Epidermal Wound Research Articles

Systemic cell cycle activation is induced following complex tissue injury in axolotl

Activation of progenitor cells is crucial to promote tissue repair following injury in adult animals. In the context of successful limb regeneration following amputation, progenitor cells residing within the stump must re-enter the cell cycle to promote regrowth of the missing limb. We demonstrate that in axolotls, amputation is sufficient to induce cell-cycle activation in both the amputated limb and the intact, uninjured contralateral limb. Activated cells were found throughout all major tissue populations of the intact contralateral limb, with internal cellular populations (bone and soft tissue) the most affected. Further, activated cells were additionally found within the heart, liver, and spinal cord, suggesting that amputation induces a common global activation signal throughout the body. Among two other injury models, limb crush and skin excisional wound, only limb crush injuries were capable of inducing cellular responses in contralateral uninjured limbs but did not achieve activation levels seen following limb loss. We found this systemic activation response to injury is independent of formation of a wound epidermis over the amputation plane, suggesting that injury-induced signals alone can promote cellular activation. In mammals, mTOR signaling has been shown to promote activation of quiescent cells following injury, and we confirmed a subset of activated contralateral cells is positive for mTOR signaling within axolotl limbs. These findings suggest that conservation of an early systemic response to injury exists between mammals and axolotls, and propose that a distinguishing feature in species capable of full regeneration is converting this initial activation into sustained and productive growth at the site of regeneration.

Disinfection and healing effects of 222-nm UVC light on methicillin-resistant Staphylococcus aureus infection in mouse wounds

UVC radiation is known to be highly germicidal. However, exposure to 254-nm-UVC light causes DNA lesions such as cyclobutane pyrimidine dimers (CPD) in human cells, and can induce skin cancer after long-term repeated exposures. It has been reported that short wavelength UVC is absorbed by proteins in the membrane and cytosol, and fails to reach the nucleus of human cells. Hence, irradiation with 222-nm UVC might be an optimum combination of effective disinfection and biological safety to human cells. In this study, the biological effectiveness of 222-nm UVC was investigated using a mouse model of a skin wound infected with methicillin-resistant Staphylococcus aureus (MRSA). Irradiation with 222-nm UVC significantly reduced bacterial numbers on the skin surface compared with non-irradiated skin. Bacterial counts in wounds evaluated on days 3, 5, 8 and 12 after irradiation demonstrated that the bactericidal effect of 222-nm UVC was equal to or more effective than 254-nm UVC. Histological analysis revealed that migration of keratinocytes which is essential for the wound healing process was impaired in wounds irradiated with 254-nm UVC, but was unaffected in 222-nm UVC irradiated wounds. No CPD-expressing cells were detected in either epidermis or dermis of wounds irradiated with 222-nm UVC, whereas CPD-expressing cells were found in both epidermis and dermis irradiation with 254-nm UVC. These results suggest that 222-nm UVC light may be a safe and effective way to reduce the rate of surgical site and other wound infections.

Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration.

Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration.

Opposing Roles of Epidermal Integrins α3β1 and α9β1 in Regulation of mTLD/BMP-1–Mediated Laminin-γ2 Processing during Wound Healing

Proteolytic processing of the laminin-γ2 chain is a hallmark of basement membrane maturation in the skin. Integrin α3β1, a major receptor for epidermal adhesion to laminin-332, is critical for proper basement membrane organization during skin development and wound healing. Previously, we identified a role for α3β1 in promoting the processing of laminin-γ2 in cultured keratinocytes invitro and in wound epidermis invivo. In this study we identify the Bmp1 gene, which encodes variants of the mTLD/BMP-1 metalloproteases, as a critical regulator of α3β1-dependent laminin-γ2 processing, thereby expanding the role of this integrin in controlling the secretion by the epidermis of factors that modulate the tissue microenvironment. Because our previous studies identified another epidermal integrin, α9β1, as a suppressive regulator of α3β1-dependent wound angiogenesis, we investigated whether α9β1 has a similar cross-suppressive effect on the ability of α3β1 to promote basement membrane organization. Here, we show that, rather than a cross-suppressive role, α9β1 has an opposing role in basement membrane assembly/maturation through reduced laminin-γ2 processing via mTLD/BMP-1. Although α3β1 promotes this process during wound healing, α9β1 has an inhibitory role, suggesting that regulation of basement membrane assembly requires a complex interplay between these distinct epidermal integrins.

