Enhance Tissue Regeneration Research Articles

Cytokine Modulation with Biomaterials: Engineering the Immune Response for Tissue Repair and Regeneration

Cytokines are key regulators of the immune response and play critical roles in tissue repair and regeneration. Modulating the cytokine environment at sites of injury can improve healing by reducing chronic inflammation and promoting tissue remodeling. Biomaterials have emerged as effective platforms for delivering cytokines in a controlled and localized manner, providing spatial and temporal regulation of the immune response. This review discusses various biomaterial-based strategies for cytokine modulation, including hydrogels, nanoparticles, and scaffolds, each designed to influence immune cell behavior and enhance tissue regeneration. Biomaterials can be engineered to deliver specific cytokines, such as interleukin-10 (IL-10) or transforming growth factor-beta (TGF-β), which help suppress inflammation and promote the differentiation of stem cells or other progenitor cells. Applications of cytokine-modulating biomaterials in wound healing, bone regeneration, cardiac repair, and nerve regeneration are explored. Additionally, challenges such as achieving precise cytokine release, maintaining cytokine stability, and the complexity of immune regulation are addressed. Advances in biomaterial design hold great potential for developing “smart” systems capable of adjusting cytokine delivery based on the evolving tissue environment. The use of cytokine-modulating biomaterials represents a promising approach to improving clinical outcomes in regenerative medicine and tissue engineering. Keywords: Cytokine modulation, Biomaterial, Tissue regeneration, Immune response, Inflammation, Controlled release

Silk fibroin/gelatin electrospun nanofibrous dressing loaded with roxadustat accelerates wound healing in diabetic rats via HIF-1α stabilization

Diabetes presents significant health risks, including diabetic foot ulcers, which can lead to amputation or death if left untreated. It has been acknowledged that hypoxia-inducible factor-1 alpha (HIF-1α) dysfunction in diabetic wounds delays healing and tissue repair. This study explores the therapeutic potential of roxadustat (ROX), a novel HIF-1α stabilizer, within an optimized electrospun gelatin/silk fibroin nanofibrous dressing (ROX-NF). The dressing's properties were optimized using a 22 full factorial design, with varied gelatin (GN) and silk fibroin (SF) amounts as factors and responses including degradation, porosity, swelling, drug release, and drug content percentages. In vitro characterization of the optimized ROX-NF showed a degradation rate of 90.24 ± 1.29 %, porosity of 84.41 ± 3.04 %, cumulative drug release of 93.13 ± 3.05 % over 48 h, and a high drug content of 99.12 ± 0.02 %, with structural analyses confirming successful ROX integration within the nanofibrous matrix. Additionally, in vivo evaluation of the optimized ROX-NF in diabetic rat models demonstrated accelerated wound closure, achieving 5.17 ± 1.2 % wound area by day 14 compared to 19.3 ± 2.8 % with ROX dispersion. Histological analysis revealed enhanced collagen deposition, neovascularization, and organized epidermal and dermal layers. Furthermore, PCR, ELISA, and Western blot assays demonstrated a marked upregulation of key wound healing markers, including HIF-1α, TGF-β, VEGF, and collagen I, in the ROX-NF group relative to ROX dispersion. These findings underscore the efficacy of combining ROX with nanofiber characteristics, offering a promising approach for diabetic wound management through accelerated wound closure and enhanced tissue regeneration.

Collagen and chitosan-based biogenic sprayable gel of silver nanoparticle for advanced wound care.

Silver nanoparticles have gained significant attention recently due to their unique antibacterial properties, making them promising candidates for wound care applications. This study proposes a novel approach for advanced wound care using a silver nanoparticle-impregnated biogenic spray hydrogel supplemented with collagen and chitosan. Silver nanoparticles were incorporated into the hydrogel (optimized by a QbD approach) to impart antimicrobial activity, crucial for combating wound infections and promoting faster healing. The study assessed the physical and chemical properties of the biogenic hydrogel, including its viscosity, pH, and nanoparticle dispersion characteristics. In vitro, antimicrobial efficacy against common wound pathogens and in vivo studies using chronic wound models in small animals portrayed the immense potential of the developed biogenic hydrogel in effectively reducing the bacterial load of broad-spectrum pathogens. The hydrogel exhibited excellent biocompatibility, supporting cell proliferation and tissue repair without toxic effects. It accelerated wound healing, improved collagen deposition, and enhanced tissue regeneration in the tested animals by reducing proinflammatory cytokines, ROS, and NF-kb levels. Overall, this innovative silver nanoparticle-impregnated biogenic spray hydrogel of collagen and chitosan presents a uniform spray pattern that proved efficient, showing a promising solution for advanced wound care. Its biocompatibility, safety, anti-inflammatory, antimicrobial efficacy, and wound healing properties hold great potential for improving the management of complex wounds, opening new avenues in wound care and regenerative medicine.

