oxidation of polymer chains and a bimodal molecular weight distribution of polymer. Specifically, alginates were partially oxidized to a theoretical extent of 1% with sodium periodate, which created acetal groups susceptible to hydrolysis. The ratio of low MW to high MW alginates used to form gels was also varied, while maintaining the gel forming ability of the polymer. The rate of degradation was found to be controlled by both the oxidation and the ratio of high to low MW alginates, as monitored by the reduction of mechanical properties and corresponding number of crosslinks, dry weight loss, and molecular weight decrease. It was subsequently examined whether these modifications would lead to reduced biocompatibility by culturing C2C12 myoblast on these gels. Myoblasts adhered, proliferated, and differentiated on the modified gels at a comparable rate as those cultured on the unmodified gels. Altogether, this data indicates these hydrogels exhibit tunable degradation rates and provide a powerful material system for tissue engineering.
Murphy WL, Hsiong S, Richardson TP, Simmons CA, Mooney DJ. Effects of a bone-like mineral film on phenotype of adult human mesenchymal stem cells in vitro. Biomaterials. 2005;26 (3) :303-10.Abstract
Multipotent cell types are rapidly becoming key components in a variety of tissue engineering schemes, and mesenchymal stem cells (MSCs) are emerging as an important tool in bone tissue regeneration. Although several soluble signals influencing osteogenic differentiation of MSCs in vitro are well-characterized, relatively little is known about the influence of substrate signals. This study was aimed at elucidating the effects of a bone-like mineral (BLM), which is vital in the process of bone bonding to orthopedic implant materials, on the osteogenic differentiation of human MSCs in vitro. Growth of a BLM film (carbonate apatite, Ca/P = 1.55) on poly(lactide-co-glycolide) (PLG) substrates was achieved via surface hydrolysis and subsequent incubation in a modified simulated body fluid. The BLM film demonstrated significantly increased adsorption of fibronectin, and supported enhanced proliferation of human mesenchymal stem cells (hMSCs) relative to PLG substrates. In the absence of osteogenic supplements hMSCs did not display a high expression of osteogenic markers on BLM or PLG. In the presence of osteogenic supplements hMSCs exhibited greater expression of osteogenic markers on PLG substrates than on BLM substrates, as measured by alkaline phosphatase activity and osteocalcin production. Taken together, these data support the concept that substrate signals significantly influence MSC growth and differentiation, highlighting the importance of carrier material composition in stem cell-based tissue engineering schemes.
Mooney DJ, Boontheekul T, Chen R, Leach K. Actively regulating bioengineered tissue and organ formation. Orthod Craniofac Res. 2005;8 (3) :141-4.Abstract
OBJECTIVES: Describe current and future approaches to tissue engineering, specifically in the area of bone regeneration. These approaches will allow one to actively regulate the cellular populations participating in this process. DESIGN: Many approaches to actively regulate cellular phenotype are under exploration, and these typically exploit known signal transduction pathways via presentation of specific receptor-binding ligands, and may also deliver mechanical information via the physical bridge formed by the receptor-ligand interactions. Cellular gene expression may also be directly modulated utilizing gene therapy approaches to control tissue regeneration. CONCLUSIONS: Significant progress has been made to date in bone regeneration using inductive molecules and transplanted cells, and FDA approved therapies have resulted. While approaches to date have focused on delivery of single stimuli (e.g. one growth factor), future efforts will likely attempt to more closely mimic developmental processes by the delivery of multiple inputs to the cells in spatially and temporally regulated fashions.
Huang Y-C, Simmons C, Kaigler D, Rice KG, Mooney DJ. Bone regeneration in a rat cranial defect with delivery of PEI-condensed plasmid DNA encoding for bone morphogenetic protein-4 (BMP-4). Gene Ther. 2005;12 (5) :418-26.Abstract
Gene therapy approaches to bone tissue engineering have been widely explored. While localized delivery of plasmid DNA encoding for osteogenic factors is attractive for promoting bone regeneration, the low transfection efficiency inherent with plasmid delivery may limit this approach. We hypothesized that this limitation could be overcome by condensing plasmid DNA with nonviral vectors such as poly(ethylenimine) (PEI), and delivering the plasmid DNA in a sustained and localized manner from poly(lactic-co-glycolic acid) (PLGA) scaffolds. To address this possibility, scaffolds delivering plasmid DNA encoding for bone morphogenetic protein-4 (BMP-4) were implanted into a cranial critical-sized defect for time periods up to 15 weeks. The control conditions included no scaffold (defect left empty), blank scaffolds (no delivered DNA), and scaffolds encapsulating plasmid DNA (non-condensed). Histological and microcomputed tomography analysis of the defect sites over time demonstrated that bone regeneration was significant at the defect edges and within the defect site when scaffolds encapsulating condensed DNA were placed in the defect. In contrast, bone formation was mainly confined to the defect edges within scaffolds encapsulating plasmid DNA, and when blank scaffolds were used to fill the defect. Histomorphometric analysis revealed a significant increase in total bone formation (at least 4.5-fold) within scaffolds incorporating condensed DNA, relative to blank scaffolds and scaffolds incorporating uncondensed DNA at each time point. In addition, there was a significant increase both in osteoid and mineralized tissue density within scaffolds incorporating condensed DNA, when compared with blank scaffolds and scaffolds incorporating uncondensed DNA, suggesting that delivery of condensed DNA led to more complete mineralized tissue regeneration within the defect area. This study demonstrated that the scaffold delivery system encapsulating PEI-condensed DNA encoding for BMP-4 was capable of enhancing bone formation and may find applications in other tissue types.
Drury JL, Boontheekul T, Boontheeku T, Mooney DJ. Cellular cross-linking of peptide modified hydrogels. J Biomech Eng. 2005;127 (2) :220-8.Abstract
Peptide modification of hydrogel-forming materials is being widely explored as a means to regulate the phenotype of cells immobilized within the gels. Alternatively, we hypothesized that the adhesive interactions between cells and peptides coupled to the gel-forming materials would also enhance the overall mechanical properties of the gels. To test this hypothesis, alginate polymers were modified with RGDSP-containing peptides and the resultant polymer was used to encapsulate C2C12 myoblasts. The mechanical properties of these gels were then assessed as a function of both peptide and cell density using compression and tensile tests. Overall, it was found that above a critical peptide and cell density, encapsulated myoblasts were able to provide additional mechanical integrity to hydrogels composed of peptide-modified alginate. This occurred presumably by means of cell-peptide cross-linking of the alginate polymers, in addition to the usual Ca++ cross-linking. These results are potentially applicable to other polymer systems and important for a range of tissue engineering applications.
Huang Y-C, Kaigler D, Rice KG, Krebsbach PH, Mooney DJ. Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration. J Bone Miner Res. 2005;20 (5) :848-57.Abstract
UNLABELLED: Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions. INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation. MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT. RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition. CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics.

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