Spindle‐Like Zinc Silicate Nanoparticles Accelerating Innervated and Vascularized Skin Burn Wound Healing

Abstract Macrophages (Mϕs) critically contribute to wound healing by coordinating inflammatory, proliferative, and angiogenic processes. A proper switch from proinflammatory M1 to anti‐inflammatory M2 dominant Mϕs accelerates the wound healing processes leading to favorable wound‐care outcomes. Herein, an exosome‐guided cell reprogramming technique is proposed to directly convert M1 to M2 Mϕs for effective wound management. The M2 Mϕ‐derived exosomes (M2‐Exo) induce a complete conversion of M1 to M2 Mϕs in vitro. The reprogrammed M2 Mϕs turn Arginase (M2‐marker) and iNOS (M1‐marker) on and off, respectively, and exhibit distinct phenotypic and functional features of M2 Mϕs. M2‐Exo has not only Mϕ reprogramming factors but also various cytokines and growth factors promoting wound repair. After subcutaneous administration of M2‐Exo into the wound edge, the local populations of M1 and M2 Mϕs are markedly decreased and increased, respectively, showing a successful exosome‐guided switch to M2 Mϕ polarization. The direct conversion of M1 to M2 Mϕs at the wound site accelerates wound healing by enhancing angiogenesis, re‐epithelialization, and collagen deposition. The Mϕ phenotype switching induced by exosomes possessing the excellent cell reprogramming capability and innate biocompatibility can be a promising therapeutic approach for various inflammation‐associated disorders by regulating the balance between pro‐ versus anti‐inflammatory Mϕs.

Introduction How transcription factors control cellular plasticity and maintain differentiation is currently of great interest, inspired by the success of experimental reprogramming, where remarkable phenotypic transitions can be induced by enforced expression of fate determining factors (Zhou and Melton, 2008). These findings raise a key question: to what extent are natural transitions in the state of differentiated cells also governed by specific transcription factors? Such phenotypic transitions are seen in tumorigenesis, dedifferentiation and transdifferentiation. They are also fundamental to tissue repair and regeneration, and in regenerative systems, a major focus of work is identification of gene programs that are selectively activated after injury and which impact the repair process. The striking regenerative capacity of the PNS rests on the surprising plasticity of Schwann cells, and the ability of these cells to switch between differentiation states, a feature that is highly unusual in mammals (Jessen and Mirsky, 2005, 2008; Jopling et al., 2011). In a process reminiscent of the radical injury responses of zebrafish cardiomyocytes or pigment cells of the newt iris, nerve injury, and loss of axonal contact causes mammalian Schwann cells to lose their differentiated morphology, downregulate myelin genes, upregulate markers of immature Schwann cells, and re-enter the cell cycle. This radical process of natural dedifferentiation has few if any parallels in mammalian systems. At the same time as Schwann cells dedifferentiate, they upregulate genes implicated in promoting axon growth, neuronal survival, and macrophage invasion, and activate mechanisms to break down their myelin sheaths and transform morphologically into cells with long, parallel processes. This allows them to form uninterrupted regeneration tracks (Bands of Bungner) that guide axons back to their targets (Chen et al., 2007; Vargas and Barres, 2007; Gordon et al., 2009). Collectively, these events together with the axonal death that triggers them are called Wallerian degeneration. This response transforms the normally growth-hostile environment of intact nerves to a growth supportive terrain, and endows the PNS with its remarkable and characteristic regenerative potential. To complete the repair process, Schwann cells envelop the regenerated axons and transform again to generate myelin and nonmyelinating (Remak) cells. Little is known about the transcriptional control of changes in adult differentiation states, including natural dedifferentiation and transdifferentiation, in any system (Jopling et al., 2011). In line with this, although Wallerian degeneration including the Schwann cell injury response are key to repair, the molecular mechanisms that control these processes are not understood (Chen et al., 2007; Jessen and Mirsky, 2008). Conceptually also, the nature of the Schwann cell injury response