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Biological behavior of mesenchymal stem cells on poly-ε-caprolactone filaments and a strategy for tissue engineering of segments of the peripheral nerves.

Carrier-Ruiz A, Evaristo-Mendonça F, Mendez-Otero R, Ribeiro-Resende VT - Stem Cell Res Ther (2015)

Bottom Line: Neurites grew and extended over the surface of PCL filaments, reaching greater distances when over MSC-plated filaments.Axons showed more organized and myelinized fibers and reinnervated significantly more muscle fibers when they were previously implanted with MSC-covered PLC filaments.We provide evidence for the interaction among MSC, Schwann cells and PCL filaments, and we also demonstrate that this system can constitute a stable and permissive support for regeneration of segments of the peripheral nerves.

View Article: PubMed Central - PubMed

Affiliation: Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Laboratório de Neuroquímica, Centro de Ciências da Saúde Bl. C, Cidade Universitária, 21949-900, Rio de Janeiro, RJ, Brazil. acruiz@biof.ufrj.br.

ABSTRACT

Introduction: Peripheral nerves may fail to regenerate across tube implants because these lack the microarchitecture of native nerves. Bone marrow mesenchymal stem cells (MSC) secrete soluble factors that improve the regeneration of the peripheral nerves. Also, microstructured poly-caprolactone (PCL) filaments are capable of inducing bands of Büngner and promote regeneration in the peripheral nervous system (PNS). We describe here the interaction between PCL filaments and MSC, aiming to optimize PNS tubular implants.

Methods: MSC were plated on PCL filaments for 48 h and the adhesion profile, viability, proliferation and paracrine capacity were evaluated. Also, Schwann cells were plated on PCL filaments covered with MSC for 24 h to analyze the feasibility of the co-culture system. Moreover, E16 dorsal root ganglia were plated in contact with PCL filaments for 4 days to analyze neurite extension. Right sciatic nerves were exposed and a 10 mm nerve segment was removed. Distal and proximal stumps were reconnected inside a 14-mm polyethylene tube, leaving a gap of approximately 13 mm between the two stumps. Animals then received phosphate-buffered saline 1×, PCL filaments or PCL filaments previously incubated with MSC and, after 12 weeks, functional gait performance and histological analyses were made. Statistical analyses were made using Student's unpaired t-test, one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni post-test.

Results: MSC were confined to lateral areas and ridges of PCL filaments, aligning along the longitudinal. MSC showed high viability (90 %), and their proliferation and secretion capabilities were not completely inhibited by the filaments. Schwann cells adhered to filaments plated with MSC, maintaining high viability (90 %). Neurites grew and extended over the surface of PCL filaments, reaching greater distances when over MSC-plated filaments. Axons showed more organized and myelinized fibers and reinnervated significantly more muscle fibers when they were previously implanted with MSC-covered PLC filaments. Moreover, animals with MSC-covered filaments showed increased functional recovery after 12 weeks.

Conclusions: We provide evidence for the interaction among MSC, Schwann cells and PCL filaments, and we also demonstrate that this system can constitute a stable and permissive support for regeneration of segments of the peripheral nerves.

No MeSH data available.


Related in: MedlinePlus

Cell proliferation and fibroblast growth factor-2 (FGF-2) expression in the presence of poly-caprolactone (PCL) filaments. a–f Confocal microscopy optical sections of enhanced green fluorescent protein (EGFP)-mesenchymal stem cells (MSC) cultured for 48 h on a coverslip (a–c) or on PCL filaments (d–f) immunolabeled for KI-67 (red) (a, b, d, e) or FGF-2 (c, f). Cell nuclei were stained with To-Pro (blue). g Quantitative analysis showing the percentage of KI-67+ MSC on a coverslip or PCL filaments. h Quantitative analysis of the FGF-2 concentration (pg/mL) detected by ELISA assay in the conditioned medium from MSC cultures on a coverslip or with PCL filaments. Scale bars: a–f = 100 μm. ***p < 0.0001, **p < 0.001, Mann-Whitney
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Fig3: Cell proliferation and fibroblast growth factor-2 (FGF-2) expression in the presence of poly-caprolactone (PCL) filaments. a–f Confocal microscopy optical sections of enhanced green fluorescent protein (EGFP)-mesenchymal stem cells (MSC) cultured for 48 h on a coverslip (a–c) or on PCL filaments (d–f) immunolabeled for KI-67 (red) (a, b, d, e) or FGF-2 (c, f). Cell nuclei were stained with To-Pro (blue). g Quantitative analysis showing the percentage of KI-67+ MSC on a coverslip or PCL filaments. h Quantitative analysis of the FGF-2 concentration (pg/mL) detected by ELISA assay in the conditioned medium from MSC cultures on a coverslip or with PCL filaments. Scale bars: a–f = 100 μm. ***p < 0.0001, **p < 0.001, Mann-Whitney

Mentions: To assess the cellular behavior of MSC plated on PLC filaments, the proliferation and expression of FGF-2, a trophic factor, was evaluated (Fig. 3). The number of cells that were positive for Ki-67 was used to quantify the proliferating cells. When MSC were cultured alone on coverslips, approximately 35 % of the cells were positive for Ki-67 (Fig. 3a,b,f). When the same cells were co-cultured with PCL filaments, the number of proliferating cells was reduced to approximately 10 % (Fig. 3d,e,g; p < 0.0001, Mann-Whitney).Fig. 3


Biological behavior of mesenchymal stem cells on poly-ε-caprolactone filaments and a strategy for tissue engineering of segments of the peripheral nerves.

