Advertisement

Evaluation of the Induced Membrane for Neurotrophic Factors

Published:December 02, 2021DOI:https://doi.org/10.1016/j.jhsa.2021.08.023

      Purpose

      Despite gaining popularity as a bridge for small and moderate nerve gaps, an acellular nerve allograft (ANA) lacks many of the neurotrophic characteristics of a nerve autograft. Pseudomembranes induced to form around temporary skeletal spacers are rich in growth factors. Induced membranes may have beneficial neurotrophic factors which could support ANA.

      Methods

      Twenty-two male Sprague-Dawley rats underwent resection of 2 cm of the sciatic nerve. A silicone rod was inset in the defect of 11 experimental rats, and marking sutures only were placed in the nerve stumps of the remaining 11 control rats. After allowing 4 weeks for tissue maturation, tissue samples harvested from the induced membrane (experimental group) and the tissue bed (control group) were analyzed using Luminex multiplex assay to quantify differences in detectable levels of the following neurotrophic factors: nerve growth factor, glial-derived nerve factor, vascular endothelial growth factor, and transforming growth factor ß (TGF-ß) 1, 2, and 3, interleukin-1ß, and monocyte chemoattractant protein 1.

      Results

      No difference was detected between the control and experimental groups in levels of vascular endothelial growth factor. Higher levels of TGF-ß1, TGF-ß2, TGF-ß3, glial-derived nerve factor, nerve growth factor, monocyte chemoattractant protein 1, and interleukin-1ß were detected in the experimental group.

      Conclusions

      In the setting of peripheral nerve injury, an induced membrane has higher levels of several neurotrophic factors that may support nerve regeneration compared to wound bed cicatrix.

      Clinical relevance

      This investigation provides impetus for further study examining the utility of using a staged induced membrane technique in conjunction with delayed nerve grafting in reconstruction of some peripheral nerve defects.

