Alsberg E, Anderson KW, Albeiruti A, Rowley JA, Mooney DJ. Engineering growing tissues. Proc Natl Acad Sci U S A. 2002;99 (19) :12025-30.Abstract
Regenerating or engineering new tissues and organs may one day allow routine replacement of lost or failing tissues and organs. However, these engineered tissues must not only grow to fill a defect and integrate with the host tissue, but often they must also grow in concert with the changing needs of the body over time. We hypothesized that tissues capable of growing with time could be engineered by supplying growth stimulus signals to cells from the biomaterial used for cell transplantation. In this study, chondrocytes and osteoblasts were cotransplanted on hydrogels modified with an RGD-containing peptide sequence to promote cell multiplication. New bone tissue was formed that grew in mass and cellularity by endochondral ossification in a manner similar to normal long-bone growth. Transplanted cells organized into structures that morphologically and functionally resembled growth plates. These engineered tissues could find utility in treating diseases and injuries of the growth plate, testing the effect of experimental drugs on growth-plate function and development, and investigating the biology of long-bone growth. Furthermore, this concept of promoting the growth of engineered tissues could find great utility in engineering numerous tissue types by way of the transplantation of a small number of precursor cells.
Peters MC, Polverini PJ, Mooney DJ. Engineering vascular networks in porous polymer matrices. J Biomed Mater Res. 2002;60 (4) :668-78.Abstract
Enhanced vascularization is critical to the treatment of ischemic tissues and the engineering of new tissues and organs. We have investigated whether sustained and localized delivery of vascular endothelial growth factor (VEGF) combined with transplantation of human microvascular endothelial cells (HMVECs) can be used to engineer new vascular networks. VEGF was incorporated and released in a sustained manner from porous poly(lactic-co-glycolic acid) (PLG) matrices to promote angiogenesis at the transplantation site. VEGF could be incorporated and released in a biologically active form from PLG matrices, with the majority of VEGF release (64%) occurring within 2 weeks. These matrices promoted a 260% increase in the density of host SCID mouse-derived capillaries invading the matrices after 7 days of implantation, confirming the activity of the released VEGF. HMVECs were transplanted into SCID mice on PLG matrices, and organized to form immature human-derived vessels within 3 days. Functional vessels were observed within 7 days. Importantly, when HMVECs were transplanted on VEGF-releasing matrices, a 160% increase in the density of human-derived blood vessels was observed after 14 days. These findings suggest that combining elements of vasculogenesis and angiogenesis provides a viable and novel approach to enhancing local vascularization.
Cunningham JJ, Nikolovski J, Linderman JJ, Mooney DJ. Quantification of fibronectin adsorption to silicone-rubber cell culture substrates. Biotechniques. 2002;32 (4) :876, 878, 880 passim.Abstract
As the role of mechanical force in cellular signaling gained recognition, investigators designed a number of devices to deliver controlled regimens of mechanical force to cultured cells. One type of device uses thin silicone-rubber membranes to support monolayer cell adhesion and to transmit mechanical force in the form of biaxial strain. We have observed that cell attachment and spreading are impaired on these membranes compared to polystyrene, even when both are passively coated with identical amounts of extracellular matrix. The purpose of these studies was to quantify the efficiency and stability of passive matrix adsorption onto commercially available elastic culture substrates. A theoretically saturating density (1 microg/cm2) of fibronectin was added to each well, and the initial efficiency of adsorption to the walls and elastic membranes was found to be 31 +/- 2% of the protein added. Strikingly, when the protein adsorbed specifically to the membranes was quantified after seven days, only 10-26 ng/cm2 fibronectin were present, revealing that most of the adsorption is to the sides of the wells. These results indicate that the adsorption of matrix proteins to silicone-rubber substrates is relatively inefficient and that investigators who use these systems must be aware of this fact and design their experiments accordingly.
