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Healing power: The mammalian macrophage in skeletal regeneration, scar formation, and regenerative medicine
... Macrophages play a significant role in the initial inflammatory phase [35] and orchestrate the tissue microenvironment at the wound site [36]. They undergo polarization into two distinct phenotypes: the antimicrobial and proinflammatory M1-macrophages, and the anti-inflammatory and pro-regenerative M2-macrophages [37]. ...
Despite reductions in bacterial infection and enhanced success rate, the widespread use of systemic antibiotic prophylaxis in implant dentistry is controversial. This use has contributed to the growing problem of antimicrobial resistance, along with creating significant health and economic burdens. The basic mechanisms that cause implant infection can be targeted by new prevention and treatment methods which can also lead to the reduction of systemic antibiotic exposure and its associated adverse effects. This review aims to summarize advanced biomaterial strategies applied to implant components based on anti-pathogenic mechanisms and immune balance mechanisms. It emphasizes that modifying the dental implant surface and regulating the early immune response are promising strategies, which may further prevent or slow the development of peri-implant infection, and subsequent failure.
... differentiated tissues is impossible after damage [4]. The defects of these tissues heal by replacement with fibrotic scars (regeneration by substitution) instead of epimorphic (complete) reparative regeneration [5][6][7]. This leads to the development of hypertrophic and keloid scars of the skin, cicatricial changes in tendons, osteoarthrosis, urethral strictures, and tracheal stenosis [8][9][10][11]. ...
Control over endogenous reparative mechanisms is the future of regenerative medicine. The rabbit ear defect is a rare model which allows the observation of the epimorphic regeneration of elastic cartilage. However, the mechanisms of phenotypical restoration of this highly differentiated tissue have not been studied. We modelled circular ear defects of different sizes (4, 6, and 8 mm in diameter) in 12 laboratory rabbits, and observed them during 30, 60, 90, and 120 day periods. Excised tissues were processed and analyzed by standard histological methods and special histochemical reactions for senescence associated-β-galactosidase and lectin markers. We demonstrated that larger defects caused significant elevation of senescence associated-β-galactosidase in chondrocytes. The fullness of epimorphic regeneration of elastic cartilage depended on the activation of cellular senescence and synthesis of elastic fibers. Further investigation into the role of cells with senescence-associated secretory phenotype in damaged tissues can present new targets for controlled tissue regeneration.
... Experts estimate that the mortality rate of liverrelated diseases will significantly rise in the upcoming years (Mikulic and Mrzljak, 2020). Fibrosis may be characterized a dysregulated wound healing process, resulting in the generation of tissue scars (Simkin et al., 2020). The presence of bridging fibers between portal regions is the signs of advanced stages of liver fibrosis (Chang and Li, 2020). ...
Liver fibrosis is a major cause of morbidity and mortality worldwide due to chronic liver damage and leading to cirrhosis, liver cancer, and liver failure. To date, there is no effective and specific therapy for patients with hepatic fibrosis. As a result of their various advantages such as biocompatibility, imaging contrast ability, improved tissue penetration, and superparamagnetic properties, magnetic nanoparticles have a great potential for diagnosis and therapy in various liver diseases including fibrosis. In this review, we focus on the molecular mechanisms and important factors for hepatic fibrosis and on potential magnetic nanoparticles-based therapeutics. New strategies for the diagnosis of liver fibrosis are also discussed, with a summary of the challenges and perspectives in the translational application of magnetic nanoparticles from bench to bedside.
... In this initial inflammatory phase, macrophages are widely involved [25], even if the injury is chronic and there is persistent inflammation [26]. Hence today macrophages are considered important to guide the tissue microenvironment at the site of the wound [27]. Macrophages polarize into two phenotypes: the antimicrobial and proin-flammatory M1-macrophages, and the anti-inflammatory and pro-regenerative M2-macrophages [28]. ...
There is a complex interaction between titanium dental implants, bone, and the immune system. Among them, specific immune cells, macrophages play a crucial role in the osseointegration dynamics. Infiltrating macrophages and resident macrophages (osteomacs) contribute to achieving an early pro-regenerative peri-implant environment. Also, multinucleated giant cells (MNGCs) in the bone-implant interface and their polarization ability, maintain a peri-implant immunological balance to preserve osseointegration integrity. However, dental implants can display cumulative levels of antigens (ions, nano and microparticles and bacterial antigens) at the implant–tissue interface activating an immune-inflammatory response. If the inflammation is not resolved or reactivated due to the stress signals and the immunogenicity of elements present, this could lead implants to aseptic loosening, infections, and subsequent bone loss. Therefore, to maintain osseointegration and prevent bone loss of implants, a better understanding of the osteoimmunology of the peri-implant environment would lead to the development of new therapeutic approaches. In this line, depicting osteoimmunological mechanisms, we discuss immunomodulatory strategies to improve and preserve a long-term functional integration between dental implants and the human body.
Scientific field of dental science: implant dentistry.
While mammals tend to repair injuries, other adult vertebrates like salamanders and fish regenerate damaged tissue. One prominent hypothesis offered to explain an inability to regenerate complex tissue in mammals is a bias during healing toward strong adaptive immunity and inflammatory responses. Here we directly test this hypothesis by characterizing part of the immune response during regeneration in spiny mice (Acomys cahirinus and Acomys percivali) vs. fibrotic repair in Mus musculus. By directly quantifying cytokines during tissue healing, we found that fibrotic repair was associated with a greater release of pro-inflammatory cytokines (i.e., IL-6, CCL2, and CXCL1) during acute inflammation in the wound microenvironment. However, reducing inflammation via COX-2 inhibition was not sufficient to reduce fibrosis or induce a regenerative response, suggesting that inflammatory strength does not control how an injury heals. Although regeneration was associated with lower concentrations of many inflammatory markers, we measured a comparatively larger influx of T cells into regenerating ear tissue and detected a local increase in the T cell associated cytokines IL-12 and IL-17 during the proliferative phase of regeneration. Taken together, our data demonstrate that a strong adaptive immune response is not antagonistic to regeneration and that other mechanisms likely explain the distribution of regenerative ability in vertebrates.
