Kimberly A. Woodhouse

Synthesis and characterization of degradable polyurethane elastomers containing an amino acid-based chain extender

Degradable polyurethane elastomers were synthesized using a diester chain extender. The chain extender was synthesized by a diesterification reaction between L-phenylalanine and 1,4-cyclohexane dimethanol to yield a diester, diamine. Soft segment chemistry (polycaprolactone diol, PCL and polyethylene oxide, PEO) and molecular weight were varied and the impact on polyurethane physicochemical and degradation characteristics was evaluated. It was found that the PEO containing polyurethanes absorbed large amounts of water while the PCL containing ones did not, indicating a large difference in bulk hydrophilicity. The rate of water vapor permeance (WVP) through the polyurethane films generally followed the water absorption trends. However, soft segment crystallinity, noted by DSC, for the PCL containing polyurethanes served to reduce WVP values with increasing PCL molecular weight. Polyurethane surface characterization was carried out by water contact angles and XPS. The PEO containing polyurethanes exhibited low contact angles in comparison with the PCL ones. In addition, angle-resolved XPS demonstrated soft segment surface enrichment in all cases typical for phase segregated materials. Significant variation in the physicochemical properties of the experimental polyurethanes was observed indicating potential use in a variety of biomaterials applications. An in vitro degradation study was carried out by incubating the polymers in 0.1 M TBS at 37 degrees C, pH 8.0 for up to 56 days. Degradation was followed by measuring mass loss, change in molecular weight by GPC and surface alteration by scanning electron microscopy. The polyurethane containing PEO was found to exhibit substantial mass and molecular weight loss over 56 days resulting in a porous material of little strength. In contrast, the PCL containing polyurethane displayed modest mass and molecular weight loss after 56 days. This polyurethane retained its strength and displayed little surface alteration after 56 days in buffer. It was hypothesized that differences in polyurethane hydrophilicity as well as initial molecular weight may have been responsible for the dramatic difference in degradation rate observed here.

22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model.

Previous studies on the reconstruction of porcine bladder using bladder acellular matrix allograft (BAMA) have indicated positive preliminary results with respect to graft shrinkage and cellular repopulation. The current study was conducted to investigate the feasibility of using BAMA in a similar model of bladder reconstruction out to longer time frames (22 weeks). At predetermined time points, the macroscopic, histological and mechanical properties of explanted native and BAMA tissues were evaluated and compared. Macroscopically, contracture of the BAMA was observed. The peripheral regions of the grafts experienced extensive cellular repopulation. Towards the centre however, all grafts were consistently devoid of organized smooth muscle bundles and a well-developed urothelium. An alteration in both the amount and organization of collagen was also observed within this region. Significant differences (p < 0.05) in the rupture strain and the elastic modulus of the BAMA compared to native bladder tissue appear to correlate with macroscopic graft contracture as well as the fibroproliferative tissue response of the matrix.

Self-aggregation characteristics of recombinantly expressed human elastin polypeptides.

Elastin is an extracellular matrix protein found in tissues requiring extensibility and elastic recoil. Monomeric elastin has the ability to aggregate into fibrillar structures in vitro, and has been suggested to participate in the organization of its own assembly into a polymeric matrix in vivo. Although hydrophobic sequences in elastin have been suggested to be involved in this process of self-organization, the contributions of specific hydrophobic and crosslinking domains to the propensity of elastin to self-assemble have received less attention. We have used a series of defined, recombinant human elastin polypeptides to investigate the factors contributing to elastin self-assembly. In general, coacervation temperature of these polypeptides, used as a measure of their propensity to self-assemble, was influenced both by salt concentration and polypeptide concentration. In addition, hydrophobic domains appeared to be essential for the ability of these polypeptides to self-assemble. However, neither overall molecular mass, number of hydrophobic domains nor general hydropathy of the polypeptides provided a complete explanation for differences in coacervation temperature, suggesting that the specific nature of the sequences of these hydrophobic domains are an important determinant of the ability of elastin polypeptides to self-assemble.

Culture on electrospun polyurethane scaffolds decreases atrial natriuretic peptide expression by cardiomyocytes in vitro.