Hyaluronate likely contributes to the immunesuppression of the regenerating tail blastema in lizards: Implications for organ regeneration in amniotes

AbstractThe regenerating tail blastema of lizards appears immunesuppressed, and the distribution of hyaluronate, a possible agent of immunesuppression, is evaluated. Transcriptome data show upregulation of genes stimulating hyaluronate production in the tail and limb blastemas. The distribution of hyaluronate in the tail and limb blastemas has been studied using alcian blue at pH 2.5 and 1.0 and by immunohistochemistry for hyaluronate receptors. Both tail and limb blastemas initially contain hyaluronate, but this glycosaminoglycan disappears in the scarring limb blastema at 25‐ to 30‐day postamputation after intense inflammation. The regenerating tail tip moves distally during regeneration but conserves hyaluronate that is replaced by sulphated glycosaminoglycans and glycoproteins in proximal regions of the regenerating tail. Hyaluronate receptors are diffuse among blastema cells and ependyma of the tail, intense in wound epidermis but low to absent in the scarring limb. The electron microscope shows that an amorphous extracellular matrix likely containing hyaluronate surrounds mesenchymal cells, ependyma and wound epidermis in the tail but is scarce to absent in the limb, whereas collagen fibrils prevail. Extracellular matrix containing hyaluronate may shield blastema cells from circulating immune cells preventing deleterious immune reactions in the tail but not in the limb, a process leading to scarring.

Immunohistochemical and western blot analysis suggest that the soluble forms of FGF1-2 and FGFR1-2 sustain tail regeneration in the lizard

Fibroblast Growth Factors 1-2 (FGF1-2) stimulate tail regeneration in lizards and therefore the distribution of their receptors, FGFR1-2, in the regenerating tail of the lizard. Podarcis muralis has been studied using immunofluorescence and western blotting. Immunoreactive protein bands at 15–16kDa for FGF1-2 in addition to those at 50–65kDa are detected in the regenerating epidermis, but weak bands at 35, 45 and 50kDa appear from the regenerating connective tissues. Strongly immunolabeled bands for FGFR1 at 32, 60, and 80kDa and less intense for FGFR2 only appear in the regenerating tail. In normal tail epidermis and dermis, higher MW forms are present at 80 and 115–140kDa, respectively, but they disappear in the regenerating epidermis and dermis where low MW forms of FGFR1-2 are found at 50–70kDa. Immunolocalization confirms that most FGFR1-2 are present in the wound epidermis, Apical Epidermal Peg, ependymal tube while immunolabeling lowers in regenerating muscles, blastema cells, cartilage and connectives tissues. The likely release of FGFs from the Apical Epidermal Peg and ependyma and the presence of their receptors in these tissues may determine the autocrine stimulation of proliferation and a paracrine stimulation of the blastema cells through their FGF Receptors.

FGFs Treatment on Amputated Lizard Limbs Stimulate the Regeneration of Long Bones, Opening New Avenues for Limb Regeneration in Amniotes: A Morphological Study

