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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 adhesion and viability in the presence of poly-caprolactone (PCL) filaments. a, b Confocal microscopy optical sections of mesenchymal stem cells (MSC) incubated at medium density (2 × 106 cells/mL) for 48 h on PCL filaments immunolabeled for CD90. b Orthogonal projections of Z-stack in high magnification, showing an individual PCL filament covered by two MCS. c Three-dimensional reconstruction by confocal microscopy of a single MSC (surface view) on an isolated PCL filament. d Quantitative analysis of the number of CD-90+ cells in contact with the PCL filaments and e the percentage of MSC aligned with the PCL filaments. f Percentage of viable cells in the different experimental conditions. Scale bars: a = 100 μm; b, c = 50 μm. ***p < 0.0001 by ANOVA. SC Schwann cells
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Fig2: Cell adhesion and viability in the presence of poly-caprolactone (PCL) filaments. a, b Confocal microscopy optical sections of mesenchymal stem cells (MSC) incubated at medium density (2 × 106 cells/mL) for 48 h on PCL filaments immunolabeled for CD90. b Orthogonal projections of Z-stack in high magnification, showing an individual PCL filament covered by two MCS. c Three-dimensional reconstruction by confocal microscopy of a single MSC (surface view) on an isolated PCL filament. d Quantitative analysis of the number of CD-90+ cells in contact with the PCL filaments and e the percentage of MSC aligned with the PCL filaments. f Percentage of viable cells in the different experimental conditions. Scale bars: a = 100 μm; b, c = 50 μm. ***p < 0.0001 by ANOVA. SC Schwann cells

Mentions: To study the interaction between MSC and PCL filaments, a custom culture system was developed, consisting of a bundle of filaments deposited on a microscope slide in a Petri dish (Fig. 1e). Using this system, we first evaluated the interactions of MSC with the PCL filaments. After 48 h in culture, numerous CD90-positive cells were found in contact with the filaments (Fig. 2a), mainly over the ridges and longitudinally aligned, as seen in orthogonal planes with confocal microscopy (Fig. 2b). Furthermore, three-dimensional reconstruction of optical planes revealed that, although the cell nuclei were located on the ridges, the cell bodies and processes extended toward the grooves (Fig. 2c), thereby enlarging the area covered by the cells on the filaments.Fig. 2


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 adhesion and viability in the presence of poly-caprolactone (PCL) filaments. a, b Confocal microscopy optical sections of mesenchymal stem cells (MSC) incubated at medium density (2 × 106 cells/mL) for 48 h on PCL filaments immunolabeled for CD90. b Orthogonal projections of Z-stack in high magnification, showing an individual PCL filament covered by two MCS. c Three-dimensional reconstruction by confocal microscopy of a single MSC (surface view) on an isolated PCL filament. d Quantitative analysis of the number of CD-90+ cells in contact with the PCL filaments and e the percentage of MSC aligned with the PCL filaments. f Percentage of viable cells in the different experimental conditions. Scale bars: a = 100 μm; b, c = 50 μm. ***p < 0.0001 by ANOVA. SC Schwann cells
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522087&req=5

Fig2: Cell adhesion and viability in the presence of poly-caprolactone (PCL) filaments. a, b Confocal microscopy optical sections of mesenchymal stem cells (MSC) incubated at medium density (2 × 106 cells/mL) for 48 h on PCL filaments immunolabeled for CD90. b Orthogonal projections of Z-stack in high magnification, showing an individual PCL filament covered by two MCS. c Three-dimensional reconstruction by confocal microscopy of a single MSC (surface view) on an isolated PCL filament. d Quantitative analysis of the number of CD-90+ cells in contact with the PCL filaments and e the percentage of MSC aligned with the PCL filaments. f Percentage of viable cells in the different experimental conditions. Scale bars: a = 100 μm; b, c = 50 μm. ***p < 0.0001 by ANOVA. SC Schwann cells
Mentions: To study the interaction between MSC and PCL filaments, a custom culture system was developed, consisting of a bundle of filaments deposited on a microscope slide in a Petri dish (Fig. 1e). Using this system, we first evaluated the interactions of MSC with the PCL filaments. After 48 h in culture, numerous CD90-positive cells were found in contact with the filaments (Fig. 2a), mainly over the ridges and longitudinally aligned, as seen in orthogonal planes with confocal microscopy (Fig. 2b). Furthermore, three-dimensional reconstruction of optical planes revealed that, although the cell nuclei were located on the ridges, the cell bodies and processes extended toward the grooves (Fig. 2c), thereby enlarging the area covered by the cells on the filaments.Fig. 2

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