Gasdermin D unlocks metabolic pathways to enhance tissue regeneration.

Gasdermin D unlocks metabolic pathways to enhance tissue regeneration.

3D‐Printed In Situ Growth of Bilayer MOF Hydrogels for Accelerated Osteochondral Defect Repair

AbstractRepairing osteochondral (OC) defect presents a significant challenge due to the intricate structural requirements and the unpredictable differentiation pathways of bone marrow mesenchymal stem cells (BMSCs). To address this challenge, a novel biomimetic OC hydrogel scaffold is developed that features a structure of soft and hard components. This scaffold incorporates bilayer metal–organic frameworks (MOFs), specifically ZIF‐67 in the upper layer and ZIF‐8 in the lower layer, achieved through an in situ printing process. This configuration enables the spatial and temporal modulation of BMSC differentiation by controlling the release of Co2⁺ and Zn2⁺. The results demonstrate that the bilayer MOF hydrogels significantly outperform hydrogels that either lack MOFs or contain a single type of MOF in enhancing repair outcomes in rabbit models of knee OC defects. The improved regenerative efficacy is attributed to the distinct chondrogenic and osteogenic differentiation cues provided by the bilayer MOFs, effectively guiding BMSCs toward enhanced tissue regeneration. This customizable biomimetic OC hydrogel scaffold not only opens new avenues for innovative therapeutic strategies but also holds great promise for widespread clinical applications.

Amniotic Membrane Transplantation: Clinical Applications in Enhancing Wound Healing and Tissue Regeneration.

Chronic wounds and non-healing tissue defects pose significant clinical challenges, necessitating innovative therapeutic approaches. A comprehensive literature review of amniotic membrane transplantation for wound healing and tissue repair evaluates the efficacy and safety of amniotic membrane transplantation in enhancing wound healing and tissue repair. Amniotic membranes promote wound closure and reduce inflammation and scarring via abundant growth factors, cytokines, and extracellular matrix components, which foster conducive environments for tissue regeneration. Amniotic membrane transplantation is effective in various medical disciplines, including ophthalmology, dermatology, and orthopedics. Low immunogenicity and anti-microbial properties ensure their safe application. Amniotic membrane transplantation offers a promising therapeutic approach for wound healing and tissue repair, and further research is warranted to explore its regenerative potential fully.

Capsaicin/silica-infused polygalacturonic acid/polyvinyl alcohol nano-matrix for enhanced wound healing in skin injuries

Wound healing is a complex physiological process, demanding advanced strategies for efficient tissue regeneration. To address this, we developed a novel nanofibrous matrix composed of polygalacturonic acid (PGA), polyvinyl alcohol (PVA), capsaicin, and zinc-doped mesoporous silica (Zn/MCM-41). This copolymeric matrix offers enhanced mechanical stability, controlled drug release, and improved cellular adhesion and proliferation, leading to effective tissue regeneration. Infusing capsaicin/Zn-MCM-41 confers synergistic advantages, including accelerated wound closure, diminished inflammation, and enhanced tissue regeneration, culminating in superior wound healing outcomes. Comprehensive physicochemical characterization of the nanofiber was conducted, employing techniques such as EDS, EDX, XRD, FESEM, HRTEM, FTIR, BET, TGA, DSC and Zeta potential, confirming the successful synthesis of Zn/MCM-41 at the nanoscale, exhibiting uniform porosity, colloidal stability, and thermal resilience. In vivo quantitative assessment demonstrates a significant acceleration in wound healing facilitated by the composite nanofibers. Furthermore, the incorporation of capsaicin into Zn/MCM-41 augments the wound healing process, as corroborated by histological evaluations. In summary, our investigation introduces an advanced composite nanofiber formulation that promotes accelerated wound healing quantitatively through the synergistic amalgamation of PVA, PGA, capsaicin, and Zn/MCM-41. The demonstrated efficacy of the nanofibers underscores their potential in translational regenerative medicine and wound healing applications.

Advanced Biomaterials for Lacrimal Tissue Engineering: A Review.