Regeneration is rare in mammals, but spiny mice (Acomys spp.) naturally regenerate skin and ear holes. Inflammation is thought to inhibit regeneration during wound healing, but aspects of inflammation contribute to both regeneration and pathogen defense. We compared neutrophil traits among uninjured, regeneration-competent (Acomys: A. cahirinus, A. kempi, A. percivali) and -incompetent (Mus musculus: Swiss Webster, wild-caught strains) murids to test for constitutive differences in neutrophil quantity and function between these groups. Neutrophil quantity differed significantly among species. In blood, Acomys had lower percentages of circulating neutrophils than Mus; and in bone marrow, Acomys had higher percentages of band neutrophils and lower percentages of segmented neutrophils. Functionally, Acomys and Mus neutrophils did not differ in their ability to migrate or produce reactive oxygen species, but Acomys neutrophils phagocytosed more fungal zymosan. Despite this enhanced phagocytosis activity, Acomys neutrophils were not more effective than Mus neutrophils at killing Escherichia coli. Interestingly, whole blood bacteria killing was dominated by serum in Acomys versus neutrophils only or neutrophils and serum in Mus, suggesting that Acomys primarily rely on serum to kill bacteria whereas Mus do not. These subtle differences in neutrophil traits may allow regeneration-competent species to offset damaging effects of inflammation without compromising pathogen defense.
Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.
We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.
We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.
Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.
Hypertrophic scars are pathological scars that result from abnormal responses to trauma, and could cause serious functional and cosmetic disability. To date, no optimal treatment method has been established. A variety of cell types are involved in hypertrophic scar formation after wound healing, but the underlying molecular mechanisms and cellular origins of hypertrophic scars are not fully understood. Macrophages are major effector cells in the immune response after tissue injury that orchestrates the process of wound healing. Depending on the local microenvironment, macrophages undergo marked phenotypic and functional changes at different stages during scar pathogenesis. This review intends to summarize the direct and indirect roles of macrophages during hypertrophic scar formation. The in vivo depletion of macrophages or blocking their signaling reduces scar formation in experimental models, thereby establishing macrophages as positive regulatory cells in the skin scar formation. In the future, a significant amount of attention should be given to molecular and cellular mechanisms that cause the phenotypic switch of wound macrophages, which may provide novel therapeutic targets for hypertrophic scars.
Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of
epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry
and sequential in-vivo microcomputed tomography (μCT). Mammalian regeneration is restricted to amputations transecting the distal region
of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation.
The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous
ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals,
and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol
uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of
regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the
use of sequential in vivo μCT to create high resolution images to examine morphological changes after amputation, as well as quantify volume
and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both
epimorphic and tissue regenerative responses in mammals.
Several abnormalities have been reported in the peripheral blood mononuclear cells of keloid-forming patients and particularly in the monocyte cell fraction. The goal of this in vitro study was to determine whether monocytes from keloid-prone patients contribute to the keloid phenotype in early developing keloids, and whether monocyte differentiation is affected by the keloid microenvironment. Therefore, keloid-derived keratinocytes and fibroblasts were used to reconstruct a full thickness, human, in vitro keloid scar model. The reconstructed keloid was co-cultured with monocytes from keloid-forming patients and compared to reconstructed normal skin co-cultured with monocytes from non-keloid-formers. The reconstructed keloid showed increased contraction, dermal thickness (trend) and α-SMA+ staining, but co-culture with monocytes did not further enhance the keloid phenotype. After 2-week culture, all monocytes switched from a CD11chigh/CD14high/CD68low to a CD11chigh/CD14low/CD68high phenotype. However, only monocytes co-cultured with either reconstructed keloid scar or normal skin models skewed towards the more fibrotic M2-macrophage phenotype. There was negligible fibroblast and fibrocyte differentiation in mono- and co-cultured monocytes. These results indicate that monocytes differentiate into M2 macrophages when in the vicinity of early regenerating and repairing tissue, independent of whether the individual is prone to normal or keloid scar formation.
During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.
A major goal of regenerative medicine is to stimulate tissue regeneration after traumatic injury. We previously discovered that treating digit amputation wounds with BMP2 in neonatal mice stimulates endochondral ossification to regenerate the stump bone. Here we show that treating the amputation wound with BMP9 stimulates regeneration of a synovial joint that forms an articulation with the stump bone. Regenerated structures include a skeletal element lined with articular cartilage and a synovial cavity, and we demonstrate that this response requires the Prg4 gene. Combining BMP2 and BMP9 treatments in sequence stimulates the regeneration of bone and joint. These studies provide evidence that treatment of growth factors can be used to engineer a regeneration response from a non-regenerating amputation wound.
Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here, we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1⁺SiglecF⁺ transitional macrophages localized to the fibrotic niche and had a profibrotic effect in vivo. Human orthologs of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.
