Activated Schwann Cell-Like Cells on Aligned Fibrin-Poly(Lactic-Co-Glycolic Acid) Structures: A Novel Construct for Application in Peripheral Nerve Regeneration.

Tissue engineering approaches in nerve regeneration search for ways to support gold standard therapy (autologous nerve grafts) and to improve results by bridging nerve defects with different kinds of conduits. In this study, we describe electrospinning of aligned fibrin-poly(lactic-co-glycolic acid) (PLGA) fibers in an attempt to create a biomimicking tissue-like material seeded with Schwann cell-like cells (SCLs) in vitro for potential use as an in vivo scaffold. Rat adipose-derived stem cells (rASCs) were differentiated into SCLs and evaluated with flow cytometry concerning their differentiation and activation status [S100b, P75, myelin-associated glycoprotein (MAG), and protein 0 (P0)]. After receiving the proliferation stimulus forskolin, SCLs expressed S100b and P75; comparable to native, activated Schwann cells, while cultured without forskolin, cells switched to a promyelinating phenotype and expressed S100b, MAG, and P0. Human fibrinogen and thrombin, blended with PLGA, were electrospun and the alignment and homogeneity of the fibers were proven by scanning electron microscopy. Electrospun scaffolds were seeded with SCLs and the formation of Büngner-like structures in SCLs was evaluated with phalloidin/propidium iodide staining. Carrier fibrin gels containing rASCs acted as a self-shaping matrix to form a tubular structure. In this study, we could show that rASCs can be differentiated into activated, proliferating SCLs and that these cells react to minimal changes in stimulus, switching to a promyelinating phenotype. Aligned electrospun fibrin-PLGA fibers promoted the formation of Büngner-like structures in SCLs, which also rolled the fibrin-PLGA matrix into a tubular scaffold. These in vitro findings favor further in vivo testing.

Vascular endothelial growth factor and fibroblast growth factor-2 incorporation in starch-based bone tissue-engineered constructs promote the in vivo expression of neovascularization mediators.

The ideal bone tissue-engineered (TE) construct remains to be found, although daily discoveries significantly contribute to improvements in the field and certainly have valuable long-term outcomes. In this work, different TE elements, aiming at bone TE applications, were assembled and its effect on the expression of several vascularization/angiogenesis mediators analyzed. Starch/polycaprolactone (SPCL) scaffolds, obtained by two different methodologies, were combined with fibrin sealant (Baxter(®)), human adipose-derived stem cells (hASCs), and growth factors (vascular endothelial growth factor [VEGF] or fibroblast growth factor-2 [FGF-2]), and implanted in vascular endothelial growth factor receptor-2 (VEGFR2)-luc transgenic mice. The expression of VEGFR2 along the implantation of the designed constructs was followed using a luminescence device (Xenogen(®)) and after 2 weeks, the explants were retrieved to perform histological analysis and reverse transcriptase-polymerase chain reaction for vascularization (VEGF and VEGFR1) and inflammatory (tumor necrosis factor-alpha, interleukin-4, and interferon-gamma) markers. It was showed that SPCL scaffolds obtained by wet spinning and by fiber bonding constitute an adequate support for hASCs. The assembled TE constructs composed by fibrin sealant, hASCs, VEGF, and FGF-2 induce only a mild inflammatory reaction after 2 weeks of implantation. Additionally, the release of VEGF and FGF-2 from the constructs enhanced the expression of VEGFR2 and other important mediators in neovascularization (VEGF and VEGFR1). These results indicate the potential of VEGF or FGF-2 within a bone TE construct composed by wet-spun SPCL, fibrin sealant, and hASCs in promoting the vascularization of newly formed tissue.

BMP-2 but not VEGF or PDGF in fibrin matrix supports bone healing in a delayed-union rat model.

Treatment of delayed bone healing and non-unions after fractures, osteotomies or arthrodesis still is a relevant clinical challenge. Artificially applied growth factors can increase bone healing and progressively gain importance in clinical routine. The aim of this study was to determine the effects of rhPDGF-BB, rhVEGF-165, and rhBMP-2 in fibrin matrix on bone healing in a delayed-union rat model. Thirty-seven rats underwent a first operation where a standardized femoral critical size defect was created. A silicone spacer was implanted to impair vascularization within the defect. At 4 weeks the spacer was removed in a second operation and rhPDGF-BB, rhVEGF-165, or rhBMP-2 were applied in a fibrin clot. Animals in a fourth group received a fibrin clot without growth factors. At 8 weeks fibrin bound rhBMP-2 treated animals showed a significantly increased union rate and bone volume within the defect compared to the other groups. Single application of fibrin bound rhPDGF-BB and rhVEGF-165 failed to increase bone healing in our atrophic non-union model.

Enhanced reporter gene assay for the detection of osteogenic differentiation.

Detection of osteogenic differentiation is crucial for bone tissue engineering. Despite established standard end point assays, there is increasing demand for methods allowing noninvasive kinetic differentiation monitoring. Reporter gene assays employing tissue-specific promoters and suitable reporter genes fulfill these requirements. Many promoters, however, exhibit only weak cis-activating potential, thus limiting their application to generate sensitive reporter gene assays. Therefore, the aim of this study was to design a reporter gene assay employing elements of the murine osteocalcin promoter coupled to a viral enhancer for signal amplification. Additionally, the system's practicability was enhanced by introducing a secreted luciferase as a quantifiable reporter gene. The constructs were tested in C2C12 cells stimulated with recombinant human bone morphogenetic protein 2 for osteogenic differentiation in two-dimensional and three-dimensional culture. Osteogenic differentiation was confirmed by standard assays for osteogenesis. The reporter gene signal was detected through a secreted luciferase or fluorescence microscopy for enhanced yellow fluorescent protein. The constructs exhibited strong activation upon treatment with recombinant human bone morphogenetic protein 2. Weak background expression was observable in negative controls, attributed to the pan-active viral enhancer. In conclusion, a novel enhancer/tissue-specific promoter combination allows specific signal-amplified, kinetic monitoring of osteogenic differentiation in a nonsample-destructive manner.