although the peripheral nervous system has the ability to regenerate axons following injury , when nerve gaps are larger than 12 cm , bridging strategies are required for repair . in such cases , autologous nerve grafts have been used as the gold standard ( deumens et al . , 2010 ; pfister et al . ,
. however , there are limited numbers of suitable sites for harvesting nerve grafts , because of the adverse effects to donor sites , such as the loss of motor and sensory function .
in order to overcome these issues , scaffold bridging materials that may be synthetic , absorbable or non - absorbable have been studied vigorously . through these studies ,
several criteria were suggested to be necessary for better results , as follows : 1 ) the bridging conduit is necessary to maintain adequate mechanical support of separated nerve ends , in addition to preventing the diffusion of neurotrophic and neurotropic factors secreted by transected stumps ( deumens et al . , 2010 ) ; 2 ) nerve scaffolds should be biocompatible in order to eliminate undesirable influences on neural cells / tissues , such as immune rejection ( avitable et al . , 2001 ) ;
3 ) it should be degradable and/or absorbable in the appropriate duration in vivo ( de ruiter et al . , 2009 ) ; 4 ) it must have sufficient permeability for nutrient and gas exchange ( she et al .
, 2008 ) ; and 5 ) it must have a biomechanical flexibility to allow bending at the graft site , without kinking the transected nerve stumps ( gu et al . , 2011 ) . for longer nerve gap injuries ,
scaffolds with a single hollow lumen represent the basic structure , but this has been refined using various biosynthetic materials in order to mimic the nerve - graft structure and to facilitate axonal re - extension .
the multi - luminal substructure ( de ruiter et al . , 2008b ) and the filament - filled structure ( bunting et al .
; hu et al . , 2008 ) are typical cases ; the former shows no significant benefit , but the latter shows improved nerve regeneration , as compared to the single lumen scaffold .
intraluminal surface coating with extracellular matrix ( ecm ) components , such as laminin , fibronectin and collagen , has been also attempted to promote active axonal re - growth .
first , this was attempted using a single hollow conduit , and several positive effects were obtained ( kauppila et al . , 1993 ;
the use of conduits of biological origin , such as acellular conduits , which have preserved ecm components , but are decellularized and immunosuppressed , was another approach ( frerichs et al .
, 2002 ; hudson et al . , 2004a ) . however , use of this hollow - type conduit shows non - significant effects on nerve regeneration across long gaps ( pfister et al . , 2011 ) .
in contrast , conduits including laminin - coated collagen fibers were able to support the regeneration of long sciatic nerve gaps of over 80 mm in dogs ( matsumoto et al . , 2000 ) .
at this point , the filament - filled conduit with laminin coating is likely to be the best alternative method .
importantly , these efforts have been performed to expect alternative effects of laminin - coated fibers in the conduit , similar to the basal lamina sheet in the individual endoneurium of the healthy nerve fibers .
laminin coating may exert supportive effects for the migration , proliferation and orientation of preserved schwann cells , similarly to formation of bands of bungner , which normally occurs in the case of axonotmesis ( deumens et al .
furthermore , application of growth factors into the conduit is another method to mimic native conditions .
for example , supply of several factors into the conduit , such as nerve growth factor ( ngf ) , brain - derived neurotrophic factor ( bdnf ) , glial cell line - derived neurotrophic factor ( gdnf ) , ciliary neurotrophic factor ( cntf ) , basic fibroblast growth factor ( bfgf ) and insulin - like growth factor-1 ( igf-1 ) , has also been performed , and favorable effects on axonal growth and nerve regeneration have been confirmed ( rich et al . , 1989 ; nachemson et al . , 1990 ; fine et al . , 2002 ;
all of the applications described above were based on the notion of simulating the healthy nerve graft condition . in this
regard , transplantation of schwann cells with the conduit is considered to be the best method as the alternative for nerve grafting .
acellular conduits have thus been used with several cell sources , including schwann cells and/or schwann - like cells induced from cultivated bone morrow stromal cells ( dezawa et al . , 2005 ) , olfactory ensheathing cells ( radtke et al . , 2011 ) and adipose tissue - derived cells ( kingham et al . , 2007 ) , but it is unlikely that these cells would match or exceed the performance of auto nerve grafts .