While mammals cannot regenerate amputated limbs, mice and humans have regenerative ability restricted to amputations transecting the digit tip, including the terminal phalanx (P3). In mice the regeneration process is epimorphic and mediated by the formation of a blastema comprised of undifferentiated proliferating cells that differentiate to regenerate the amputated structures. Blastema formation distinguishes the regenerative response from a scar‐forming healing response. The mouse digit tip serves as a pre‐clinical model to investigate mammalian blastema formation and endogenous regenerative capabilities. We report that P3 blastema formation initiates prior to epidermal closure and concurrent with the bone histolytic response. In this early healing response, proliferation and cells entering the early stages of osteogenesis are localized to the periosteal and endosteal bone compartments. After the completion of stump bone histolysis, epidermal closure is completed and cells associated with the periosteal and endosteal compartments blend to form the blastema proper. Osteogenesis associated with the periosteum occurs as a polarized progressive wave of new bone formation that extends from the amputated stump and restores skeletal length. Bone patterning is restored along the proximal‐distal and medial digit axes, but is imperfect in the dorsal‐ventral axis with the regeneration of excessive new bone that accounts for the enhanced regenerated bone volume noted in previous studies. Periosteum depletion studies show that this compartment is required for the regeneration of new bone distal to the original amputation plane. These studies provide evidence that blastema formation initiates early in the healing response and that the periosteum is an essential tissue for successful epimorphic regeneration in mammals.
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Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.
Following severe tissue injury, patients are exposed to various danger- and microbe-associated molecular patterns, which provoke a strong activation of the neutrophil defense system. Neutrophils trigger and modulate the initial posttraumatic inflammatory response and contribute critically to subsequent repair processes. However, severe trauma can affect central neutrophil functions, including circulation half-life, chemokinesis, phagocytosis, cytokine release, and respiratory burst. Alterations in neutrophil biology may contribute to trauma-associated complications, including immune suppression, sepsis, multiorgan dysfunction, and disturbed tissue regeneration. Furthermore, there is evidence that neutrophil actions depend on the quality of the initial stimulus, including trauma localization and severity, the micromilieu in the affected tissue, and the patient’s overall inflammatory status. In the present review, we describe the effects of severe trauma on the neutrophil phenotype and dysfunction and the consequences for tissue repair. We particularly concentrate on the role of neutrophils in wound healing, lung injury, and bone fractures, because these are the most frequently affected tissues in severely injured patients.
ELife digest
The cells of the immune system are essential to defend an organism from disease. In addition, some of them are also thought to play an important role in helping injured tissues heal or even regrow. For example, when an animal is injured, immune cells such as macrophages rush to the wounded site to clear debris and help repair the damage. Macrophages come in different forms and subtypes, and express different protein markers on their surface, depending on where in the body they reside.
Few mammals can completely renew or regrow a damaged tissue – a process known as tissue regeneration. Instead, humans and most other mammals repair injuries by producing scar tissue, which has different properties compared to the original tissue it replaces. One exception is the African spiny mouse, which, unlike other rodents studied, can regrow skin and fur, nerves, muscles, and even cartilage. It has been shown that in highly regenerative animals such as salamanders and zebrafish, macrophages are necessary to initiate tissue regeneration. Documented cases of tissue regeneration in mammals are rare and therefore less understood. Until now, it was not clear why two species as closely related as spiny mice and house mice would heal identical injuries in different ways.
To better understand how new tissue regenerates, Simkin et al. compared the healing abilities of spiny mice and house mice after they received an injury to their ear and showed that macrophages appeared to be important for both the regeneration of new tissue and the formation of scar tissue. When Simkin et al. removed all macrophages in the ear of spiny mice, their ear tissue could not heal and regrow. When the macrophages were allowed to re-invade the injured site, the tissue in the ear regenerated. Further experiments showed that during tissue regeneration and scarring, different subtypes of macrophages appeared to be active.
The findings suggest that specific subtypes of macrophages could be a key element in helping tissue to regenerate. An important next step will be to further explore the different types of macrophages and whether the injury site determines what types of cells are active. A deeper understanding of how tissues can regrow in mammals will be essential to advancing our ability to stimulate tissue regeneration in humans.
DOI: http://dx.doi.org/10.7554/eLife.24623.002
In mammals, macrophages are known to play a major role in tissue regeneration. These cells contribute to inflammation, histolysis, re-epithelialization, re-vascularization and cell proliferation. While macrophages have been shown to be essential for regeneration in salamanders and fish, their role has not been elucidated in mammalian epimorphic regeneration. Here, using the regenerating mouse digit tip as a mammalian model, we demonstrate that macrophages are essential for the regeneration process. Using cell depletion strategies, we show that regeneration is completely inhibited; bone histolysis does not occur, wound re-epithelization is inhibited and the blastema does not form. While rescue of epidermal wound closure, in the absence of macrophages, promotes blastema accumulation it does not rescue cell differentiation indicating that macrophages play a key role in the re-differentiation of the blastema. Further, we provide additional evidence that while bone degradation is a part of the regenerative process, it is not essential to the overall regenerative process. These findings show that macrophages play an essential role in coordinating the epimorphic regenerative response in mammals.
Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloiddefective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta (il1b). Myeloid cells are considered to be the principal source of Il1b, but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response, and that its resolution by macrophages is necessary for survival of regenerative cells. We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes. Our study reveals that proper levels of Il1b signaling and tissue inflammation, which are tuned by macrophages, play a crucial role in tissue regeneration.
