Tuning Macrophage Phenotype to Mitigate Skeletal Muscle Fibrosis.

Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-ß1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of Tgfb1 abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-ß1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.

Characterizing the Circulating Cell Populations in Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) occurs secondary to trauma, causing pain and functional limitations. Identification of the cells that contribute to HO is critical to the development of therapies. Given that innate immune cells and mesenchymal stem cells are known contributors to HO, we sought to define the contribution of these populations to HO and to identify what, if any, contribution circulating populations have to HO. A shared circulation was obtained using a parabiosis model, established between an enhanced green fluorescent protein-positive/luciferase+ donor and a same-strain nonreporter recipient mouse. The nonreporter mouse received Achilles tendon transection and dorsal burn injury to induce HO formation. Bioluminescence imaging and immunostaining were performed to define the circulatory contribution of immune and mesenchymal cell populations. Histologic analysis showed circulating cells present throughout each stage of the developing HO anlagen. Circulating cells were present at the injury site during the inflammatory phase and proliferative period, with diminished contribution in mature HO. Immunostaining demonstrated that most early circulatory cells were from the innate immune system; only a small population of mesenchymal cells were present in the HO. We demonstrate the time course of the participation of circulatory cells in trauma-induced HO and identify populations of circulating cells present in different stages of HO. These findings further elucidate the relative contribution of local and systemic cell populations to HO.

Analysis of Bone-Cartilage-Stromal Progenitor Populations in Trauma Induced and Genetic Models of Heterotopic Ossification.

Heterotopic ossification (HO), the formation of extra-skeletal bone in soft tissues, is a pathologic process occurring after substantial burns or trauma, or in patients with type I bone morphogenetic protein (BMP) receptor hyperactivating mutations. Identifying the cells responsible for de novo bone formation during adulthood is of critical importance for therapeutic and regenerative purposes. Using a model of trauma-induced HO with hind limb Achilles' tenotomy and dorsal burn injury and a genetic nontrauma HO model (Nfatc1-Cre/caAcvr1(fl/wt) ), we demonstrate enrichment of previously defined bone-cartilage-stromal progenitor cells (BCSP: AlphaV+/CD105+/Tie2-/CD45-/Thy1-/6C3-) at the site of HO formation when compared with marrow isolated from the ipsilateral hind limb, or from tissue of the contralateral, uninjured hind limb. Upon transplantation into tenotomy sites soon after injury, BCSPs isolated from neonatal mice or developing HO incorporate into the developing lesion in cartilage and bone and express chondrogenic and osteogenic transcription factors. Additionally, BCSPs isolated from developing HO similarly incorporate into new HO lesions upon transplantation. Finally, adventitial cells, but not pericytes, appear to play a supportive role in HO formation. Our findings indicate that BCSPs contribute to de novo bone formation during adulthood and may hold substantial regenerative potential. Stem Cells 2016;34:1692-1701.

Hair follicle specific ACVR1/ALK2 critically affects skin morphogenesis and attenuates wound healing.

The bone morphogenic protein signaling (BMP) is intricately involved in the quiescence and regulation of stem cells through activation of BMP receptors. Hair follicle stem cells play a critical role in cutaneous homeostasis and regeneration. Here, we utilize a novel mouse model with targeted overexpression of the BMP receptor ALK2/ACVR1 in hair follicle stem cells, to characterize its role in skin development and postnatal wound healing. Initial histologic evaluation demonstrated significant dysregulation in hair follicle morphogenesis in mutant mice. These demonstrated increased numbers of individual hair follicles with altered morphology and localization. Mutant follicles were found to exhibit elevated proliferative activity as well as increased prevalence of CD34 and ITGA6 positive follicle stem cells. Interestingly, constitutive overexpression of ALK2 resulted in attenuation of cutaneous wound healing. These findings demonstrate that hair follicle specific ALK2 is intricately involved in maintenance of the stem cell niche and wound healing.

Lymphatic Contribution to the Cellular Niche in Heterotopic Ossification.