Murphy WL, Dennis RG, Kileny JL, Mooney DJ. Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds. Tissue Eng. 2002;8 (1) :43-52.Abstract
Macroporous scaffolds composed of biodegradable polymers have found extensive use as three-dimensional substrates either for in vitro cell seeding followed by transplantation, or as conductive substrates for direct implantation in vivo. Methods abound for creation of macroporous scaffolds for tissue engineering, and common methods typically employ a solid porogen within a three-dimensional polymer matrix to create a well-defined pore size, pore structure, and total scaffold porosity. This study describes an approach to impart improved pore interconnectivity to polymer scaffolds for tissue engineering by partially fusing the solid porogen together prior to creation of a continuous polymer matrix. Three dimensional, porous scaffolds of the copolymer 85:15 poly(lactide-co-glycolide) were fabricated via either a solvent casting/particulate leaching process, or a gas foaming/particulate leaching process. Prior to creation of a continuous polymer matrix the NaCl crystals, which serve as the solid porogen, are partially fused via treatment in 95% humidity. Scanning electron micrographs clearly display fused salt crystals and an enhancement in pore interconnectivity in the salt fused scaffolds prepared via both solvent casting and gas foaming, and the extent of pore interconnectivity is enhanced with longer treatment times. Fusion of salt crystal for 24 h increased the radius of curvature of salt crystals, and led to a twofold increase in the compressive modulus of solvent cast scaffolds (total porosity of 97 +/- 1%). Fusion of NaCl crystals prior to gas foaming resulted in a decrease in scaffold compressive modulus from 277 +/- 60k Pa to 187 +/- 30k Pa (total porosity of 94 +/- 1%). The resulting highly interconnected scaffolds have implications for facilitated cell migration, abundant cell-cell interaction, and potentially improved neural and vascular growth within tissue engineering scaffolds.
Lee KY, Bouhadir KH, Mooney DJ. Evaluation of chain stiffness of partially oxidized polyguluronate. Biomacromolecules. 2002;3 (6) :1129-34.Abstract
The chain stiffness of macromolecules is considered critical in their design and applications. This study utilizes polyguluronate derived from alginate, a typical polysaccharide widely utilized in many biomedical applications, as a model macromolecule to investigate how the chain stiffness can be tightly regulated by partial oxidation. Alginate has a backbone of inherently rigid alpha-L-guluronate (i.e., polyguluronate) and more flexible beta-D-mannuronate. The chain stiffness of the polyguluronate was specifically studied in this paper, as this component plays a critical role in the formation of alginate hydrogels with divalent cations and is the dominant factor in determining the chain stiffness of alginate. We have utilized size-exclusion chromatography, equipped with refractive index, viscosity, and light-scattering detectors, to determine the intrinsic viscosity and the weight-average molecular weight of each fraction of samples. The chain stiffness of partially oxidized polyguluronate was then evaluated from the exponent of the Mark-Houwink equation and the persistence length. We have found that partial oxidation can be used to tightly regulate the steric hindrance and stiffness of the polyguluronate backbone. This approach to control the chain stiffness of inherently rigid polysaccharides by partial oxidation may find many applications in biomedical utilization of these materials.
Cunningham JJ, Linderman JJ, Mooney DJ. Externally applied cyclic strain regulates localization of focal contact components in cultured smooth muscle cells. Ann Biomed Eng. 2002;30 (7) :927-35.Abstract
Mechanical signals are critical regulators of cellular gene expression, yet little is understood of the mechanism whereby cells sense mechanical forces. In this study we have tested the hypothesis that mechanical strain applied to populations of cells via their adhesion substrate rapidly alters the cellular distribution of focal contact proteins. Focal contact-associated components (vinculin, a-actinin, paxillin) were assayed by immunofluorescence microscopy and quantitative western blotting. Application of a single step increase in strain in multiple experiments caused overall a small change in focal contact-associated vinculin. In contrast, cyclic strain induced a large and very reproducible increase in detergent-insoluble vinculin (52% relative to static) after just 1 min of strain. Insoluble paxillin was transiently enriched with a similar time course, whereas insoluble a-actinin did not change significantly in response to cyclic strain. Rhodamine-labeled chicken vinculin added to permeabilized cells preferentially localized to focal contacts in response to cyclic strain, but not a single step increase in strain. These findings establish that insoluble levels of focal contact components are altered rapidly following application of an appropriate number of mechanical perturbations, and suggest that at least one component of the mechanism does not involve soluble intermediates.