Regeneration of mammalian limbs is restricted to amputation of the distal digit tip, the terminal phalanx (P3). The adjacent skeletal element, the middle phalanx (P2), has emerged as a model system to investigate regenerative failure and as a site to test approaches aimed at enhancing regeneration. We report that exogenous application of BMP2 stimulates the formation of a transient cartilaginous callus distal to the amputation plane that mediates the regeneration of the amputated P2 bone. BMP2 initiates a significant regeneration response during the periosteal-derived cartilaginous healing phase of P2 bone repair, yet fails to induce regeneration in the absence of periosteal tissue, or after boney callus formation. We provide evidence that a temporal component exists in the induced-regeneration of P2 that we define as the “regeneration window”. In this window, cells are transiently responsive to BMP2 after the amputation injury. Simple re-injury of the healed P2 stump acts to reinitiate endogenous bone repair, complete with periosteal chondrogenesis, thus re-opening the “regeneration window” and thereby recreating a regeneration-permissive environment that is responsive to exogenous BMP2 treatment. This article is protected by copyright. All rights reserved
In salamanders, grafting of a left limb blastema onto a right limb stump yields regeneration of three limbs, the normal limb and two 'supernumerary' limbs. This experiment and other research have shown that the juxtaposition of anterior and posterior limb tissue plus innervation are necessary and sufficient to induce complete limb regeneration in salamanders. However, the cellular and molecular basis of the requirement for anterior-posterior tissue interactions were unknown. Here we have clarified the molecular basis of the requirement for both anterior and posterior tissue during limb regeneration and supernumerary limb formation in axolotls (Ambystoma mexicanum). We show that the two tissues provide complementary cross-inductive signals that are required for limb outgrowth. A blastema composed solely of anterior tissue normally regresses rather than forming a limb, but activation of hedgehog (HH) signalling was sufficient to drive regeneration of an anterior blastema to completion owing to its ability to maintain fibroblast growth factor (FGF) expression, the key signalling activity responsible for blastema outgrowth. In blastemas composed solely of posterior tissue, HH signalling was not sufficient to drive regeneration; however, ectopic expression of FGF8 together with endogenous HH signalling was sufficient. In axolotls, FGF8 is expressed only in the anterior mesenchyme and maintenance of its expression depends on sonic hedgehog (SHH) signalling from posterior tissue. Together, our findings identify key anteriorly and posteriorly localized signals that promote limb regeneration and show that these single factors are sufficient to drive non-regenerating blastemas to complete regeneration with full elaboration of skeletal elements.
Why mammals have poor regenerative ability has remained a long-standing question in biology. In regenerating vertebrates, injury can induce a process known as epimorphic regeneration to replace damaged structures. Using a 4-mm ear punch assay across multiple mammalian species, here we show that several Acomys spp. (spiny mice) and Oryctolagus cuniculus completely regenerate tissue, whereas other rodents including MRL/MpJ 'healer' mice heal similar injuries by scarring. We demonstrate ear-hole closure is independent of ear size, and closure rate can be modelled with a cubic function. Cellular and genetic analyses reveal that injury induces blastema formation in Acomys cahirinus. Despite cell cycle re-entry in Mus musculus and A. cahirinus, efficient cell cycle progression and proliferation only occurs in spiny mice. Together, our data unite blastema-mediated regeneration in spiny mice with regeneration in other vertebrates such as salamanders, newts and zebrafish, where all healthy adults regenerate in response to injury.
Regeneration of amputated structures is severely limited in humans and mice, with complete regeneration restricted to the distal portion of the terminal phalanx (P3). Here, we investigate the dynamic tissue repair response of the second phalangeal element (P2) post amputation in the adult mouse, and show that the repair response of the amputated bone is similar to the proximal P2 bone fragment in fracture healing. The regeneration-incompetent P2 amputation response is characterized by periosteal endochondral ossification resulting in the deposition of new trabecular bone, corresponding to a significant increase in bone volume; however, this response is not associated with bone lengthening. We show that cells of the periosteum respond to amputation and fracture by contributing both chondrocytes and osteoblasts to the endochondral ossification response. Based on our studies, we suggest that the amputation response represents an attempt at regeneration that ultimately fails due to the lack of a distal organizing influence that is present in fracture healing.
While regeneration occurs in a number of taxonomic groups across the Metazoa, there are very few reports of regeneration in mammals, which generally respond to wounding with fibrotic scarring rather than regeneration. A recent report described skin shedding, skin regeneration and extensive ear punch closure in two rodent species, Acomys kempi and Acomys percivali. We examined these striking results by testing the capacity for regeneration of a third species, Acomys cahirinus, and found a remarkable capacity to repair full thickness circular punches in the ear pinna. Four-millimeter diameter wounds closed completely in 2 months in 100% of ear punches tested. Histology showed extensive formation of elastic cartilage, adipose tissue, dermis, epidermis and abundant hair follicles in the repaired region. Furthermore, we demonstrated abundant angiogenesis and unequivocal presence of both muscle and nerve fibers in the reconstituted region; in contrast, similar wounds in C57BL/6 mice simply healed the borders of the cut by fibrotic scarring. Our results confirm the regenerative capabilities of Acomys, and suggest this model merits further attention. This article is protected by copyright. All rights reserved
The mechanisms by which macrophages control the inflammatory response, wound healing, biomaterial-interactions, and tissue regeneration appear to be related to their activation/differentiation states. Studies of macrophage behavior in vitro can be useful for elucidating their mechanisms of action, but it is not clear to what extent the source of macrophages affects their apparent behavior, potentially affecting interpretation of results. Although comparative studies of macrophage behavior with respect to cell source have been conducted, there has been no direct comparison of the three most commonly used cell sources: murine bone marrow, human monocytes from peripheral blood (PB), and the human leukemic monocytic cell line THP1, across multiple macrophage phenotypes. In this study, we used multivariate discriminant analysis to compare the in vitro expression of 34 genes commonly chosen to assess macrophage phenotype across all three sources of macrophages, as well as those derived from induced pluripotent stem cells (iPSCs), that were polarized towards four distinct phenotypes using the same differentiation protocols: M(LPS,IFN) (aka M1), M(IL4,IL13) (aka M2a), M(IL10) (aka M2c), and M(-) (aka M0) used as control. Several differences in gene expression trends were found among the sources of macrophages, especially between murine bone marrow-derived and human blood-derived M(LPS,IFN) and M(IL4,IL13) macrophages with respect to commonly used phenotype markers like CCR7 and genes associated with angiogenesis and tissue regeneration like FGF2 and MMP9. We found that the genes with the most similar patterns of expression among all sources were CXCL-10 and CXCL-11 for M(LPS,IFN) and CCL17 and CCL22 for M(IL4,IL13). Human PB-derived macrophages and human iPSC-derived macrophages showed similar gene expression patterns among the groups and genes studied here, suggesting that iPSC-derived monocytes have the potential to be used as a reliable cell source of human macrophages for in vitro studies. These findings could help select appropriate markers when testing macrophage behavior in vitro and highlight those markers that may confuse interpretation of results from experiments employing macrophages from different sources.
Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-β induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.
In the mouse, digit tip regeneration progresses through a series of discreet stages that include inflammation, histolysis, epidermal closure, blastema formation and redifferentiation. Recent studies reveal how each regenerative stage influences subsequent stages to establish a blastema that directs the successful regeneration of a complex mammalian structure. The focus of this review is on early events of healing and how an amputation wound transitions into a functional blastema. The stepwise formation of a mammalian blastema is proposed to provide a model for how specific targeted treatments can enhance regenerative performance in humans.This article is protected by copyright. All rights reserved.
Mammalian digit regeneration progresses through consistent stages: histolysis, inflammation, epidermal closure, blastema formation and finally redifferentiation. What we do not yet know is how each stage can affect others. Questions of stage timing, tissue interactions, and micro-environmental states are becoming increasingly important as we look toward solutions for whole limb regeneration. This study focuses on the timing of epidermal closure which, in mammals, is delayed if compared to more regenerative animals like the axolotl. We use a standard wound closure device, Dermabond, to induce earlier epidermal closure, and we evaluate the effect of fast epidermal closure on histolysis, blastema formation and redifferentiation. We find that fast epidermal closure is reliant upon a hypoxic micro-environment. Additionally, early epidermal closure eliminates the histolysis stage and results in a regenerate that more closely replicates the amputated structure. We show that tools like Dermabond and oxygen are able to independently influence the various stages of regeneration enabling us to uncouple histolysis, wound closure, and other regenerative events. With this study, we start to understand how each stage of mammalian digit regeneration is controlled.This article is protected by copyright. All rights reserved.
The regenerating mouse digit tip is a unique model for investigating blastema formation and epimorphic regeneration in mammals. The blastema is characteristically avascular and we previously reported that blastema expression of a known anti-angiogenic factor gene, Pedf, correlated with a successful regenerative response (Yu, L., Han, M., Yan, M., Lee, E. C., Lee, J., Muneoka, K., 2010. BMP signaling induces digit regeneration in neonatal mice. Development. 137, 551–9). Here we show that during regeneration Vegfa transcripts are not detected in the blastema but are expressed at the onset of differentiation. Treating the amputation wound with VEGF enhances angiogenesis but inhibits regeneration. We next tested BMP9, another known mediator of angiogenesis, and found that BMP9 is also a potent inhibitor of digit tip regeneration. BMP9 induces Vegfa expression in the digit stump suggesting that regenerative failure was mediated by enhanced angiogenesis. Finally, we show that BMP9 inhibition of regeneration is completely rescued by treatment with PEDF. These studies show that precocious angiogenesis is inhibitory for regeneration, and provide compelling evidence that the regulation of angiogenesis is a critical factor in designing therapies aimed at stimulating mammalian regeneration.This article is protected by copyright. All rights reserved.
Neutrophils and macrophages, as key mediators of inflammation, have defined functionally important roles in mammalian tissue repair. Although recent evidence suggests that similar cells exist in zebrafish and also migrate to sites of injury in larvae, whether these cells are functionally important for wound healing or regeneration in adult zebrafish is unknown. To begin to address these questions, we first tracked neutrophils (lyzC(+), mpo(+)) and macrophages (mpeg1(+)) in adult zebrafish following amputation of the tail fin, and detailed a migratory timecourse that revealed conserved elements of the inflammatory cell response with mammals. Next, we used transgenic zebrafish in which we could selectively ablate macrophages, which allowed us to investigate whether macrophages were required for tail fin regeneration. We identified stage-dependent functional roles of macrophages in mediating fin tissue outgrowth and bony ray patterning, in part through modulating levels of blastema proliferation. Moreover, we also sought to detail molecular regulators of inflammation in adult zebrafish and identified Wnt/β-catenin as a signaling pathway that regulates the injury microenvironment, inflammatory cell migration and macrophage phenotype. These results provide a cellular and molecular link between components of the inflammation response and regeneration in adult zebrafish.
The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.
Some organisms, such as the Mexican axolotl, have the capacity to regenerate complicated biological structures throughout their lives. Which molecular pathways are sufficient to induce a complete endogenous regenerative response in injured tissue is an important question that remains unanswered. Using a gain-of-function regeneration assay, known as the Accessory Limb Model (ALM), we and others have begun to identify the molecular underpinnings of the three essential requirements for limb regeneration; wounding, neurotrophic signaling, and the induction of pattern from cells that retain positional memory. We have previously shown that treatment of Mexican axolotls with exogenous retinoic acid (RA) is sufficient to induce the formation of complete limb structures from blastemas that were generated by deviating a nerve bundle into an anterior-located wound site on the limb. Here we show that these ectopic structures are capable of regenerating and inducing new pattern to form when grafted into new anterior-located wounds. We additionally found that the expression of Alx4 decreases, and Shh expression increases in these anterior located blastemas, but not in the mature anterior tissues, supporting the hypothesis that RA treatment posteriorizes blastema tissue. Based on these and previous observations, we used the ALM assay to test the hypothesis that a complete regenerative response can be generated by treating anterior-located superficial limb wounds with a specific combination of growth factors at defined developmental stages. Our data shows that limb wounds that are first treated with a combination of FGF-2, FGF-8, and BMP-2, followed by RA treatment of the resultant mid-bud stage blastema, will result in the generation of limbs with complete proximal/distal and anterior/posterior limb axes. Thus, the minimal signaling requirements from the nerve and a positional disparity are achieved with the application of this specific combination of signaling molecules.