      Key words

      Segmental peripheral nerve injuries cause significant morbidity and offer unique reconstructive challenges. Current repair methods involve the use of an allograft or autograft. Acellular nerve allograft (ANA) is an “off the shelf” bridging tool for overcoming small and medium gaps in peripheral nerve surgery. With the same 3-dimensional architecture as a nerve autograft, ANA provides an internal scaffold to support and guide axonal regeneration without donor site morbidity. However, in longer length grafts, unsatisfactory axonal ingrowth is generally attributed to degradation of the neurotrophic environment within the ANA.
      • Saheb-Al-Zamani M.
      • Yan Y.
      • Farber S.J.
      • et al.
      Limited regeneration in long acellular nerve allografts is associated with increased Schwann cell senescence.
      Many strategies have been pursued to improve the clinical effectiveness of ANA. Based on normal physiology, the number of possible therapeutic targets is extensive. Following peripheral nerve transection, through Wallerian degeneration, Schwann cells dedifferentiate, remove myelin debris, and secrete trophic factors that recruit macrophages and promote axon regeneration.
      • Chen P.
      • Piao X.
      • Bonaldo P.
      Role of macrophages in Wallerian degeneration and axonal regeneration after peripheral nerve injury.
      Macrophages in turn also remove debris in the distal stump and release factors promoting axon regeneration.
      • Chen P.
      • Piao X.
      • Bonaldo P.
      Role of macrophages in Wallerian degeneration and axonal regeneration after peripheral nerve injury.
      Dedifferentiated Schwann cells recruit macrophages with secretion of interleukin-1ß (IL-1ß), chemokine (C-C motif) ligand 2 (CCL2) (also known as monocyte chemoattractant protein 1 [MCP-1]), and transforming growth factor ß (TGF-ß), among other factors. Recruited macrophages also express IL-1ß, TGF-ß, MCP-1, and vascular endothelial growth factor (VEGF).
      • Atkins S.
      • Smith K.G.
      • Loescher A.R.
      • Boissonade F.M.
      • Ferguson M.W.
      • Robinson P.P.
      The effect of antibodies to TGF-beta1 and TGF-beta2 at a site of sciatic nerve repair.
      • Einheber S.
      • Hannocks M.J.
      • Metz C.N.
      • Rifkin D.B.
      • Salzer J.L.
      Transforming growth factor-beta 1 regulates axon/Schwann cell interactions.
      • Taskinen H.S.
      • Ruohonen S.
      • Jagodic M.
      • Khademi M.
      • Olsson T.
      • Roytta M.
      Distinct expression of TGF-beta1 mRNA in the endo- and epineurium after nerve injury.
      • Gordon T.
      • Sulaiman O.
      • Boyd J.G.
      Experimental strategies to promote functional recovery after peripheral nerve injuries.
      • Perrin F.E.
      • Lacroix S.
      • Aviles-Trigueros M.
      • David S.
      Involvement of monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha and interleukin-1 beta in Wallerian degeneration.
      • Shamash S.
      • Reichert F.
      • Rotshenker S.
      The cytokine network of Wallerian degeneration: tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-1 beta.
      • Tofaris G.K.
      • Patterson P.H.
      • Jessen K.R.
      • Mirsky R.
      Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF.
      • Banner L.R.
      • Patterson P.H.
      Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia.
      • Sugiura S.
      • Lahav R.
      • Han J.
      • et al.
      Leukaemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro.
      • Kurek J.B.
      • Austin L.
      • Cheema S.S.
      • Bartlett P.F.
      • Murphy M.
      Up-regulation of leukaemia inhibitory factor and interleukin-6 in transected sciatic nerve and muscle following denervation.
      • Chen P.
      • Cescon M.
      • Megighian A.
      • Bonaldo P.
      Collagen VI regulates peripheral nerve myelination and function.
      • Mokarram N.
      • Merchant A.
      • Mukhatyar V.
      • Patel G.
      • Bellamkonda R.V.
      Effect of modulating macrophage phenotype on peripheral nerve repair.
      • Chen P.
      • Cescon M.
      • Zuccolotto G.
      • et al.
      Collagen VI regulates peripheral nerve regeneration by modulating macrophage recruitment and polarization.
      Axonal regeneration is dependent on, and contiguous with, the process of Wallerian degeneration and is associated with several additional neurotrophic factors, such as nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF), also secreted by Schwann cells and macrophages.
      • Zhang J.Y.
      • Luo X.G.
      • Xian C.J.
      • Liu Z.H.
      • Zhou X.F.
      Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents.
      • Funakoshi H.
      • Frisen J.
      • Barbany G.
      • et al.
      Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve.
      • Boyd J.G.
      • Gordon T.
      Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo.
      • Huang E.J.
      • Reichardt L.F.
      Neurotrophins: roles in neuronal development and function.
      • Li R.
      • Wu J.
      • Lin Z.
      • et al.
      Single injection of a novel nerve growth factor coacervate improves structural and functional regeneration after sciatic nerve injury in adult rats.
      • Takemura Y.
      • Imai S.
      • Kojima H.
      • Katagi M.
      • Yamakawa I.
      • Kasahara T.
      • et al.
      Brain-derived neurotrophic factor from bone marrow-derived cells promotes post-injury repair of peripheral nerve.
      • Yan Q.
      • Matheson C.
      • Lopez O.T.
      In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons.
      • Ghayemi N.
      • Haghighat A.
      • Amini K.
      • Mohammadi R.
      Functional effect of local administration of glial derived neurotrophic factor combined with inside-out artery graft on sciatic nerve regeneration in rat.
      The induced membrane technique described by Masquelet et al
      • Masquelet A.C.
      • Fitoussi F.
      • Begue T.
      • Muller G.P.
      Reconstruction of the long bones by the induced membrane and spongy autograft.
      is an accepted, reproducible, staged method of spanning osseous defects.
      • Morelli I.
      • Drago L.
      • George D.A.
      • Gallazzi E.
      • Scarponi S.
      • Romano C.L.
      Masquelet technique: myth or reality? A systematic review and meta-analysis.
      The success of this technique
      • Morelli I.
      • Drago L.
      • George D.A.
      • Gallazzi E.
      • Scarponi S.
      • Romano C.L.
      Masquelet technique: myth or reality? A systematic review and meta-analysis.
      has been attributed, at least in part, to the high concentration of growth factors such as VEGF and TGF-ß that have been identified within the membrane at 4 weeks.
      • Wang X.
      • Wei F.
      • Luo F.
      • Huang K.
      • Xie Z.
      Induction of granulation tissue for the secretion of growth factors and the promotion of bone defect repair.
      ,
      • Gruber H.E.
      • Ode G.
      • Hoelscher G.
      • Ingram J.
      • Bethea S.
      • Bosse M.J.
      Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects.
      Based on the well-established application in tendon surgery, Zadegan et al
      • Zadegan S.A.
      • Firouzi M.
      • Nabian M.H.
      • Zanjani L.O.
      • Ashtiani A.M.
      • Kamrani R.S.
      Two-stage nerve graft in severe scar: a time-course study in a rat model.
      ,
      • Zadegan S.A.
      • Firouzi M.
      • Nabian M.H.
      • Zanjani L.O.
      • Kamrani R.S.
      Two-stage nerve graft using a silicone tube.
      introduced the concept of 2-stage nerve reconstruction using a silastic rod to induce a vascularized and scar-free pseudomembrane tunnel for delayed nerve grafting. Histologically, increased vascularity was noted in rats treated with this 2-stage technique compared with delayed grafting in an induced scar bed model, though their evaluation did not assess either growth factor levels or axon histomorphology. Brief delays in nerve grafting may be clinically relevant and allow demarcation of the injured nerve tissue requiring resection prior to repair, and similar to the application of this technique in tendon surgery, 2-staged nerve reconstruction may offer additional benefits in limiting perineural scarring.
      There were 3 goals for our experiment: (1) to induce a membrane around a silicone rod in a segmental nerve defect in an animal model; (2) to use high throughput technology (ie, Luminex) to evaluate levels of neurotrophic factors in the induced membrane; and (3) to compare the levels of neurotrophic factors between the induced membrane tissue and a control scar tissue bed. We hypothesized that an induced membrane within a nerve defect would be reliably generated and would exhibit higher levels of neurotrophic factors compared to control tissue.

