Publications

2004
Riddle KW, Mooney DJ. Role of poly(lactide-co-glycolide) particle size on gas-foamed scaffolds. J Biomater Sci Polym Ed. 2004;15 (12) :1561-70.Abstract
Macroporous polymeric scaffolds are frequently used in tissue engineering to allow for cell seeding and host cell invasion of the scaffold following implantation. The process of gas foaming/particulate leaching (GF/PL) is one method to form porous three dimensional scaffolds from particulate poly(lactide-co-glycolide) (PLG). The current study was designed to test the hypothesis that the size of the polymer particles used in this process will control the properties of the scaffolds. Scaffolds were prepared from PLG particles of various sizes (less than 75 microm, 75-106 microm, 106-250 microm and 250-425 microm) and subsequently analyzed. Scaffolds formed from large particles (250-425 microm) displayed significantly decreased compressive moduli, as compared to scaffolds fabricated from smaller particles. In addition, these scaffolds have a pore structure that is less interconnected and contains closed pores. Analysis of tissue in-growth, utilizing a novel computer-aided method, demonstrated that scaffolds formed from smaller particle sizes (less than 106 microm) have significantly more tissue penetration than those formed from larger particle sizes (greater than 106 microm). These results indicate that using small PLG particles (less than 106 microm) leads to high elastic moduli, provides a more interconnected pore structure and promotes greater tissue penetration into the scaffolds in vivo.
2003
Lee S-H, Kim B-S, Kim SH, Choi SW, Jeong SI, Kwon IK, Kang SW, Nikolovski J, Mooney DJ, Han Y-K, et al. Elastic biodegradable poly(glycolide-co-caprolactone) scaffold for tissue engineering. J Biomed Mater Res A. 2003;66 (1) :29-37.Abstract
Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, and it would be necessary for the development of the elastic scaffolds if one wishes to engineer SM tissues under cyclic mechanical loading. This study reports on the development of an elastic scaffold fabricated from a biodegradable polymer. Biodegradable poly(glycolide-co-caprolactone) (PGCL) copolymer was synthesized from glycolide and epsilon-caprolactone in the presence of stannous octoate as catalyst. The copolymer was characterized by (1)H-NMR, gel permeation chromatography and differential scanning calorimetry. Scaffolds for tissue engineering applications were fabricated from PGCL copolymer using the solvent-casting and particle-leaching technique. The PGCL scaffolds produced in this fashion had open pore structures (average pore size = 250 microm) without the usual nonporous skin layer on external surfaces. Mechanical testing revealed that PGCL scaffolds were far more elastic than poly(lactic-co-glycolic acid) (PLGA) scaffolds fabricated using the same method. Tensile mechanical tests indicated that PGCL scaffolds could withstand an extension of 250% without cracking, which was much higher than withstood by PLGA scaffolds (10-15%). In addition, PGCL scaffolds achieved recoveries exceeding 96% at applied extensions of up to 230%, whereas PLGA scaffolds failed (cracked) at an applied strain of 20%. Dynamic mechanical tests showed that the permanent deformation of the PGCL scaffolds in a dry condition produced was less than 4% of the applied strain, when an elongation of 20% at a frequency of 1 Hz (1 cycle per second) was applied for 6 days. Moreover, PGCL scaffolds in a buffer solution also had permanent deformations less than 5% of the applied strain when an elongation of 10% at a frequency of 1 Hz was applied for 2 days. The usefulness of the PGCL scaffolds was demonstrated by engineering SM tissues in vivo. This study shows that the elastic PGCL scaffolds produced in this study could be used to engineer SM-containing tissues (e.g. blood vessels and bladders) in mechanically dynamic environments.
Polverini PJ, Nör JE, Peters MC, Mooney DJ. Growth of human blood vessels in severe combined immunodeficient mice. A new in vivo model system of angiogenesis. Methods Mol Med. 2003;78 :161-77.