The lacrimal gland (LG) is vital for ocular health, producing tears that lubricate and protect the eye. Dysfunction of the LG leads to aqueous-deficient dry eye disease (DED), significantly impacting quality of life. Current treatments mainly address symptoms rather than the underlying LG dysfunction, highlighting the need for regenerative therapies. Tissue engineering offers a promising solution, with biomaterials playing crucial roles in scaffolding and supporting cell growth for LG regeneration. This review focuses on recent advances in biomaterials used for tissue engineering of the lacrimal gland. We discuss both natural and synthetic biomaterials that mimic the extracellular matrix and provide structural support for cell proliferation and differentiation. Natural biomaterials, such as Matrigel, decellularized extracellular matrices, chitosan, silk fibroin hydrogels, and human amniotic membrane are evaluated for their biocompatibility and ability to support lacrimal gland cells. Synthetic biomaterials, like polyethersulfone, polyesters, and biodegradable polymers (PLLA and PLGA), are assessed for their mechanical properties and potential to create scaffolds that replicate the complex architecture of the LG. We also explore the integration of growth factors and stem cells with these biomaterials to enhance tissue regeneration. Challenges such as achieving proper vascularization, innervation, and long-term functionality of engineered tissues are discussed. Advances in 3D bioprinting and scaffold fabrication techniques are highlighted as promising avenues to overcome current limitations.

EXOSOME-ENHANCED HYDROGEL DRESSINGS FOR ACCELERATED ACUTE SKIN WOUND HEALING

Background and Objectives: Acute skin wounds present significant challenges, necessitating effective therapeutic strategies. This study investigates the potential of extra­cellular vesicles (EVs) in combination with hydrogel matrices for improving wound healing outcomes. Materials and Methods: A literature review was conduc­ted following PRISMA guidelines to identify relevant studies. Inclusion criteria comprised prospective in-vivo trials utilizing standardized acute skin wounds on healthy subjects. Key treatments involved topical application of EVs incorporated into hydrogel matrices. Electronic literature searches were conducted in PubMed, Science Direct, and Cochrane Library using specific keywords. Results: Among 541 initially identified publications, 37 studies met inclusion criteria. After full-text analysis, six studies were included in the review. Findings revealed significant advancements in wound healing outcomes when EVs were combined with hydrogel matrices. These combinations demonstrated accelerated wound closure, enhanced tissue regeneration, and increased collagen deposition compared to controls. Conclusions: The integration of EVs with hydrogel matri­ces represents a promising approach for improving acute skin wound healing. Synergistic effects observed in the reviewed studies underscore the potential of this com­bination therapy. However, further research is needed to standardize protocols, optimize dosage and delivery mechanisms, and evaluate long-term safety and efficacy. Overall, this study highlights the potential of EVs and hydrogel matrices in addressing the unmet clinical needs associated with novel acute skin wound management.

Barbaloin loaded chitosan-gum kondagogu polyelectrolyte complex based biocomposites film for enhanced antibacterial, antioxidant and wound healing activity

This study developed an advanced biocomposites film by combining chitosan (CS) with gum kondagogu (GKG) to form a polyelectrolyte complex (PEC) and incorporating zinc oxide nanoparticles (ZnO NP) and barbaloin (BB) to boost wound healing. In brief, the PEC formed between CS and GKG was confirmed through FTIR analysis. The integration of BB into the PEC polymeric matrix resulted in a uniform distribution of BB and loss of crystallinity. As well, the optimized CS/GKG/BB-3 biocomposite film displayed good mechanical strength (8.9 ± 0.30 MPa), high water absorption (451.1 ± 6.02 %), and good water retention potential. The optimized biocomposite film exhibited notable antioxidant properties and effective antimicrobial activity against Staphylococcus aureus and Escherichia coli. Moreover, in vivo studies demonstrated that this biocomposite film facilitated rapid and complete re-epithelialization, enhancing tissue regeneration with minimal scarring, outperforming other biocomposite film formulations. Here, the controlled degradation profile and excellent moisture retention of the CS/GKG/BB-3 biocomposite film created an ideal environment for wound healing. In conclusion, the prepared CS/GKG/BB-3 biocomposite film offers the potential as a superior alternative to traditional wound dressings, emphasizing the synergistic benefits of CS, GKG, ZnO NP, and BB in developing advanced biomaterials for effective wound care and tissue repair.

Conditioned medium of human umbilical cord-mesenchymal stem cells cultivated with human cord blood serum enhances stem cell stemness and secretome profiles.