Ennett AB, Mooney DJ. Tissue engineering strategies for in vivo neovascularisation. Expert Opin Biol Ther. 2002;2 (8) :805-18.Abstract
Neovascularisation is a promising alternative therapeutic approach to re-establish blood flow in ischaemic tissues of patients suffering from coronary artery or peripheral artery disease. Often, these patients are not suitable candidates for current revascularisation procedures such as coronary angioplasty or bypass surgery. Several strategies are presently under investigation to induce vascularisation by stimulating the body's natural processes of vasculogenesis, angiogenesis and arteriogenesis. These strategies involve transplantation of various cell types into the ischaemic site and the delivery of recombinant angiogenic agents through direct protein administration or gene transfer. We will examine the basic approaches for these neovacularisation strategies and their therapeutic potential as demonstrated in animal models and human trials to date.
Chang SC, Rowley JA, Tobias G, Genes NG, Roy AK, Mooney DJ, Vacanti CA, Bonassar LJ. Injection molding of chondrocyte/alginate constructs in the shape of facial implants. J Biomed Mater Res. 2001;55 (4) :503-11.Abstract
Over one million patients per year undergo some type of procedure involving cartilage reconstruction. Polymer hydrogels, such as alginate, have been shown to be effective carriers for chondrocytes in subcutaneous cartilage formation. The goal of our current study was to develop a method to create complex structures (nose bridge, chin, etc.) with good dimensional tolerance to form cartilage in specific shapes. Molds of facial implants were prepared using Silastic ERTV. Suspensions of chondrocytes in 2% alginate were gelled by mixing with CaSO(4) (0.2 g/mL) and injected into the molds. Constructs of various cell concentrations (10, 25, and 50 million/mL) were implanted in the dorsal aspect of nude mice and harvested at times up to 30 weeks. Analysis of implanted constructs indicated progressive cartilage formation with time. Proteoglycan and collagen constructs increased with time to approximately 60% that of native tissue. Equilibrium modulus likewise increased with time to 15% that of normal tissue, whereas hydraulic permeability decreased to 20 times that of native tissue. Implants seeded with greater concentrations of cells increased proteoglycan content and collagen content and equilibrium and decreased permeability. Production of shaped cartilage implants by this technique presents several advantages, including good dimensional tolerance, high sample-to-sample reproducibility, and high cell viability. This system may be useful in the large-scale production of precisely shaped cartilage implants.
Shea LD, Mooney DJ. Nonviral DNA delivery from polymeric systems. Methods Mol Med. 2001;65 :195-207.Abstract
Gene therapy holds great promise for the treatment of disease by delivering genes encoding for therapeutic proteins. Although it was originally devised for the treatment of inherited genetic disorders, such as cystic fibrosis, recent work has expanded the applications of gene therapy to develop strategies for HIV, cancer (1), and wound healing applications (2). The challenge of gene therapy is to develop safe and efficient gene delivery systems (1). Most studies have focused on the use of viral vectors because of their potentially high efficiencies; however, the safety and ease of manufacturing of nonviral vectors may make them the preferred choice in the future.
Bouhadir KH, Mooney DJ. Promoting angiogenesis in engineered tissues. J Drug Target. 2001;9 (6) :397-406.Abstract
There is a tremendous need for organs and tissues to replace those lost due to diseases or trauma. In theory, transplanting cells on biomaterial matrices can create functional tissue. A critical question, however, is how to supply cells embedded within large cell-polymer constructs with sufficient oxygen and nutrients to sustain their survival and proliferation, and allow for the integration of the developing tissue with the surrounding tissue. A rapid and high level of vascularization of transplanted polymer-cell matrices is essential in tissue engineering approaches to meet these challenges. This review summarizes the current approaches and materials under development in our laboratory to promote angiogenesis in engineered tissues.