Lee KY, Peters MC, Mooney DJ. Comparison of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in SCID mice. J Control Release. 2003;87 (1-3) :49-56.Abstract
Therapeutic angiogenesis is a promising approach to treat patients with cardiovascular disease, and will likely be critical to engineering large tissues. Many growth factors have been found to play significant roles in angiogenesis, and vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the most extensively investigated angiogenic factors to date. However, the appropriate dose to obtain a desired response and the effectiveness of each factor, relative to the other, in promoting angiogenesis at a specific site in the body remains unclear. We have used alginate hydrogels as localized delivery vehicles for VEGF and bFGF, and compared the ability of these factors to promote new blood vessel formation in the subcutaneous tissue of severe combined immunodeficient (SCID) mice. We have found that the thickness of a granulation tissue layer formed around the gel and the number of blood vessels in the layer increased with the dose of VEGF in the gel, but the density of new blood vessels remained relatively constant. Sustained and localized delivery of bFGF from the gels, while similarly leading to an increase in the density of blood vessels in the granulation tissue, did not lead to as high of a blood vessel density as VEGF. The results of this study support previous studies demonstrating the utility of both VEGF and bFGF in promoting angiogenesis, and suggest VEGF is more appropriate for creating a dense bed of new blood vessels in this model.
Simmons CA, Matlis S, Thornton AJ, Chen S, Wang CY, Mooney DJ. Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway. J Biomech. 2003;36 (8) :1087-96.Abstract
Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.
Nikolovski J, Kim B-S, Mooney DJ. Cyclic strain inhibits switching of smooth muscle cells to an osteoblast-like phenotype. FASEB J. 2003;17 (3) :455-7.Abstract
Ectopic calcification is commonly associated with cardiovascular disease, injury, aging, and biomaterial implantation. We hypothesized that the normal mechanical environment of smooth muscle cells (SMCs) inhibits a phenotypic switch to a pattern of gene expression more typical for bone and inducive for calcification. This hypothesis was tested using a 3-D engineered smooth muscle tissue model subjected to cyclic mechanical strain. This simplified model maintained a 3-D tissue architecture while eliminating systemic effects as can be seen with in vivo models. All engineered tissues were found to express bone-associated genes (osteopontin, matrix gla protein, alkaline phosphatase, and the transcription factor CBFA-1). Strikingly, however, expression of these genes was down-regulated in tissues exposed to cyclic strain at all time points ranging from 5 to 150 days. Furthermore, long-term strain played a protective role in regard to calcification, as unstrained tissues exhibited increased calcium deposition with respect to strained tissues. The results of this study suggest that without an appropriate mechanical environment, SMCs in 3-D culture undergo a phenotypic conversion to an osteoblast-like pattern of gene expression. This finding has significant implications for the mechanisms underlying a variety of cardiovascular diseases and indicates the broad utility of engineered tissue models in basic biology studies.
Kong H, Smith MK, Mooney DJ. Designing alginate hydrogels to maintain viability of immobilized cells. Biomaterials. 2003;24 (22) :4023-9.Abstract
Hydrogel-forming materials have been widely utilized as an immobilization matrix and transport vehicle for cells. Success in these applications is dependent upon maintaining cell viability through the gel preparation process. We hypothesized that the high viscosity of pre-gelled solutions typically used in these applications may decrease cell viability due to the high shear forces required to mix cells with these solutions. Further, we proposed this harmful effect could be mediated by decreasing the molecular weight (Mw) of the polymer used to form the gel, while maintaining its gel-forming ability. To investigate this hypothesis, alginate was used as model system, as this copolymer consists of cross-linkable guluronic acid (G) blocks and non-cross-linkable blocks. Decreasing the Mw of alginate using irradiation (e.g., irradiating at dose of 2 Mrad) decreased the low shear viscosity of 2% (w/w) pre-gelled solutions from 1000 to 4 cP, while maintaining high elastic moduli, once cross-linked to form a gel. Importantly, the immobilization of cells with these polymer hydrogels increased cell viability from 40% to 70%, as compared to using high Mw polymer chains to form the gels. Furthermore, the solids concentration of gels formed with the low Mw alginate could be raised to further increase the moduli of gels without significantly deteriorating the viability of immobilized cells. This was likely due to the limited increase in the viscosity of these solutions. This material design approach may be useful with a variety of synthetic or naturally occurring block copolymers used to immobilize cells.