The proteins secreted by human umbilical cord mesenchymal stem cells (hUC-MSCs) may enhance tissue regeneration and wound healing. Traditional hUC-MSC cultures may not be enough since they undergo recurrent cellular senescence during large-scale production. This decreases the therapeutic ability of hUC-MSCs by altering genes and proteins that control stemness, proliferation, and protein release. Human cord blood serum (CBS) and the middle-density technique were used to evaluate hUC-MSC regeneration ability. To evaluate early-passage hMSCs for secretome-based therapies, they were expanded and secreted in vitro. After 4 days, hUC-MSCs cultivated at 3000 cells/cm2 and supplemented with 1 ng/ml CBS showed increased growth, cell proliferation, and a much lower population doubling time. CBS treatment reduced CD34, CD45, and HLA-DR levels in human umbilical cord mesenchymal stem cells (hUC-MSCs) by less than 2 %. Positive markers such CD73, CD90, and CD105 were found at >97 %, like control hUC-MSCs. Over extended culture, this combination culture can increase survival, proliferation, and stemness and postpone cell death and hUC-MSC senescence. The protein profile and hUC-MSC secretion were improved to make MSC secretion protein therapeutic. This improves cell-free treatment, proliferation, and wound healing in human skin cells. To improve cell-based transplantation or cosmeceutical manufacturing, this technique can boost hUC-MSC regeneration capacity.

Polyphenols in wound healing: unlocking prospects with clinical applications.

Wound healing is a multifaceted, complex process that factors like aging, metabolic diseases, and infections may influence. The potentiality of polyphenols, natural compounds, has shown anti-inflammatory and antimicrobial properties in promoting wound healing and their potential applications in wound management. The studies reviewed indicate that polyphenols have multiple mechanisms that promote wound healing. This involves enhancing antioxidant defenses, reducing oxidative stress, modulating inflammatory responses, improving healing times, reducing infection rates, and enhancing tissue regeneration in clinical trials and in vivo and in vitro studies. Polyphenols have been proven to be effective in managing hard-to-heal wounds, especially in diabetic and elderly populations. Polyphenols have shown significant benefits in promoting angiogenesis and stimulating collagen synthesis. Polyphenol treatment has been demonstrated to have therapeutic effects in wound healing and chronic wound management. Their ability to regulate key healing processes makes them suitable for new wound care products and treatments. Future research should enhance formulations and delivery methods to optimize polyphenols' bioavailability and therapeutic efficacy in wound management approaches.

Fish collagen sponge with human umbilical cord mesenchymal stem cells for diabetic wound repair in rats.

Stem cell therapy offers a novel approach to treating difbetic foot ulcers. Fish skin decellularized matrix, a type I collagen, provides a promising carrier for stem cells, creating a supportive microenvironment that enhances cell survival and therapeutic potential. This study aims to investigate the effects and mechanisms of human umbilical cord mesenchymal stem cells (HUCMSCs) loaded onto a fish collagen sponge for wound healing in diabetic rats. The study evaluates stem cell-loading efficiency with fish collagen sponge in vitro, assesses material distribution on diabetic rat wounds, and establishes a wound model. Rats are divided into the Self-healing group, Fish collagen sponge group, and Sponge loaded with HUCMSCs group. Therapeutic effects are evaluated through various analyses, including histopathology and reverse transcription polymerase chain reaction for collagen-related gene expression levels. Compared to the self-healing group, both the fish collagen group and the composite group show faster wound repair and improved healing outcomes. The composite group exhibits superior wound healing quality, with fish collagen contributing to enhanced tissue regeneration through collagen regulation at the wound site. Loading HUCMSCs onto a fish collagen sponge shows promise for treating diabetic wounds by addressing nutrient deficiency and cell supply issues, offering potential benefits for patients undergoing treatment.

