npj Regenerative Medicine最新文献

Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex-specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later-stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile, including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

Tendons are essential, frequently injured connective tissues that transmit forces from muscle to bone. Their unique highly ordered, matrix-rich structure is critical for proper function. While adult mammalian tendons heal after acute injuries, endogenous tendon cells, or tenocytes, fail to respond appropriately, resulting in the formation of disorganized fibrovascular scar tissue with impaired function and increased propensity for re-injury. Here, we show that, unlike mammals, adult zebrafish tenocytes activate upon injury and fully regenerate the tendon. Using a full tear injury model in the adult zebrafish craniofacial tendon, we defined the hallmark stages and cellular basis of tendon regeneration through multiphoton imaging, lineage tracing, and transmission electron microscopy approaches. Remarkably, we observe that zebrafish tendons regenerate and restore normal collagen matrix ultrastructure by 6 months post-injury (mpi). Tendon regeneration progresses in three main phases: inflammation within 24 h post-injury (hpi), cellular proliferation and formation of a cellular bridge between the severed tendon ends at 3-5 days post-injury (dpi), and re-differentiation and matrix remodeling beginning from 5 dpi to 6 mpi. Importantly, we demonstrate that pre-existing tenocytes are the main cellular source of regeneration, proliferating and migrating upon injury to ultimately bridge the tendon ends. Finally, we show that TGF-β signaling is required for tenocyte recruitment and bridge formation. Collectively, our work debuts and aptly positions the adult zebrafish tendon as an invaluable comparative system to elucidate regenerative mechanisms that may inspire new therapeutic strategies.

After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene legumain. Analysis of legumain mutant zebrafish shows a requirement for early ECM remodeling and efficient ligament regeneration. Our study establishes a new model of adult scar-free ligament regeneration and highlights roles of immune-mesenchyme cross-talk in ECM remodeling that initiates regeneration.

Legg-Calvé-Perthes disease is juvenile idiopathic osteonecrosis of the femoral head (ONFH) that has no effective clinical treatment. Previously, local injection of bone morphogenetic protein-2 (BMP2) for ONFH treatment showed a heterogeneous bone repair and a high incidence of heterotopic ossification (HO) due to the BMP2 leakage. Here, we developed a BMP2-hydrogel treatment via a transphyseal bone wash and subsequential injection of BMP2-loaded hydrogel. In vitro studies showed that a hydrogel of gelatin-heparin-tyramine retained the BMP2 for four weeks. The injection of the hydrogel can efficiently prevent leakage. With the bone wash, the injected hydrogel had a broad distribution in the head. In vivo studies on pigs revealed that the BMP2-hydrogel treatment produced a homogeneous bone regeneration without HO. It preserved the subchondral contour and restored the subchondral endochondral ossification, although it increased growth plate fusions. In summary, the study demonstrated a promising BMP2-hydrogel treatment for ONFH treatment, especially for teenagers.

Non-healing wounds have a negative impact on quality of life and account for many cases of amputation and even early death among patients. Diabetic patients are the predominate population affected by these non-healing wounds. Despite the significant clinical demand, treatment with biologics has not broadly impacted clinical care. Interleukin-4 (IL-4) is a potent modulator of the immune system, capable of skewing macrophages towards a pro-regeneration phenotype (M2) and promoting angiogenesis, but can be toxic after frequent administration and is limited by its short half-life and low bioavailability. Here, we demonstrate the design and characterization of an engineered recombinant interleukin-4 construct. We utilize this collagen-binding, serum albumin-fused IL-4 variant (CBD-SA-IL-4) delivered in a hyaluronic acid (HA)-based gel for localized application of IL-4 to dermal wounds in a type 2 diabetic mouse model known for poor healing as proof-of-concept for improved tissue repair. Our studies indicate that CBD-SA-IL-4 is retained within the wound and can modulate the wound microenvironment through induction of M2 macrophages and angiogenesis. CBD-SA-IL-4 treatment significantly accelerated wound healing compared to native IL-4 and HA vehicle treatment without inducing systemic side effects. This CBD-SA-IL-4 construct can address the underlying immune dysfunction present in the non-healing wound, leading to more effective tissue healing in the clinic.