Aframian DJ, Zheng C, Goldsmith CM, Nikolovski J, Cukierman E, Yamada KM, Mooney DJ, Birkedal-Hansen H, Baum BJ. Using HSV-thymidine kinase for safety in an allogeneic salivary graft cell line. Tissue Eng. 2001;7 (4) :405-13.Abstract
Extreme salivary hypofunction is a result of tissue damage caused by irradiation therapy for cancer in the head and neck region. Unfortunately, there is no currently satisfactory treatment for this condition that affects up to 40,000 people in the United States every year. As a novel approach to managing this problem, we are attempting to develop an orally implantable, fluid-secreting device (an artificial salivary gland). We are using the well-studied HSG salivary cell line as a potential allogeneic graft cell for this device. One drawback of using a cell line is the potential for malignant transformation. If such an untoward response occurred, the device could be removed. However, in the event that any HSG cells escaped, we wished to provide additional patient protection. Accordingly, we have engineered HSG cells with a hybrid adeno-retroviral vector, AdLTR.CMV-tk, to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene as a novel safety factor. Cells were grown on plastic plates or on poly-L-lactic acid disks and then transduced with different multiplicities of infection (MOIs) of the hybrid vector. Thereafter, various concentrations of ganciclovir (GCV) were added, and cell viability was tested. Transduced HSG cells expressed HSV-tk and were sensitive to GCV treatment. Maximal effects were seen at a MOI of 10 with 50 microM of GCV, achieving 95% cell killing on the poly-L-lactic acid substrate. These results suggest that engineering the expression of a suicide gene in an allogeneic graft cell may provide additional safety for use in an artificial salivary gland device.
Putnam AJ, Schultz K, Mooney DJ. Control of microtubule assembly by extracellular matrix and externally applied strain. Am J Physiol Cell Physiol. 2001;280 (3) :C556-64.Abstract
A number of studies have suggested that externally applied mechanical forces and alterations in the intrinsic cell-extracellular matrix (ECM) force balance equivalently induce changes in cell phenotype. However, this possibility has never been directly tested. To test this hypothesis, we directly investigated the response of the microtubule (MT) cytoskeleton in smooth muscle cells to both mechanical signals and alterations in the ECM. A tensile force that resulted in a positive 10% step change in substrate strain increased MT mass by 34 +/- 10% over static controls, independent of the cell adhesion ligand and tyrosine phosphorylation. Conversely, a compressive force that resulted in a negative 10% step change in substrate strain decreased MT mass by 40 +/- 6% over static controls. In parallel, increasing the density of the ECM ligand fibronectin from 50 to 1,000 ng/cm(2) in the absence of any applied force increased the amount of polymeric tubulin in the cell from 59 +/- 11% to 81 +/- 13% of the total cellular tubulin. These data are consistent with a model in which MT assembly is, in part, controlled by forces imposed on these structures, and they suggest a novel control point for MT assembly by altering the intrinsic cell-ECM force balance and applying external mechanical forces.
Alsberg E, Hill EE, Mooney DJ. Craniofacial tissue engineering. Crit Rev Oral Biol Med. 2001;12 (1) :64-75.Abstract
There is substantial need for the replacement of tissues in the craniofacial complex due to congenital defects, disease, and injury. The field of tissue engineering, through the application of engineering and biological principles, has the potential to create functional replacements for damaged or pathologic tissues. Three main approaches to tissue engineering have been pursued: conduction, induction by bioactive factors, and cell transplantation. These approaches will be reviewed as they have been applied to key tissues in the craniofacial region. While many obstacles must still be overcome prior to the successful clinical restoration of tissues such as skeletal muscle and the salivary glands, significant progress has been achieved in the development of several tissue equivalents, including skin, bone, and cartilage. The combined technologies of gene therapy and drug delivery with cell transplantation will continue to increase treatment options for craniofacial cosmetic and functional restoration.
Lee KY, Alsberg E, Mooney DJ. Degradable and injectable poly(aldehyde guluronate) hydrogels for bone tissue engineering. J Biomed Mater Res. 2001;56 (2) :228-33.Abstract
Degradable and injectable hydrogels may be ideal for bone-tissue engineering, especially in the craniofacial region because of the ease of access for injection. Alginate hydrogels potentially could be used as injectable cell delivery vehicles, but they exhibit a limited range of mechanical properties and uncontrollable disintegration time. Therefore we synthesized new hydrogels, composed of poly(aldehyde guluronate) (PAG) and adipic acid dihydrazide, that have a wide range of mechanical stiffness and controllable degradation rate. MC3T3-E1 cells adhered and multiplied on PAG hydrogels in vitro. When primary rat calvarial osteoblasts were mixed with PAG hydrogels and subcutaneously injected into the backs of mice, mineralized bone tissues were formed 9 weeks following implantation. These hydrogels may find wide utility as an injectable delivery system for bone precursor cells as well as for other applications in tissue engineering.
Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ. Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol Prog. 2001;17 (5) :945-50.Abstract
Alginate has been widely used in a variety of biomedical applications including drug delivery and cell transplantation. However, alginate itself has a very slow degradation rate, and its gels degrade in an uncontrollable manner, releasing high molecular weight strands that may have difficulty being cleared from the body. We hypothesized that the periodate oxidation of alginate, which cleaves the carbon-carbon bond of the cis-diol group in the uronate residue and alters the chain conformation, would result in promoting the hydrolysis of alginate in aqueous solutions. Alginate, oxidized to a low extent (approximately 5%), degraded with a rate depending on the pH and temperature of the solution. This polymer was still capable of being ionically cross-linked with calcium ions to form gels, which degraded within 9 days in PBS solution. Finally, the use of these degradable alginate-derived hydrogels greatly improved cartilage-like tissue formation in vivo, as compared to alginate hydrogels.
Nör JE, Peters MC, Christensen JB, Sutorik MM, Linn S, Khan MK, Addison CL, Mooney DJ, Polverini PJ. Engineering and characterization of functional human microvessels in immunodeficient mice. Lab Invest. 2001;81 (4) :453-63.Abstract
SUMMARY: Current model systems used to investigate angiogenesis in vivo rely on the interpretation of results obtained with nonhuman endothelial cells. Recent advances in tissue engineering and molecular biology suggest the possibility of engineering human microvessels in vivo. Here we show that human dermal microvascular endothelial cells (HDMEC) transplanted into severe combined immunodeficient (SCID) mice on biodegradable polymer matrices differentiate into functional human microvessels that anastomose with the mouse vasculature. HDMEC were stably transduced with Flag epitope or alkaline phosphatase to confirm the human origin of the microvessels. Endothelial cells appeared dispersed throughout the sponge 1 day after transplantation, became organized into empty tubular structures by Day 5, and differentiated into functional microvessels within 7 to 10 days. Human microvessels in SCID mice expressed the physiological markers of angiogenesis: CD31, CD34, vascular cellular adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). Human endothelial cells became invested by perivascular smooth muscle alpha-actin-expressing mouse cells 21 days after implantation. This model was used previously to demonstrate that overexpression of the antiapoptotic protein Bcl-2 in HDMEC enhances neovascularization, and that apoptotic disruption of tumor microvessels is associated with apoptosis of surrounding tumor cells. The proposed SCID mouse model of human angiogenesis is ideally suited for the study of the physiology of microvessel development, pathologic neovascular responses such as tumor angiogenesis, and for the development and investigation of strategies designed to enhance the neovascularization of engineered human tissues and organs.
Bouhadir KH, Alsberg E, Mooney DJ. Hydrogels for combination delivery of antineoplastic agents. Biomaterials. 2001;22 (19) :2625-33.Abstract
The systemic delivery of anticancer agents has been widely investigated during the past decade but localized delivery may offer a safer and more effective delivery approach. We have designed and synthesized a novel hydrogel to locally deliver antineoplastic agents, and demonstrate the different types of release that can be achieved from these hydrogels using three model drugs: methotrexate, doxorubicin, and mitoxantrone. Alginate was chemically modified into low molecular weight oligomers and cross-linked with a biodegradable spacer (adipic dihydrazide) to form biodegradable hydrogels. The model antineoplastic agents were loaded into the hydrogel via three different mechanisms. Methotrexate was incorporated within the pores of the hydrogel and was released by diffusion into the surrounding medium. Doxorubicin was covalently attached to the polymer backbone via a hydrolytically labile linker and was released following the chemical hydrolysis of the linker. Mitoxantrone was ionically complexed to the polymer and was released after the dissociation of this complex. These three release mechanisms could potentially be used to deliver a wide selection of antineoplastic agents, based on their chemical structure. This novel delivery system allows for the release of single or combinations of antineoplastic agents, and may find utility in localized antineoplastic agent delivery.