OBJECTIVE: The objective of this study was to determine the contribution of lymphatic tissue to heterotopic ossification (HO). BACKGROUND: HO is the pathologic development of ectopic bone within soft tissues often following severe trauma. Characterization of the tissue niche supporting HO is critical to identifying therapies directed against this condition. Lymphangiogenesis is upregulated during incidents of trauma, thereby coincident with the niche supportive of HO. We hypothesized that lymphatic tissues play a critical role in HO formation. METHODS: Mice underwent hindlimb Achilles' tendon transection and dorsal burn injury (burn/tenotomy) to induce HO. The popliteal and inguinal lymph nodes were excised ipsilateral to the tenotomy site. Flow cytometry and immunostaining were used to quantify and localize lymphoendothelium. MicroCT was used to quantify HO. RESULTS: Enrichment of mature lymphatic tissues was noted 2 weeks after injury at the tendon transection sites when compared with the contralateral, intact tendon based on LYVE1+ tubules (10.9% vs 0.8%, P < 0.05). Excision of the inguinal and popliteal nodes with draining popliteal lymphatic vessel significantly decreased the presence of mature lymphoendothelium 2 weeks after injury (10.9% vs 3.3%, P < 0.05). Bone-cartilage-stromal progenitor cells (CD105+/AlphaV+/Tie2-/CD45-/CD90-/BP1-) were also significantly decreased after lymph node excision (10.2% vs 0.5%, P < 0.05). A significant decrease was noted in the volume of de novo HO present within the soft tissues (0.12 mm vs 0.02 mm). CONCLUSION: These findings suggest that lymphatic vessels are intimately linked with the de novo formation bone within soft tissues following trauma, and their presence may facilitate bone formation.

Tracking the elusive fibrocyte: identification and characterization of collagen-producing hematopoietic lineage cells during murine wound healing.

Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45- and Cd11b-, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing.

Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

OBJECTIVE: To investigate how epithelial mechanotransduction pathways impact wound repair. BACKGROUND: Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. METHODS: A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. RESULTS: Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. CONCLUSIONS: Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

Paracrine mechanism of angiogenesis in adipose-derived stem cell transplantation.

INTRODUCTION: Adipose-derived stem cells (ASCs) have shown potential for cell-based therapy in the field of plastic surgery. However, the fate of ASCs after transplantation and the mechanism(s) of their biologic capabilities remain unclear. METHODS: We isolated and cultured ASCs from transgenic mice that express both luciferase and green fluorescent protein and injected the cells into the inguinal fat pads of wild-type mice. We tested 4 experimental groups, namely, ischemic fat pads with/without ASCs and control fat pads with/without ASCs. RESULTS: Transplanted ASCs were tracked with bioluminescence imaging. The luminescence gradually decreased over 28 days, indicating cell death after transplantation. More ASCs were retained in ischemic fat pads on day 7 compared to control fat pads. On day 14, adipose tissue vascular density was higher in the ASC transplantation groups compared to those without ASCs. On day 28, there was decreased atrophy of adipose tissue in ASC-treated ischemic fat pads. Transplanted ASCs were detected as nonproliferating green fluorescent protein-positive cells, whereas native endothelial cells adjacent to the transplanted ASCs were proliferative. Protein analysis demonstrated higher expression of hepatocyte growth factor and vascular endothelial growth factor in the ASC transplantation groups, suggesting a paracrine mechanism, which was confirmed by in vitro experiments with conditioned media from ASCs. CONCLUSIONS: Transplanted ASCs are preferentially retained in ischemic adipose tissue, although most of the cells eventually undergo cell death. They exert an angiogenic effect on adipose tissue mainly through a paracrine mechanism. Increased understanding of these effects will help develop ASCs as a tool for cell-based therapy.

A Review of Laser Therapies for the Treatment of Scarring and Vascular Anomalies.

Significance: Laser use has become part of the gold standard of treatment as an effective adjuvant in multimodal therapy for pathologic scarring caused by burns, trauma, acne, and surgery, as well as vascular anomalies. Understanding indications and applications for laser therapy is essential for physicians to improve patient outcomes. Recent Advances: Since the 1980s, the medical use of lasers has continuously evolved with improvements in technology. Novel lasers and fractionated technologies are currently being studied in the hopes to improve treatment efficacy, while reducing complications. Recent advancements include acne treatment with novel picosecond lasers, new hypertrophic scar therapies with simultaneous laser and intense pulsed light use, and novel systems such as lasers with intralesional optical fiber delivery devices. In addition, optimizing the timing of laser therapy and its use in multimodal treatments continue to advance the field of photothermolysis. Critical Issues: Selecting the correct laser for a given indication is the fundamental decision when choosing a laser balancing effective treatment with minimal complications. This article covers the principles of laser therapy, the preferred lasers used for the treatment of scarring and vascular anomalies, and discusses the current evidence behind these laser choices. Future Directions: To optimize laser therapy, larger randomized control trials and split scar studies are needed. Continued advancement through better randomized controlled studies will help to improve patient outcomes on a broader scale.

Phosphorylation of IWS1 by AKT maintains liposarcoma tumor heterogeneity through preservation of cancer stem cell phenotypes and mesenchymal-epithelial plasticity.

