Current concepts of cell migration were established in regular two-dimensional (2D) cell culture, but the roles of topography are poorly understood for cells migrating in an oriented 3D fibrillar extracellular matrix (ECM). We use a novel micropatterning technique termed microphotopatterning (μPP) to identify functions for 1D fibrillar patterns in 3D cell migration. In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome. We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.

We have assessed the biocompatibility of a new composite bone graft consisting of calcium phosphate cement (CPC) and poly(1actide-co-glycolide) (PLGA) microspheres (approximate diameter of 0.184.36 mm) using cell culture techniques. CPC powder is mixed with PLGA microspheres and water to yield a workable paste that could be sculpted to fit the contours of a wound. The cement then hardens into a matrix of hydroxyapatite microcrystals containing PLGA microspheres. The rationale for this design is that the microspheres will initially stabilize the graft but can then degrade to leave behind macropores for colonization by osteoblasts. The CPC matrix could then be resorbed and replaced with new bone. In the present study, osteoblast-like cells (MC3T3-El cells) were seeded onto graft specimens and evaluated with fluorescence microscopy, environmental scanning electron microscopy and the Wst-1 assay (an enzymatic assay for mitochondria1 dehydrogenase activity). Cells were able to adhere, attain a normal morphology, proliferate and remain viable when cultured on the new composite graft (CPC-PLGA) or on a control graft (CPC alone). These results suggest that our new cement consisting of CPC and PLGA microspheres is biocompatible. This is the first time that a 'polymer-in-mineral' (PLGA microspheres dispersed in a CPC matrix) cement has been formulated that is moldable, resorbable and that can form macropores after the cement has set.

To improve the effectiveness of calcium phosphate cement (CPC), we have developed a method to seed osteoblasts into the cement. CPC powder is mixed with water to form a paste that can be shaped to fit a bone defect in situ. The paste hardens in 30 min, reacts to form hydroxyapatite, and is replaced with new bone. Reacted CPC is biocompatible but unreacted CPC paste was found to have toxic effects when placed on cell monolayers (MC3T3-E1 cells). In contrast, when cells were indirectly exposed to CPC paste using a porous membrane or by placing a coverslip containing adherent cells onto a bed of CPC paste, the unreacted CPC was nontoxic. These results suggested that gel encapsulation of the cells might protect them from the CPC paste. Thus, cells were encapsulated in alginate beads (3.6-mm diameter), mixed with CPC paste, and incubated overnight. Both vital staining (calcein-AM and ethidium homodimer-1) and the Wst-1 assay (measures dehydrogenase activity) showed that cell survival in alginate beads that were mixed with CPC was similar to survival in untreated control beads. These results suggest that gel encapsulation could be used as a mechanism to protect cells for seeding into CPC.

No abstract

The introduction of tissue engineering strategies for the repair and replacement of human body components extends the application and importance of biomaterials. Implanted biomaterials frequently evoke inflammatory responses that are complex and not well understood at present. The goals of this work were to develop improved measurement methods for the quantification of cellular inflammatory responses to biomaterials and obtain data that lead to an enhanced understanding of the ways in which the body responds to the introduction of biomaterials. To evaluate the biocompatibility of materials, we established a system that allows for the analysis and quantitation of cellular inflammatory responses in vitro. In this study, the inflammatory responses of murine macrophages (RAW 264.7) were analyzed. The cells were incubated with polymethylmethacrylate (PMMA) microspheres in the presence and absence of lipopolysaccharide (LPS) at 8 and 18 h. The analysis of the genetic material obtained from the cells was quantitated using real-time reverse transcription polymerase chain reaction (RT-PCR). The cell populations treated with LPS or PMMA microspheres singly resulted in an elevation of cytokine levels compared to the untreated control. LPS resulted in a 258-fold increase, while PMMA resulted in an 87.9-fold increase at 8 h. RAW 264.7 cells incubated with LPS and PMMA particles demonstrated a synergistic effect by producing a marked increase in the level of cytokine expression, 336-fold greater than that of the untreated control at 8 h. Fluorescence microscopy studies that assessed cellular viability were also performed and are consistent with the RT-PCR results.