Nör JE, Christensen J, Liu J, Peters M, Mooney DJ, Strieter RM, Polverini PJ. Up-Regulation of Bcl-2 in microvascular endothelial cells enhances intratumoral angiogenesis and accelerates tumor growth. Cancer Res. 2001;61 (5) :2183-8.Abstract
Vascular endothelial growth factor (VEGF) has been shown to be a potent mediator of angiogenesis that functions as a survival factor for endothelial cells by up-regulating Bcl-2 expression. We have recently reported that human dermal microvascular endothelial cells (HDMECs) seeded in biodegradable sponges and implanted into severe combined immunodeficient (SCID) mice organize into functional human microvessels that transport mouse blood cells. In this study, we implanted sponges seeded with OSCC-3 (oral squamous cell carcinoma) or SLK (Kaposi's sarcoma) together with endothelial cells into SCID mice to generate human tumors vascularized with human microvessels. This model system was used to examine the role of both endothelial cell Bcl-2 and the proangiogenic chemokine interleukin-8 (IL-8) on tumor growth and intratumoral microvascular density. Coimplantation of HDMECs overexpressing Bcl-2 (HDMEC-Bcl-2) and tumor cells resulted in a 3-fold enhancement of tumor growth when compared with the coimplantation of control HDMECs and tumor cells. This was associated with increased intratumoral microvascular density and enhanced endothelial cell survival. To determine whether the enhanced neovascularization mediated by Bcl-2 overexpression in endothelial cells was influenced by the synthesis of endogenous mediators of angiogenesis, we screened these cells for expression of VEGF, basic fibroblast growth factor (bFGF), and IL-8 by ELISA. HDMEC-Bcl-2 cells and VEGF-treated HDMECs exhibited a 15-fold and 4-fold increase, respectively, in the expression of the proangiogenic chemokine IL-8 in vitro, whereas the expression of VEGF and bFGF remained unchanged. Transfection of antisense Bcl-2 into HDMECs blocked VEGF-mediated induction of IL-8. Conditioned media from HDMEC-Bcl-2 induced proliferation and sprouting of endothelial cells in vitro and neovascularization in rat corneas. Anti-IL-8 antibody added to HDMEC-Bcl-2 conditioned media markedly reduced the potency of these responses. SCID mice bearing VEGF-producing tumor implants that were treated with anti-lL-8 antibody exhibited a 43% reduction in microvessel density and a 50% reduction in tumor weight compared with treatment with a nonspecific antibody. These results demonstrate that the up-regulation of Bcl-2 expression in endothelial cells that constitute tumor microvessels enhances intratumoral microvascular survival and density and accelerates tumor growth. Furthermore, endothelial cells that overexpress Bcl-2 have more angiogenic potential than control cells, and IL-8-neutralizing antibodies attenuate their angiogenic activity in vitro and in vivo.
Alsberg E, Anderson KW, Albeiruti A, Franceschi RT, Mooney DJ. Cell-interactive alginate hydrogels for bone tissue engineering. J Dent Res. 2001;80 (11) :2025-9.Abstract
There is significant interest in the development of injectable carriers for cell transplantation to engineer bony tissues. In this study, we hypothesized that adhesion ligands covalently coupled to hydrogel carriers would allow one to control pre-osteoblast cell attachment, proliferation, and differentiation. Modification of alginate with an RGD-containing peptide promoted osteoblast adhesion and spreading, whereas minimal cell adhesion was observed on unmodified hydrogels. Raising the adhesion ligand density increased osteoblast proliferation, and a minimum ligand density (1.5-15 femtomoles/cm2) was needed to elicit this effect. MC3T3-E1 cells demonstrated increased osteoblast differentiation with the peptide-modified hydrogels, as confirmed by the up-regulation of bone-specific differentiation markers. Further, transplantation of primary rat calvarial osteoblasts revealed statistically significant increases of in vivo bone formation at 16 and 24 weeks with G4RGDY-modified alginate compared with unmodified alginate. These findings demonstrate that biomaterials may be designed to control bone development from transplanted cells.
Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chem Rev. 2001;101 (7) :1869-79.