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New fibrin conduit for peripheral nerve repair
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
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2009 (English)In: Journal of reconstructive microsurgery, ISSN 0743-684X, E-ISSN 1098-8947, Vol. 25, no 1, 27-33 p.Article in journal (Refereed) Published
Abstract [en]

An ideal substitute to treat a nerve gap has not been found. Initially, silicone conduits were employed. Later, conduits were fabricated from collagen or polyesters carbonates. More recently, it has been shown that a bioresorbable material, poly-3-hydroxybutyrate (PHB), can enhance nerve repair. The present investigation shows the use of fibrin as a conduit to guide nerve regeneration and bridge nerve defects. In this study we prepared and investigated a novel nerve conduit made from fibrin glue. Using a rodent sciatic nerve injury model (10-mm gap), we compared the extent of nerve regeneration through the new fibrin conduits versus established PHB conduits. After 2 and 4 weeks, conduits containing proximal and distal stumps were harvested. We evaluated the initial axon and Schwann cell stimulation using immunohistochemistry. The conduits presented full tissue integration and were completely intact. Axons crossed the gap after 1 month. Immunohistochemistry using the axonal marker PGP 9.5 showed a superior nerve regeneration distance in the fibrin conduit compared with PHB (4.1 mm versus 1.9 mm). Schwann cell intrusion (S100 staining) was similarly enhanced in the fibrin conduits, both from the proximal (4.2 mm versus 2.1 mm) and distal ends (3.2 mm versus 1.7 mm). These findings suggest an advantage of the new fibrin conduit for the important initial phase of peripheral nerve regeneration. The use of fibrin glue as a conduit is a step toward a usable graft to bridge peripheral nerve lesions. This might be clinically interesting, given the widespread acceptance of fibrin glue among the surgical community.

Place, publisher, year, edition, pages
2009. Vol. 25, no 1, 27-33 p.
Keyword [en]
Peripheral nerve regeneration, nerve gap, fibrin glue, Tisseel
National Category
Surgery
Research subject
Surgery
Identifiers
URN: urn:nbn:se:umu:diva-19232DOI: 10.1055/s-0028-1090619PubMedID: 18925549OAI: oai:DiVA.org:umu-19232DiVA: diva2:201603
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Nerve gap repair by the use of artificial conduits and cultured cells
Open this publication in new window or tab >>Nerve gap repair by the use of artificial conduits and cultured cells
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Peripheral nerve injuries are often associated with loss of nerve tissue and require autologous nerve grafts to provide a physical substrate for axonal growth. This thesis investigates the use of fibrin as both a tubular conduit to guide nerve regeneration and also as a matrix material to suspend various regenerative cell types within/on poly-3-hydroxybutyrate (PHB) nerve conduits. Adipose derived stem cells (ASC) are found in abundant quantities. In this thesis the ability of rat ASC to differentiate into Schwann cells was determined and a preliminary study of the neurotrophic potential of human ASC was also investigated.

Rat sciatic nerve axotomy was performed proximally in the thigh to create a 10-mm gap between the nerve stumps and the gap was bridged using the various conduits.  At early time points the nerve grafts were harvested and investigated for axonal and Schwann cell markers.  After 16 weeks the regenerative response from sensory and motor neurons was also evaluated following retrograde labelling with Fast Blue fluorescent tracer. Stem cells were treated with a mixture of glial growth factors and after 2 weeks in vitro the expression of Schwann cell markers was analysed by immunocytochemistry and Western blotting.  ASC were cocultured with the NG108-15 neuronal cell line to determine their ability to promote neurite outgrowth.  Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  RT-PCR was used to investigate the expression of neurotrophic factors.

Immunohistochemistry showed a superior nerve regeneration distance in the fibrin conduit compared with PHB. The fibrin conduit promoted regeneration of 60% of sensory neurones and 52% of motor neurones when compared with an autograft group at 16 weeks. The total number of myelinated axons in the distal nerve stump in the fibrin-conduit group reached 86% of the graft and the weight of gastrocnemius and soleus muscles recovered to 82% and 89% of the controls, respectively. In vitro studies showed that rat ASC could be differentiated to a Schwann cell phenotype. These treated cells enhanced both the number of NG108-15 cells expressing neurites and neurite length. In the same coculture model system, human superficial fat layer ASC induced significantly enhanced neurite outgrowth when compared with the deep layer fat cells. RT-PCR analysis showed ASC isolated from both layers expressed neurotrophic factors.