Previous studies indicated that Fibroblast Growth Factors (FGFs) are present during tail and early limb regeneration in lizards, but FGFs disappear in the limb that turns into a scar and does not regenerate at 25–40 days post-amputation. Based on these indications, the aim of the present study was to evaluate the influence of administered FGFs on limb regeneration in lizards by injections of FGF1–2 into amputated hind-limbs that were studied between 40 and 70 days post-amputation. Outgrowths of 2.0 to 3.5 mm were produced but they did not develop an autopodium during this period. The skin remained most un-scaled, resembling that of a tail blastema. Four hours before sacrifice, the animals were injected with 5BrdU to study cell proliferation using microscopic and immunofluorescent methods. Histological examination of the outgrowths at 40–70 days of regeneration showed the presence a rod of cartilage (femur), or partially or completely sub-divided into two parts likely corresponding to a tibia and fibula. The regenerated cartilage was in continuity with the transected long bones and was surrounded by a perichondrium and a dense connective tissue, sparse nerves while muscles were reduced or absent. Qualitative observations on 5BrdU-immunolabeling indicated that most proliferating cells were present in the apical wound epidermis, the apical-most perichondrium and in the regenerating scales at 40–60 days post-amputation, but decreased at 70 days. Few 5BrdU-labeled cells were seen in other tissues, including in the regenerated cartilages. The present study indicates that FGF1-2 treatment in lizards mainly stimulate cartilage regeneration and the formation of a thick epidermis with an Apical Epidermal Peg, the epidermal micro-region that favors regeneration. In summary, these results suggest that FGFs treatments on amputated limbs could also be attempted in others amniotes, including mammals. However FGFs are not capable to induce an autopodium, which requires further signaling factors for its formation.

Effects of seawater immersion on the inflammatory response and oxygen free radical injury of rats with superficial partial-thickness scald at early stage

Objective: To study the effects of seawater immersion on the inflammatory response and oxygen free radical injury of rats with superficial-thickness scald at early stage. Methods: Seventy Wistar rats were divided into healthy control group (HC, n=7), pure scald group (PS, n=21), scald+ fresh water immersion group (SF, n=21), and scald+ seawater immersion group (SS, n=21) according to the random number table. Rats in group HC did not receive any treatment, while 5% total body surface area superficial partial-thickness scald was made on the back of rats in the latter three groups. Rats in group PS lived freely immediately post burn, while wounds on the back of rats in groups SF and SS were immersed into fresh water and seawater, respectively. Serum and full-thickness skin tissue in the center of wounds on the back of 7 rats in groups PS, SF, and SS at post immersion (injury) hour (PIH) 2, 4, and 6 were collected, respectively, while serum and full-thickness skin tissue at the same position of the 7 rats in group HC were collected at PIH 6 of rats in other groups. Morphology of skin tissue was observed with HE staining; tumor necrosis factor-alpha (TNF-α) content in serum and skin tissue was determined by enzyme-linked immunosorbent assay; superoxide dismutase (SOD) content in serum and skin tissue was determined by hydroxylamine method; malondialdehyde content in serum and skin tissue was determined by thiobarbituric acid method. Data were processed with analysis of variance of factorial design, one-way analysis of variance, Welch test, LSD test, and Tamhane test. Results: (1) Epidermal cells of skin tissue of rats in group HC arranged in order and continuously, and the dermis tissue and accessory structures were clear and complete. The skin layer and epidermis of wounds of rats in group PS had no significant change, but the edema of epidermis and dermis and infiltration of inflammatory cells enhanced over time at PIH 2, 4, and 6. The horny layer of epidermis of wounds of rats in group SF reduced, and the edema of epidermis and dermis and infiltration of inflammatory cells enhanced over time at PIH 2, 4, and 6; some epidermal cells disintegrated at PIH 6. The horny layer of epidermis of wounds of rats in group SS significantly reduced, along with the increase in disintegration of epidermal cells, the significant enhancement of edema of epidermis and dermis, and infiltration of a large number of inflammatory cells over time at PIH 2, 4, and 6. (2) Compared with (247±27) pg/mL in group HC, the serum content of TNF-α of rats in group PS significantly increased at PIH 2 and 4 [respectively (675±122) and (367±54) pg/mL, P<0.05 or P<0.01] but significantly decreased at PIH 6 [(147±27) pg/mL, P<0.01]; the serum content of TNF-α of rats in group SF significantly decreased at PIH 6 [(90±24) pg/mL, P<0.01]; the serum content of TNF-α of rats in group SS significantly increased at PIH 2, 4, and 6 [respectively (1 646±58), (2 086±114), and (2 951±58) pg/mL, with P values below 0.01]. Compared with (364±123) U/mL in group HC, the serum content of SOD of rats in group PS significantly increased at PIH 2 and 4 [respectively (489±13) and (447±14) U/mL, with P values below 0.05]; the serum content of SOD of rats in group SF significantly decreased at PIH 6 [(282±13) U/mL, P<0.05]; the serum content of SOD of rats in group SS significantly increased at PIH 2 [(461±23) U/mL, P<0.05] but significantly decreased at PIH 4 and 6 [respectively (226±8) and (205±10) U/mL, with P values below 0.01]. Compared with that in group HC, the serum content of malondialdehyde of rats in groups PS, SF, and SS significantly increased at PIH 2, 4, and 6 (with P values below 0.01). (3) Compared with that in group HC, the TNF-α content in wound tissue of rats in groups PS and SS significantly increased at PIH 2, 4, and 6 (P<0.05 or P<0.01), and the TNF-α content in wound tissue of rats in group SF significantly increased at PIH 2 and 4 (with P values below 0.01). Compared with that in group HC, the SOD content in wound tissue of rats in groups PS and SF significantly increased at PIH 2, 4, and 6 (P<0.05 or P<0.01), and the SOD content in wound tissue of rats in group SS significantly increased at PIH 2 and 4 (with P values below 0.01). Compared with that in group HC, the malondialdehyde content in wound tissue of rats in groups PS, SF, and SS significantly increased at PIH 2, 4, and 6 (with P values below 0.01). Conclusions: Seawater immersion can enhance the inflammatory response and oxygen free radical injury of wounds and the whole body of rats with superficial partial-thickness scald at early stage.

Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing

Cell–cell fusion is widely observed during development and disease, and imposes a dramatic change on participating cells. Cell fusion should be tightly controlled, but the underlying mechanism is poorly understood. Here, we found that the JAK/STAT pathway suppressed cell fusion during wound healing in the Drosophila larval epidermis, restricting cell fusion to the vicinity of the wound. In the absence of JAK/STAT signaling, a large syncytium containing a 3-fold higher number of nuclei than observed in wild-type tissue formed in wounded epidermis. The JAK/STAT ligand-encoding genes upd2 and upd3 were transcriptionally induced by wounding, and were required for suppressing excess cell fusion. JNK (also known as Basket in flies) was activated in the wound vicinity and activity peaked at ∼8 h after injury, whereas JAK/STAT signaling was activated in an adjoining concentric ring and activity peaked at a later stage. Cell fusion occurred primarily in the wound vicinity, where JAK/STAT activation was suppressed by fusion-inducing JNK signaling. JAK/STAT signaling was both necessary and sufficient for the induction of βPS integrin (also known as Myospheroid) expression, suggesting that the suppression of cell fusion was mediated at least in part by integrin protein.

Review: Biological and Molecular Differences between Tail Regeneration and Limb Scarring in Lizard: An Inspiring Model Addressing Limb Regeneration in Amniotes.