      Materials and Methods

      After study protocol approval by our institutional animal care and use committee, 22 male Sprague-Dawley rats were used in accordance with the guidelines of the authors’ institution, the National Institutes of Health, and any national law on the care and use of laboratory animals.
      Sample size determination was based on previous protein quantifying studies of “Masquelet-type” membranes in rabbits.
      • Pelissier P.
      • Masquelet A.C.
      • Bareille R.
      • Pelissier S.M.
      • Amedee J.
      Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration.
      Though effect size and SD varied for different proteins, a statistically significant difference for levels of VEGF and TGF-ß1
      • Zadegan S.A.
      • Firouzi M.
      • Nabian M.H.
      • Zanjani L.O.
      • Kamrani R.S.
      Two-stage nerve graft using a silicone tube.
      allowed an a priori sample size estimate with the following input parameters: single tail, normal parent distribution, effect size 1.5, alpha error probability 0.05, and power 0.95.
      Under inhaled isoflurane anesthesia and using aseptic technique, the right sciatic nerve was exposed via a standard bicep femoris-semitendinosus muscle splitting approach. Two centimeters of sciatic nerve were excised in all 22 animals. In the experimental group (n = 11), a sterile 6-F silicone pediatric Foley catheter (Medline) was placed in the nerve defect and secured to the adjacent epineurium with 8-0 nylon suture. In the control group (n = 11), after nerve resection, the cut nerve ends were tagged with the same sutures to mark the scar bed. The skin and subcutaneous tissue were sutured close. All animals received analgesia after surgery and were monitored per institution protocol.
      Four weeks later, the hind limb was reopened, and the induced membrane from the experimental group and connective tissue from the center of the scar bed of the excised sciatic nerve in the control rats were harvested using the previously placed sutures as a guide. Tissue was immediately stored in sterile sealed DNase-, RNase-free tubing that was flash frozen and stored in -80 °C freezer. In preparation for analysis, the tissue was both physically and chemically lysed using liquid nitrogen chilled mortar and pestle (Scienceware Liquid Nitrogen Cooled Mini Mortar & Pestle Set) and Luminex-compatible buffer (Invitrogen tissue extraction reagent with cOmplete, Mini, EDTA-free protease inhibitor cocktail), then centrifuged. Supernatant was used for analysis.
      In order to verify that our 2-step lysis procedure yielded sufficient protein content and to subsequently determine the appropriate dilution of supernatant, the prepared tissue underwent rat-specific enzyme-linked immunosorbent assays for NGF, GDNF, brain-derived neurotrophic factor, and VEGF (Sigma Aldrich RAB0883, RAB1144, RAB1138, and RAB0512, respectively).
      Processed tissue from the control and experimental samples was then analyzed for the presence and quantities of the following neurotrophic factors using Luminex assays: TGF ß-1, 2, 3, and GDNF (11 matched samples tested); and IL-1B, VEGF, MCP-1, and NGF (10 matched samples tested based on available quantities of reagents). The following Emmanuel Merck, Darmstadt Millipore MILLIPLEX MAP plates were used: HADCYMAG-61K containing antihuman NGF; HNDG4MAG-36K containing antihuman GDNF; RECYTMAG-65K containing antirat VEGF, MCP-1, IL-1ß; TGFBMAG-64K-03 for antihuman TGF-ß 1, 2, and 3. Quantifying and comparing levels of these factors were completed using commercially available software (xPONENT 3.1 and Milliplex Analyst 5.1). These programs allowed for raw fluorescent intensity per bead to be recorded and quantified.
      • Breen E.J.
      • Tan W.
      • Khan A.
      The statistical value of raw fluorescence signal in Luminex xMAP based multiplex immunoassays.
      The concentration values and detection limits were determined from the standard curves generated from each analyte kit standard using a 5PL weighted curve fitting procedure.
      Levels of factors were not normally distributed; therefore, the statistical comparisons of levels were performed using nonparametric tests (Wilcoxon signed-rank test for 2 groups). Values below the limits of detection were assigned a value equivalent to the lower limit of detection. A P value of .05 or lower was considered significant.

      Results

      At 4 weeks postimplantation, the formation of a uniform, identifiable membrane around the silicone rod was observed in all 11 experimental specimens (Fig. 1). The induced membrane (experimental tissue) and tissue from the deep wound bed (control tissue) were excised in similar volumes as recorded by weight.
      Figure thumbnail gr1
      Figure 1A Demonstrates the membrane induced around a silicone rod. The silicone rod is labeled with an black asterisk and the membrane tissue is marked with a black arrow. B Another example of the induced membrane (white arrows) around a silicone rod (white asterisk).
      There were significantly higher levels of TGF-ß1, TGF-ß2, TGF-ß3, GDNF, IL-1ß, NGF, and MCP-1 in the induced membrane tissue compared with control tissue (Fig. 2). No statistically significant difference (P = .153) was detected between the control and experimental groups for levels of VEGF.
      Figure thumbnail gr2
      Figure 2Comparison of target growth factors measured in induced membrane versus scar tissue (∗P < .05).