Macrophages are well recognized for their dual roles in orchestrating inflammatory responses and regulating tissue repair. In almost all acutely inflamed tissues, 2 main subclasses of macrophages coexist. These include embryonically derived resident tissue macrophages and BM-derived recruited macrophages. While it is clear that macrophage subsets categorized in this fashion display distinct transcriptional and functional profiles, whether all cells within these categories and in the same inflammatory microenvironment share similar functions or whether further specialization exists has not been determined. To investigate inflammatory macrophage heterogeneity on a more granular level, we induced acute lung inflammation in mice and performed single cell RNA sequencing of macrophages isolated from the airspaces during health, peak inflammation, and resolution of inflammation. In doing so, we confirm that cell origin is the major determinant of alveolar macrophage (AM) programing, and, to our knowledge, we describe 2 previously uncharacterized, transcriptionally distinct subdivisions of AMs based on proliferative capacity and inflammatory programing.
Mice are intrinsically capable of regenerating the tips of their digits after amputation. Mouse digit tip regeneration is reported to be a peripheral nerve-dependent event. However, it is presently unknown what types of nerves and Schwann cells innervate the digit tip, and to what extent these cells regenerate in association with the regenerative response. Given the necessity of peripheral nerves for mammalian regeneration, we investigated the neuroanatomy of the unamputated, regenerating, and regenerated mouse digit tip. Using immunohistochemistry for β-III-tubulin (β3T) or neurofilament H (NFH), substance P (SP), tyrosine hydroxylase (TH), myelin protein zero (P0), and glial fibrillary acidic protein (GFAP), we identified peripheral nerve axons (sensory and sympathetic), and myelinating- and non-myelinating-Schwann cells. Our findings show that the digit tip is innervated by two digital nerves that each bifurcate into a bone marrow (BM) and connective tissue (CT) branch. The BM branches are composed of sympathetic axons that are ensheathed by non-myelinating-Schwann cells whereas the CT branches are composed of sensory and sympathetic axons and are ensheathed by myelinating- and non-myelinating-Schwann cells. The regenerated digit neuroanatomy differs from unamputated digit in several key ways. First, there is 7.5 fold decrease in CT branch axons in the regenerated digit compared to the unampuated digit. Second, there is a 5.6 fold decrease in myelinating-Schwann cells in the regenerated digit compared to the unamputated digit that is consistent with the decrease in CT branch axons. Importantly, we also find that the central portion of the regenerating digit blastema is aneural, with axons and Schwann cells restricted to peripheral and distal blastema regions. Finally, we show that even with impaired innervation, digits maintain the ability to regenerate after re-amputation. Taken together, these data indicate that nerve regeneration is impaired in the context of mouse digit tip regeneration.
How the axolotl makes a new limb
Unlike most vertebrate limbs, the axolotl limb regenerates the skeleton after amputation. Dermal and interstitial fibroblasts have been thought to provide sources for skeletal regeneration, but it has been unclear whether preexisting stem cells or dedifferentiation of fibroblasts formed the blastema. Gerber et al. developed transgenic reporter animals to compare periskeletal cell and fibroblast contributions to regeneration. Callus-forming periskeletal cells extended existing bone, but fibroblasts built new limb segments. Single-cell transcriptomics and Brainbow-based lineage tracing revealed the lack of a preexisting stem cell. Instead, the heterogeneous population of fibroblasts lost their adult features to form a multipotent skeletal progenitor expressing the embryonic limb program.
Science , this issue p. eaaq0681
Studying regeneration in animals where and when it occurs is inherently interesting and a challenging research topic within developmental biology. Historically, vertebrate regeneration has been investigated in animals that display enhanced regenerative abilities and we have learned much from studying organ regeneration in amphibians and fish. From an applied perspective, while regeneration biologists will undoubtedly continue to study poikilothermic animals (i.e., amphibians and fish), studies focused on homeotherms (i.e., mammals and birds) are also necessary to advance regeneration biology. Emerging mammalian models of epimorphic regeneration are poised to help link regenerative biology and regenerative medicine. The regenerating rodent digit tip, which parallels human fingertip regeneration, and the regeneration of large circular defects through the ear pinna in spiny mice and rabbits, provide tractable, experimental systems where complex tissue structures are regrown through blastema formation and morphogenesis. Using these models as examples, we detail similarities and differences between the mammalian blastema and its classical counterpart to arrive at a broad working definition of a vertebrate regeneration blastema. This comparison leads us to conclude that regenerative failure is not related to the availability of regeneration-competent progenitor cells, but is most likely a function of the cellular response to the microenvironment that forms following traumatic injury. Recent studies demonstrating that targeted modification of this microenvironment can restrict or enhance regenerative capabilities in mammals helps provide a roadmap for eventually pushing the limits of human regeneration.
The mechanistic details of keloid formation are still not understood. Given that the immune system is engaged in skin lesion repair, we examined the CD14+ macrophages and CD3+ T cells in keloid tissues and in the normal skin. Compared to the normal skin, keloid tissues presented significantly elevated infiltration by CD14+ macrophages. Moreover, the transcription and protein expression of iNOS, IL-12, IL-10, and TGF-β were significantly higher in keloid macrophages than in normal skin macrophages, in which the expression of M2-associated genes were further elevated compared to M1-associated genes in keloid. We also observed that keloid tissues presented higher infiltration by CD3+ T cells, of which the majority was CD4+ T cells. Notably, the frequency of Foxp3+ regulatory T cells (Tregs) in keloid tissues was significantly higher compared to that in the peripheral blood. Furthermore, macrophages from keloid tissues possessed potent capacity to induce Foxp3 expression in circulating CD3+ T cells. Together, this study suggested that macrophages in keloid tissues presented high activation status and were polarized toward the M2 subtype; moreover, these macrophages could promote Treg differentiation by upregulating Foxp3 expression.
