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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

Neurite outgrowth from dorsal root ganglia (DRG) explants in the different culture conditions. a–e Confocal microscopy images of E16 rat DRG explants placed on a coverslip (a), on poly-caprolactone (PCL) filaments (c), on PCL filaments + msenshymal stem cells (MSC) (d), and on PCL filaments + MSC + Schwann cells (SC) (e) and cultured for 72 h. Neurites are identified by immunolabeling with NF-200 (green) and cell nuclei with To-Pro (blue). b Nomarski DIC image showing the organization of PCL filaments in these experiments. Dashed circles illustrate the area where DRGs were placed (b–e). f–h High-magnification confocal optical sections showing enhanced green fluorescent protein (EGFP) + MSCs (green) on PCL filaments (f) 72 h after incubation with DRG (thick arrows indicate MSC-EGFP and thin arrows indicate migrated non-MSC on PCL filaments). In (g), neurites were immunolabeled for NF-200 (red) and the merged image reveals neurites growing aligned in close association with MSC (h, arrows). i,j Histograms of quantitative analysis of maximum neurite extension (i) and neurite density on PCL filaments at 500 μm from DRG (j) after 72 h of incubation. Scale bars: a = 400 μm; b–e = 200 μm; f–h = 50 μm. *p < 0.01, **p < 0.001, ***p < 0.0001, ANOVA
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Fig4: Neurite outgrowth from dorsal root ganglia (DRG) explants in the different culture conditions. a–e Confocal microscopy images of E16 rat DRG explants placed on a coverslip (a), on poly-caprolactone (PCL) filaments (c), on PCL filaments + msenshymal stem cells (MSC) (d), and on PCL filaments + MSC + Schwann cells (SC) (e) and cultured for 72 h. Neurites are identified by immunolabeling with NF-200 (green) and cell nuclei with To-Pro (blue). b Nomarski DIC image showing the organization of PCL filaments in these experiments. Dashed circles illustrate the area where DRGs were placed (b–e). f–h High-magnification confocal optical sections showing enhanced green fluorescent protein (EGFP) + MSCs (green) on PCL filaments (f) 72 h after incubation with DRG (thick arrows indicate MSC-EGFP and thin arrows indicate migrated non-MSC on PCL filaments). In (g), neurites were immunolabeled for NF-200 (red) and the merged image reveals neurites growing aligned in close association with MSC (h, arrows). i,j Histograms of quantitative analysis of maximum neurite extension (i) and neurite density on PCL filaments at 500 μm from DRG (j) after 72 h of incubation. Scale bars: a = 400 μm; b–e = 200 μm; f–h = 50 μm. *p < 0.01, **p < 0.001, ***p < 0.0001, ANOVA

Mentions: DRGs were cultured over MSC/filaments to investigate a possible effect on neurite outgrowth. Neurites were immunostained for NF-200, and their maximum length was evaluated in the different culture conditions (Fig. 4). When DRGs were plated on coverslips, we observed a halo of NF-200-positive neurites extending radially from the explant. When DRGs were plated on filaments (Fig. 4b,c) we observed a preferential outgrowth of the neurites following the filaments (Fig. 4c). Most of the neurites were aligned with the filaments, and the maximum distance of neurite growth from the explant was twice that observed in DRGs plated on coverslips (Fig. 4a; p < 0.05, ANOVA). When the filaments were previously incubated with MSC (Fig. 4d) or with MSC + SC (Fig. 4e), the neurites extended over even longer distances compared with the DRG plated on coverslips (fourfold; p < 0.0001, ANOVA) or plated on PCL filaments alone (twofold; p < 0.001, ANOVA). There was no statistical difference between MSC and MSC + SC (Fig. 4i). We also counted the number of neurites at a distance of 500 μm from the explant (Fig. 4j). The largest number of neurites was observed in the presence of filaments plus MSC and SC (Fig. 4j). At higher magnification, we observed that DRG neurons extended neurites over migrated SC (GFP−, thick arrow) on the filaments, and were also in direct contact with the MSC (GFP+, thin arrow) on the filaments (Fig. 4f–h).Fig. 4