      Discussion

      We achieved our goal of inducing a membrane around a silicone rod placed in a peripheral nerve and successfully demonstrated higher levels of NGF, GDNF, TGF-ß1, TGF-ß2, TGF-ß3, IL-1ß, and MCP-1 in this tissue. Elevations in VEGF levels were not found to be statistically significant compared with the normal tissue bed. We focused on these specific growth factors based on their potential neurotrophic properties previously documented in the literature. Silicone was chosen as a flexible, practical, and clinically relevant implant material, and the 4-week end point was chosen by extrapolating data from studies demonstrating optimal growth factor levels in polymethylmethacrylate (PMMA)-induced membranes at this time point.
      • Pelissier P.
      • Masquelet A.C.
      • Bareille R.
      • Pelissier S.M.
      • Amedee J.
      Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration.
      ,
      • Aho O.M.
      • Lehenkari P.
      • Ristiniemi J.
      • Lehtonen S.
      • Risteli J.
      • Leskela H.V.
      The mechanism of action of induced membranes in bone repair.
      Nerve growth factor is involved in neural survival, development, and function in peripheral cells and is implicated in regulating Schwann cell differentiation and axon remyelination.
      • Huang E.J.
      • Reichardt L.F.
      Neurotrophins: roles in neuronal development and function.
      In a rat sciatic nerve crush injury model, administration of NGF improved motor function after 30 days, reduced atrophy of the target muscle, and increased the number of myelinated axons compared to control.
      • Li R.
      • Wu J.
      • Lin Z.
      • et al.
      Single injection of a novel nerve growth factor coacervate improves structural and functional regeneration after sciatic nerve injury in adult rats.
      Acellular nerve allograft soaked in NGF resulted in higher axon counts when implanted in a rodent model; one study noting improved motor regeneration
      • Yu H.
      • Peng J.
      • Guo Q.
      • et al.
      Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor.
      and a separate study noting improved sensory regeneration.
      • Ovalle Jr., F.
      • Patel A.
      • Pollins A.
      • et al.
      A simple technique for augmentation of axonal ingrowth into chondroitinase-treated acellular nerve grafts using nerve growth factor.
      Glial-derived neurotrophic factor is associated with promotion of survival and function of neuronal populations in the peripheral nervous system.
      • Yan Q.
      • Matheson C.
      • Lopez O.T.
      In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons.
      In a rat sciatic nerve injury model, defects treated with GDNF demonstrated faster recovery of regenerated axons compared to controls as seen by improved functional assessment; increased nerve conduction velocity; increased target muscle weight; and greater nerve fiber, axon diameter, and myelin sheath thickness.
      • Ghayemi N.
      • Haghighat A.
      • Amini K.
      • Mohammadi R.
      Functional effect of local administration of glial derived neurotrophic factor combined with inside-out artery graft on sciatic nerve regeneration in rat.
      Specifically, a sustained GDNF delivery system applied around an implanted ANA resulted in a more robust axonal regeneration.
      • Tajdaran K.
      • Gordon T.
      • Wood M.D.
      • Shoichet M.S.
      • Borschel G.H.
      A glial cell line-derived neurotrophic factor delivery system enhances nerve regeneration across acellular nerve allografts.
      The cytokine IL-1ß and chemokine MCP-1 are associated with increased expression after rat sciatic nerve injury.
      • Perrin F.E.
      • Lacroix S.
      • Aviles-Trigueros M.
      • David S.
      Involvement of monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha and interleukin-1 beta in Wallerian degeneration.
      In one study, IL-1ß mRNA was not detected in intact sciatic rat nerves but was detected in increasing amounts shortly following injury.
      • Shamash S.
      • Reichert F.
      • Rotshenker S.
      The cytokine network of Wallerian degeneration: tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-1 beta.
      When IL-1ß and MCP-1 antibodies were infused near the site of cut rat sciatic nerve, reduced recruitment and activation of macrophages as well as delayed myelin phagocytosis was observed.
      • Perrin F.E.
      • Lacroix S.
      • Aviles-Trigueros M.
      • David S.
      Involvement of monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha and interleukin-1 beta in Wallerian degeneration.
      Macrophage chemotaxis was also decreased in the presence of MCP-1 antibodies in vitro.
      • Tofaris G.K.
      • Patterson P.H.
      • Jessen K.R.
      • Mirsky R.
      Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF.
      Vascular endothelial growth factor has a well-defined role in angiogenesis, the development of new blood vessels, as well as vessel maintenance.
      • Welti J.
      • Loges S.
      • Dimmeler S.
      • Carmeliet P.
      Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer.
      • Lee S.
      • Chen T.T.
      • Barber C.L.
      • et al.
      Autocrine VEGF signaling is required for vascular homeostasis.
      • Moens S.
      • Goveia J.
      • Stapor P.C.
      • Cantelmo A.R.
      • Carmeliet P.
