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Nerve gap repair by the use of artificial conduits and cultured cells
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery.
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 [en]
adipose stem cells, cell matrix, fibrin, nerve conduit, nerve gap
National Category
Surgery
Research subject
Surgery
Identifiers
URN: urn:nbn:se:umu:diva-35582ISBN: 978-91-7459-038-8 (print)OAI: oai:DiVA.org:umu-35582DiVA: diva2:345223
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
List of papers
1. 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, 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.

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: 2017-12-13Bibliographically approved
2. 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, E-ISSN 1532-1959, 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: 2017-12-12Bibliographically 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. Schwann cell strip for peripheral nerve repair
Open this publication in new window or tab >>Schwann cell strip for peripheral nerve repair
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2008 (English)In: Journal of Hand Surgery - British and European Volume, ISSN 0266-7681, E-ISSN 1532-2211, Vol. 33, no 5, 587-594 p.Article in journal (Refereed) Published
Abstract [en]

Many strategies have been investigated to provide an ideal substitute to treat a nerve gap injury. Initially, silicone conduits were used and more recently conduits fabricated from natural materials such as poly-3-hydroxybutyrate (PHB) showed good results but still have their limitations. Surgically, a new concept optimising harvested autologous nerve graft has been introduced as the single fascicle method. It has been shown that a single fascicle repair of nerve grafting is successful. We investigated a new approach using a PHB strip seeded with Schwann cells to mimic a small nerve fascicle. Schwann cells were attached to the PHB strip using diluted fibrin glue and used to bridge a 10-mm sciatic nerve gap in rats. Comparison was made with a group using conventional PHB conduit tubes filled with Schwann cells and fibrin glue. After 2 weeks, the nerve samples were harvested and investigated for axonal and Schwann cell markers. PGP9.5 immunohistochemistry showed a superior nerve regeneration distance in the PHB strip group versus the PHB tube group (> 10 mm, crossed versus 3.17+/- 0.32 mm respectively, P<0.05) as well as superior Schwann cell intrusion (S100 staining) from proximal (> 10 mm, crossed versus 3.40+/- 0.36 mm, P<0.01) and distal (> 10 mm, crossed versus 2.91+/- 0.31 mm, P<0.001) ends. These findings suggest a significant advantage of a strip in rapidly connecting a nerve gap lesion and imply that single fascicle nerve grafting is advantageous for nerve repair in rats.

National Category
Surgery
Research subject
handkirurgi
Identifiers
urn:nbn:se:umu:diva-19241 (URN)10.1177/1753193408090755 (DOI)18977829 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2017-12-13Bibliographically approved
5. Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro.
Open this publication in new window or tab >>Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro.
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2007 (English)In: Exp Neurol, ISSN 0014-4886, Vol. 207, no 2, 267-74 p.Article in journal (Refereed) Published
Abstract [en]

Experimentally, peripheral nerve repair can be enhanced by Schwann cell transplantation but the clinical application is limited by donor site morbidity and the inability to generate a sufficient number of cells quickly. We have investigated whether adult stem cells, isolated from adipose tissue, can be differentiated into functional Schwann cells. Rat visceral fat was enzymatically digested to yield rapidly proliferating fibroblast-like cells, a proportion of which expressed the mesenchymal stem cell marker, stro-1, and nestin, a neural progenitor protein. Cells treated with a mixture of glial growth factors (GGF-2, bFGF, PDGF and forskolin) adopted a spindle-like morphology similar to Schwann cells. Immunocytochemical staining and western blotting indicated that the treated cells expressed the glial markers, GFAP, S100 and p75, indicative of differentiation. When co-cultured with NG108-15 motor neuron-like cells, the differentiated stem cells enhanced the number of NG108-15 cells expressing neurites, the number of neurites per cell and the mean length of the longest neurite extended. Schwann cells evoked a similar response whilst undifferentiated stem cells had no effect. These results indicate adipose tissue contains a pool of regenerative stem cells which can be differentiated to a Schwann cell phenotype and may be of benefit for treatment of peripheral nerve injuries.

Keyword
Adipose Tissue/*cytology, Adult Stem Cells/drug effects/*physiology, Animals, Cell Count, Cell Differentiation/drug effects/*physiology, Cell Proliferation, Coculture Techniques/methods, Glial Fibrillary Acidic Protein/metabolism, Myelin Proteins/metabolism, Nerve Tissue Proteins/metabolism, Neuregulin-1/pharmacology, Neurites/drug effects/*physiology, Neuroblastoma/pathology, Neurons/*cytology, Rats, Rats; Sprague-Dawley, Schwann Cells/*physiology, Time Factors
Identifiers
urn:nbn:se:umu:diva-8120 (URN)10.1016/j.expneurol.2007.06.029 (DOI)17761164 (PubMedID)
Available from: 2008-01-21 Created: 2008-01-21 Last updated: 2016-02-22Bibliographically approved
6. Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat
Open this publication in new window or tab >>Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat
(English)Manuscript (preprint) (Other academic)
Abstract [en]

New approaches to the clinical treatment of traumatic nerve injuries may one day utilize stem cells to enhance nerve regeneration.  Adipose derived stem cells (ASC) are found in abundant quantities and can be harvested by minimally invasive procedures which should facilitate their use in such regenerative applications.  In this study, we have analyzed the properties of human ASC isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  Cells from the superficial layer proliferated significantly faster than those from the deep layer. Both in the deep and superficial layers, ASC expressed the pluripotent stem cell markers oct4 and nanog and also the stro-1 cell surface antigen.  Superficial layer ASC induced significantly enhanced neurite outgrowth from NG108-15 motor neuron like cells when compared with the deep layer cells.  However, RT-PCR analysis showed that ASC isolated from both layers expressed similar levels of the neurotrophic factors NGF, BDNF, GDNF and NT-3.  These results indicate that human ASC have promising potential for the treatment of traumatic nerve injuries and that superficial layer ASC might represent the more optimal cell type for such applications.

National Category
Surgery
Research subject
Surgery
Identifiers
urn:nbn:se:umu:diva-35583 (URN)
Available from: 2010-08-24 Created: 2010-08-24 Last updated: 2010-08-30Bibliographically approved

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