Osteal macrophages (osteomacs) contribute to bone homeostasis and regeneration. To further distinguish their functions from osteoclasts, which share many markers and growth factor requirements, we developed a rapid, enzyme-free osteomac enrichment protocol that permitted characterization of minimally manipulated osteomacs by flow cytometry. Osteomacs differ from osteoclasts in expression of Siglec1 (CD169). This distinction was confirmed using the CD169-diphtheria toxin (DT) receptor (DTR) knock-in model. DT treatment of naïve CD169-DTR mice resulted in selective and striking loss of osteomacs, whilst osteoclasts and trabecular bone area were unaffected. Consistent with a previously-reported trophic interaction, osteomac loss was accompanied by a concomitant and proportionately striking reduction in osteoblasts. The impact of CD169⁺ macrophage depletion was assessed in two models of bone injury that heal via either intramembranous (tibial injury) or endochondral (internally-plated femoral fracture model) ossification. In both models, CD169⁺ macrophage, including osteomac depletion compromised bone repair. Importantly, DT treatment in CD169-DTR mice did not affect osteoclast frequency in either model. In the femoral fracture model, the magnitude of callus formation correlated with the number of F4/80⁺ macrophages that persisted within the callus. Overall these observations provide compelling support that CD169⁺ osteomacs, independent of osteoclasts, provide vital pro-anabolic support to osteoblasts during both bone homeostasis and repair.
Cellular senescence has been recently linked to the promotion of age-related pathologies, including a decline in regenerative capacity. While such capacity deteriorates with age in mammals, it remains intact in species such as salamanders, which have an extensive repertoire of regeneration and can undergo multiple episodes through their lifespan. Here we show that, surprisingly, there is a significant induction of cellular senescence during salamander limb regeneration, but that rapid and effective mechanisms of senescent cell clearance operate in normal and regenerating tissues. Furthermore, the number of senescent cells does not increase upon repetitive amputation or ageing, in contrast to mammals. Finally, we identify the macrophage as a critical player in this efficient senescent cell clearance mechanism. We propose that effective immunosurveillance of senescent cells in salamanders supports their ability to undergo regeneration throughout their lifespan.
Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.
The immune system mediates tissue growth and homeostasis and is the first responder to injury or biomaterial implantation. Recently, it has been appreciated that immune cells play a critical role in wound healing and tissue repair and should thus be considered as potentially beneficial particularly in the context of scaffolds for regenerative medicine. Here, we present a flow cytometric analysis of cellular recruitment to tissue-derived extracellular matrix scaffolds where we quantitatively describe the infiltration and polarization of several immune subtypes including macrophages, dendritic cells, neutrophils, monocytes, T cells, and B cells. We define a specific scaffold-associated macrophage (SAM) that expresses CD11b+F4/80+CD11c+/-CD206hiCD86+MHCII+ that are characteristic of an M2-like cell (CD206hi) with high antigen presentation capabilities (MHCII+). Adaptive immune cells tightly regulate the phenotype of a mature SAM. These studies provide a foundation for detailed characterization of the scaffold immune microenvironment (SIM) of a given biomaterial scaffold to determine the effect of scaffold changes on immune response and subsequent therapeutic outcome of that material.
Age potentiates neurodegeneration and impairs recovery from spinal cord injury (SCI). Previously, we observed that age alters the balance of destructive (M1) and protective (M2) macrophages, however, the age-related pathophysiology in SCI is poorly understood. NADPH oxidase (NOX) contributes to reactive oxygen species (ROS)-mediated damage and macrophage activation in neurotrauma. Further, NOX/ROS increase with CNS age. Here, we found significantly higher ROS generation in 14 vs. 4-month-old (MO) mice after contusion SCI. Notably, NOX2 increased in 14 MO ROS-producing macrophages suggesting that macrophages and NOX contribute to SCI oxidative stress. Indicators of lipid peroxidation, a downstream cytotoxic effect of ROS accumulation, were significantly higher in 14 vs. 4 MO SCI mice. We also detected a higher percentage of ROS-producing M2 (Arginase-1-positive) macrophages in 14 vs. 4 MO mice, a previously unreported SCI phenotype, and increased M1 (CD16/32-positive) macrophages with age. Thus, NOX and ROS are age-related mediators of SCI pathophysiology and normally protective M2 macrophages may potentiate secondary injury through ROS generation in the aged injured spinal cord.
Urodele amphibians have a remarkable organ regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in limb and tail regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful limb regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired limb regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ regeneration ability.
