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Biosynthetic conduits and cell transplantation for neural repair
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Anatomy)
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 [en]
Spinal cord injury; Peripheral nerve injury; Nerve graft; Biosynthetic conduit; Nerve tissue engineering; Neural prosthesis
National Category
Neurosciences
Research subject
Neurology; Neurosurgery
Identifiers
URN: urn:nbn:se:umu:diva-42440ISBN: 978-91-7459-160-6OAI: oai:DiVA.org:umu-42440DiVA: diva2:409263
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
List of papers
1. Biodegradable poly-beta-hydroxybutyrate scaffold seeded with Schwann cells to promote spinal cord repair
Open this publication in new window or tab >>Biodegradable poly-beta-hydroxybutyrate scaffold seeded with Schwann cells to promote spinal cord repair
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2008 (English)In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 29, no 9, 1198-1206 p.Article in journal (Refereed) Published
Abstract [en]

Cavity formation is an important obstacle impeding regeneration after spinal cord injury and bridging strategies are essential to provide physical substrate allowing axons to grow across the lesion site. In this study we evaluated effects of biodegradable tubular conduit made from poly-beta-hydroxybutyrate (PHB) scaffold with predominantly unidirectional fiber orientation and supplemented with cultured adult Schwann cells on axonal regeneration after cervical spinal cord injury in adult rats. After transplantation into the injured spinal cord, plain PHB conduit was well-integrated into posttraumatic cavity and induced modest astroglial reaction. Regenerating axons were found mainly outside the PHB with only single fibers crossing the host-graft interface. No host Schwann cells migrated into the graft. In contrast, when suspension of adult Schwann cells was added to the PHB during transplantation, neurofilament-positive axons filled the conduit and became associated with the implanted cells. Although rubrospinal fibers did not enter the PHB, numerous raphaespinal and CGRP-positive axons were found within the conduit. Modification of PHB surface with fibronectin, laminin or collagen significantly increased Schwann cell attachment and proliferation in vitro. However, transplantation of PHB conduit pre-coated with fibronectin and seeded with Schwann cells did not alter axonal growth response. The results demonstrate that a PHB scaffold promotes attachment, proliferation and survival of adult Schwann cells and supports marked axonal regeneration within the graft.

Keyword
Animal model; Spinal cord injury; Nerve tissue engineering; Neural prosthesis; Transplantation
Identifiers
urn:nbn:se:umu:diva-8116 (URN)10.1016/j.biomaterials.2007.11.033 (DOI)18083223 (PubMedID)
Available from: 2008-01-21 Created: 2008-01-21 Last updated: 2011-04-28Bibliographically approved
2. Labeling of olfactory ensheathing glial cells with fluorescent tracers for neurotransplantation
Open this publication in new window or tab >>Labeling of olfactory ensheathing glial cells with fluorescent tracers for neurotransplantation
2010 (English)In: Brain Research Bulletin, ISSN 0361-9230, E-ISSN 1873-2747, Vol. 81, no 1, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

Development of cell-based treatment strategies for repair of the injured nervous system requires cell tracing techniques to follow the fate of transplanted cells and their interaction with the host tissue. The present study investigates the efficacy of fluorescent cell tracers Fast Blue, PKH26, DiO and CMFDA for long-term labeling of olfactory ensheathing glial cells (OEC) in culture and following transplantation into the rat spinal cord. All tested dyes produced very efficient initial labeling of p75-positive OEC in culture. The number of Fast Blue-positive cells remained largely unchanged during the first 4 weeks but only about 21% of the cells retained tracer 6 weeks after labeling. In contrast, the number of cells labeled with PKH26 and DiO was reduced to 51-55% after 2 weeks in culture and reached 8-12% after 4-6 weeks. CMFDA had completely disappeared from the cells 2 weeks after labeling. AlamarBlue assay showed that among four tested tracers only CMFDA reduced proliferation rate of the OEC. After transplantation into spinal cord, Fast Blue-labeled OEC survived for at least 8 weeks but demonstrated very limited migration from the injection sites. Additional immunostaining with glial and neuronal markers revealed signs of dye leakage from the transplanted cells resulted in weak labeling of microglia and spinal neurons. The results show that Fast Blue is an efficient cell marker for cultured OEC. However, transfer of the dye from the transplanted cells to the host tissue should be considered and correctly interpreted.

Keyword
Olfactory ensheathing cells; Spinal cord; Transplantation; Fluorescent tracers; Long-term labeling
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-41359 (URN)10.1016/j.brainresbull.2009.10.005 (DOI)000274101500020 ()19828127 (PubMedID)
Available from: 2011-03-23 Created: 2011-03-23 Last updated: 2013-10-24Bibliographically approved
3. Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit
Open this publication in new window or tab >>Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit
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2008 (English)In: Journal of plastic, reconstructive and aesthetic surgery, ISSN 1878-0539, Vol. 61, no 6, 669-675 p.Article in journal (Refereed) Published
Abstract [en]

Peripheral nerve injury presents with specific problems of neuronal reconstructions, and from a clinical viewpoint a tissue engineering approach would facilitate the process of repair and regeneration. We have previously used artificial nerve conduits made from bioresorbable poly-3-hydroxybutyrate (PHB) in order to refine the ways in which peripheral nerves are repaired and reconnected to the target muscles and skin. The addition of Schwann cells (SC) or differentiated mesenchymal stem cells (dMSC) to the conduits enhances regeneration. In this study, we have used a matrix based on fibrin (Tisseel) to fill optimally the nerve-conduits with cells. In vitro analysis showed that both SC and MSC adhered significantly better to PHB in the presence of fibrin and cells continued to maintain their differentiated state. Cells were more optimally distributed throughout the conduit when seeded in fibrin than by delivery in growth medium alone. Transplantation of the nerve conduits in vivo showed that cells in combination with fibrin matrix significantly increased nerve regeneration distance (using PGP9.5 and S100 distal and proximal immunohistochemistry) when compared with empty PHB conduits. This study shows the beneficial combinatory effect of an optimised matrix, cells and conduit material as a step towards bridging nerve gaps which should ultimately lead to improved functional recovery following nerve injury.

