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.

The optimal carrier for BMP-2: a comparison of collagen versus fibrin matrix.

INTRODUCTION: The aim of our study was to investigate in vitro and in a new in vivo rat model for impaired bone healing whether a low dose BMP-2 preparation in fibrin would be equivalent or better than the combination of collagen and a high dose of BMP-2 which is currently in clinical use. MATERIALS AND METHODS: In a 14 day period we compared the in vitro release kinetics of an absorbable collagen sponge (ACS) with 72 µg rhBMP-2 in the BMPC group and fibrin matrix with 10 µg rhBMP-2 in the BMPF group. In our in vivo experiment a critical sized osteotomy was performed in the rat femur, which was filled with a spacer, inhibiting bone formation for a period of 4 weeks. In a second operation this spacer was removed and the test item was applied into the defect. We compared the BMPF and BMPC groups with the ACS alone, FIBRIN alone and the EMPTY (4w/8w) control groups. 4 and 8 weeks after the second operation, specimens were analysed by X-ray and µCT imaging. Mechanically stable femurs were biomechanically evaluated. RESULTS: Cumulative BMP-2 release was five times higher in the BMPF group than in the BMPC group during the observation period. µCT analysis revealed that both the extent of bone union and the bone volume were significantly higher in the group with a lower dose of BMP-2 in fibrin matrix than in the groups without BMP-2 treatment. However there was no statistically significant difference between the BMPF and BMPC groups. CONCLUSION: We conclude that fibrin matrix is an excellent carrier for BMP-2 and that it provides equivalent results with a sevenfold lower dose of BMP-2 compared with ACS.

Sustained (rh)VEGF(165) release from a sprayed fibrin biomatrix induces angiogenesis, up-regulation of endogenous VEGF-R2, and reduces ischemic flap necrosis.

This study investigated (1) the release of recombinant human vascular endothelial growth factor ([rh]VEGF(165)) from an in vitro fibrin matrix, (2) the effects of (rh)VEGF(165) released from an in vivo fibrin matrix on ischemic flap necrosis in the rat dorsal skin flap model, and (3) the effects of (rh)VEGF(165) released from an in vivo fibrin matrix on VEGF-R2 expression in transgenic VEGF-R2/luc mice. In vitro fibrin matrices were spiked with (rh)VEGF(165) and demonstrated (rh)VEGF(165) release over 88 hours with 66% recovery. Ischemic dorsal flaps were treated with a fibrin sealant (FS), FS spiked with (rh)VEGF(165), or left untreated. Flaps treated with FS spiked with (rh)VEGF(165) showed greater viability than controls as measured by planimetric analysis. Immunohistochemical analyses revealed stronger neovascularization than that exhibited by controls. Transgenic mice implanted with FS spiked with (rh)VEGF(165) had significant increases in VEGF-R2 expression relative to controls at days 5-13 after implantation. Conclusions drawn from this work are that (1) (rh)VEGF(165) is released from an in vitro fibrin matrix at clinically appropriate times, (2) (rh)VEGF(165) increases the viability of tissue flaps in vivo, and (3) (rh)VEGF(165) induces the expression of VEGF-R2 expression. This work demonstrates the clinical ability of sprayed FS to locally deliver growth factors to ischemic tissue of patients.

Fabrication of electrospun poly(D,L lactide-co-glycolide)80/20 scaffolds loaded with diclofenac sodium for tissue engineering.

BACKGROUND: Adaptation of nanotechnology into materials science has also advanced tissue engineering research. Tissues are basically composed of nanoscale structures hence making nanofibrous materials closely resemble natural fibers. Adding a drug release function to such material may further advance their use in tissue repair. METHODS: In the current study, bioabsorbable poly(D,L lactide-co-glycolide)80/20 (PDLGA80/20) was dissolved in a mixture of acetone/dimethylformamide. Twenty percent of diclofenac sodium was added to the solution. Nanofibers were manufactured using electrospinning. The morphology of the obtained scaffolds was analyzed by scanning electron microscopy (SEM). The release of the diclofenac sodium was assessed by UV/Vis spectroscopy. Mouse fibroblasts (MC3T3) were seeded on the scaffolds, and the cell attachment was evaluated with fluorescent microscopy. RESULTS: The thickness of electrospun nanomats was about 1 mm. SEM analysis showed that polymeric nanofibers containing drug particles formed very interconnected porous nanostructures. The average diameter of the nanofibers was 500 nm. Drug release was measured by means of UV/Vis spectroscopy. After a high start peak, the release rate decreased considerably during 11 days and lasted about 60 days. During the evaluation of the release kinetics, a material degradation process was observed. MC3T3 cells attached to the diclofenac sodium-loaded scaffold. CONCLUSIONS: The nanofibrous porous structure made of PDLGA polymer loaded with diclofenac sodium is feasible to develop, and it may help to improve biomaterial properties for controlled tissue repair and regeneration.

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.