Human macrophage polarisation and regulation of angiogenesis and osteogenesis is dependent on culture extracellular matrix and dimensionality

The immune system plays a crucial role in tissue repair and regeneration. Macrophages have been identified as master regulators of the early immune response and healing outcome, by orchestrating the temporal nature of the initial inflammation phase and coordinating the fate of stem/progenitor cells involved in regeneration. However, traditional in-vitro models for the study of macrophages often fail to fully replicate the complexity of the in-vivo microenvironment, therefore generating models which do not fully capture the extensive spectrum of macrophage behaviour seen in native tissues. To this end, we used a hematoma-mimetic 3D fibrin matrix characteristic of early injured tissues to generate a 3D in-vitro model mirroring the local macrophage microenvironment. Leveraging this framework, we demonstrated significant effects of extracellular matrix and dimensionality on macrophage basal signalling and polarisation, achieving more pronounced regenerative phenotypes upon stimulation with the M2a polarisation factors compared to traditional 2D tissue culture conditions. Moreover, this enhanced physiological macrophage behaviour corresponded to increased coordination of angiogenesis and osteogenesis, better mirroring the healing processes seen in-vivo. Taken together, this study demonstrates the critical importance of integrating tissue composition and 3D architecture when investigating the macrophage behaviour in-vitro, establishing a powerful tool that overcomes known limitations associated with traditional 2D culture on plastic, and can be used to identify and validate novel immunomodulation strategies to enhance tissue regeneration.

Immunomodulatory Biomaterials: Tailoring Surface Properties to Mitigate Foreign Body Reaction and Enhance Tissue Regeneration.

The immune cells have demonstrated the ability to promote tissue repair by removing debris, breaking down the extracellular matrix, and regulating cytokine secretion profile. If the behavior of immune cells is not well directed, chronic inflammation and foreign body reaction (FBR) will lead to scar formation and loss of biomaterial functionality. The immunologic response toward tissue repair or chronic inflammation after injury and implantation can be modulated by manipulating the surface properties of biomaterials. Tailoring surface properties of biomaterials enables the regulation of immune cell fate such as adhesion, proliferation, recruitment, polarization, and cytokine secretion profile. This review begins with an overview of the role of immune cells in tissue healing and their interactions with biomaterials. It then discusses how the surface properties of biomaterials influence immune cell behavior. The core focus is reviewing surface modification methods to create innovative materials that reduce foreign body reactions and enhance tissue repair and regeneration by modulating immune cell activities. The review concludes with insights into future advancements in surface modification techniques and the associated challenges.

Intelligent Hydrogel with Physiologically Dependent Capacities of Photothermal Conversion and Nanocatalytic Medicine to Integratively Inhibit Bacteria and Inflammation for On-Demand Treatment of Infected Wound.

Although chemodynamic therapy (CDT) and photothermal therapy (PTT) based on a variety of nanoparticles have been developed to achieve effective anti-bacterial therapy, the limited therapeutic efficiency of CDT alone, as well as the undifferentiated damage of PTT to both bacteria and surrounding healthy tissue are still challenges for their clinical application of infected wounds treatments. In addition, during the CDT and PTT-mediated antimicrobial processes, the endogenous macrophages would be easily converted to pro-inflammatory macrophages (M1 phenotype) under local ROS and hyperthermia to promote inflammation, resulting in unexpected suppression of tissue regeneration and possible wound deterioration. To address these problems, a biodegradable sodium alginate/hyaluronic acid hydrogel loaded with functional CeO2-Au nano-alloy (AO@ACP) is fabricated to not only achieve precise and efficient antibacterial activity through infection-environment dependent photothermal-chemodynamic therapy but also rapidly eliminate the excess reactive oxygens (ROS) in the M1 type macrophage at the infected area to induce their polarization to M2 type for significant inhibition of inflammation and remarkable enhancement of tissue regeneration, hopefully developing an effective strategy to treat infected wound.

Quantitative Characterization of Zebrafish Caudal Fin Regeneration Based on Mueller Matrix OCT In Vivo.

Zebrafish serves as a valuable model for studying tissue regeneration due to their comprehensive regenerative abilities, particularly in bone tissue. In this study, a Mueller matrix optical coherence tomography (OCT) system was applied to monitor the regenerative processes of zebrafish caudal fins in vivo. The analysis focused on evaluating the thickness of the caudal fin tip and the distribution of internal bone tissue during the regenerative process. Subsequently, the effect of ectoine solution on the regeneration process was observed and discussed. Our findings revealed that the caudal fin blastema did not exhibit phase-induced polarization characteristics in the Mueller matrix OCT images. Statistical analyses indicated that the caudal fins did not fully regenerate to their original state within 21 days. Furthermore, the results suggested that ectoine solution could enhance tissue regeneration. This approach provides a method for quantifying zebrafish caudal fin regeneration and advances observation techniques for biomedical and clinical applications.

Localized Administration of Mesenchymal Stem Cell-Derived Exosomes for the Treatment of Refractory Perianal Fistula in Crohn's Disease Patients: A Phase II Clinical Trial.

