Paul A. DiMilla

In vitro analysis of biodegradable polymer blend/hydroxyapatite composites for bone tissue engineering

Blends of biodegradable polymers, poly(caprolactone) and poly(D, L-lactic-co-glycolic acid), have been examined as scaffolds for applications in bone tissue engineering. Hydroxyapatite granules have been incorporated into the blends and porous discs were prepared. Mechanical properties and degradation rates in vitro of the composites were determined. The discs were seeded with rabbit bone marrow or cultured bone marrow stromal cells and incubated under physiological conditions. Polymer/ceramic scaffolds supported cell growth throughout the scaffold for 8 weeks. Scanning and transmission electron microscopy, and histological analyses were used to characterize the seeded composites. This study suggests the feasibility of using novel polymer/ceramic composites as scaffold in bone tissue engineering applications.

Characterization of osteoblast-like behavior of cultured bone marrow stromal cells on various polymer surfaces

The creation of novel bone substitutes requires a detailed understanding of the interaction between cells and materials. This study was designed to test certain polymers, specifically poly(caprolactone) (PCL), poly(D,L-lactic-CO-glycolic acid) (PLGA), and combinations of these polymers for their ability to support bone marrow stromal cell proliferation and differentiation. Bone marrow stromal cells were cultured from New Zealand White rabbits and were seeded onto glass slides coated with a thin layer of PCL, PLGA, and combinations of these two polymers in both a 40:60 and a 10:90 ratio. Growth curves were compared. At the end of 2 weeks, the cells were stained for both matrix mineralization and alkaline phosphatase activity. There was no statistically significant difference in growth rate of the cells on any polymer or polymer combination. However, there was a striking difference in Von Kossa staining and alkaline phosphatase staining. Cells on PCL did not show Von Kossa staining or alkaline phosphatase staining. However, in the 40:60 and 10:90 blends, there was both positive Von Kossa and alkaline phosphatase staining. These data indicate that PCL alone may not be a satisfactory material for the creation of a bone substitute. However, it may be used in combination with PLGA for the creation of a bone substitute material.

Application of fluid mechanic and kinetic models to characterize mammalian cell detachment in a radial-flow chamber

The strength of adhesion and dynamics of detachment of murine 3T3 fibroblasts from self-assembled monolayers were measured in a radial-flow chamber (RFC) by applying models for fluid mechanics, adhesion strength probability distributions, and detachment kinetics. Four models for predicting fluid mechanics in a RFC were compared to evaluate the accuracy of each model and the significance of inlet effects. Analysis of these models indicated an outer region at large radial positions consistent with creeping flow, an intermediate region influenced by inertial dampening, and an inner region dominated by entrance effects from the axially-oriented inlet. In accompanying experiments patterns of the fraction of cells resisting detachment were constructed for individual surfaces as a function of the applied shear stress and evaluated by comparison with integrals of both a normal and a log-normal distribution function. The two functions were equally appropriate, yielding similar estimates of the mean strength of adhesion. Further, varying the Reynolds number in the inlet, Re(d), between 630 and 1480 (corresponding to volumetric flow rates between 0.9 and 2.1 mL/s) did not affect the mean strength of adhesion. For these same experiments, analysis of the dynamics of detachment revealed three temporal phases: 1) rapid detachment of cells at the onset of flow, consistent with a first-order homogeneous kinetic model; 2) time-dependent rate of detachment during the first 30 sec. of exposure to hydrodynamic shear, consistent with the first-order heterogeneous kinetic model proposed by Dickinson and Cooper (1995); and 3) negligible detachment, indicative of pseudo-steady state after 60 sec. of flow. Our results provide rigorous guidelines for the measurement of adhesive interactions between mammalian cells and prospective biomaterial surfaces using a RFC. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 616-629, 1997.

Novel three Dimensional Biodegradable Scaffolds for Bone Tissue Engineering

We have constructed osteogenic scaffolds using solid freeform fabrication techniques. Blends of biodegradable polymers, polycaprolactone and poly(D,L-lactic-co-glycolic acid), have been examined as scaffolds for applications in bone tissue engineering. Hydroxyapatite granules were incorporated into the blends and porous discs were prepared. Mechanical properties and degradation rates of the composites were determined. The discs were seeded with rabbit bone marrow or cultured bone marrow stromal cells and in vitro studies were conducted. Electron microscopy and histological analysis revealed an osteogenic composite that supports bone cell growth not only on the surface but throughout the 1 mm thick scaffold as well. Seeded and unseeded discs were mechanically assembled in layers and implanted in a rabbit rectus abdominis muscle. Bone growth was evident after eight weeks in vivo . Electron microscopy and histological analyses indicate vascularization and primitive bone formation throughout the seeded composite, and also a “fusion” of the layers to form a single, solid construct. Finally, we have begun to incorporate the growth factor IGF-I into the scaffold to enhance osteogenicity and/or as an alternative to cell seeding.

Effect of cell-cell interactions on the observable strength of adhesion of sheets of cells.

AbstractPrevious research in cellular adhesion has focused primarily on studying isolated cells under conditions where cells do not interact with each other. However, in vivo cells form sheets where both cell–substratum and cell–cell interactions contribute to the overall adhesive behavior. Our understanding of how cell–cell and cell–substratum interactions affect the overall process of cell adhesion in these situations is limited. To address this problem, we developed a systematic approach to evaluate how cell–cell and cell–substratum interactions affect the critical shear stress for detachment for semiconfluent and confluent sheets of cells. Our studies were based on subjecting cultures of adherent cells to a defined hydrodynamic flow in a radial-flow chamber with a gap height of 140 μm. Using phase-contrast microscope imaging and analysis we measured shear-dependent patterns of detachment as a function of the extent of cell confluency. Our results show that the critical shear stress for detachment is maximum at intermediate extents of confluency of 10%–40%. These results have important implications for sodding vascular grafts and tissue engineering.

Adhesion and Traction Forces in Migration: Insights From Mathematical Models and Experiments

The migration of mammalian tissue and white blood cells plays a critical role in a diverse array of physiological and pathological phenomena, including the proper development and repair of organs, inflammation, angiogenesis, and metastasis (Trinkaus, 1984). The phenomenon of migration is complex, depending on coordinated interactions among many underlying biochemical and biophysical processes. Much information has been obtained at the molecular level about the identity and properties of components involved in these processes, especially receptor-mediated adhesion (Buck and Horwitz, 1987; Hynes, 1992) and cytoskeletal force generation (Stossel et al., 1985; Bray and White, 1988), as well as at the cell behavioral level (Lackie, 1986; Singer and Kupfer, 1986; Devreotes and Zigmond, 1988; Heath and Holifield, 1991).