      The multifaceted activity of VEGF in angiogenesis - Implications for therapy responses.
      Delayed axonal regeneration has been associated with slow revascularization of ANA.
      • Zhu Z.
      • Huang Y.
      • Zou X.
      • et al.
      The vascularization pattern of acellular nerve allografts after nerve repair in Sprague-Dawley rats.
      Vascular endothelial growth factor improved vascularization in ANA
      • Sondell M.
      • Lundborg G.
      • Kanje M.
      Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts.
      and increased axonal regeneration in a proximal nerve graft.
      • Rovak J.M.
      • Mungara A.K.
      • Aydin M.A.
      • Cederna P.S.
      Effects of vascular endothelial growth factor on nerve regeneration in acellular nerve grafts.
      Axon growth enhancement was marginal in a VEGF-treated 20 mm ANA.
      • Hoben G.
      • Yan Y.
      • Iyer N.
      • et al.
      Comparison of acellular nerve allograft modification with Schwann cells or VEGF.
      Vascular endothelial growth factor is consistently increased within pseudomembranes induced by PMMA implanted for osseus defects,
      • Aho O.M.
      • Lehenkari P.
      • Ristiniemi J.
      • Lehtonen S.
      • Risteli J.
      • Leskela H.V.
      The mechanism of action of induced membranes in bone repair.
      ,
      • Christou C.
      • Oliver R.A.
      • Yu Y.
      • Walsh W.R.
      The Masquelet technique for membrane induction and the healing of ovine critical sized segmental defects.
      so the lack of increased levels within the silicone rod induced membrane was unexpected. Pseudomembrane formation (and presumably growth factor content) is influenced by implant material and the subsequent tissue bed reaction.
      • Gaio N.
      • Martino A.
      • Toth Z.
      • Watson J.T.
      • Nicolaou D.
      • McBride-Gagyi S.
      Masquelet technique: the effect of altering implant material and topography on membrane matrix composition, mechanical and barrier properties in a rat defect model.
      ,
      • Toth Z.
      • Roi M.
      • Evans E.
      • Watson J.T.
      • Nicolaou D.
      • McBride-Gagyi S.
      Masquelet technique: effects of spacer material and micro-topography on factor expression and bone regeneration.
      Silicone may not induce the same tissue response as PMMA, though in the only comparative study identified in the literature, nonirradiated silicone-induced membranes and PMMA-induced membranes had similarly weak VEGF levels on Western-blot, and microscopic vessel quantification between the 2 groups were not statistically significant (P value not published).
      • de Mones E.
      • Schlaubitz S.
      • Oliveira H.
      • et al.
      Comparative study of membranes induced by PMMA or silicone in rats, and influence of external radiotherapy.
      Additionally, VEGF has been shown to be elevated in tissue healing in general, which would affect the levels detected in the tissue bed harvested for analysis, even in our control group.
      • Wilgus T.A.
      Vascular endothelial growth factor and cutaneous scarring.
      Transforming TGF-ß is a cytokine associated with a variety of functions including bone metabolism, extracellular matrix synthesis, angiogenesis, macrophage activation, and scar tissue.
      • Atkins S.
      • Smith K.G.
      • Loescher A.R.
      • Boissonade F.M.
      • Ferguson M.W.
      • Robinson P.P.
      The effect of antibodies to TGF-beta1 and TGF-beta2 at a site of sciatic nerve repair.
      • Einheber S.
      • Hannocks M.J.
      • Metz C.N.
      • Rifkin D.B.
      • Salzer J.L.
      Transforming growth factor-beta 1 regulates axon/Schwann cell interactions.
      • Taskinen H.S.
      • Ruohonen S.
      • Jagodic M.
      • Khademi M.
      • Olsson T.
      • Roytta M.
      Distinct expression of TGF-beta1 mRNA in the endo- and epineurium after nerve injury.
      ,
      • O'Kane S.
      • Ferguson M.W.
      Transforming growth factor beta s and wound healing.
      Transforming TGF-ß has multiple isoforms (1, 2, and 3), and TGF-ß1 is secreted by macrophages and Schwann cells.
      • Atkins S.
      • Smith K.G.
      • Loescher A.R.
      • Boissonade F.M.
      • Ferguson M.W.
      • Robinson P.P.
      The effect of antibodies to TGF-beta1 and TGF-beta2 at a site of sciatic nerve repair.
      Transforming TGF-ß is not only secreted by macrophages, but also attracts macrophages.
      • Atkins S.
      • Smith K.G.
      • Loescher A.R.
      • Boissonade F.M.
      • Ferguson M.W.
      • Robinson P.P.
      The effect of antibodies to TGF-beta1 and TGF-beta2 at a site of sciatic nerve repair.
      Following nerve injury, TGF-ß1 levels increase and are associated with inducing expression of neurotrophic factors in Schwann cells.
      • Einheber S.
      • Hannocks M.J.
      • Metz C.N.
      • Rifkin D.B.
      • Salzer J.L.
      Transforming growth factor-beta 1 regulates axon/Schwann cell interactions.
      ,
      • Taskinen H.S.
      • Ruohonen S.
      • Jagodic M.
      • Khademi M.
      • Olsson T.
      • Roytta M.
      Distinct expression of TGF-beta1 mRNA in the endo- and epineurium after nerve injury.
      One study incubated chronically denervated Schwann cells in TGF-ß and found that those treated with TGF-ß promoted more than twice the number of motoneurons to regenerate axons across a bridge between proximal and distal nerve stumps compared to the control group.