Carrier-Ruiz A, Evaristo-Mendonça F, Mendez-Otero R, Ribeiro-Resende VT - Stem Cell Res Ther (2015)

Cell proliferation and fibroblast growth factor-2 (FGF-2) expression in the presence of poly-caprolactone (PCL) filaments. a–f Confocal microscopy optical sections of enhanced green fluorescent protein (EGFP)-mesenchymal stem cells (MSC) cultured for 48 h on a coverslip (a–c) or on PCL filaments (d–f) immunolabeled for KI-67 (red) (a, b, d, e) or FGF-2 (c, f). Cell nuclei were stained with To-Pro (blue). g Quantitative analysis showing the percentage of KI-67+ MSC on a coverslip or PCL filaments. h Quantitative analysis of the FGF-2 concentration (pg/mL) detected by ELISA assay in the conditioned medium from MSC cultures on a coverslip or with PCL filaments. Scale bars: a–f = 100 μm. ***p < 0.0001, **p < 0.001, Mann-Whitney
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4522087&req=5

Fig3: Cell proliferation and fibroblast growth factor-2 (FGF-2) expression in the presence of poly-caprolactone (PCL) filaments. a–f Confocal microscopy optical sections of enhanced green fluorescent protein (EGFP)-mesenchymal stem cells (MSC) cultured for 48 h on a coverslip (a–c) or on PCL filaments (d–f) immunolabeled for KI-67 (red) (a, b, d, e) or FGF-2 (c, f). Cell nuclei were stained with To-Pro (blue). g Quantitative analysis showing the percentage of KI-67+ MSC on a coverslip or PCL filaments. h Quantitative analysis of the FGF-2 concentration (pg/mL) detected by ELISA assay in the conditioned medium from MSC cultures on a coverslip or with PCL filaments. Scale bars: a–f = 100 μm. ***p < 0.0001, **p < 0.001, Mann-Whitney
Mentions: To assess the cellular behavior of MSC plated on PLC filaments, the proliferation and expression of FGF-2, a trophic factor, was evaluated (Fig. 3). The number of cells that were positive for Ki-67 was used to quantify the proliferating cells. When MSC were cultured alone on coverslips, approximately 35 % of the cells were positive for Ki-67 (Fig. 3a,b,f). When the same cells were co-cultured with PCL filaments, the number of proliferating cells was reduced to approximately 10 % (Fig. 3d,e,g; p < 0.0001, Mann-Whitney).Fig. 3

Bottom Line: Neurites grew and extended over the surface of PCL filaments, reaching greater distances when over MSC-plated filaments.Axons showed more organized and myelinized fibers and reinnervated significantly more muscle fibers when they were previously implanted with MSC-covered PLC filaments.We provide evidence for the interaction among MSC, Schwann cells and PCL filaments, and we also demonstrate that this system can constitute a stable and permissive support for regeneration of segments of the peripheral nerves.

View Article: PubMed Central - PubMed

Affiliation: Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Laboratório de Neuroquímica, Centro de Ciências da Saúde Bl. C, Cidade Universitária, 21949-900, Rio de Janeiro, RJ, Brazil. acruiz@biof.ufrj.br.

ABSTRACT

Introduction: Peripheral nerves may fail to regenerate across tube implants because these lack the microarchitecture of native nerves. Bone marrow mesenchymal stem cells (MSC) secrete soluble factors that improve the regeneration of the peripheral nerves. Also, microstructured poly-caprolactone (PCL) filaments are capable of inducing bands of Büngner and promote regeneration in the peripheral nervous system (PNS). We describe here the interaction between PCL filaments and MSC, aiming to optimize PNS tubular implants.

Methods: MSC were plated on PCL filaments for 48 h and the adhesion profile, viability, proliferation and paracrine capacity were evaluated. Also, Schwann cells were plated on PCL filaments covered with MSC for 24 h to analyze the feasibility of the co-culture system. Moreover, E16 dorsal root ganglia were plated in contact with PCL filaments for 4 days to analyze neurite extension. Right sciatic nerves were exposed and a 10 mm nerve segment was removed. Distal and proximal stumps were reconnected inside a 14-mm polyethylene tube, leaving a gap of approximately 13 mm between the two stumps. Animals then received phosphate-buffered saline 1×, PCL filaments or PCL filaments previously incubated with MSC and, after 12 weeks, functional gait performance and histological analyses were made. Statistical analyses were made using Student's unpaired t-test, one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni post-test.

Results: MSC were confined to lateral areas and ridges of PCL filaments, aligning along the longitudinal. MSC showed high viability (90 %), and their proliferation and secretion capabilities were not completely inhibited by the filaments. Schwann cells adhered to filaments plated with MSC, maintaining high viability (90 %). Neurites grew and extended over the surface of PCL filaments, reaching greater distances when over MSC-plated filaments. Axons showed more organized and myelinized fibers and reinnervated significantly more muscle fibers when they were previously implanted with MSC-covered PLC filaments. Moreover, animals with MSC-covered filaments showed increased functional recovery after 12 weeks.

Conclusions: We provide evidence for the interaction among MSC, Schwann cells and PCL filaments, and we also demonstrate that this system can constitute a stable and permissive support for regeneration of segments of the peripheral nerves.

No MeSH data available.


Related in: MedlinePlus