has remained uncertain, since the generation of the denervated Schwann cell is commonly referred to either as dedifferentiation or as activation. These terms highlight two distinct aspects of the process, namely loss of the differentiated Schwann cell phenotypes of normal nerves and gain of the regeneration promoting phenotype, respectively, without providing a framework for analysis and comparison with other regenerative models. Here, we use mice with selective inactivation of the transcription factor c-Jun in Schwann cells to show that c-Jun is a global regulator of the Schwann cell injury response that specifies the characteristic gene expression, structure, and function of the denervated Schwann cell, a cell that is essential for nerve repair. Consequently, axonal regeneration and functional repair are strikingly compromised or absent when Schwann cell c-Jun is inactivated. Notably, the effects of c-Jun are injury specific, since c-Jun inactivation has no significant effects on nerve development or adult nerve function. These observations provide a molecular basis for understanding Schwann cell plasticity, show that c-Jun is a key regulator of Wallerian degeneration, and offer conclusive support for the notion that glial cells control repair in the PNS. They also show that the Schwann cell injury response has much in common with transdifferentiation, since it represents the generation, by dedicated transcriptional controls, of a distinct Schwann cell repair phenotype, specialized for supporting axon growth and neuronal survival in injured nerves. Because these cells form the regeneration tracks called Bungner’s bands, we will refer to them as Bungner cells. Results c-Jun Controls the Molecular Phenotype of the Denervated Schwann Cell Earlier, we found that neonatal mice with conditional deletion of c-Jun in Schwann cells (c-Jun mutant mice) show delayed loss of myelin proteins and mRNA after nerve injury (Parkinson et al., 2008). This suggested that Schwann cell c-Jun might play an important role in specifying the phenotype of denervated Schwann cells. To test this comprehensively, we used Affymetrix whole-genome microarray to examine gene expression in the sciatic nerve of adult c-Jun mutant mice and control (WT) littermates and compared this with gene expression in denervated cells in the distal stump of transected nerves without regenerating axons, to avoid the complicating effects of axon-induced redifferentiation (Figure 1). We chose 7 days after injury since in regenerating mouse nerves this is near the mid-point of active axonal regrowth. Seven day denervated cells therefore represent the terrain that confronts regenerating axons in WT and mutant nerves. Before injury, the nerves of adult c-Jun mutant mice were normal on the basis of a number of criteria. Thus, the numbers of myelinated and unmyelinated axons (see Figures 4E and 4F), myelinating Schwann cells and Remak bundles (see Table S1 available online), g-ratios (Figure S1), sciatic functional index (SFI) (see Figure 7E), motor performance in a rotarod test (unpublished), and responses to heat and light touch (see Figures 7B and 7C) were similar to WT controls. While c-Jun was excised from almost all Schwann cells (Parkinson et al., 2008), c-Jun expression in neurons, macrophages, and fibroblasts was normal, and the rate of axonal disintegration after cut was similar in WT and mutants (Figures S2 and S3). The close similarity between WT and mutant nerves was confirmed by the Affymetrix screen (Figure 1), since only two genes (keratin 8 and desmoplakin) were differentially expressed. Furthermore, following injury, a comparable number of genes changed expression in WT and c-Jun mutants (Figure 1A). Importantly, however, comparison of the distal stumps of WT and c-Jun mutants revealed 172 significant differences in gene expression (Figure 1 and Tables S2 and S3). The differentially regulated genes included genes which have been implicated in regeneration and trophic support such as BDNF, GDNF, Artn, Shh, and GAP-43 that failed to upregulate after injury, together with genes that failed to downregulate normally after injury such as the myelin genes Mpz, Mbp, and Cdh1 (also known as E-cadherin). Gene ontology analysis indicated that known functions of these 172 genes were particularly related to neuronal growth and regeneration (Figure 1C). We selected 32 of the 172 disregulated genes for further analysis by RT-QPCR. In every case this confirmed the