      • Gordon T.
      • Sulaiman O.
      • Boyd J.G.
      Experimental strategies to promote functional recovery after peripheral nerve injuries.
      The use of the Luminex system allowed the measurement of multiple proteins of interest within the same tissue. Luminex is a commercially available bead-based flow cytometric multiplex array system that offers the ability for high throughput, simultaneous analysis of multiple analytes with a single, low-volume sample in less time and with increased reproducibility compared to traditional immunoassays.
      • Leng S.X.
      • McElhaney J.E.
      • Walston J.D.
      • Xie D.
      • Fedarko N.S.
      • Kuchel G.A.
      ELISA and multiplex technologies for cytokine measurement in inflammation and aging research.
      • Purohit S.
      • Sharma A.
      • She J.X.
      Luminex and other multiplex high throughput technologies for the identification of, and host response to, environmental triggers of type 1 diabetes.
      • Yu J.
      • Lin J.
      • Kim K.H.
      • Benjamin Jr., W.H.
      • Nahm M.H.
      Development of an automated and multiplexed serotyping assay for Streptococcus pneumoniae.
      A traditional enzyme-linked immunosorbent assay test requires that each sample can only be run against a single analyte at a time. In contrast, a multiplex assay allows for each sample to be run against multiple analytes concurrently. Each Luminex bead has 2 identifiers: a unique color profile and a unique biomarker/reporter molecule (eg, antibody) that allows proteins to bind with the bead and is coupled with a fluorescent marker. This technology allows for multiplex assays (multiple specimens run against multiple analytes concurrently) on a standard 96-well plate to be completed in a few hours. For some proteins in this study, antihuman markers were used when antirat markers were not available. This type of multiplex assay cross-reactivity has been previously reported across species.
      • Duran M.C.
      • Willenbrock S.
      • Muller J.M.
      • Nolte I.
      • Feige K.
      • Murua Escobar H.
      Establishment and evaluation of a bead-based luminex assay allowing simultaneous quantification of equine IL-12 and IFN- γ.
      Conservation of structure and immunological properties between human and rodent NGF, GDNF, TGF-ß, and IL-1ß has been previously demonstrated,
      • Ibanez C.F.
      • Hallbook F.
      • Soderstrom S.
      • Ebendal T.
      • Persson H.
      Biological and immunological properties of recombinant human, rat, and chicken nerve growth factors: a comparative study.
      • Oo T.F.
      • Kholodilov N.
      • Burke R.E.
      Regulation of natural cell death in dopaminergic neurons of the substantia nigra by striatal glial cell line-derived neurotrophic factor in vivo.
      • Moses H.L.
      • Roberts A.B.
      • Derynck R.
      The discovery and early days of TGF-β: a historical perspective.
      • Owyang A.M.
      • Issafras H.
      • Corbin J.
      • et al.
      XOMA 052, a potent, high-affinity monoclonal antibody for the treatment of IL-1beta-mediated diseases.
      so the cross-reactivity between antihuman markers for rat proteins should be high. This was, however, not validated in our study, and these results should be considered as relative differences and not absolute values.
      While our study is able to evaluate and describe the level of neurotrophic factors in an induced membrane model for peripheral nerve defects, there are several limitations. First, this is a pilot study with a small sample size, and although adequately powered, the variability in protein concentrations (ie, levels of neurotrophic factors) within tissue samples would be improved with larger sample sizes. Second, this is an animal study and may not accurately reflect similar biologic mechanisms in humans. Third, we did use markers for antihuman NGF, GDNF, TGF-ß 1, 2, and 3, and IL-1ß. Follow-up validation studies evaluating cross-reactivity for these markers with the assay system are necessary.
      There are important implications for further study based on the findings in our pilot study. While a membrane was induced reliably around a silicone rod placed in a peripheral nerve defect in rats at 4 weeks, the ideal time of membrane “maturity” is unknown, and the ideal size and material of the implant might vary depending on the clinical scenario. For this study, we used a 6-F (2 mm outer diameter) Foley catheter, which was appropriately larger than the average diameter of an adult rat sciatic nerve (approximately 1 mm to 1.5 mm). Although there is a significant elevation in several neurotrophic factors in the induced membrane tissue, the threshold level required to promote nerve regeneration as well as dynamic interaction of these factors in axonal regeneration is unknown. For example, supraphysiological levels of GDNF attract axons, though these axons become trapped in the area of “high GDNF” and do not continue to regenerate.
      • Ee X.
      • Yan Y.
      • Hunter D.A.
      • et al.
      Transgenic SCs expressing GDNF-IRES-DsRed impair nerve regeneration within acellular nerve allografts.
      Most importantly, there is impetus for further study using the induced membrane technique in a peripheral nerve injury model with autograft or allograft reconstruction.