Putnam AJ, Cunningham JJ, Pillemer BBL, Mooney DJ. External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly. Am J Physiol Cell Physiol. 2003;284 (3) :C627-39.Abstract
Transmission of externally applied mechanical forces to the interior of a cell requires coordination of biochemical signaling pathways with changes in cytoskeletal assembly and organization. In this study, we addressed one potential mechanism for this signal integration by applying uniform single external mechanical strains to aortic smooth muscle cells (SMCs) via their adhesion substrate. A tensile strain applied to the substrate for 15 min significantly increased microtubule (MT) assembly by 32 +/- 7%, with no apparent effect on the cells' focal adhesions as revealed by immunofluorescence and quantitative analysis of Triton X-100-insoluble vinculin levels. A compressive strain decreased MT mass by 24 +/- 9% but did not influence the level of vinculin in focal adhesions. To understand the decoupling of these two cell responses to mechanical strain, we examined a redistribution of the small GTPases RhoA and Rac. Tensile strain was found to decrease the amount of membrane-associated RhoA and Rac by 70 +/- 9% and 45 +/- 11%, respectively, compared with static controls. In contrast, compressive strain increased membrane-associated RhoA and Rac levels by 74 +/- 17% and 36 +/- 13%, respectively. Disruption of the MT network by prolonged treatments with low doses of either nocodazole or paclitaxel before the application of strain abolished the redistribution of RhoA and Rac in response to the applied forces. Combined, these results indicate that the effects of externally applied mechanical strain on the distribution and activation of the Rho family GTPases require changes in the state of MT polymerization.
Kaigler D, Krebsbach PH, Polverini PJ, Mooney DJ. Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells. Tissue Eng. 2003;9 (1) :95-103.Abstract
One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.
Richardson TP, Murphy WL, Mooney DJ. Selective adipose tissue ablation by localized, sustained drug delivery. Plast Reconstr Surg. 2003;112 (1) :162-70.Abstract
The reduction of adipose depots is widely considered to be the optimal approach to limit pathologies associated with obesity. While many current antiobesity strategies are centered on regulating satiety, these approaches typically attempt an overall weight loss and are unable to target distinct adipose depots specifically associated with disease risk. The authors report a novel therapeutic modality utilizing localized and sustained delivery of drugs to provide for the selective ablation of adipose tissue. Using the epididymal fat pad of Sprague-Dawley rats as a model, they injected into the tissue poly(lactide-co-glycolide) microspheres encapsulating tumor necrosis factor-alpha, a well-known regulator of adipose tissue mass. The utility of this approach was investigated in vivo by measuring the fat pad mass relative to the contralateral control within the same animal (n = 4 at each time point) and in vitro by measuring apoptosis in adipose organ cultures. The authors demonstrated control over the localization of tumor necrosis factor-alpha by performing blood analysis. This is the first report of localized drug delivery for adipose tissue ablation, and these results indicate the potential utility of the general tissue ablation approach for treatment of numerous pathologies.
Kong H, Mooney DJ. The effects of poly(ethyleneimine) (PEI) molecular weight on reinforcement of alginate hydrogels. Cell Transplant. 2003;12 (7) :779-85.Abstract
Alginate hydrogels are widely used for cell encapsulation and transplantation, and they are frequently surface reinforced with secondary polymers to enhance their mechanical rigidity and stability. We hypothesized that the molecular weight (MW) of the polymer utilized to reinforce alginate would be an important factor in their stability, particularly when the gel network was homogeneously reinforced with the polymer. This hypothesis was investigated with alginate hydrogels cross-linked with Ca2+, and reinforced throughout the bulk of the gel with poly(ethyleneimine) (PEI) having different MWs. Interactions between the two polymers became significant following gelation, leading to higher elastic moduli (E) than gels with no PEI. The decrease in E of gels incubated in isotonic salt solutions over time, utilized as an indication of gel break down, was ameliorated with an increase in the MW of the PEI. In addition, the dependencies of the moduli and viscoelasticity on the temperature also became smaller with the use of high MW PEI. This is likely due to the limited mobility of high MW PEI, leading to a higher energy for dissociation. The stable interactions between the alginate and PEI prevented alterations of the pore structure in the gels, and slowed the deterioration of gel properties even under continuous agitation in a bioreactor. The results of this study will likely be useful in designing alginate encapsulation strategies for various applications.
Huang Y-C, Connell M, Park Y, Mooney DJ, Rice KG. Fabrication and in vitro testing of polymeric delivery system for condensed DNA. J Biomed Mater Res A. 2003;67 (4) :1384-92.Abstract
Polyethylenimine (PEI) was combined with plasmid DNA and freeze dried following the addition of sucrose as a lyoprotectant and pore-forming agent. Freeze-dried PEI DNA condensates were dry mixed with granular polylactideglycolic acid (PLGA) then compression molded and sponged to encapsulated PEI DNA. A measurement of the elastic modulus indicated that 91 wt% sucrose substituted for 95 wt% sodium chloride as a porogen, resulting in PLGA sponges with a mechanical modulus of 100 kPa. The PEI DNA was retained (80%) within PLGA sponges prepared with sucrose during the leaching and subsequent 2-week release studies, whereas sodium chloride PLGA sponges caused the premature release (100%) of PEI DNA within 2 days. In vitro gene transfer studies with PEI DNA PLGA sponges established that adherent and infiltrating fibroblasts expressed reporter gene for 15 days compared with the short, 3-day expression mediated by direct gene of PEI DNA on cells in culture. The results demonstrate an approach to encapsulate condensed DNA in a PLGA sponge for the purpose of retaining DNA within the matrices and creating efficient gene transfer during tissue engineering.
Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials. 2003;24 (24) :4337-51.Abstract
Polymer scaffolds have many different functions in the field of tissue engineering. They are applied as space filling agents, as delivery vehicles for bioactive molecules, and as three-dimensional structures that organize cells and present stimuli to direct the formation of a desired tissue. Much of the success of scaffolds in these roles hinges on finding an appropriate material to address the critical physical, mass transport, and biological design variables inherent to each application. Hydrogels are an appealing scaffold material because they are structurally similar to the extracellular matrix of many tissues, can often be processed under relatively mild conditions, and may be delivered in a minimally invasive manner. Consequently, hydrogels have been utilized as scaffold materials for drug and growth factor delivery, engineering tissue replacements, and a variety of other applications.
Chen RR, Mooney DJ. Polymeric growth factor delivery strategies for tissue engineering. Pharm Res. 2003;20 (8) :1103-12.Abstract
PURPOSE: Tissue engineering seeks to replace and regrow damaged or diseased tissues and organs from either cells resident in the surrounding tissue or cells transplanted to the tissue site. The purpose of this review is to present the application of polymeric delivery systems for growth factor delivery in tissue engineering. METHODS: Growth factors direct the phenotype of both differentiated and stem cells, and methods used to deliver these molecules include the development of systems to deliver the protein itself, genes encoding the factor, or cells secreting the factor. RESULTS: Results in animal models and clinical trials indicate that these approaches may be successfully used to promote the regeneration of numerous tissue types. CONCLUSIONS: Controlling the dose, location, and duration of these factors through polymeric delivery strategies will dictate their utility in tissue regeneration.
Boontheekul T, Mooney DJ. Protein-based signaling systems in tissue engineering. Curr Opin Biotechnol. 2003;14 (5) :559-65.Abstract
Tissue engineering aims to replace damaged tissues or organs using either transplanted cells or host cells recruited to the target site. Protein signaling is crucial to regulate cell phenotype and thus engineered tissue structure and function. Biomaterial vehicles are being designed to incorporate and locally deliver various molecules involved in this signaling, including both growth factors and peptides that mimick whole proteins. Controlling the concentration, local duration and spatial distribution of these factors is key to their utility and efficacy. Recent advances have been made in the development of polymeric delivery systems intended to achieve this control.
Alsberg E, Kong HJ, Hirano Y, Smith MK, Albeiruti A, Mooney DJ. Regulating bone formation via controlled scaffold degradation. J Dent Res. 2003;82 (11) :903-8.Abstract
It is widely assumed that coupling the degradation rate of polymers used as cell transplantation carriers to the growth rate of the developing tissue will improve its quantity or quality. To test this hypothesis, we developed alginate hydrogels with a range of degradation rates by gamma-irradiating high-molecular-weight alginate to yield polymers of various molecular weights and structures. Decreasing the size of the polymer chains increased the degradation rate in vivo, as measured by implant retrieval rates, masses, and elastic moduli. Rapidly and slowly degrading alginates, covalently modified with RGD-containing peptides to control cell behavior, were then used to investigate the effect of biodegradation rate on bone tissue development in vivo. The more rapidly degrading gels led to dramatic increases in the extent and quality of bone formation. These results indicate that biomaterial degradability is a critical design criterion for achieving optimal tissue regeneration with cell transplantation.
2002
Murphy WL, Mooney DJ. Molecular-scale biomimicry. Nat Biotechnol. 2002;20 (1) :30-1.