Keyword
Nerve gap; PHB conduit; Modified mesenchymal stem cells; Tisseel®
National Category
Surgery
Research subject
Human Anatomy
Identifiers
urn:nbn:se:umu:diva-26613 (URN)10.1016/j.bjps.2007.12.015 (DOI)18218346 (PubMedID)
Available from: 2009-10-16 Created: 2009-10-16 Last updated: 2011-04-28Bibliographically approved
4. New fibrin conduit for peripheral nerve repair
Open this publication in new window or tab >>New fibrin conduit for peripheral nerve repair
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2009 (English)In: Journal of reconstructive microsurgery, ISSN 0743-684X, 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.

Keyword
Peripheral nerve regeneration, nerve gap, fibrin glue, Tisseel
National Category
Surgery
Research subject
Surgery
Identifiers
urn:nbn:se:umu:diva-19232 (URN)10.1055/s-0028-1090619 (DOI)18925549 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2011-04-28Bibliographically approved
5. Biodegradable fibrin conduit promotes long-term regeneration after peripheral nerve injury in adult rats
Open this publication in new window or tab >>Biodegradable fibrin conduit promotes long-term regeneration after peripheral nerve injury in adult rats
2010 (English)In: Journal of Plastic, Reconstructive & Aesthetic Surgery, ISSN 1748-6815, Vol. 63, no 11, 1893-1899 p.Article in journal (Refereed) Published
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. Biosynthetic neural conduits could be an alternative treatment strategy in such injuries. The present study investigates the long-term effects of a tubular fibrin conduit on neuronal regeneration, axonal sprouting and recovery of muscle weight following peripheral nerve injury and repair in adult rats. Sciatic axotomy was performed proximally in the thigh to create a 10-mm gap between the nerve stumps. The injury gap was bridged by using a 14-mm-long fibrin glue conduit, entubulating 2mm of the nerve stump at each end. A reversed autologous nerve graft was used as a control. The regenerative response from sensory and motor neurones was evaluated following retrograde labelling with Fast Blue fluorescent tracer. In control experiments, at 16 weeks following peripheral nerve grafting, 5184 (+/-574 standard error of mean (SEM)) sensory dorsal root ganglion neurones and 1001 (+/-37 SEM) spinal motor neurones regenerated across the distal nerve-graft interface. The fibrin conduit promoted regeneration of 60% of sensory neurones and 52% of motor neurones when compared to the control group. The total number of myelinated axons in the distal nerve stump in the fibrin-conduit group reached 86% of the control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89% of the controls, respectively. The present results suggest that a tubular fibrin conduit can be used to promote neuronal regeneration following peripheral nerve injury.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
Biosynthetic conduit, Dorsal root ganglion, Fluorescent tracer, Motor neurone, Nerve injury, Nerve graft
National Category
Neurosciences
Research subject
Surgery
Identifiers
urn:nbn:se:umu:diva-35218 (URN)10.1016/j.bjps.2009.11.024 (DOI)000282944600034 ()20005193 (PubMedID)
Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2013-10-24Bibliographically approved
6. Muscle recovery after repair of short and long peripheral nerve gaps using fibrin conduits
Open this publication in new window or tab >>Muscle recovery after repair of short and long peripheral nerve gaps using fibrin conduits
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2011 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 500, no 1, 41-46 p.Article in journal (Refereed) Published
Abstract [en]

Peripheral nerve injuries with loss of nervous tissue are a significant clinical problem and are currently treated using autologous nerve transplants. To avoid the need for donor nerve, which results in additional morbidity such as loss of sensation and scarring, alternative bridging methods have been sought. Recently we showed that an artificial nerve conduit moulded from fibrin glue is biocompatible to nerve regeneration. In this present study, we have used the fibrin conduit or a nerve graft to bridge either a 10 mm or 20 mm sciatic nerve gap and analyzed the muscle recovery in adult rats after 16 weeks. The gastrocnemius muscle weights of the operated side were similar for both gap sizes when treated with nerve graft. In contrast, muscle weight was 48.32 ± 4.96% of the contra-lateral side for the 10 mm gap repaired with fibrin conduit but only 25.20 ± 2.50% for the 20 mm gap repaired with fibrin conduit. The morphology of the muscles in the nerve graft groups showed an intact, ordered structure, with the muscle fibers grouped in fascicles whereas the 20 mm nerve gap fibrin group had a more chaotic appearance. The mean area and diameter of fast type fibers in the 20 mm gap repaired with fibrin conduits were significantly (P < 0.01) worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nervegraft showed similar fiber size. Furthermore, the 10 mm gaps repaired with either nerve graft or fibrin conduit showed similar muscle fiber size. These results indicate that the fibrin conduit can effectively treat short nerve gaps but further modification such as the inclusion of regenerative cells may be required to attain the outcomes of nerve graft for long gaps.

Place, publisher, year, edition, pages
Amsterdam: , 2011
Keyword
autograft; biosynthetic conduit; muscle fiber; nerve injury; regeneration
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-42426 (URN)10.1016/j.neulet.2011.06.002 (DOI)
Available from: 2011-04-07 Created: 2011-04-07 Last updated: 2016-02-22Bibliographically approved

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