Macrophage gene expression determines phagocyte responses and effector functions. Macrophage plasticity has been mainly addressed in in vitro models that do not account for the environmental complexity observed in vivo. In this study, we show that microarray gene expression profiling revealed a highly dynamic landscape of transcriptomic changes of Ly6C(pos)CX3CR1(lo) and Ly6C(neg)CX3CR1(hi) macrophage populations during skeletal muscle regeneration after a sterile damage. Systematic gene expression analysis revealed that the time elapsed, much more than Ly6C status, was correlated with the largest differential gene expression, indicating that the time course of inflammation was the predominant driving force of macrophage gene expression. Moreover, Ly6C(pos)/Ly6C(neg) subsets could not have been aligned to canonical M1/M2 profiles. Instead, a combination of analyses suggested the existence of four main features of muscle-derived macrophages specifying important steps of regeneration: 1) infiltrating Ly6C(pos) macrophages expressed acute-phase proteins and exhibited an inflammatory profile independent of IFN-γ, making them damage-associated macrophages; 2) metabolic changes of macrophages, characterized by a decreased glycolysis and an increased tricarboxylic acid cycle/oxidative pathway, preceded the switch to and sustained their anti-inflammatory profile; 3) Ly6C(neg) macrophages, originating from skewed Ly6C(pos) cells, actively proliferated; and 4) later on, restorative Ly6C(neg) macrophages were characterized by a novel profile, indicative of secretion of molecules involved in intercellular communications, notably matrix-related molecules. These results show the highly dynamic nature of the macrophage response at the molecular level after an acute tissue injury and subsequent repair, and associate a specific signature of macrophages to predictive specialized functions of macrophages at each step of tissue injury/repair.
Engineering a healing immune response
Infections, surgeries, and trauma can all cause major tissue damage. Biomaterial scaffolds, which help to guide regenerating tissue, are an exciting emerging therapeutic strategy to promote tissue repair. Sadtler et al. tested how biomaterial scaffolds interact with the immune system in damaged tissue to promote repair (see the Perspective by Badylak). Scaffolds derived from cardiac muscle and bone extracellular matrix components trigger a tissue-reparative T cell immune response in mice with injured muscles.
Science , this issue p. 366 ; see also p. 298
In contrast to the lab mouse, Mus musculus, several species of spiny mouse, Acomys, can regenerate epidermis, dermis, hairs, sebaceous glands with smooth muscle erector pili muscles and skeletal muscle of the panniculus carnonsus after full thickness skin wounding. Here, we have compared the responses of these scarring and nonscarring organisms concentrating on the immune cells and wound cytokines, cell proliferation, and the collagenous components of the wound bed and scar. The blood of Acomys is very neutropenic but there are greater numbers of mast cells in the Acomys wound than the Mus wound. Most importantly there are no F4/80 macrophages in the Acomys wound and many proinflammatory cytokines are either absent or in very low levels which we suggest may be primarily responsible for the excellent regenerative properties of the skin of this species. There is little difference in cell proliferation in the two species either in the epidermis or mesenchymal tissues but the cell density and matrix composition of the wound is very different. In Mus there are 8 collagens which are up-regulated at least 5-fold in the wound creating a strongly trichrome-positive matrix whereas in Acomys there are very few collagens present and the matrix shows only light trichrome staining. The major component of the Mus matrix is collagen XII which is up-regulated between 10 and 30-fold after wounding. These results suggest that in the Acomys wound the absence of many cytokines resulting in the lack of macrophages is responsible for the failure to up-regulate fibrotic collagens, a situation which permits a regenerative response within the skin rather than the generation of a scar.
Significance
Although full mammalian limbs do not regenerate after amputation, the fingertips of select mammalian species do. Understanding digit tip regeneration at the molecular level can potentially provide insight into designing translational therapies for regrowing greater portions of the limbs and other nonregenerative tissues. The nail is known to be critical for digit tip regeneration, at least in part through a mechanism dependent on Wnt signaling. Here, we identify a cell population expressing a mediator of Wnt signaling, Lgr6 (leucine-rich repeat-containing G protein-coupled receptor 6), as key stem cells for the nail. Moreover, we find that Lgr6 is required for proper digit tip regeneration.
With the explosion of knowledge from molecular biology and the burgeoning interest in generating or regenerating tissues or organs through various bioengineering or stem cell approaches, many scientists and students have shown a renewed interest in the phenomenon of regeneration. Because relatively few have had the luxury of being able to approach the phenomenon of regeneration from a broad biological perspective, Dr. Carlson has produced a book that outlines the fundamental principles of regeneration biology. Subject matters focus principally on regeneration in vertebrate systems, but also invertebrate regeneration. In order to manipulate regenerative processes, it is important to understand the underlying principles of regeneration.Principles of Regnerative Biologyis the key introductory reference for all developmental biologists, geneticists, and tissue and stem cell researchers. * Creates a general understanding of one of the most fascinating and complex phenomenas in biology * Discusses the the ability and diversity of regeneration in various organisms * Explains the history and origins of cells in regenerating systems * Includes information on stem cells and its important role in regeneration.
Macrophages are responsive to local tissue signals and alter their phenotypes accordingly. In disease tissues this means that macrophage phenotypes may change with disease progression, exacerbating or facilitating the resolution of the pathology.
Macrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogramed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the microenvironment, along with pioneer factors in orchestrating identity and plasticity.
Copyright © 2014 Elsevier Inc. All rights reserved.
Macrophages reside in essentially all tissues of the body and play key roles in innate and adaptive immune responses. Distinct populations of tissue macrophages also acquire context-specific functions that are important for normal tissue homeostasis. To investigate mechanisms responsible for tissue-specific functions, we analyzed the transcriptomes and enhancer landscapes of brain microglia and resident macrophages of the peritoneal cavity. In addition, we exploited natural genetic variation as a genome-wide "mutagenesis" strategy to identify DNA recognition motifs for transcription factors that promote common or subset-specific binding of the macrophage lineage-determining factor PU.1. We find that distinct tissue environments drive divergent programs of gene expression by differentially activating a common enhancer repertoire and by inducing the expression of divergent secondary transcription factors that collaborate with PU.1 to establish tissue-specific enhancers. These findings provide insights into molecular mechanisms by which tissue environment influences macrophage phenotypes that are likely to be broadly applicable to other cell types.
Copyright © 2014 Elsevier Inc. All rights reserved.
The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80+Mac-2+) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80+Mac-2neg), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window.