Mara L. Macdonald

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

Tunable dual growth factor delivery from polyelectrolyte multilayer films

A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein-2) and angiogenic rhVEGF₁₆₅ (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(β-amino ester)/polyanion/growth factor/polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF₁₆₅ eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF₁₆₅ upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF₁₆₅ PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.

Release of a model protein from biodegradable self assembled films for surface delivery applications.

Layer-by-layer (LbL) films have multiple features that make them attractive for drug delivery, including the potential to sequentially deliver growth factors from implantable medical devices or tissue engineering scaffolds. To date, however, characterization has been lacking for protein delivery from such films. Here, LbL polyelectrolyte films constructed with the model protein lysozyme and a hydrolytically degradable and biocompatible synthetic polycation are characterized. Milligram/cm(2) scale linear or power law release profiles can be achieved over 2 to 34 days, and control over loading and release are demonstrated through parameters such as tuning the degradability of the synthetic polycation, changing the number of layers used, or changing the polysaccharide polyanion. Functionality is maintained at nearly 100%, underscoring mild processing conditions apt to preserve fragile protein function. LbL films thus have promise as a tool for exploring protein modulation of the interaction between implanted surfaces and the cells they contact.

Characterization of tunable FGF-2 releasing polyelectrolyte multilayers.

Fibroblast growth factor 2 (FGF-2) is a potent mediator of stem cell differentiation and proliferation. Although FGF-2 has a well-established role in promoting bone tissue formation, flaws in its delivery have limited its clinical utility. Polyelectrolyte multilayer films represent a novel system for FGF-2 delivery that has promise for local, precisely controlled, and sustained release of FGF-2 from surfaces of interest, including medical implants and tissue engineering scaffolds. In this work, the loading and release of FGF-2 from synthetic hydrolytically degradable multilayer thin films of various architectures is explored; drug loading was tunable using at least three parameters (number of nanolayers, counterpolyanion, and type of degradable polycation) and yielded values of 7-45 ng/cm(2) of FGF-2. Release time varied between 24 h and approximately five days. FGF-2 released from these films retained in vitro activity, promoting the proliferation of MC3T3 preosteoblast cells. The use of biologically derived counterpolyanions heparin sulfate and chondroitin sulfate in the multilayer structures enhanced FGF-2 activity. The control over drug loading and release kinetics inform future in vivo bone and tissue regeneration models for the exploration of clinical relevance of LbL growth factor delivery films.