This study evaluated the in vitro cytotoxicity of poly(propylene fumarate) (PPF). PPF is an aliphatic biodegradable polymer that has been well characterized for use in bone tissue engineering scaffolds. Four different cell types, human mesenchymal stem cells (hMSC), fibroblasts (L929), pre-osteoblasts (MC3T3), and canine mesenchymal stem cells (cMSC), were used to evaluate the cytotoxicity of PPF. These cell types represent the tissues that PPF would interact with in vivo as a bone tissue scaffold. The sol fraction of the PPF films was measured and then utilized to estimate crosslinking density. Cytotoxicity was evaluated using XTT assay and fluorescence imaging. Results showed that PPF supported similar cell metabolic activities of hMSC, L929, MC3T3 and cMSC compared to the non-cytotoxic control, high density polyethylene (HDPE) and were statistically different than those cultured with the cytotoxic control, a polyurethane film containing 0.1% zinc diethyldithiocarbamate (ZCF). Results showed differing cellular responses to ZCF, the cytotoxic control. The L929 cells had the lowest cell metabolic activity levels after exposure to ZCF compared to the cell metabolic activity levels of the MC3T3, hMSC or cMSC cells. Qualitative verification of the results using fluorescence imaging demonstrated no change in cell morphology, vacuolization, or detachment when cultured with PPF compared to HDPE or blank media cultures. Overall the cytotoxicity response of the cells to PPF was demonstrated to be similar to the cytotoxic response of cells to known non-cytotoxic materials (HDPE).

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of such tissue engineered bone constructs can succeed, tactics to promote neovascularization need to be strengthened. We have previously demonstrated that the tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cells (hMSC). Here, we devised a strategy to address the need for a functional microvasculature by designing an in vitro coculture system that simultaneously cultures osteogenic differentiating hMSCs with endothelial cells (ECs). We utilized the TPS bioreactor as a dynamic coculture environment, which we hypothesize will encourage prevascularization of endothelial cells and early formation of bone tissue and could aid in anastomosis of the graft with the host vasculature after patient implantation. To evaluate the effect of different natural scaffolds for this coculture system, the cells were encapsulated in alginate and/or collagen hydrogel scaffolds. We discovered the necessity of cell-to-cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. We discovered increased osteogenic and angiogenic potential as seen by amplified gene and protein expression of ALP, BMP-2, VEGF, and PECAM. The TPS bioreactor further augmented these expressions, indicating a synergistic effect between coculture and applied shear stress. The development of this dynamic coculture platform for the prevascularization of engineered bone, emphasizing the importance of the construct microenvironments and will advance the clinical use of tissue engineered constructs.

Within the field of tissue engineering and regenerative medicine, the fabrication of tissue grafts of any significant size--much less a whole organ or tissue--remains a major challenge. Currently, tissue-engineered constructs cultured in vitro have been restrained in size primarily due to the diffusion limit of oxygen and nutrients to the center of these grafts. Previously, we developed a novel tubular perfusion system (TPS) bioreactor, which allows the dynamic culture of bead-encapsulated cells and increases the supply of nutrients to the entire cell population. More interestingly, the versatility of TPS bioreactor allows a large range of engineered tissue volumes to be cultured, including large bone grafts. In this study, we utilized alginate-encapsulated human mesenchymal stem cells for the culture of a tissue-engineered bone construct in the size and shape of the superior half of an adult human femur (∼ 200 cm(3)), a 20-fold increase over previously reported volumes of in vitro engineered bone grafts. Dynamic culture in TPS bioreactor not only resulted in high cell viability throughout the femur graft, but also showed early signs of stem cell differentiation through increased expression of osteogenic genes and proteins, consistent with our previous models of smaller bone constructs. This first foray into full-scale bone engineering provides the foundation for future clinical applications of bioengineered bone grafts.

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Professor of Mechanical Engineering, University of MarylandMethods for determining the stress intensity factor, K, from isochromatic fringe loops obtained from photoelastic models with sharp cracks are briefly reviewed. A new three-parameter method is introduced which follows the twoparameter approach developed previously by Irwin. In the three-parameter method, three different terms in an analytical solution are adjusted to obtain a suitable match between the theoretical and experimental isochromatic loops. The three parameters are: K / G , which describes the crack-tip singularity, (b) P K J , z to account for variations in the stress field removed from the crack tip and (c) uox = d K / G a , which is added to the u, component of the stress field to account for the biaxiality of the far-field stresses.A relationship is derived for the maximum shear stress, r,,,, in terms of the three fitting parameters ( K , m, and P, and the two geometric parameters (r, and 0,) associated with the isochromatic fringe loops. A method of solution based on measurements of rm and 0, from any two independent fringe loops is given.The accuracy of the three-parameter method was determined by using exact results from the central crack problem for a standard. It was found that the error was within k 1 per cent for 6 9 O < 0, < 1 4 5 O . Comparison with the twoparameter method shows that the three-parameter method is more accurate and is applicable over a wider range of