disregulation shown by the microarray data (Figures 1D–1F and Table S3). Six of the thirty-two genes were then analyzed in purified Schwann cell cultures. Comparison of c-Jun mutant and WT cells confirmed the regulation seen in the distal stumps. Furthermore, as predicted, enforced c-Jun expression in c-Jun mutant cells, by adenoviral gene transfer, activated c-Jun, BDNF, and GDNF expression but suppressed Chd1, Mpz, and Mbp expression (Figure 2A). These results show directly that c-Jun regulates these genes in Schwann cells, demonstrates that this control is independent of the nerve environment, and confirms results obtained by microarray and RT-QPCR. Lastly, we found that three proteins implicated in regeneration, N-cadherin, p75NTR, and NCAM, were disregulated in cut mutant nerves, although their mRNAs were normally expressed. Injured mutant nerves expressed strongly reduced N-cadherin and p75NTR but elevated levels of NCAM (Figures 2B and 2C). Sox2 protein, which, like c-Jun, is upregulated in WT Schwann cells of injured nerves (Parkinson et al., 2008), remained normally upregulated in injured nerves of c-Jun mutants (Figure S4). Denervated Schwann cells in injured adult nerves are often considered similar to immature Schwann cells in developing nerves. However, the immature cells for instance do not share the axon guidance, myelin breakdown and macrophage recruitment functions of denervated cells, and these cells differ in molecular expression (Jessen and Mirsky, 2008). To explore the idea that the denervated cell represents a distinct Schwann cell phenotype regulated by c-Jun, we examined three genes, Olig1, Shh, and GDNF, which showed strong, c-Jun-dependent activation in denervated cells (Figure 1D). Using RT-QPCR and in situ hybririsization we confirmed strong expression of these genes in WT adult denervated cells, but found that they were not (Olig1 and Shh) or borderline (GDNF) detectable in immature Schwann cells (from WT embryo day 18 nerve). They were also essentially absent from uncut nerves (Figures 2D and 2E and Table S4). This supports the notion that denervated adult Schwann cells and immature Schwann cells in perinatal nerves represent distinct cell types. It shows also that c-Jun takes part in controlling the distinctive molecular profile of the adult denervated cell. The response of neonatal cells to injury remains to be determined. Together these results show that c-Jun controls the molecular reprogramming that transforms mature Schwann cells to the denervated cell phenotype following injury. This includes the regulation of genes that differentiate denervated from immature cells and extends to the posttranscriptional control of protein expression. c-Jun Controls the Structure of Regeneration Tracks (Bands of Bungner) Denervated Schwann cells form cellular columns that replace the axon-Schwann cell units of intact nerves and serve as substrate for growing axons. We examined these structures by electron microscopy in the distal stump 4 weeks after cut. Because these cells have been without axonal contact for 4 weeks they are comparable to the cells encountered by growing axons in distal parts of crushed nerves in the c-Jun mutant where regeneration is delayed beyond the normal 3–4 week period, while at this time WT nerves have just reached their targets. We found that the structure of these regeneration tracks is strikingly abnormal in c-Jun mutants (Figure 3A). There are many fewer cell profiles per column, indicating reduced process formation (Figure 3B) and the cells have flattened as confirmed by reduction in the roundness index of cell profiles in vivo (Figure 3C). Flattening and paucity of processes are also seen even in c-Jun −/− cells from neonatal nerves in vitro (Figures 3D and 3E). Therefore, this is a robust phenotype that does not depend on long term denervation in vivo. Thus, c-Jun is an cell-intrinsic determinant of Schwann cell morphology that controls the structure of the essential regeneration tracks that guide growing axons back to correct targets. In c-Jun Mutants, Injury Results in Extensive Death of Sensory (DRG) Neurons c-Jun specification of gene expression and morphology of denervated cells suggested that Schwann cell c-Jun might exert a decisive control over nerve repair. Because survival of injured neurons is the basis for repair, we measured the survival of small and large dorsal root sensory (DRG) neurons following sciatic nerve crush at the sciatic notch. We