Chemotherapy remains the mainstay of treatment for patients with advanced liposarcoma (LPS), but response rates are only 25% and the overall survival at 5 years is dismal at 20-34%. Translation of other therapies have not been successful and there has been no significant improvement in prognosis for nearly 20 years. The aberrant activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the aggressive clinical behavior LPS and in resistance to chemotherapy, but the precise mechanism remains elusive and efforts to target AKT clinically have failed. Here we show that the AKT-mediated phosphorylation of the transcription elongation factor IWS1, promotes the maintenance of cancer stem cells in both cell and xenograft models of LPS. In addition, phosphorylation of IWS1 by AKT contributes to a "metastable" cell phenotype, characterized by mesenchymal/epithelial plasticity. The expression of phosphorylated IWS1 also promotes anchorage-dependent and independent growth, cell migration, invasion, and tumor metastasis. In patients with LPS, IWS1 expression is associated with reduced overall survival, increased frequency of recurrence, and shorter time to relapse after resection. These findings indicate that IWS1-mediated transcription elongation is an important regulator of human LPS pathobiology in an AKT-dependent manner and implicate IWS1 as an important molecular target to treat LPS.

CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor ß1 (TGF-ß1) signaling.

Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-ß1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

Wnt-ß-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.

BACKGROUND & AIMS: Ischemia and reperfusion injury are common causes of oxidative tissue damage associated with many liver diseases and hepatic surgery. The Wnt-ß-catenin signaling pathway is an important regulator of hepatic development, regeneration, and carcinogenesis. However, the role of Wnt signaling in the hepatocellular response to ischemia-reperfusion (I/R) injury has not been determined. METHODS: Hepatic injury following ischemia or I/R was investigated in hepatocyte-specific, ß-catenin-deficient mice, as well as Wnt1-overexpressing and wild-type (control) mice. RESULTS: Wnt-ß-catenin signaling was affected by the cellular redox balance in hepatocytes. Following ischemia or I/R, mice with ß-catenin-deficient hepatocytes were significantly more susceptible to liver injury. Conversely, mice that overexpressed Wnt1 in hepatocytes were resistant to hepatic I/R injury. Hypoxia inducible factor (HIF)-1α signaling was reduced in ß-catenin-deficient liver but increased in hepatocytes that overexpressed Wnt1 under hypoxia and following I/R, indicating an interaction between ß-catenin and HIF-1α signaling in the liver. The mechanism by which Wnt signaling protects against liver injury involves the role of ß-catenin as a transcriptional coactivator of HIF-1α signaling, which promotes hepatocyte survival under hypoxic conditions. CONCLUSIONS: Cellular redox balance affects Wnt-ß-catenin signaling, which protects against hypoxia and I/R injury. These findings might be used to develop strategies for protection of hepatocytes, regeneration of liver, and inhibition of carcinogenesis.

Mechanical force prolongs acute inflammation via T-cell-dependent pathways during scar formation.

Mechanical force significantly modulates both inflammation and fibrosis, yet the fundamental mechanisms that regulate these interactions remain poorly understood. Here we performed microarray analysis to compare gene expression in mechanically loaded wounds vs. unloaded control wounds in an established murine hypertrophic scar (HTS) model. We identified 853 mechanically regulated genes (false discovery rate <2) at d 14 postinjury, a subset of which were enriched for T-cell-regulated pathways. To substantiate the role of T cells in scar mechanotransduction, we applied the HTS model to T-cell-deficient mice and wild-type mice. We found that scar formation in T-cell-deficient mice was reduced by almost 9-fold (P < 0.001) with attenuated epidermal (by 2.6-fold, P < 0.01) and dermal (3.9-fold, P < 0.05) proliferation. Mechanical stimulation was highly associated with sustained T-cell-dependent Th2 cytokine (IL-4 and IL-13) and chemokine (MCP-1) signaling. Further, T-cell-deficient mice failed to recruit systemic inflammatory cells such as macrophages or monocytic fibroblast precursors in response to mechanical loading. These findings indicate that T-cell-regulated fibrogenic pathways are highly mechanoresponsive and suggest that mechanical forces induce a chronic-like inflammatory state through immune-dependent activation of both local and systemic cell populations.

Macrophage TGF-ß signaling is critical for wound healing with heterotopic ossification after trauma.