Tissue regeneration in lizards represents a unique model of regeneration and scarring in amniotes. The tail and limb contain putative stem cells but also dedifferentiating cells contribute to regeneration. Following tail amputation, inflammation is low and cell proliferation high, leading to regeneration while the intense inflammation in the limb leads to low proliferation and scarring. FGFs stimulate tail and limb regeneration and are present in the wound epidermis and blastema while they disappear in the limb wound epidermis 2-3 weeks postamputation in the scarring outgrowth. FGFs localize in the tail blastema and the apical epidermal peg (AEP), an epidermal microregion that allows tail growth but is absent in the limb. Inflammatory cells invade the limb blastema and wound epidermis, impeding the formation of an AEP. An embryonic program of growth is activated in the tail, dominated by Wnt-positive and -negative regulators of cell proliferation and noncoding RNAs, that represent the key regenerative genes. The balanced actions of these regulators likely impede the formation of a tumor in the tail tip. Genes for FACIT and fibrillar collagens, protease inhibitors, and embryonic keratins are upregulated in the regenerating tail blastema. A strong downregulation of genes for both B and T-lymphocyte activation suggests the regenerating tail blastema is a temporal immune-tolerated organ, whereas a scarring program is activated in the limb. Wnt inhibitors, pro-inflammatory genes, negative regulators of cell proliferation, downregulation of myogenic genes, proteases, and oxidases favoring scarring are upregulated. The evolution of an efficient immune system may be the main limiting barrier for organ regeneration in amniotes, and the poor regeneration of mammals and birds is associated with the efficiency of their mature immune system. This does not tolerate embryonic antigens formed in reprogrammed embryonic cells (as for neoplastic cells) that are consequently eliminated impeding the regeneration of lost organs.

Communicating Science through a Novel Type of Journal.

Communicating Science through a Novel Type of Journal.

Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

AbstractThe immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.

Regulation of the Glycerol Transporter, Aquaporin-3, by Histone Deacetylase-3 and p53 in Keratinocytes

Aquaporin- (AQP) 3, a water and glycerol channel, plays an important role in epidermal function, with studies showing its involvement in keratinocyte proliferation, differentiation, and migration and in epidermal wound healing and barrier repair. Increasing speculation about the use of histone deacetylase (HDAC) inhibitors to treat skin diseases led us to investigate HDAC's role in the regulation of AQP3. The broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid induced AQP3 mRNA and protein expression in a dose- and time-dependent manner in normal keratinocytes. The SAHA-induced increase in AQP3 levels resulted in enhanced [3H]glycerol uptake in normal but not in AQP3-knockout keratinocytes, confirming that the expressed AQP3 was functional. Use of HDAC inhibitors with different specificities limited our exploration of the responsible HDAC member to HDAC1, HDAC2, or HDAC3. Cre-recombinase-mediated knockdown and overexpression of HDAC3 suggested a role for HDAC3 in suppressing AQP3 expression basally. Further investigation implicated p53 as a transcription factor involved in regulating HDAC inhibitor-induced AQP3 expression. Thus, our study supports the regulation of AQP3 expression by HDAC3 and p53. Because suberoylanilide hydroxamic acid is already approved to treat cutaneous T-cell lymphoma, it could potentially be used as a therapy for skin diseases like psoriasis, where AQP3 is abnormally expressed.

Wounding induces dedifferentiation of epidermal Gata6+ cells and acquisition of stem cell properties.

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

Yorkie regulates epidermal wound healing in Drosophila larvae independently of cell proliferation and apoptosis

Yorkie (Yki), the transcriptional co-activator of the Hippo signaling pathway, has well-characterized roles in balancing apoptosis and cell division during organ growth control. Yki is also required in diverse tissue regenerative contexts. In most cases this requirement reflects its well-characterized roles in balancing apoptosis and cell division. Whether Yki has repair functions outside of the control of cell proliferation, death, and growth is not clear. Here we show that Yki and Scalloped (Sd) are required for epidermal wound closure in the Drosophila larval epidermis. Using a GFP-tagged Yki transgene we show that Yki transiently translocates to some epidermal nuclei upon wounding. Genetic analysis strongly suggests that Yki interacts with the known wound healing pathway, Jun N-terminal kinase (JNK), but not with Platelet Derived Growth Factor/Vascular-Endothelial Growth Factor receptor (Pvr). Yki likely acts downstream of or parallel to JNK signaling and does not appear to regulate either proliferation or apoptosis in the larval epidermis during wound repair. Analysis of actin structures after wounding suggests that Yki and Sd promote wound closure through actin regulation. In sum, we found that Yki regulates an epithelial tissue repair process independently of its previously documented roles in balancing proliferation and apoptosis.