counted axons in L4 dorsal roots (Coggeshall et al., 1997) and the tibial nerve, and neuronal somas and nucleoli in DRGs. Comparable results were obtained using all methods. Axon counts in WT dorsal roots showed that 20%–25% of the unmyelinated axons were lost following crush, as expected (Coggeshall et al., 1997). In contrast, 55%–60% of these axons were lost in c-Jun mutants, showing increased death of small DRG neurons in the mutant. This was confirmed by corrected (Abercrombie, 1946) counts of B neuron profiles in DRGs, showing 25%–30% loss in WT but 45%–65% loss in the mutants (Figures 4A and 4B). The number of myelinated axons in dorsal roots remained unchanged in injured WT mice as expected (Coggeshall et al., 1997). But surprisingly, in the mutants the number of myelinated axons was reduced by 30%–35%, indicating death of large DRG cells. In confirmation, the corrected number of large A cell profiles in DRGs was reduced by about 40% in the mutants. The number of these profiles did not change significantly in injured WT (Figures 4C and 4D). We also carried out counts on DRG sections using nucleoli as the counted entity, an approach that theoretically provides increased accuracy. Nucleoli in A type DRG neurons from uncut WT (n = 3), 10 week cut WT (n = 3), and 10 week cut c-Jun mutant (n = 3) mice were counted, corrected (Abercrombie, 1946), and expressed as percentage of uncut WT. This showed a 12% reduction in cut WT, (not significant; p > 0.40) but a 50% reduction in the c-Jun mutant (highly significant; p  150 μm2) foamy macrophages bloated with debris was elevated 3-fold in mutant nerves at this time point. Macrophage numbers in the distal stump were strongly elevated in both WT and c-Jun mutant nerves after injury. Three days after cut, their number close to the injury site was significantly higher in WT mice, and a migration assay using Boyden chambers showed that WT nerves attracted more macrophages than mutant nerves (Figures S5C and S5D). But at 1 and 6 weeks after cut, the number of macrophages was similar in WT and mutants, and at 4 and 14 days after crush, macrophage numbers were not significantly different (Table S7). RT-QPCR of cytokines in the distal stumps 36 hr after cut did not reveal significant differences between WT and mutants (Figure S5E). This indicates that c-Jun mutants do not suffer from a major failure in macrophage recruitment. Reduced numbers shortly after injury in the mutant might relate to lower Schwann cell numbers rather than to significant disturbance in the expression of macrophage attractants by individual cells. These results show that injured c-Jun mutant nerves develop substantial problems with myelin clearance. This is evident not only in Schwann cells but also in macrophages, an observation that suggests a role for Schwann cells in the control of macrophage activation and myelin degradation. Functional Recovery Fails in the Absence of c-Jun in Schwann Cells Previous sections show that injury-activated Schwann cell c-Jun controls the generation of the denervated Schwann cell, and controls key cellular interactions during Wallerian degeneration and nerve repair. The end result of this process is functional recovery. This is remarkably effective in rodents, where full recovery is seen 3–4 weeks after crush. We found that this essential feature of peripheral nerves was abolished or strikingly compromised in c-Jun mutant mice. To measure sensory function, we used the following: (1) toe pinching (pressure), (2) Von Frey hairs (light touch), and (3) the Hargreaves test (temperature). In WT and c-Jun mutants, sciatic nerve crush abolished the response to toe pinching and decreased the responses to light touch and heat to the minimum measurable by these assays. Control mice recovered in 3–4 weeks as expected, using the toe-pinching test. c-Jun mutants, however, showed minimal recovery, even after extensive (up to 70 day) periods (Figure 7A). Similarly, even 70 days after crush mutant mice showed no recovery in the Hargreaves and Von Frey tests, although control mice recovered fully (Figures 7B and 7C). To measure motor function we used the toe-spreading reflex. We found that toe extension, abolished by nerve crush, recovered on schedule in WT controls but failed to recover even at 70 days in mutants (Figure 7D). To measure sensory motor coordination we measured the sciatic functional index (SFI; Inserra et al., 1998). We found the expected reduction in SFI following sciatic nerve crush in WT and c-Jun mutants, but a permanent failure of recovery in mutants only (Figure 7E). These experiments show that Schwann cell c-Jun is a necessary driver of functional recovery of injured peripheral nerves. c-Jun Is Sufficient to Generate a Growth-Supportive Nerve Phenotype Having shown that c-Jun activation is necessary for the conversion of injured nerves to an environment that supports repair, we tested whether c-Jun activation alone was sufficient for this critical transformation. We took advantage of Wld s mice, in which axons degenerate slowly after cut, and Schwann cells therefore remain differentiated and unsupportive of repair (Coleman and Freeman, 2010). We confirmed delayed regeneration in these mice compared to WT controls using the nerve pinch test and counting galanin-positive fibers distal to nerve crush (Figures 7F and 7H). Previously we showed that Schwann cells in Wld s nerves fail to activate c-Jun after injury (Jessen and Mirsky, 2008). We therefore used adenoviral infection to enforce c-Jun expression in crushed Wld s nerves (Figure S6). Remarkably, this converted the inhospitable Wld s nerves to a terrain that supported regeneration as effectively as WT nerves (Figures 7F–7H). This shows that c-Jun is not only necessary but also sufficient for the generation of a growth supporting environment in injured nerves. This observation also confirms that regeneration failure in Wld s mice is caused by a failure of timely Schwann cell injury response in these animals. Discussion Identification of transcription factors that define cell type, control transit between differentiation states, and enable tissues to repair is a central issue in regenerative biology. The response of Schwann cells to injury provides an exceptionally striking example of a phenotypic transition by adult, differentiated cells. This process is also the basis for the singular regenerative power of peripheral nerves. We show that the Schwann cell injury response represents a c-Jun dependent natural reprogramming of differentiated Schwann cells to generate the repair cell, a distinct Schwann cell state (Bungner cell, since they form Bungner’s bands) specialized to promote regeneration. In mice without c-Jun in Schwann cells, activation of the repair program fails. The disregulated repair cell formed in these mutants is unable to support normal axonal regeneration, neuronal survival, myelin clearance, and macrophage activity. The result is a striking failure of functional recovery. In addition to the c-Jun mutant, Wld s mice represent another situation where Schwann cell c-Jun is not activated during attempted regeneration after injury (Jessen and Mirsky, 2008). In this case also, the result is substantial reduction in regeneration. We find that enforced c-Jun expression in injured Wld s nerves is sufficient to restore axonal regeneration rates to WT values, lending significant support to our model. It is important to note that although the Bungner cells generated in the mutants are dysfunctional, other Schwann cell functions are normal. Thus, mutant cells remyelinated those axons that regenerated, Schwann cell development appeared normal, and Schwann cells and nerve function in uninjured adults were normal. Although 172 genes were disregulated in the distal stump of the c-Jun mutants, the large majority of the ∼4,000 genes regulated in injured WT nerves remained normally regulated. Therefore, the absence of c-Jun does not have a general impact on the Schwann cell phenotype. Instead, c-Jun appears to have a specific function in adult cells, where it is required for activation of the repair program and timely suppression of the myelin program. The Schwann Cell Injury Response Resembles Transdifferentiation The Schwann cell response to injury is commonly referred to as dedifferentiation, implying that adult denervated cells revert to an earlier stage resembling the immature Schwann cells of perinatal nerves (Harrisingh et al., 2004; Jessen and Mirsky, 2005; Chen et al., 2007; Woodhoo et al., 2009). It is becoming clear, however, that this view is incomplete. These cells have a different structure, molecular profile, and function. Therefore, the immature cells, generated from Schwann cell precursors during development, and Bungner cells generated in response to adult nerve injury, represent two distinct differentiation states. In injured nerves, myelinating Schwann cells, that are specialized to support fast conduction of action potentials, transform to Bungner cells that are specialized for the unrelated task of organizing nerve repair. This represents an unambiguous change of function, brought about by the combination of dedifferentiation and activation of an alternative differentiation program, the c-Jun dependent Schwann cell repair program. Transitions that share this set of features have been described in other systems, where they are generally referred to as transdifferentiation (Jopling et al., 2011). c-Jun Is a Global Regulator of a Schwann Cell Repair Program The regeneration defects in the c-Jun mutant are substantially more severe than those reported for other mouse mutants, in spite of the fact that the genetic defect is restricted to Schwann cells. The likely reason is the number and diversity of the molecules controlled by this single transcription factor. Among the 172 molecules that are abnormally expressed in the mutant are growth factors, adhesion molecules, growth-associated proteins, and transcription factors. This allows c-Jun to integrate a broad collection of functions that support nerve regeneration, and therefore to act as a global regulator of the Schwann cell repair program. This program involves regulation of molecules that have been directly implicated in repair such as the surface proteins N-cadherin, p75NTR, and NCAM, and the signaling molecules GDNF, artemin, sonic hedgehog, and BDNF. It also includes the morphogenetic processes that change myelinating cells to process bearing cells forming regeneration tracks, and the conversion of Schwann cells to cells equipped to rapidly clear myelin from injured nerves (Stoll et al., 2002; Chen et al., 2007; Vargas and Barres, 2007; Wang et al., 2008; Gordon et al., 2009; Höke and Brushart, 2010; Angeloni et al., 2011). The exceptional repair potential of peripheral nerves is likely due to the coordinated functions of the repair program. Yet individual factors can also be presumed to play a prominent role, as exemplified by the enhanced regeneration seen when GDNF and artemin levels are increased in c-Jun mutant facial nerves (Fontana et al., 2012). Activation of c-Jun and Interaction with Other Transcriptional Regulators c-Jun is absent from Schwann cell precursors, expressed in immature cells in vivo and in cultured Schwann cells, suppressed by Krox-20 on myelination, but rapidly re-expressed at high levels in Schwann cells of injured nerves (Parkinson et al., 2004, 2008; D.K.W., unpublished). Among potential intracellular activators of c-Jun is the AP-1 transcription complex, of which c-Jun is a key component. AP-1 activity, in turn, is controlled by numerous signals, including the major MAPK pathways Erk1/2, JNK, and p38. These are all activated in injured nerves and therefore potential upstream regulators of c-Jun (Sheu et al., 2000; Myers et al., 2003; Harrisingh et al., 2004; Jessen and Mirsky, 2008: Parkinson et al., 2008; Napoli et al., 2012; Yang et al., 2012). Genetically, the transcription factor Sox2 is not downstream of c-Jun, since Sox2 remains normally upregulated in injured c-Jun mutant nerves (Figure S4). We described previously that c-Jun shows cross-inhibitory interactions with the pro-myelin transcription factor Krox20 (Parkinson et al., 2008). Mirroring the function of c-Jun in denervated cells, Krox20 is involved in the regulation of 100–200 genes in myelinating Schwann cells (P. Topilko, personal communication) and is required for the normal activation of the myelin program. We therefore suggest that Krox20/c-Jun are central components of a cross-inhibitory switch that regulates cell fate in injured and regenerating nerves. Macrophage Recruitment and Activation The long term persistence of Schwann cell lipid droplets and large multivacuolated (foamy) macrophages in transected mutant nerves suggests problems with lipid clearance and macrophage activation and exit. Recent evidence indicates that failure of lipid breakdown may delay regeneration (Winzeler et al., 2011). The reduced macrophage numbers in the mutant early after injury is unlikely to contribute substantially to the regeneration problems, a conclusion supported by the microfluidic chamber experiments, where axon growth fails in the presence of mutant Schwann cells, even in the absence of macrophages. Even severe depletion of invading macrophages has no effect on the number of myelinated axons in dorsal roots following nerve injury (Barrette et al., 2008). There Is