These results indicate that a tubular fibrin conduit can be used to promote neuronal regeneration following peripheral nerve injury. There was also a beneficial effect of using a fibrin matrix to seed cells within/on PHB conduits which should ultimately lead to improved functional recovery following nerve injury.  There might also be an advantage to use a simple strip of PHB rather than a conventional tube-like structure implying that single fascicle nerve grafting could be advantageous for nerve repair.  The results of in vitro experiments indicate adipose tissue contains a pool of regenerative stem cells which can be differentiated to a Schwann cell phenotype and given that human ASC express a range of neurotrophic factors they are likely to be of clinical benefit for treatment of peripheral nerve injuries.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2010. 54 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1369
Keyword
adipose stem cells, cell matrix, fibrin, nerve conduit, nerve gap
National Category
Surgery
Research subject
Surgery
Identifiers
urn:nbn:se:umu:diva-35582 (URN)978-91-7459-038-8 (ISBN)
Public defence
2010-09-20, BiA201, Biologihuset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2010-08-30 Created: 2010-08-24 Last updated: 2010-08-30Bibliographically approved
2. Biosynthetic conduits and cell transplantation for neural repair
Open this publication in new window or tab >>Biosynthetic conduits and cell transplantation for neural repair
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Spinal cord injury results in complete failure of the central neurons to regenerate and is associated with cyst formation and enlargement of the trauma zone. In contrast to the spinal cord, axons in the injured peripheral nerve have the capacity to undergo some spontaneous regeneration. However, significant post-traumatic loss of nervous tissue causing long nerve gap is one of the main reasons for the poor restoration of function following microsurgical repair of injured nerves. The present thesis investigates the effects of biodegradable conduits prepared from fibrin glue and poly-beta-hydroxybutyrate (PHB) in combination with cultured Schwann cells, mesenchymal stem cells and extracellular matrix molecules on regeneration after spinal cord and peripheral nerve injury in adult rats.

At 4-8 weeks after transplantation into the injured spinal cord, the PHB conduit was well integrated into the cavity but regenerating axons were found mainly outside the PHB. When suspension of BrdU-labeled Schwann cells was added to the PHB, regenerating axons filled the conduit and became associated with the implanted cells. Modification of the PHB surface with extracellular matrix molecules significantly increased Schwann cell attachment and proliferation but did not alter axonal regeneration. To improve the labeling technique of the transplanted cells, the efficacy of fluorescent cell tracers Fast Blue, PKH26, Vibrant DiO and Cell Tracker™ Green CMFDA was evaluated. All tested dyes produced very efficient initial labeling of olfactory ensheathing glial cells in culture. The number of Fast Blue-labeled cells remained largely unchanged during the first 4 weeks whereas the number of cells labeled with other tracers was significantly reduced after 2 weeks. After transplantation into the spinal cord, Fast Blue-labeled glial cells survived for 8 weeks but demonstrated very limited migration from the injection sites. Additional immunostaining with glial and neuronal markers demonstrated transfer of the dye from the transplanted cells to the host tissue.

In a sciatic nerve injury model, the extent of axonal regeneration through a 10mm gap bridged with tubular PHB conduit was compared with a fibrin glue conduit. At 2 weeks after injury, the fibrin conduit supported similar axonal regeneration and migration of the host Schwann cells compared with the PHB conduit augmented with a diluted fibrin matrix and GFP-labeled Schwann cells or mesenchymal stem cells. The long-term regenerative response was evaluated using retrograde neuronal labeling. The fibrin glue conduit promoted regeneration of 60% of sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed more chaotic appearance and the mean area and diameter of fast type fibers were significantly worse than those of the corresponding 10mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size.

In summary, these results show that a PHB conduit promotes attachment, proliferation and survival of adult Schwann cells and supports marked axonal growth after transplantation into the injured spinal cord. The data suggest an advantage of the fibrin conduit for the important initial phase of peripheral nerve regeneration and demonstrate potential of the conduit to promote long-term neuronal regeneration and muscle recovery.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2011. 61 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1408
Keyword
Spinal cord injury; Peripheral nerve injury; Nerve graft; Biosynthetic conduit; Nerve tissue engineering; Neural prosthesis
National Category
Neurosciences
Research subject
Neurology; Neurosurgery
Identifiers
urn:nbn:se:umu:diva-42440 (URN)978-91-7459-160-6 (ISBN)
Public defence
2011-05-19, BiA201, Biologihuset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2011-04-28 Created: 2011-04-07 Last updated: 2011-04-28Bibliographically approved

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