      Acknowledgments

      The authors acknowledge Geetanjali Bendale, PhD, and Satya Mallu, MD. This study was supported by a 2018 American Foundation for Surgery of the Hand Resident/Fellow Fast Track Grant.

      References

        • Saheb-Al-Zamani M.
        • Yan Y.
        • Farber S.J.
        • et al.
        Limited regeneration in long acellular nerve allografts is associated with increased Schwann cell senescence.
        Exp Neurol. 2013; 247C: 165-177
        • Chen P.
        • Piao X.
        • Bonaldo P.
        Role of macrophages in Wallerian degeneration and axonal regeneration after peripheral nerve injury.
        Acta Neuropathol. 2015; 130: 605-618
        • Atkins S.
        • Smith K.G.
        • Loescher A.R.
        • Boissonade F.M.
        • Ferguson M.W.
        • Robinson P.P.
        The effect of antibodies to TGF-beta1 and TGF-beta2 at a site of sciatic nerve repair.
        J Peripher Nerv Syst. 2006; 11: 286-293
        • Einheber S.
        • Hannocks M.J.
        • Metz C.N.
        • Rifkin D.B.
        • Salzer J.L.
        Transforming growth factor-beta 1 regulates axon/Schwann cell interactions.
        J Cell Biol. 1995; 129: 443-458
        • Taskinen H.S.
        • Ruohonen S.
        • Jagodic M.
        • Khademi M.
        • Olsson T.
        • Roytta M.
        Distinct expression of TGF-beta1 mRNA in the endo- and epineurium after nerve injury.
        J Neurotrauma. 2004; 21: 969-975
        • Gordon T.
        • Sulaiman O.
        • Boyd J.G.
        Experimental strategies to promote functional recovery after peripheral nerve injuries.
        J Peripher Nerv Syst. 2003; 8: 236-250
        • Perrin F.E.
        • Lacroix S.
        • Aviles-Trigueros M.
        • David S.
        Involvement of monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha and interleukin-1 beta in Wallerian degeneration.
        Brain. 2005; 128: 854-866
        • Shamash S.
        • Reichert F.
        • Rotshenker S.
        The cytokine network of Wallerian degeneration: tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-1 beta.
        J Neurosci. 2002 15; 22: 3052-3060
        • Tofaris G.K.
        • Patterson P.H.
        • Jessen K.R.
        • Mirsky R.
        Denervated Schwann cells attract macrophages by secretion of leukemia inhibitory factor (LIF) and monocyte chemoattractant protein-1 in a process regulated by interleukin-6 and LIF.
        J Neurosci. 2002; 22: 6696-6703
        • Banner L.R.
        • Patterson P.H.
        Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia.
        Proc Natl Acad Sci U S A. 1994; 91: 7109-7113
        • Sugiura S.
        • Lahav R.
        • Han J.
        • et al.
        Leukaemia inhibitory factor is required for normal inflammatory responses to injury in the peripheral and central nervous systems in vivo and is chemotactic for macrophages in vitro.
        Eur J Neurosci. 2000; 12: 457-466
        • Kurek J.B.
        • Austin L.
        • Cheema S.S.
        • Bartlett P.F.
        • Murphy M.
        Up-regulation of leukaemia inhibitory factor and interleukin-6 in transected sciatic nerve and muscle following denervation.
        Neuromuscul Disord. 1996; 6: 105-114
        • Chen P.
        • Cescon M.
        • Megighian A.
        • Bonaldo P.
        Collagen VI regulates peripheral nerve myelination and function.
        FASEB J. 2014; 28: 1145-1156
        • Mokarram N.
        • Merchant A.
        • Mukhatyar V.
        • Patel G.
        • Bellamkonda R.V.
        Effect of modulating macrophage phenotype on peripheral nerve repair.
        Biomaterials. 2012; 33: 8793-8801
        • Chen P.
        • Cescon M.
        • Zuccolotto G.
        • et al.
        Collagen VI regulates peripheral nerve regeneration by modulating macrophage recruitment and polarization.
        Acta Neuropathol. 2015; 129: 97-113
        • Zhang J.Y.
        • Luo X.G.
        • Xian C.J.
        • Liu Z.H.
        • Zhou X.F.
        Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents.
        Eur J Neurosci. 2000; 12: 4171-4180
        • Funakoshi H.
        • Frisen J.
        • Barbany G.
        • et al.
        Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve.
        J Cell Biol. 1993; 123: 455-465
        • Boyd J.G.
        • Gordon T.
        Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo.
        Exp Neurol. 2003; 183: 610-619
        • Huang E.J.
        • Reichardt L.F.
        Neurotrophins: roles in neuronal development and function.
        Annu Rev Neurosci. 2001; 24: 677-736
        • Li R.
        • Wu J.
        • Lin Z.
        • et al.
        Single injection of a novel nerve growth factor coacervate improves structural and functional regeneration after sciatic nerve injury in adult rats.
        Exp Neurol. 2017; 288: 1-10
        • Takemura Y.
        • Imai S.
        • Kojima H.
        • Katagi M.
        • Yamakawa I.
        • Kasahara T.
        • et al.
        Brain-derived neurotrophic factor from bone marrow-derived cells promotes post-injury repair of peripheral nerve.
        PLoS One. 2012; 7e44592
        • Yan Q.
        • Matheson C.
        • Lopez O.T.
        In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons.
        Nature. 1995; 373: 341-344
        • Ghayemi N.
        • Haghighat A.
        • Amini K.
        • Mohammadi R.
        Functional effect of local administration of glial derived neurotrophic factor combined with inside-out artery graft on sciatic nerve regeneration in rat.
        Int J Surg. 2014; 12: 457-463
        • Masquelet A.C.
        • Fitoussi F.
        • Begue T.
        • Muller G.P.
        Reconstruction of the long bones by the induced membrane and spongy autograft.
        Ann Chir Plast Esthet. 2000; 45: 346-353
        • Morelli I.
        • Drago L.
        • George D.A.
        • Gallazzi E.
        • Scarponi S.
        • Romano C.L.
        Masquelet technique: myth or reality? A systematic review and meta-analysis.
        Injury. 2016; 47: S68-S76
        • Wang X.
        • Wei F.
        • Luo F.
        • Huang K.
        • Xie Z.
        Induction of granulation tissue for the secretion of growth factors and the promotion of bone defect repair.
        J Orthop Surg Res. 2015; 10: 147
        • Gruber H.E.
        • Ode G.
        • Hoelscher G.
        • Ingram J.
        • Bethea S.
        • Bosse M.J.
        Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects.
        Bone Joint Res. 2016; 5: 106-115
        • Zadegan S.A.
        • Firouzi M.
        • Nabian M.H.
        • Zanjani L.O.
        • Ashtiani A.M.
        • Kamrani R.S.
        Two-stage nerve graft in severe scar: a time-course study in a rat model.