although schwann cells play a central role in peripheral nerve regeneration , the formation of endoneurium and/or perineurium by endoneurial fibroblasts and perineurial cells is also important because of their protective role in axons with schwann cells and myelin sheath . in particular
, the perineurium plays an important role in preventing the passage of large molecules from the epineurium into perineurial fascicles , which is also known as the  blood - nerve - barrier  system ( weerasuriya and mizisin , 2011 ) .
furthermore , reconstitution of vascular networks is also an inevitable factor in tissue reconstruction . in this
regard , cell sources that can give rise to schwann cells and cells associated with the formation of perineurium / endoneurium and blood vessels are considered to be the best source for peripheral nerve regeneration .
recently , we reported preferential and comprehensive reconstitution of severely damaged sciatic nerve using 7-day cultivated murine skeletal muscle - derived multipotent stem cells ( sk - mscs ; tamaki et al . , 2014 ) .
in this treatment , engrafted donor cells preferentially differentiated into schwann cells and perineurial / endoneurial cells , and formed myelin sheath and perineurium / endoneurium , encircling the regenerated axons .
donor cell - derived perineurium also had tight - junctions , which play a key role in the  blood - nerve - barrier  system ; thus , suggesting functional reconstitution .
stem cells showing suitable multipotency to cover all peripheral nerve support cells in damaged nerve - specific niche have not been reported previously , although cultivated bone morrow stromal cells ( mbcss ; dezawa et al . , 2005 ) , olfactory ensheathing cells ( radtke et al . , 2011 ) and adipose tissue - derived cells ( kingham et al . , 2007 )
we directly compared the contribution to damaged nerve reconstitution among cultivated sk - mscs and bmscs , and freshly isolated cells derived from damaged sciatic nerve ( sndc - d ) ( tamaki et al . ,
the results showed that sk - mscs have a significantly greater engraftment ratio when compared with the other two groups , and the values in the latter two groups were similar .
in addition , there was no definitive formation of peripheral nerve supporting cells or incorporation into blood vessels in bmsc transplantation , whereas sk - mscs and sndc - d showed typical differentiation into all support cells .
in addition , engrafted sk - mscs contributed to increased vascular formation , which is favorable for blood supply and waste product excretion .
facilitation of blood vessel formation in sk - mscs was also confirmed in damaged skeletal muscle ( tamaki et al .
, 2005 ; 2007b ; 2013 ) and urethra ( hoshi et al . , 2008 ) , as well as in and around the bladder wall ( nitta et al . , 2010
expression of key neurotrophic and nerve / vascular growth factor mrnas was also confirmed ; in particular , ngf ( rich et al . , 1989 ) , bdnf ( lewin et al . , 2009 ) , gdnf ( fine et al . , 2002 ) , galectin-1 ( horie et al . , 1999 ) , ninjurin ( araki and milbrandt , 1996 ) , cntf ( dubovy et al . , 2011 ) , lif ( wang et al . , 2009 ) , sox10 ( bremer et al . , 2011 ; britsch et al . , 2001 ; finzsch et al . ,
2010 ) , bfgf ( timmer et al . , 2003 ) and igf-1 ( nachemson et al . , 1990 ) are important for nerve regeneration , while vgef ( ferrara , 2004 ) and hgf ( schroder et al . , 2011 ) are important for blood vessels .
some of these are redundant and/or primarily involved in the recipient nerve regeneration process ( campbell , 2008 ) ; however , sufficient expression of these factors in the damaged / transplantation site may induce paracrine effects on both recipient and donor cells as an adjuvant for nerve regeneration .
using sk - mscs , we examined the therapeutic capacity in a long transected nerve gap on the mouse sciatic nerve via an acellular conduit ( 1.52.0  10 cells / nerve ) , and demonstrated 94% reconstitution of the number of axons , 60% recovery of myelin , and a 9.1-fold increase in blood vessels through the conduit ( tamaki et al . , 2014 ) .
features and immunohistochemical properties of regenerated nerve at 8 weeks after bridging with sk - mscs transplantation are shown in figure 1 .