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGFβ- and BMP-signaling, where inhibition of TGFβ- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGFβ-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGFβ- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

Cell-based therapeutics are promising interventions to repair ischemic cardiac tissue. However, no single cell type has yet been found to be both specialized and versatile enough to heal the heart. The synergistic effects of two regenerative cell types including endothelial colony forming cells (ECFC) and first-trimester human umbilical cord perivascular cells (FTM HUCPVC) with endothelial cell and pericyte properties respectively, on angiogenic and regenerative properties were tested in a rat model of myocardial infarction (MI), in vitro tube formation and Matrigel plug assay. The combination of FTM HUCPVCs and ECFCs synergistically reduced fibrosis and cardiomyocyte apoptosis, while promoting favorable cardiac remodeling and contractility. These effects were in part mediated by ANGPT2, PDGF-β, and VEGF-C. PDGF-β signaling-dependent synergistic effects on angiogenesis were also observed in vitro and in vivo. FTM HUCPVCs and ECFCs represent a cell combination therapy for promoting and sustaining vascularization following ischemic cardiac injury.

Red blood cell (RBC) generation from human pluripotent stem cells (PSCs) offers potential for innovative cell therapy in regenerative medicine as well as developmental studies. Ex vivo erythropoiesis from PSCs is currently limited by the low efficiency of functional RBCs with β-globin expression in culture systems. During induction of β-globin expression, the absence of a physiological microenvironment, such as a bone marrow niche, may impair cell maturation and lineage specification. Here, we describe a simple and reproducible culture system that can be used to generate erythroblasts with β-globin expression. We prepared a two-dimensional defined culture with ferric citrate treatment based on definitive hemogenic endothelium (HE). Floating erythroblasts derived from HE cells were primarily CD45⁺CD71⁺CD235a⁺ cells, and their number increased remarkably upon Fe treatment. Upon maturation, the erythroblasts cultured in the presence of ferric citrate showed high transcriptional levels of β-globin and enrichment of genes associated with heme synthesis and cell cycle regulation, indicating functionality. The rapid maturation of these erythroblasts into RBCs was observed when injected in vivo, suggesting the development of RBCs that were ready to grow. Hence, induction of β-globin expression may be explained by the effects of ferric citrate that promote cell maturation by binding with soluble transferrin and entering the cells.Taken together, upon treatment with Fe, erythroblasts showed advanced maturity with a high transcription of β-globin. These findings can help devise a stable protocol for the generation of clinically applicable RBCs.

Mitochondrial dysfunction and subsequent accumulation of reactive oxygen species (ROS) are key contributors to the pathology of ischemic cerebrovascular disease. Therefore, elimination of ROS and damaged mitochondria is crucial for the effective treatment of this disease. For this purpose, we designed an inhalation nanotherapeutic agent, P/D@Mn/Co₃O₄, to treat ischemic cerebrovascular disease. Mn/Co₃O₄ effectively removed excess ROS from cells, reduced acute cellular oxidative stress, and protected neural cells from apoptosis. Furthermore, it depleted the H⁺ surrounding mitochondria and depolarized the mitochondrial membrane potential, inducing mitophagy and eliminating abnormal mitochondria, thereby avoiding the continuous overproduction of ROS by eliminating the source of ROS regeneration. On intranasal administration, Mn/Co₃O₄ encapsulated by platelet membranes and 2,3-(dioxy propyl)-trimethylammonium chloride can bypass the blood-brain barrier, enter the brain through the trigeminal and olfactory pathways, and target inflammatory regions to remove ROS and damaged mitochondria from the lesion area. In rat models of stroke and vascular dementia, P/D@Mn/Co₃O₄ effectively inhibited the symptoms of acute and chronic cerebral ischemia by scavenging ROS and damaged mitochondria in the affected area. Our findings indicate that the nanotherapeutic agent developed in this study can be used for the effective treatment of ischemic cerebrovascular disease.