Aframian DJ, Redman RS, Yamano S, Nikolovski J, Cukierman E, Yamada KM, Kriete MF, Swaim WD, Mooney DJ, Baum BJ. Tissue compatibility of two biodegradable tubular scaffolds implanted adjacent to skin or buccal mucosa in mice. Tissue Eng. 2002;8 (4) :649-59.Abstract
Radiation therapy for cancer in the head and neck region leads to a marked loss of salivary gland parenchyma, resulting in a severe reduction of salivary secretions. Currently, there is no satisfactory treatment for these patients. To address this problem, we are using both tissue engineering and gene transfer principles to develop an orally implantable, artificial fluid-secreting device. In the present study, we examined the tissue compatibility of two biodegradable substrata potentially useful in fabricating such a device. We implanted in Balb/c mice tubular scaffolds of poly-L-lactic acid (PLLA), poly-glycolic acid coated with PLLA (PGA/PLLA), or nothing (sham-operated controls) either beneath the skin on the back, a site widely used in earlier toxicity and biocompatibility studies, or adjacent to the buccal mucosa, a site quite different functionally and immunologically. At 1, 3, 7, 14, and 28 days postimplantation, implant sites were examined histologically, and systemic responses were assessed by conventional clinical chemistry and hematology analyses. Inflammatory responses in the connective tissue were similar regardless of site or type of polymer implant used. However, inflammatory reactions were shorter and without epithelioid and giant cells in sham-operated controls. Also, biodegradation proceeded more slowly with the PLLA tubules than with the PGA/PLLA tubules. No significant changes in clinical chemistry and hematology were seen due to the implantation of tubular scaffolds. These results indicate that the tissue responses to PLLA and PGA/PLLA scaffolds are generally similar in areas subjacent to skin in the back and oral cavity. However, these studies also identified several potentially significant concerns that must be addressed prior to initiating any clinical applications of this device.
Rowley JA, Mooney DJ. Alginate type and RGD density control myoblast phenotype. J Biomed Mater Res. 2002;60 (2) :217-23.Abstract
Alginates are being increasingly used for cell encapsulation and tissue engineering applications; however, these materials cannot specifically interact with mammalian cells. We have covalently modified alginates of varying monomeric ratio with RGD-containing cell adhesion ligands using carbodiimide chemistry to initiate cell adhesion to these polymers. We hypothesized that we could control the function of cells adherent to RGD-modified alginate hydrogels by varying alginate polymer type and cell adhesion ligand density, and we have addressed this possibility by studying the proliferation and differentiation of C2C12 skeletal myoblasts adherent to these materials. RGD density on alginates of varying monomeric ratio could be controlled over several orders of magnitude, creating a range of surface densities from 1-100 fmol/cm(2). Myoblast adhesion to these materials was specific to the RGD ligand, because adhesion could be competed away with soluble RGD in a dose-dependent manner. Myoblast proliferation and differentiation could be regulated by varying the alginate monomeric ratio and the density of RGD ligands at the substrate surface, and specific combinations of alginate type and RGD density were required to obtain efficient myoblast differentiation on these materials.
Murphy WL, Mooney DJ. Bioinspired growth of crystalline carbonate apatite on biodegradable polymer substrata. J Am Chem Soc. 2002;124 (9) :1910-7.Abstract
Mineralization in biological systems is a widespread, yet incompletely understood phenomenon involving complex interactions at the biomacromolecule-mineral nucleus interface. This study was aimed at understanding and controlling mineral formation in a poly(alpha-hydroxy ester) model system, to gain insight into biological mineralization processes and to develop biomaterials for orthopaedic tissue regeneration. We specifically hypothesized that providing a high surface density of anionic functional groups would enhance nucleation and growth of bonelike mineral following exposure to simulated body fluids (SBF). Polymer surface functionalization was achieved via hydrolysis of 85:15 poly(lactide-co-glycolide) (PLG) films. This treatment led to an increase in surface carboxylic acid and hydroxyl groups, resulting in a substantial increase in polymer surface energy from 42 to 49 dynes/cm2. Treated polymers exhibited a 3-fold increase in heterogeneous mineral grown and growth of a continuous mineral film on the polymer surface. The mineral grown on PLG surfaces is a carbonate apatite, the major mineral component of vertebrate bone tissue. Mineral crystal size and morphology were dependent on the solution characteristics but unaffected by the degree of surface prehydrolysis. The mechanism of heterogeneous carbonate apatite growth was examined via ion binding assays, which indicated that calcium binding is mediated independently by the presence of soluble phosphate counterions and surface functional groups. These findings indicate that poly(alpha-hydroxy ester) materials can be readily mineralized using a biomimetic process, and that the impetus for mineral nucleation in this system appears more complicated than the simple electrostatic interactions proposed in previous biomineralization theory.

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