thick gfp tissues were evident throughout the conduit ( figure 1a ) , and a large number of gfp multi - luminal substructures , such as perineurium / endoneurium , were also apparent in histological sections ( figure 1b and c ) .
importantly , regenerated axons in the conduit were encircled by engrafted gfp perineurium / endoneurium ( figure 1b ) , having myelination ( figure 1c ) , thus suggesting that regenerating axons extended along the endoneurial / perineurial substructures with supporting schwann cells .
the bridging acellular conduit also includes a large number of blood vessels ( figure d ) ; thus , favorable blood supply and waste product excretion are expected .
these relationships between gfp tissues and regenerated axons were confirmed in longitudinal sections ( figure 1e ) , and gfp multi - luminal substructures also possessed the basal lamina sheath ( figure 1f and g ) .
these results clearly indicated that our nerve treatment method using the sk - mscs with acellular conduits meet all of the above criteria , making it useful for nerve regeneration .
in addition , we also demonstrated the greater therapeutic potential of sk - msc treatment as compared to case of healthy nerve grafts using the same mouse model ( tamaki et al . , 2014 ) .
typical structure of engrafted gfp skeletal muscle - derived multipotent stem cells ( sk - mscs ) in acellular bridging conduit at 8 weeks after transplantation .
( a ) macroscopic features of regenerated sciat - ic nerve with engrafted gfp tissues .
( b ) cross - sectional profile of the bridging conduit filled with donor - derived gfp perineurium / endoneu - rium .
( c ) cross - sectional profile of bridging conduit filled with do - nor - derived gfp perineurium / endoneurium .
( d ) same portion of panel ( c ) is stained with cd31 ( vascular endothelial cell marker ) .
gfp perineurium / endoneu - rium show continuously elongated features having axons ( red ) inside .
( f ) longitudinal view of gfp peri - neurium / endoneurium in the conduit , and ( g ) laminin staining ( red ) .
gfp perineurium / en - doneurium also has basal lamina sheets , which are detected by anti - laminin staining .
skeletal muscle is the largest organ in the body , comprising approximately 4050% of total body mass , and presumably allowing donor cells to be obtained with relative ease and safety .
therefore , sk - mscs represent a novel / suitable alternative cell source for healthy nerve autografts .
skeletal muscle - derived stem cells have been used by many researchers for various purposes ; however , our isolation method of sk - mscs differs from others ( tamaki et al . , 2002 ; 2010 ) , and
this is potentially crucial to our results after transplantation ( tamaki et al . , 2005 , 2007a , b ) .
in future research , the potential of human skeletal muscle - derived cells needs to be clarified .
we are currently investigating the optimal isolation , fractionation and expansion culture conditions for human cells in order to maximize the nerve regeneration potential .
in vivo evidence of the differentiation and reconstruction capacities of human sk - mscs is also important .
we used the acellular conduit from the wild - type mice esophageal submucous membrane ( mainly longitudinal muscle layer ) separated after 3 days of 70% ethanol dehydration . in the human case , use of an appropriate size vein may be a candidate cellular or acellular conduit .
however , from the viewpoint of minimizing invasiveness , biosynthetic tubes that are a good match for sk - mscs treatment need to be identified .
application of sk - msc treatment for the total regeneration of multi - branched nerve transections may also be useful in the case of surgical nerve damage .
 long gap peripheral nerve injuries usually reulting in life - changing problems for patients . skeletal muscle derived - multipotent stem cells 
 ( sk - mscs ) can differentiate into schwann and perineurial / endoneurial cells , vascular relating pericytes , and endothelial and smooth muscle cells in the damaged peripheral nerve niche . 
 application of the sk - mscs in the bridging conduit for repairing long nerve gap injury resulted favorable axonal regeneration , which showing superior effects than gold standard therapy -- healthy nerve autograft . 
 this means that it does not need to sacrifice of healthy nerves or loss of related functions for repairing peripheral nerve injury . 
Introduction
Development of nerve bridging conduit
Combination with cell transplantation
Comprehensive regeneration with all nerve support cells/tissues
Future perspectives