Transforming growth factor-ß1 (TGF-ß1) plays a central role in normal and aberrant wound healing, but the precise mechanism in the local environment remains elusive. Here, using a mouse model of aberrant wound healing resulting in heterotopic ossification (HO) after traumatic injury, we find autocrine TGF-ß1 signaling in macrophages, and not mesenchymal stem/progenitor cells, is critical in HO formation. In-depth single-cell transcriptomic and epigenomic analyses in combination with immunostaining of cells from the injury site demonstrated increased TGF-ß1 signaling in early infiltrating macrophages, with open chromatin regions in TGF-ß1-stimulated genes at binding sites specific for transcription factors of activated TGF-ß1 (SMAD2/3). Genetic deletion of TGF-ß1 receptor type 1 (Tgfbr1; Alk5), in macrophages, resulted in increased HO, with a trend toward decreased tendinous HO. To bypass the effect seen by altering the receptor, we administered a systemic treatment with TGF-ß1/3 ligand trap TGF-ßRII-Fc, which resulted in decreased HO formation and a delay in macrophage infiltration to the injury site. Overall, our data support the role of the TGF-ß1/ALK5 signaling pathway in HO.

Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery.

BACKGROUND: Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. METHODS: In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. RESULTS: The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. CONCLUSION: The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

Surgical approaches to create murine models of human wound healing.

Wound repair is a complex biologic process which becomes abnormal in numerous disease states. Although in vitro models have been important in identifying critical repair pathways in specific cell populations, in vivo models are necessary to obtain a more comprehensive and pertinent understanding of human wound healing. The laboratory mouse has long been the most common animal research tool and numerous transgenic strains and models have been developed to help researchers study the molecular pathways involved in wound repair and regeneration. This paper aims to highlight common surgical mouse models of cutaneous disease and to provide investigators with a better understanding of the benefits and limitations of these models for translational applications.

Akt-mediated mechanotransduction in murine fibroblasts during hypertrophic scar formation.

Although numerous factors are implicated in skin fibrosis, the exact pathophysiology of hypertrophic scarring remains unknown. We recently demonstrated that mechanical force initiates hypertrophic scar formation in a murine model, potentially enhancing cellular survival through Akt. Here, we specifically examined Akt-mediated mechanotransduction in fibroblasts using both strain culture systems and our murine scar model. In vitro, static strain increased fibroblast motility, an effect blocked by wortmannin (a phosphoinositide-3-kinase/Akt inhibitor). We also demonstrated that high-frequency cyclic strain was more effective at inducing Akt phosphorylation than low frequency or static strain. In vivo, Akt phosphorylation was induced by mechanical loading of dermal fibroblasts in both unwounded and wounded murine skin. Mechanically loaded scars also exhibited strong expression of α-smooth muscle actin, a putative marker of pathologic scar formation. In vivo inhibition of Akt increased apoptosis but did not significantly abrogate hypertrophic scar development. These data suggest that although Akt signaling is activated in fibroblasts during mechanical loading of skin, this is not the critical pathway in hypertrophic scar formation. Future studies are needed to fully elucidate the critical mechanotransduction components and pathways which activate skin fibrosis.

NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma.

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFß to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.

Laser-mediated fixation of collagen-based scaffolds to dermal wounds.

BACKGROUND AND OBJECTIVE: Collagen scaffolds are popular for the reconstitution of dermal equivalents. Usually, these scaffolds are fixed with sutures or staples and in many cases these devices have to be removed in a second procedure. Laser-mediated tissue welding in a wet environment is a potential alternative for collagen scaffold fixation and may be advantageous to suture, staple, and tissue glue fixation. MATERIALS AND METHODS: Welding was performed with a continuous-wave diode laser system emitting radiation at a wavelength of 968 nm. Tensile strength after fixation to porcine skin and laser parameters were determined in vitro. In vivo, 24 excisional deep partial thickness wounds were created on flanks of two Goettingen mini pigs and covered with collagen scaffolds. These were randomized and fixated with either (1) staples, (2) fibrin glue, or (3) laser-mediated welding. Tissue biopsies for histological analysis were periodically performed and analyzed for wound healing progression, epidermal thickness, and extracellular matrix formation. RESULTS: Biomechanical stability after laser welding was time dependent. A dwell time of up to 10 seconds led to a strong bonding with a tensile strength of more than 30 g. In vivo, the wound healing process was macroscopically comparable in all groups and showed no significant differences. Microscopic analysis determined a more progressed and quicker wound closure in both the laser and staples group compared to the fibrin glue fixated scaffold. Laser-mediated fixation led to a significantly reduced epidermal thickness when compared with stapling or fibrin glue (P < 0.05). CONCLUSIONS: Laser tissue welding is a feasible approach for temporary fixation of collagen scaffolds to the wound bed. It improves wound healing properties and may lead to faster wound healing and cosmetically better scarring. Laser tissue welding is thus a very interesting and promising alternative to currently established fixation methods in a single step, no touch procedure.

Regulation of heterotopic ossification by monocytes in a mouse model of aberrant wound healing.

Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGFß1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic macrophage TGFß levels and help ameliorate HO. Our data thus implicate CD47 activation as a therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.