Immunodetection of FGF1-2 and FGFR1-2 Indicates that these Proteins Disappear in the Wound Epidermis and Blastema of the Scarring Limb in Lizard

In lizards while the tail broadly regenerates after amputation the limb generally forms short scarring outgrowths FGFs are important growth factors that are present in regenerating tissues and appear to stimulate tail regeneration in lizards In the limb immunohistochemistry at days post amputation indicates that FGFs and FGFR are present with different intensity in the wound regenerating epidermis and sparsely in connective muscle and cartilaginous cells of the healing tissues of the stump However the immunofluorescence study indicate that FGF and FGF receptors are little or no immunodetectable at days post amputation especially in the wound epidermis when the limb blastema is turning into a scarring connective tissue of the short limb outgrowth Western blot analysis shows no low MW bands for FGF and altered or degraded bands of FGFR in the limb at days post amputation when scarring is taking place The present observations indicate that the disappearance of these proteins is related to loss of cell proliferation that leads to the failure of limb regeneration in lizards The study further stress the essential role of FGFs and their receptors in the stimulation of organ regeneration in lizards and likely in other amniotes

850 After skin wounding, noncoding dsRNA coordinates prostaglandins and WNT7b to promote regeneration

In a rare example akin to organogenesis in adult mammals, large full-thickness skin wounds in mice and rabbits lead to de novo morphogenesis of hair follicles. It is still not fully understood what controls this process, known as Wound Induced Hair Neogenesis (WIHN). Previously, our group has demonstrated that double stranded RNA (dsRNA) released during wounding is both necessary and sufficient to stimulate WIHN through Toll like receptor 3 (TLR3), coincident with activation of WNT/β-catenin signaling. In other systems, prostaglandin E2 (PGE2) is an important effector of regeneration and has been shown to stimulate the WNT/β-catenin pathway. Our microarray data shows WNT7b is the most potently enriched WNT transcript induced by dsRNA (#187 out of 49,395 transcripts). Thus, we hypothesize that dsRNA and TLR3-mediated damage-sensing stimulate a PG-dependent mechanism to activate the WNT7b/β-catenin pathway, and WIHN. We find that Wnt7b expression is induced by synthetic dsRNA (poly I:C) in both the epidermis of healing mouse skin wounds and cultured normal human epidermal keratinocytes (P < 0.05, N=3). Mice lacking TLR3 and epidermal STAT3 display a marked reduction of Wnt7b expression (P < 0.05, N=8). Supportively, IL-6 rescues WIHN and Wnt7b levels in TLR3 null mice (P < 0.05, N=8). Additionally, we find the amount and kinetics of WNT7b expression induced by poly I:C closely correlated to that of prostaglandin-endoperoxide synthase 2 (PTGS2/COX2), which was also required for WNT7b protein levels based on PTGS2-siRNA treatment of human keratinocytes (P < 0.05, N=3). In vivo results show that the Ptgs2 inhibitor celecoxib reduces dsRNA-induced WIHN (P < 0.05, N=32) and Wnt7b. Exogenous PGE2 can rescue WIHN and Wnt7b (P < 0.05, N=21). Collectively, these results highlight TLR3-mediated damage sensing mechanism as a novel upstream coordinator of prostaglandin and Wnt levels in regeneration.

Erratum: Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice.

Erratum: Tissue-scale coordination of cellular behaviour promotes epidermal wound repair in live mice.

Transforming growth factor β1 regulates the expression of CCN2 in human keratinocytes via Smad-ERK signalling.