The function of the mammalian heart depends on the functional alignment of cardiomyocytes, and controlling cell alignment is an important consideration in biomaterial design for cardiac tissue engineering and research. The physical cues that guide functional cell alignment in vitro and the impact of substrate-imposed alignment on cell phenotype, however, are only partially understood. In this report, primary cardiac ventricular cells were grown on electrospun, biodegradable polyurethane (ES-PU) with either aligned or unaligned microfibers. ES-PU scaffolds supported high-density cultures and cell subpopulations remained intact over two weeks in culture. ES-PU cultures contained electrically-coupled cardiomyocytes with connexin-43 localized to points of cell:cell contact. Multi-cellular organization correlated with microfiber orientation and aligned materials yielded highly oriented cardiomyocyte groupings. Atrial natriuretic peptide, a molecular marker that shows decreasing expression during ventricular cell maturation, was significantly lower in cultures grown on ES-PU scaffolds than in those grown on tissue culture polystyrene. Cells grown on aligned ES-PU had significantly lower steady state levels of ANP and constitutively released less ANP over time indicating that scaffold-imposed cell organization resulted in a shift in cell phenotype to a more mature state. We conclude that the physical organization of microfibers in ES-PU scaffolds impacts both multi-cellular architecture and cardiac cell phenotype in vitro.

Proliferation and differentiation of adipose-derived stem cells on naturally derived scaffolds

A tissue-engineered substitute that facilitates large-volume regeneration of the subcutaneous adipose tissue layer is needed for reconstructive plastic surgery. Towards this goal, we describe the in vitro culture of primary human adipose-derived stem cells (ASC) seeded into placental decellular matrix (PDM) and cross-linked hyaluronan (XLHA) scaffolds. Specifically, we evaluated cellular proliferation and adipogenic differentiation in the PDM, XLHA, and PDM combined with XLHA scaffolds. Cellular proliferation, viability, and glucose consumption were determined prior to the induction of differentiation. Adipogenesis within each of the scaffolds was investigated through gene expression analysis using end point and real time reverse transcriptase polymerase chain reaction (RT-PCR). The results indicate that the cell-adhesive PDM scaffolds facilitated proliferation and viability, while differentiation was augmented when the cells were encapsulated in the non-adhesive XLHA gels.

Seeding Bioreactor-Produced Embryonic Stem Cell-Derived Cardiomyocytes on Different Porous, Degradable, Polyurethane Scaffolds Reveals the Effect of Scaffold Architecture on Cell Morphology

A successful regenerative therapy to treat damage incurred after an ischemic event in the heart will require an integrated approach including methods for appropriate revascularization of the infarct site, mechanical recovery of damaged tissue, and electrophysiological coupling with native cells. Cardiomyocytes are the ideal cell type for heart regeneration because of their inherent electrical and physiological properties, and cardiomyocytes derived from embryonic stem cells (ESCs) represent an attractive option for tissue-engineering therapies. An important step in developing tissue engineering-based approaches to cardiac cell therapy is understanding how scaffold architecture affects cell behavior. In this work, we generated large numbers of ESC-derived cardiomyocytes in bioreactors and seeded them on porous, 3-dimensional scaffolds prepared using 2 different techniques: electrospinning and thermally induced phase separation (TIPS). The effect of material macro-architecture on the adhesion, viability, and morphology of the seeded cells was determined. On the electrospun scaffolds, cells were elongated in shape, a morphology typical of cultured ESC-derived cardiomyocytes, whereas on scaffolds fabricated using TIPS, the cells retained a rounded morphology. Despite these gross phenotypic and physiological differences, sarcomeric myosin and connexin 43 expression was evident, and contracting cells were observed on both scaffold types, suggesting that morphological changes induced by material macrostructure do not directly correlate to functional differences.

Adipose tissue engineering with cells in engineered matrices

Tissue engineering has shown promise for the development of constructs to facilitate large volume soft tissue augmentation in reconstructive and cosmetic plastic surgery. This article reviews the key progress to date in the field of adipose tissue engineering. In order to effectively design a soft tissue substitute, it is critical to understand the native tissue environment and function. As such, the basic physiology of adipose tissue is described and the process of adipogenesis is discussed. In this article, we have focused on tissue engineering using a cell-seeded scaffold approach, where engineered extracellular matrix substitutes are seeded with exogenous cells that may contribute to the regenerative response. The strengths and limitations of each of the possible cell sources for adipose tissue engineering, including adipose-derived stem cells, are detailed. We briefly highlight some of the results from the major studies to date, involving a range of synthetic and naturally derived scaffolds. While these studies have shown that adipose tissue regeneration is possible, more research is required to develop optimized constructs that will facilitate safe, predictable, and long-term augmentation in clinical applications.

