Small pore sizes inherent to electrospun matrices can hinder efficient cellular ingrowth. To facilitate infiltration while retaining its extracellular matrix-like character, electrospinning was combined with salt leaching to produce a scaffold having deliberate, engineered delaminations. We made elegant use of a specific randomizing component of the electrospinning process, the Taylor Cone and the falling fiber beneath it, to produce a uniform, well-spread distribution of salt particles. After 3 weeks of culture, up to 4 mm of cellular infiltration was observed, along with cellular coverage of up to 70% within the delaminations. To our knowledge, this represents the first observation of extensive cellular infiltration of electrospun matrices. Infiltration appears to be driven primarily by localized proliferation rather than coordinated cellular locomotion. Cells also moved from the salt-generated porosity into the surrounding electrospun fiber matrix. Given that the details of salt deposition (amount, size, and number density) are far from optimized, the result provides a convincing illustration of the ability of mammalian cells to interact with appropriately tailored electrospun matrices. These layered structures can be precisely fabricated by varying the deposition interval and particle size conceivably to produce in vivo-like gradients in porosity such that the resulting scaffolds better resemble the desired final structure.

Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.

A novel non-toxic biodegradable lysine-di-isocyanate (LDI)-based urethane polymer was developed for use in tissue engineering applications. This matrix was synthesized with highly purified LDI made from the lysine diethylester. The ethyl ester of LDI was polymerized with glycerol to form a prepolymer. LDI-glycerol prepolymer when reacted with water foamed with the liberation of CO 2 to provide a pliable spongy urethane polymer. The LDI-glycerol matrix degraded in aqueous solutions at 100, 37, 22, and 4°C at a rate of 27.7, 1.8, 0.8, and 0.1 mM per 10 days, respectively. Its thermal stability in water allowed its sterilization by autoclaving. The degradation of the LDI-glycerol polymer yielded lysine, ethanol, and glycerol as breakdown products. The degradation products of LDI-glycerol polymer did not significantly affect the pH of the solution. The glass transition temperature (T g ) of this polymer was found to be 103.4°C. The physical properties of the polymer network were found to be adequate to support the cell growth in vitro, as evidenced by the fact that rabbit bone marrow stromal cells (BMSC) attached to the polymer matrix and remained viable on its surface. Culture of BMSC on LDI-glycerol matrix for long durations resulted in the formation of multilayered confluent cultures, a characteristic typical of bone cells. Furthermore, cells grown on LDI-glycerol matrix did not differ phenotypically from the cells grown on the tissue culture polystyrene plates as assessed by the cell growth, and expression of mRNA for collagen type I, and transforming growth factor-β1 (TGF-β1). The observations suggest that biodegradable peptide-based urethane polymers can be synthesized which may pave their way for possible use in tissue engineering applications.

The nuclear factor NF-B is a pleiotropic transcription factor whose activation results in inflammation, viral replication, and growth modulation. Due to its role in pathogenesis, NF-B is considered a key target for drug development. In the present report we show that sanguinarine (a benzophenanthridine alkaloid), a known anti-inflammatory agent, is a potent inhibitor of NF-B activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor rapidly activated NF-B, this activation was completely suppressed by sanguinarine in a dose-and time-dependent manner. Sanguinarine did not inhibit the binding of NF-B protein to the DNA but rather inhibited the pathway leading to NF-B activation. The reversal of inhibitory effects of sanguinarine by reducing agents suggests a critical sulfhydryl group is involved in NF-B activation. Sanguinarine blocked the tumor necrosis factor-induced phosphorylation and degradation of IB␣, an inhibitory subunit of NF-B, and inhibited translocation of p65 subunit to the nucleus. As sanguinarine also inhibited NF-B activation induced by interleukin-1, phorbol ester, and okadaic acid but not that activated by hydrogen peroxide or ceramide, the pathway leading to NF-B activation is likely different for different inducers. Overall, our results demonstrate that sanguinarine is a potent suppressor of NF-B activation and it acts at a step prior to IB␣ phosphorylation.

Objective. The mechanisms by which chondrocytes convert biomechanical signals into intracellular biochemical events are not well understood. In this study, we sought to determine the intracellular mechanisms of the magnitude-dependent actions of mechanical signals.Methods. Chondrocytes isolated from rabbit articular cartilage were grown on flexible membranes. Cells were subjected to cyclic tensile strain (CTS) of various magnitudes in the presence or absence of interleukin-1␤ (IL-1␤), which was used as a proinflammatory signal for designated time intervals. The regulation of NF-B was measured by reverse transcriptasepolymerase chain reaction, electrophoretic mobility shift assay, and immunofluorescence.Results. CTS of low magnitudes (4-8% equibiaxial strain) was a potent inhibitor of IL-1␤-dependent NF-B nuclear translocation. Cytoplasmic retention of NF-B and reduction of its synthesis led to sustained suppression of proinflammatory gene induction. In contrast, proinflammatory signals generated by CTS of high magnitudes (15-18% equibiaxial strain) mimicked the actions of IL-1␤ and induced rapid nuclear translocation of NF-B subunits p65 and p50.Conclusion. Magnitude-dependent signals of mechanical strain utilize the NF-B transcription factors as common elements to abrogate or aggravate proinflammatory responses. Furthermore, the intracellular events induced by mechanical overload are similar to those that are initiated by proinflammatory cytokines in arthritis.