Connective tissue growth factor (CCN2/CTGF) and transforming growth factor β1 (TGF-β1) are important regulators of skin wound healing, but controversy remains regarding their expression in epithelial cell lineages. Here, we investigate the expression of CCN2 in keratinocytes during reepithelialisation and its regulation by TGF-β1. CCN2 was detected in the epidermis of healing full-thickness porcine wounds. Human keratinocytes were incubated with or without 10 ng/ml TGF-β1, and signalling pathways were blocked with 10-μM SIS3 or 20-μM PD98059. Semi-quantitative real-time PCR was used to study CCN2 mRNA expression, and western blot was used to measure CCN2, phosphorylated-ERK1/2, ERK1/2, phosphorylated-Smad3 and Smad2/3 proteins. CCN2 was transiently expressed in neoepidermis at the leading edge of the wound in vivo. In vitro, CCN2 expression was induced by TGF-β1 at 2 hours (7·5 ± 1·9-fold mRNA increase and 3·0 ± 0·6-fold protein increase) and 12 hours (5·4 ± 1·9-fold mRNA increase and 3·3 ± 0·6-fold protein increase). Compared with inhibiting the SMAD pathway, inhibiting the mitogen-activated protein kinase (MAPK) pathway was more effective in reducing TGF-β1-induced CCN2 mRNA and protein expression. Inhibition of the MAPK pathway had minimal impact on the activity of the SMAD pathway. CCN2 is expressed in keratinocytes in response to tissue injury or TGF-β1. In addition, TGF-β1 induces CCN2 expression in keratinocytes through the ras/MEK/ERK pathway. A complete understanding of CCN2 expression in keratinocytes is critical to developing novel therapies for wound healing and cutaneous malignancy.

Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine.

Enzymatically crosslinked microporous hydrogel scaffolds that form in situ promote dermal healing Evaluation of: Griffin DR, Weaver WM, Scumpia PO, DiCarlo D, Segura T. Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat. Mater. 14, 737–744 (2015). Tunable scaffolds that respond to environmental factors could enable enhanced tissue integration and wound healing. To achieve this goal, scaffold designs have incorporated biomimetic functionality, including thrombin-activated factor XIII (FXIIIa) crosslinking [1], matrix metalloproteinase responsiveness [2] and mechanical matching [3]. Scaffolds that can alter their properties in situ in response to environmental stimuli are desired to achieve changes in stiffness, shape, release and degradation that can lead to improved tissue regeneration. In this study, Griffin et al. describe a bottom-up strategy to fabricating scaffolds that accelerate wound healing [4]. They use a microfluidic approach to form hydrogel microparticle (microgel) building blocks that can establish interconnected network structures upon FXIIIa-mediated crosslinking. These microporous annealed particles (MAPs) incorporate reactive multiarmed poly(ethylene)glycol-vinyl sulfone, the commonly used RGD cell adhesion peptide and the K and Q noncanonical transglutaminase peptide substrates with cysteine-terminated matrix metalloproteinase-sensitive peptides that allow for cell-controlled material degradation. Scaffolds are annealed by FXIIIa addition and can be injected into wound sites for in situ solidification and shape-conformation. These MAPs are mechanically robust and can be tuned to achieve storage moduli from 10 to 1000 Pa, which fall within the stiffness range of soft tissues. In vitro studies demonstrate that MAP scaffolds enable 3D cellular network formation and cell proliferation and survival after 6 days. In vivo studies in SKH1-HR and Balb/c epidermal wound healing models demonstrate integration of MAP scaffolds after 24 h, significant wound closure as compared with nonporous chemically matched controls after 5 days or physically matched control scaffolds up to 7 days. 5 days following injection of MAP scaffolds, keratin-5, keratin-14 and CD49f staining revealed re-epithelialization, and PECAM-1, NG2 and PDGFR-β staining indicated that the scaffolds promoted complex vascular development. MAP scaffolds were also able to sustain normal hair follicle formation when compared with normal tissue. The scaffolds promoted less CD11b cell infiltration as compared with nonporous controls after 5 days, which suggests that MAP scaffolds cause a lower immune response. Taken together, MAP scaffolds promote accelerated wound healing by utilizing natural enzymatic mechanisms and mimicking favorable mechanical and degradation properties to facilitate cell infiltration and proliferation. The study may have benefited from evaluating wound closure, dermal regeneration and Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine