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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

Setup for culture of PCL filaments with MSC or MSC with SC. a,b Scanning electron microscopy (ESM) photomicrographs of PCL filaments showing in low (a) and high (b) magnification the microstructured grooves formed by melting extrusion at 60 °C. c Phase-contrast photomicrograph and c’ fluorescence photomicrograph in higher magnification of mesenchymal stem cells (MSC) in culture after three passages. In c’ MSC expressed enhanced green fluorescent protein (EGFP; green), and nuclei are labeled in blue (DAPI labeling). d Phase-contrast photomicrograph of Schwann cells (SC) derived from adult sciatic nerve cultured for 10 days. d’ Fluorescence photomicrograph of SC immunostained for S-100 (red) and nuclei labeled with DAPI (blue). The vast majority of cells are positive for S-100. e Photograph illustrating the culture system used, with a bundle of PCL filaments treated with plasma-O2/poly-D-lysine/laminin and incubated with MSC or MSC plus SC for 48 h. Scale bars: a = 50 μm; b = 10 μm; c,d = 400 μm; c’,d’ = 100 μm; e = 2 cm
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Fig1: Setup for culture of PCL filaments with MSC or MSC with SC. a,b Scanning electron microscopy (ESM) photomicrographs of PCL filaments showing in low (a) and high (b) magnification the microstructured grooves formed by melting extrusion at 60 °C. c Phase-contrast photomicrograph and c’ fluorescence photomicrograph in higher magnification of mesenchymal stem cells (MSC) in culture after three passages. In c’ MSC expressed enhanced green fluorescent protein (EGFP; green), and nuclei are labeled in blue (DAPI labeling). d Phase-contrast photomicrograph of Schwann cells (SC) derived from adult sciatic nerve cultured for 10 days. d’ Fluorescence photomicrograph of SC immunostained for S-100 (red) and nuclei labeled with DAPI (blue). The vast majority of cells are positive for S-100. e Photograph illustrating the culture system used, with a bundle of PCL filaments treated with plasma-O2/poly-D-lysine/laminin and incubated with MSC or MSC plus SC for 48 h. Scale bars: a = 50 μm; b = 10 μm; c,d = 400 μm; c’,d’ = 100 μm; e = 2 cm

Mentions: The PCL filaments used in this study were kindly donated by Prof. Dr. Burkhard Schlosshauer of The Natural and Medical Sciences Institute associated with the University of Tübingen, in collaboration with the Institute of Textile Technology and Process Engineering Denkendorf, Germany. Synthetic absorbable filaments were made from PCL with a molecular weight of 50,000 g/mol (Dow Tone, P767). Long microstructured filaments were formed by a technique of melting and extrusion in a spinneret at 205 °C, using a six-leaf nozzle with 24 capillaries. The yield volume was approximately 0.4 ml/min for each capillary, and the output speed was 1000 mm/min. In this state, the diameter of each capillary was 22 μm, with 66 μm of functional circumference. The bundles of filaments were washed with distilled water, wrapped around a microscope slide to facilitate handling, and the ends sealed. Small 2 cm segments (width of a microscope slide) containing hundreds of filaments sealed at the ends were sterilized in 70 % ethanol and dried [14]. Ultrastructural analysis was carried out by bonding the specimens in brackets covered with double-sided tape and imaging in an scanning electron microscope (Jeol JSM6390LV, JEOL, Peabody, MA, USA) after sputtering with a 20 nm thick gold layer (Fig. 1a,b). The functionalization of the filaments was performed in a three-step process. Initially, the material surface was hydrophilized using a glow discharge O2 plasma for three cycles of 75 s (PELCO easiGlow™, Pelco, Redding, CA, USA). Then, the filaments were coated with poly-D-lysine (50 μg/mL H2O; Sigma-Aldrich, São Paulo, SP, Brazil) and then coated with laminin (5 μg/ml; Life Technologies, Sao Paulo, Brazil).Fig. 1