Fiber alignment and coculture with fibroblasts improves the differentiated phenotype of murine embryonic stem cell-derived cardiomyocytes for cardiac tissue engineering.

Embryonic stem cells (ESCs) are an important source of cardiomyocytes for regenerating injured myocardium. The successful use of ESC‐derived cardiomyocytes in cardiac tissue engineering requires an understanding of the important scaffold properties and culture conditions to promote cell attachment, differentiation, organization, and contractile function. The goal of this work was to investigate how scaffold architecture and coculture with fibroblasts influences the differentiated phenotype of murine ESC‐derived cardiomyocytes (mESCDCs). Electrospinning was used to process an elastomeric biodegradable polyurethane (PU) into aligned or unaligned fibrous scaffolds. Bioreactor produced mESCDCs were seeded onto the PU scaffolds either on their own or after pre‐seeding the scaffolds with mouse embryonic fibroblasts (MEFs). Viable mESCDCs attached to the PU scaffolds and were functionally contractile in all conditions tested. Importantly, the aligned scaffolds led to the anisotropic organization of rod‐shaped cells, improved sarcomere organization, and increased mESCDC aspect ratio (length‐to‐diameter ratio) when compared to cells on the unaligned scaffolds. In addition, pre‐seeding the scaffolds with MEFs improved mESCDC sarcomere formation compared to mESCDCs cultured alone. These results suggest that both fiber alignment and pre‐treatment of scaffolds with fibroblasts improve the differentiation of mESCDCs and are important parameters for developing engineered myocardial tissue constructs using ESC‐derived cardiac cells. Biotechnol. Bioeng. 2012; 109:813–822.

Adipose tissue engineering in vivo with adipose-derived stem cells on naturally derived scaffolds.

Placental decellular matrix (PDM) and PDM combined with cross-linked hyaluronan (XLHA) scaffolds, seeded with primary human adipose-derived stem cells (ASC), were investigated in a subcutaneous athymic mouse model. The in vivo response at 3 and 8 weeks was characterized using histological and immunohistochemical staining. Fibrous capsule formation was assessed and the relative number of adipocytes in each scaffold was quantified. Undifferentiated ASC were localized using immunostaining for human vimentin. Unilocular and multilocular adipocytes were identified by intracellular lipid accumulation. Staining for murine CD31 assessed implant vascularization. Both scaffolds macroscopically maintained their three-dimensional volume and supported mature adipocyte populations in vivo. There was evidence of implant integration and a host contribution to the adipogenic response. The results suggested that incorporating the XLHA had a positive effect in terms of angiogenesis and adipogenesis. Overall, the PDM and PDM with XLHA scaffolds showed great promise for adipose tissue regeneration.

Characterization of biodegradable polyurethane microfibers for tissue engineering

A polyurethane designed to be biodegradable via hydrolysis and enzyme-mediated chain cleavage, has been investigated for its use as a temporary scaffold in tissue-engineering applications. The phase-segregated nature of the polyurethane imparts elastomeric properties that are attractive for soft tissue engineering. This polyurethane has been electrospun in order to create scaffolds that incorporate several biomimetic features including small fiber diameter, large void volume, and an interconnected porous network. Material properties were evaluated via gel-permeation chromatography, differential scanning calorimetry and Raman spectroscopy before and after processing. Analysis by gel-permeation chromatography showed that the molecular weights were similar, indicating that the bulk of the polymer chains were not degraded during processing. Thermal analysis revealed that the glass transition temperature did not shift and Raman spectra of the bulk polyurethane film compared to the electrospun mat were identical, confirming that the conformation of the polymer was unaffected by the shear and electric field used in the electrospinning process. In addition, field emission scanning electron microscopy revealed that the morphology of the electrospun mats had a broad fiber diameter distribution, and mechanical analysis showed that the mats had an ultimate tensile stress of 1.33 MPa and ultimate tensile strain of 78.6%. The degradation profile was investigated in the presence of chymotrypsin. These results were compared to a previous study of thin films of this polyurethane, and it was found that the increase of surface area aided the surface-mediated erosion of the material. It is believed that an electrospun matrix of this biodegradable polyurethane shows promise for use in soft tissue engineering and regenerative medicine applications.