Crohn's disease perianal fistulae are often resistant to standard anti-TNF-α therapies. Mesenchymal stem cell (MSC)-derived exosomes are extracellular vesicles that have highly potent anti-inflammatory effects, and the previous phase of this study demonstrated their safety in the treatment of refractory perianal fistulas. To evaluate the efficacy of mesenchymal stem cell-derived exosomes for the treatment of refractory perianal fistulas. Nonrandomized, nonblinded single-center phase II clinical trial. Tertiary university hospital. Twenty-three patients were enrolled, 20 of whom completed the study. Refractory perianal fistula was defined as resistance to at least one course of treatment with anti-tumor necrosis facto-α therapy. After clinical assessment and magnetic resonance imaging, the patients were subjected to general anesthesia, and 5 mL of exosome solution was injected directly into the fistula tracts. The injections were repeated three times at 2-month intervals, and patients were followed monthly for 6 months after the last injection. Tissue samples from the tracts were obtained before each injection and subjected to immunohistopathological assessment. MRI data were obtained before and six months after the last injection. The primary outcome of this study was fistula tract closure on clinical examination and magnetic resonance imaging. The secondary outcome was an improvement in the discharge from the tracts. Fistula tracts were fully closed in 12 (60%) of the patients. Four patients showed clinical improvement, with some tracts remaining open, and four patients were completely resistant to treatment. A total of 43 fistula tracts were treated during the trial, 30 (69.7%) of which showed complete closure. Histopathological analysis revealed substantial reductions in local inflammation and signs of enhanced tissue regeneration. Immunohistochemical analysis of cluster of differentiation-68, 20 and 31 reaffirmed these results. MSC-derived exosomes are safe and effective for treating refractory perianal fistulas in patients with Crohn's disease. See Video Abstract.

Topical ABT-263 treatment reduces aged skin senescence and improves subsequent wound healing.

Senescent cells (SnC) accumulate in aging tissues, impairing their ability to undergo repair and regeneration following injury. Previous research has demonstrated that targeting tissue senescence with senolytics can enhance tissue regeneration and repair by selectively eliminating SnCs in specific aged tissues. In this study, we focused on eliminating SnC skin cells in aged mice to assess the effects on subsequent wound healing. We applied ABT-263 directly to the skin of 24-month-old mice over a 5-day period. Following topical ABT-263, aged skin demonstrated decreased gene expression of senescent markers p16 and p21, accompanied by reductions in SA-β-gal and p21-positive cells compared to DMSO controls. However, ABT-263 also triggered a temporary inflammatory response and macrophage infiltration in the skin. Bulk RNA sequencing of ABT-263-treated skin revealed prompt upregulation of genes associated with wound healing pathways, including hemostasis, inflammation, cell proliferation, angiogenesis, collagen synthesis, and extracellular matrix organization. Aged mice skin pre-treated with topical ABT-263 exhibited accelerated wound closure. In conclusion, topical ABT-263 effectively reduced several senescence markers in aged skin, thereby priming the skin for improved subsequent wound healing. This enhancement may be attributed to ABT-263-induced senolysis which in turn stimulates the expression of genes involved in extracellular matrix remodeling and wound repair pathways.

TLR9 activation in large wound induces tissue repair and hair follicle regeneration via γδT cells

The mechanisms underlying tissue repair in response to damage have been one of main subjects of investigation. Here we leverage the wound-induced hair neogenesis (WIHN) models in adult mice to explore the correlation between degree of damage and the healing process and outcome. The multimodal analysis, in combination with single-cell RNA sequencing help to explore the difference in wounds of gentle and heavy damage degrees, identifying the potential role of toll-like receptor 9 (TLR9) in sensing the injury and regulating the immune reaction by promoting the migration of γδT cells. The TLR9 deficient mice or wounds injected with TLR9 antagonist have greatly impaired healing and lower WIHN levels. Inhibiting the migration of γδT cells or knockout of γδT cells also suppress the wound healing and regeneration, which can’t be rescued by TLR9agonist. Finally, the amphiregulin (AREG) is shown as one of most important effectors secreted by γδT cells and keratinocytes both in silicon or in the laboratory, whose expression influences WIHN levels and the expression of stem cell markers. In total, our findings reveal a previously unrecognized role for TLR9 in sensing skin injury and influencing the tissue repair and regeneration by modulation of the migration of γδT cells, and identify the TLR9-γδT cells-areg axis as new potential targets for enhancing tissue regeneration.