        Arch Bone Jt Surg. 2015; 3: 82-87
        • Zadegan S.A.
        • Firouzi M.
        • Nabian M.H.
        • Zanjani L.O.
        • Kamrani R.S.
        Two-stage nerve graft using a silicone tube.
        Front Surg. 2015; 2: 12
        • Pelissier P.
        • Masquelet A.C.
        • Bareille R.
        • Pelissier S.M.
        • Amedee J.
        Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration.
        J Orthop Res. 2004; 22: 73-79
        • Breen E.J.
        • Tan W.
        • Khan A.
        The statistical value of raw fluorescence signal in Luminex xMAP based multiplex immunoassays.
        Sci Rep. 2016; 6: 26996
        • Aho O.M.
        • Lehenkari P.
        • Ristiniemi J.
        • Lehtonen S.
        • Risteli J.
        • Leskela H.V.
        The mechanism of action of induced membranes in bone repair.
        J Bone Joint Surg Am. 2013; 95: 597-604
        • Yu H.
        • Peng J.
        • Guo Q.
        • et al.
        Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor.
        Microsurgery. 2009; 29: 330-336
        • Ovalle Jr., F.
        • Patel A.
        • Pollins A.
        • et al.
        A simple technique for augmentation of axonal ingrowth into chondroitinase-treated acellular nerve grafts using nerve growth factor.
        Ann Plast Surg. 2012; 68: 518-524
        • Tajdaran K.
        • Gordon T.
        • Wood M.D.
        • Shoichet M.S.
        • Borschel G.H.
        A glial cell line-derived neurotrophic factor delivery system enhances nerve regeneration across acellular nerve allografts.
        Acta Biomater. 2016; 29: 62-70
        • Welti J.
        • Loges S.
        • Dimmeler S.
        • Carmeliet P.
        Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer.
        J Clin Invest. 2013; 123: 3190-3200
        • Lee S.
        • Chen T.T.
        • Barber C.L.
        • et al.
        Autocrine VEGF signaling is required for vascular homeostasis.
        Cell. 2007; 130: 691-703
        • Moens S.
        • Goveia J.
        • Stapor P.C.
        • Cantelmo A.R.
        • Carmeliet P.
        The multifaceted activity of VEGF in angiogenesis - Implications for therapy responses.
        Cytokine Growth Factor Rev. 2014; 25: 473-482
        • Zhu Z.
        • Huang Y.
        • Zou X.
        • et al.
        The vascularization pattern of acellular nerve allografts after nerve repair in Sprague-Dawley rats.
        Neurol Res. 2017 Nov; 39: 1014-1021
        • Sondell M.
        • Lundborg G.
        • Kanje M.
        Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts.
        Brain Res. 1999; 846: 219-228
        • Rovak J.M.
        • Mungara A.K.
        • Aydin M.A.
        • Cederna P.S.
        Effects of vascular endothelial growth factor on nerve regeneration in acellular nerve grafts.
        J Reconstr Microsurg. 2004; 20: 53-58
        • Hoben G.
        • Yan Y.
        • Iyer N.
        • et al.
        Comparison of acellular nerve allograft modification with Schwann cells or VEGF.
        Hand. 2015; 10: 396-402
        • Christou C.
        • Oliver R.A.
        • Yu Y.
        • Walsh W.R.
        The Masquelet technique for membrane induction and the healing of ovine critical sized segmental defects.
        PLoS One. 2014; 9e114122
        • Gaio N.
        • Martino A.
        • Toth Z.
        • Watson J.T.
        • Nicolaou D.
        • McBride-Gagyi S.
        Masquelet technique: the effect of altering implant material and topography on membrane matrix composition, mechanical and barrier properties in a rat defect model.
        J Biomech. 2018 27; 72: 53-62
        • Toth Z.
        • Roi M.
        • Evans E.
        • Watson J.T.
        • Nicolaou D.
        • McBride-Gagyi S.
        Masquelet technique: effects of spacer material and micro-topography on factor expression and bone regeneration.
        Ann Biomed Eng. 2019; 47: 174-189
        • de Mones E.
        • Schlaubitz S.
        • Oliveira H.
        • et al.
        Comparative study of membranes induced by PMMA or silicone in rats, and influence of external radiotherapy.
        Acta Biomater. 2015; 19: 119-127
        • Wilgus T.A.
        Vascular endothelial growth factor and cutaneous scarring.
        Adv Wound Care. 2019; 8: 671-678
        • O'Kane S.
        • Ferguson M.W.
        Transforming growth factor beta s and wound healing.
        Int J Biochem Cell Biol. 1997; 29: 63-78
        • Leng S.X.
        • McElhaney J.E.
        • Walston J.D.
        • Xie D.
        • Fedarko N.S.
        • Kuchel G.A.
        ELISA and multiplex technologies for cytokine measurement in inflammation and aging research.
        J Gerontol A Biol Sci Med Sci. 2008; 63: 879-884
        • Purohit S.
        • Sharma A.
        • She J.X.
        Luminex and other multiplex high throughput technologies for the identification of, and host response to, environmental triggers of type 1 diabetes.
        Biomed Res Int. 2015; : 326918
        • Yu J.
        • Lin J.
        • Kim K.H.
        • Benjamin Jr., W.H.
        • Nahm M.H.
        Development of an automated and multiplexed serotyping assay for Streptococcus pneumoniae.
        Clin Vaccine Immunol. 2011; 18: 1900-1907
        • Duran M.C.
        • Willenbrock S.
        • Muller J.M.
        • Nolte I.
        • Feige K.
        • Murua Escobar H.
        Establishment and evaluation of a bead-based luminex assay allowing simultaneous quantification of equine IL-12 and IFN- γ.
        Anticancer Res. 2013; 33: 1325-1336
        • Ibanez C.F.
        • Hallbook F.
        • Soderstrom S.
        • Ebendal T.
        • Persson H.
        Biological and immunological properties of recombinant human, rat, and chicken nerve growth factors: a comparative study.
        J Neurochem. 1991; 57: 1033-1041
        • Oo T.F.
        • Kholodilov N.
        • Burke R.E.
        Regulation of natural cell death in dopaminergic neurons of the substantia nigra by striatal glial cell line-derived neurotrophic factor in vivo.
        J Neurosci. 2003; 23: 5141-5148
        • Moses H.L.
        • Roberts A.B.
        • Derynck R.
        The discovery and early days of TGF-β: a historical perspective.
        Cold Spring Harb Perspect Biol. 2016; 8
        • Owyang A.M.
        • Issafras H.
        • Corbin J.
        • et al.
        XOMA 052, a potent, high-affinity monoclonal antibody for the treatment of IL-1beta-mediated diseases.
        MAbs. 2011; 3: 49-60
        • Ee X.
        • Yan Y.
        • Hunter D.A.
        • et al.
        Transgenic SCs expressing GDNF-IRES-DsRed impair nerve regeneration within acellular nerve allografts.
        Biotechnol Bioeng. 2017; 114: 2121-2130