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)

Setup for culture of PCL filaments with MSC or MSC with SC. a,b Scanning electron microscopy (ESM) photomicrographs of PCL filaments showing in low (a) and high (b) magnification the microstructured grooves formed by melting extrusion at 60 °C. c Phase-contrast photomicrograph and c’ fluorescence photomicrograph in higher magnification of mesenchymal stem cells (MSC) in culture after three passages. In c’ MSC expressed enhanced green fluorescent protein (EGFP; green), and nuclei are labeled in blue (DAPI labeling). d Phase-contrast photomicrograph of Schwann cells (SC) derived from adult sciatic nerve cultured for 10 days. d’ Fluorescence photomicrograph of SC immunostained for S-100 (red) and nuclei labeled with DAPI (blue). The vast majority of cells are positive for S-100. e Photograph illustrating the culture system used, with a bundle of PCL filaments treated with plasma-O2/poly-D-lysine/laminin and incubated with MSC or MSC plus SC for 48 h. Scale bars: a = 50 μm; b = 10 μm; c,d = 400 μm; c’,d’ = 100 μm; e = 2 cm
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Setup for culture of PCL filaments with MSC or MSC with SC. a,b Scanning electron microscopy (ESM) photomicrographs of PCL filaments showing in low (a) and high (b) magnification the microstructured grooves formed by melting extrusion at 60 °C. c Phase-contrast photomicrograph and c’ fluorescence photomicrograph in higher magnification of mesenchymal stem cells (MSC) in culture after three passages. In c’ MSC expressed enhanced green fluorescent protein (EGFP; green), and nuclei are labeled in blue (DAPI labeling). d Phase-contrast photomicrograph of Schwann cells (SC) derived from adult sciatic nerve cultured for 10 days. d’ Fluorescence photomicrograph of SC immunostained for S-100 (red) and nuclei labeled with DAPI (blue). The vast majority of cells are positive for S-100. e Photograph illustrating the culture system used, with a bundle of PCL filaments treated with plasma-O2/poly-D-lysine/laminin and incubated with MSC or MSC plus SC for 48 h. Scale bars: a = 50 μm; b = 10 μm; c,d = 400 μm; c’,d’ = 100 μm; e = 2 cm
Mentions: The PCL filaments used in this study were kindly donated by Prof. Dr. Burkhard Schlosshauer of The Natural and Medical Sciences Institute associated with the University of Tübingen, in collaboration with the Institute of Textile Technology and Process Engineering Denkendorf, Germany. Synthetic absorbable filaments were made from PCL with a molecular weight of 50,000 g/mol (Dow Tone, P767). Long microstructured filaments were formed by a technique of melting and extrusion in a spinneret at 205 °C, using a six-leaf nozzle with 24 capillaries. The yield volume was approximately 0.4 ml/min for each capillary, and the output speed was 1000 mm/min. In this state, the diameter of each capillary was 22 μm, with 66 μm of functional circumference. The bundles of filaments were washed with distilled water, wrapped around a microscope slide to facilitate handling, and the ends sealed. Small 2 cm segments (width of a microscope slide) containing hundreds of filaments sealed at the ends were sterilized in 70 % ethanol and dried [14]. Ultrastructural analysis was carried out by bonding the specimens in brackets covered with double-sided tape and imaging in an scanning electron microscope (Jeol JSM6390LV, JEOL, Peabody, MA, USA) after sputtering with a 20 nm thick gold layer (Fig. 1a,b). The functionalization of the filaments was performed in a three-step process. Initially, the material surface was hydrophilized using a glow discharge O2 plasma for three cycles of 75 s (PELCO easiGlow™, Pelco, Redding, CA, USA). Then, the filaments were coated with poly-D-lysine (50 μg/mL H2O; Sigma-Aldrich, São Paulo, SP, Brazil) and then coated with laminin (5 μg/ml; Life Technologies, Sao Paulo, Brazil).Fig. 1

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