Extensive Death of Injured Neurons in c-Jun Mutants The disregulated mutant Bungner cell not only fails to support axon regeneration, but also fails to rescue injured neurons from death. In the mutants, injured type B DRG neurons are about twice as likely to die as in WT mice. Even more notable is the death of about a third of type A neurons, because we find no death of these cells in WT animals, in agreement with previous work in mice and other species (Jiang and Jakobsen, 2004). The majority of facial motoneurons also die after facial nerve injury in the mutant (Fontana et al., 2012). The Identity of the Bungner Repair Cell The observation that denervated adult Schwann cells acquire the ability to generate melanocytes, a property of Schwann cell precursors but not of immature Schwann cells (Adameyko et al., 2009), raises an intriguing possibility. Namely that after injury, Schwann cells dedifferentiate past the immature Schwann cell stage to a cell that shares some properties in common with the Schwann cell precursor. c-Jun is not significantly expressed in Schwann cell precursors (D.K.W., unpublished). It is therefore possible that the unique identity of the Bungner repair cell in adult nerves consists of a c-Jun-activated repair program in a cell that in significant other aspects has dedifferentiated more completely than hitherto envisaged. It is clear that the transdifferentiation of myelinating cells to Bungner cells is central to nerve repair. But much remains to be learned about the twin components of this process, the dedifferentiation and repair programs, and about the molecular links that integrate them. This includes issues of practical importance such as the identification of methods to sustain expression of the repair program over the long periods required for nerve repair in humans, and the question of whether the repair program can be activated in other glial cells. Experimental Procedures Animal experiments conformed to UK Home Office guidelines. P 0- CRE + /c-Jun fl/fl mice were generated as described (Parkinson et al., 2008). P 0- CRE − /c-Jun fl/fl littermates were used as controls. c-Jun was excised from c-Jun fl/fl cells using adenovirally expressed CRE-recombinase. Experiments for which n numbers are not shown in figure legends were done at least three times. Nerve Injury Sciatic nerves of adult mice were cut or crushed at the sciatic notch. Microarray Hybridization RNA was extracted, cDNA generated and applied to Mouse 430 2.0 array (Affymetrix, MA). Significantly different genes were selected with Bayes’ t test. After control for false discovery rate, genes with a p value of less than 0.05 were filtered out. The microarray data are MIAME compliant. In Situ Hybridization This was performed as described (Lee et al., 1997). RT-QPCR QPCR was performed with Sybrgreen SYBR Green JumpStart (Sigma) and carried out using Chromo4 Real Time Detector (Bio-Rad). For primers see Table S5. Data was analyzed using Opticon monitor 3 software and fold-changes determined with the Livak method (see Supplemental Information). Adenoviral Infection Adenovirus expressing c-Jun was generated by cloning mouse c-jun sequence into pAdTrack-CMV. This was recombined into adenoviral backbone plasmid pAdEasy-1 in bacteria. Schwann cell cultures (Dong et al., 1999) were infected with purified adenoviral supernatants (Parkinson et al., 2001, 2008). Immunocytochemistry Nerve segments, spinal cords or Schwann cell cultures were fixed in paraformaldehyde (PF)/PBS for 10 min–2 hr. Sections were fixed in 2% or 4% PF/PBS for 10 min or methanol for 30 min prior to immunolabeling. Alternatively, nerves were fixed in PF/PBS for 24 hr and wax embedded. Four micrometer sections were deparaffinized and antigen retrieved prior to immunolabeling. Blocking solution was used before incubation with primary antibodies overnight followed by secondary antibodies for 30 min to 1 hr. The first layer was omitted as a control. Functional Tests The nerve pinch test was used to assess axonal regeneration distance in vivo. Sensory motor coordination was assessed using mouse footprints to calculate the sciatic functional index. Sensory function was assessed by Von Frey Hair analysis, the Hargreaves test and response to toe pinching. Motor function was analyzed by observing toe spread (see Supplemental Information). Retrograde Tracer Labeling True Blue (2 μl) was injected into the tibialis anterior muscle at three sites to label motor neurons in