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)

Neurite outgrowth from dorsal root ganglia (DRG) explants in the different culture conditions. a–e Confocal microscopy images of E16 rat DRG explants placed on a coverslip (a), on poly-caprolactone (PCL) filaments (c), on PCL filaments + msenshymal stem cells (MSC) (d), and on PCL filaments + MSC + Schwann cells (SC) (e) and cultured for 72 h. Neurites are identified by immunolabeling with NF-200 (green) and cell nuclei with To-Pro (blue). b Nomarski DIC image showing the organization of PCL filaments in these experiments. Dashed circles illustrate the area where DRGs were placed (b–e). f–h High-magnification confocal optical sections showing enhanced green fluorescent protein (EGFP) + MSCs (green) on PCL filaments (f) 72 h after incubation with DRG (thick arrows indicate MSC-EGFP and thin arrows indicate migrated non-MSC on PCL filaments). In (g), neurites were immunolabeled for NF-200 (red) and the merged image reveals neurites growing aligned in close association with MSC (h, arrows). i,j Histograms of quantitative analysis of maximum neurite extension (i) and neurite density on PCL filaments at 500 μm from DRG (j) after 72 h of incubation. Scale bars: a = 400 μm; b–e = 200 μm; f–h = 50 μm. *p < 0.01, **p < 0.001, ***p < 0.0001, ANOVA
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Neurite outgrowth from dorsal root ganglia (DRG) explants in the different culture conditions. a–e Confocal microscopy images of E16 rat DRG explants placed on a coverslip (a), on poly-caprolactone (PCL) filaments (c), on PCL filaments + msenshymal stem cells (MSC) (d), and on PCL filaments + MSC + Schwann cells (SC) (e) and cultured for 72 h. Neurites are identified by immunolabeling with NF-200 (green) and cell nuclei with To-Pro (blue). b Nomarski DIC image showing the organization of PCL filaments in these experiments. Dashed circles illustrate the area where DRGs were placed (b–e). f–h High-magnification confocal optical sections showing enhanced green fluorescent protein (EGFP) + MSCs (green) on PCL filaments (f) 72 h after incubation with DRG (thick arrows indicate MSC-EGFP and thin arrows indicate migrated non-MSC on PCL filaments). In (g), neurites were immunolabeled for NF-200 (red) and the merged image reveals neurites growing aligned in close association with MSC (h, arrows). i,j Histograms of quantitative analysis of maximum neurite extension (i) and neurite density on PCL filaments at 500 μm from DRG (j) after 72 h of incubation. Scale bars: a = 400 μm; b–e = 200 μm; f–h = 50 μm. *p < 0.01, **p < 0.001, ***p < 0.0001, ANOVA
Mentions: DRGs were cultured over MSC/filaments to investigate a possible effect on neurite outgrowth. Neurites were immunostained for NF-200, and their maximum length was evaluated in the different culture conditions (Fig. 4). When DRGs were plated on coverslips, we observed a halo of NF-200-positive neurites extending radially from the explant. When DRGs were plated on filaments (Fig. 4b,c) we observed a preferential outgrowth of the neurites following the filaments (Fig. 4c). Most of the neurites were aligned with the filaments, and the maximum distance of neurite growth from the explant was twice that observed in DRGs plated on coverslips (Fig. 4a; p < 0.05, ANOVA). When the filaments were previously incubated with MSC (Fig. 4d) or with MSC + SC (Fig. 4e), the neurites extended over even longer distances compared with the DRG plated on coverslips (fourfold; p < 0.0001, ANOVA) or plated on PCL filaments alone (twofold; p < 0.001, ANOVA). There was no statistical difference between MSC and MSC + SC (Fig. 4i). We also counted the number of neurites at a distance of 500 μm from the explant (Fig. 4j). The largest number of neurites was observed in the presence of filaments plus MSC and SC (Fig. 4j). At higher magnification, we observed that DRG neurons extended neurites over migrated SC (GFP−, thick arrow) on the filaments, and were also in direct contact with the MSC (GFP+, thin arrow) on the filaments (Fig. 4f–h).Fig. 4

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