spinal cord segments L2 to L6. Seven days later, mice were perfused. Serial 30 μm sections were collected and the number of labeled neurons was counted (Supplemental Information). Counts of DRG Neurons, Schwann Cells, and Macrophages The L4 DRG was cryosectioned. DRG neurons (nuclei) were counted as described (Puigdellívol-Sánchez et al., 2000). Ten micrometer serial sections were labeled with Neurotrace fluorescent Nissl green stain. Every sixth section was analyzed and systematic random sampling (SRS; see Supplemental Information) applied to ensure unbiased estimation of neuron numbers. A and B cells were differentiated on size and morphological criteria as described (Tandrup et al., 2000). For further confirmation, A cells in 10 week cut WT and mutant DRG were quantified by nucleolar counts (Jiang and Jakobsen, 2010). Both nuclear and nucleolar counts were corrected as described in Abercrombie (1946). Schwann cells and macrophages in injured tibial nerves were counted in whole transverse sections in the electron microscope using SRS (see Supplemental Information). Lipid Labeling Following PF fixation, 10 μm sections were treated with 2% OsO4-PBS solution overnight. Percentage stained nerve area relative to that in uninjured nerves was quantified using NIH ImageJ. Western Blotting Frozen nerve samples or cell lysates were blotted as described (Parkinson et al., 2004). Microfluidic Chambers Using a three-compartment microfuidic chamber (Taylor et al., 2005), 5,000 adult DRG neurons were plated in the central compartment in defined medium with 50 mM glucose (Dong et al., 1999). 2 × 105 WT Schwann cells, c-Jun null cells or c-Jun null cells infected with c-Jun adenovirus were plated in the side chambers. The number of axons longer than 50 μm growing into the side compartment was counted. Alternatively the area of axonal elongation into the side chambers from the microgrooves up to 300 μm beyond them was measured using ImageJ. Ultrathin Sections Thickness of Myelin Sheath (g Ratio) Photographs (15–20) were taken at 2500× of transverse ultrathin sections of tibial nerve 5 mm from the sciatic notch. The g ratio was calculated for each myelinated fiber (axon diameter divided by diameter of the axon and myelin sheath). Statistical difference was measured using Mann Whitney U test. Counts of Myelinated Axons in Adult Tibial Nerve and Dorsal and Ventral Roots after Nerve Crush or Cut A montage of ultrathin sections (×1000) was made and the number of myelinated fibers counted. For unmyelinated fibers, 30%–40% of each nerve was photographed (×5000). The ratio myelinated:unmyelinated fibers was measured and the total for each nerve/root was multiplied by total myelin fiber count, as described elsewhere (Coggeshall et al., 1997). For counts of Schwann cells and macrophages, ultrathin sections were mounted on film, nuclei counted in every third field and multiplied by 3 to generate totals. Statistical difference was measured using Mann Whitney U test. Migration Assays Macrophage migration was assessed using 6.5 mm Transwells with 5 μm pores (Corning Costar; see Supplemental Information). Statistical Analysis Data are presented as arithmetic mean ± standard error of the mean (SEM) unless otherwise stated. Statistical significance was estimated by Student’s t test, two-way ANOVA, or Mann-Whitney U-test.

A patch with the capability of avoiding wound infection and promoting tissue remolding is of great value for wound healing. In this paper, we develop a biomass chitosan microneedle array (CSMNA) patch integrated with smart responsive drug delivery for promoting wound healing. Chitosan possesses many outstanding features such as the natural antibacterial property and has been widely utilized for wound healing. Besides, the microstructure of microneedles enables the effective delivery of loaded drugs into the target area and avoids the excessive adhesion between the skin and the patch. Also, vascular endothelial growth factor (VEGF) is encapsulated in the micropores of CSMNA by temperature sensitive hydrogel. Therefore, the smart release of the drugs can be controllably realized via the temperature rising induced by the inflammation response at the site of wounds. It is demonstrated that the biomass CSMNA patch can promote inflammatory inhibition, collagen deposition, angiogenesis, and tissue regeneration during the wound closure. Thus, this versatile CSMNA patch is potentially valuable for wound healing in clinical applications.