Segment regeneration in earthworms is a remarkable example of postembryonic morphogenesis, yet its fidelity and cellular mechanisms remain incompletely understood. The present study investigated posterior segment regeneration in adult specimens of the earthworm model Eisenia andrei from wound closure to the 5th postoperative week using anatomical, histological, and ultrastructural approaches. Rapid wound closure occurred through fusion of the cut edges of the body wall and midgut without direct involvement of coelomocytes. The regeneration blastema consisted of dedifferentiated epithelial and muscle cells, innervated by fibers from the last intact ventral nerve cord ganglion. Coelomocytes accumulated in the last intact segments and were primarily involved in debris clearance. Notably, early regenerating tissues lacked collagen fibers, which appeared only after the third postoperative week and remained sparse until the fifth week, whereas original segments exhibited intense, region-specific collagen deposition. Transmission electron microscopy revealed characteristic cytological changes in distinct stages of body wall regeneration, including muscle dedifferentiation and the emergence of collagen-producing fibroblasts. These findings indicate that early cell migration, proliferation, and orientation in the blastema proceed independently of collagen and that collagen functions as a delayed structural scaffold, supporting tissue integrity without impeding regeneration. Importantly, no scar formation was observed between old and new tissues, resembling scarless fetal wound healing. Overall, we clarified previously controversial cellular mechanisms and propose a new, comprehensive model for the early stages of segment regeneration. Our results highlight that coordinated dedifferentiation, spatiotemporal extracellular remodeling, and delayed collagen deposition underlie effective, scar-free regeneration in earthworms, offering insights into conserved mechanisms of regenerative repair across metazoans and potential strategies for enhancing tissue regeneration in mammals.

The mouse fetal intrauterine wound healing model is crucial and commonly used for investigating mechanisms and evaluating potential therapies for scarless skin regeneration compared to fibrotic healing. However, traditional intrauterine surgery remains technically challenging and understudied, which is associated with high maternal mortality and pregnancy loss, prompting us to refine the surgical protocol. Here, we report how the choice of surgical and mating procedure impact outcomes obtained. Pregnant mice underwent fetal surgery at embryonic days 15.5, 16.5 (E15.5, E16.5, scarless) and 18.5 (e18.5, fibrotic). Two surgical protocols were used: traditional method involved purse-string sutures, microsurgical scissors, amniotic fluid supplementation, and suture closure (Traditional); and our modified method omitting purse-string sutures, replacing scissors with needle puncture for uterine and fetal incisions, eliminating amniotic fluid supplementation, and employing skin staples for abdominal closure (Modified). The modified protocol significantly increased the likelihood of successful pregnancy, reduced operative time, decreased abortion rates, and enabled earlier modeling compared to the traditional method. At 48h, 7days, and 9days post-surgery, E15.5 wounds healed scarlessly, displaying regenerated hair follicles and organized collagen. Conversely, E18.5 wounds formed typical fibrotic scars, characterized by dense, disorganized collagen without hair follicles. The optimized surgical protocol presented here provides a simplified, reliable fetal mouse model with improved pregnancy success, reduced fetal loss, earlier implementation, and consistent phenotypic outcomes. This refined model enhances experimental efficiency, reproducibility, and animal welfare, having a major impact on mechanistic studies and therapeutic exploration for scarless skin regeneration.

Wound healing is a complex process that, when disrupted, can result in hypertrophic scars or keloids, causing significant physical and psychological discomfort. Despite advances in understanding fibrotic scar formation, achieving scarless healing remains challenging. Inspired by fetal wound healing, this research aims to develop a viscoelastic hydrogel mimicking fetal extracellular matrix properties. The hydrogel comprises hyaluronic acid and alginate, forming reversible dynamic Schiff base and ionic bonds. Interleukin-10 (IL-10), an anti-inflammatory cytokine, is encapsulated using polyelectrolyte complex nanoparticles (PCNs), allowing sustained release and mitigating scar formation by inhibiting inflammatory responses. The results show that this hydrogel demonstrates stress relaxation and self-healing abilities, mimicking the natural characteristics of the extracellular matrix. Additionally, cross-linking with calcium ions induces spontaneous hydrogel contraction, facilitating wound closure and providing tension shielding around the wound site. Such action effectively relieves stress in the wound milieu, reducing the likelihood of fibroblasts differentiation into myofibroblasts and preventing excessive collagen deposition. The viscoelastic hydrogel significantly enhances wound healing by integrating immunomodulatory and tension-shielding properties, thereby creating an optimal environment for scarless healing.

Although hyaluronic acid (HA) has long been used for many medical applications, only in recent years has it gained greater popularity in the field of periodontics because of its biological effects during wound healing. Even today, most clinicians are not aware that more than one type of HA exists and that the extent of its biological functions may vary depending upon the particular characteristics of the biomolecule itself. To review and synthesize the current preclinical and clinical evidence on the biological effects and therapeutic applications of HA in periodontology, with a focus on its role in wound healing and regeneration. The origin and chemical structure of HA are discussed first, with a focus on the importance of its molecular weight and the possibility of modifying its structure and form. The main biological properties of HA followed by its effects on the cells of periodontal tissues are summarized and followed by the presentation of the results from preclinical studies in animals which have evaluated the effects of HA in various types of defects. Subsequently, the data from clinical studies evaluating the application of HA in nonsurgical periodontal therapy, regenerative periodontal surgery, and mucogingival surgery are summarized, and recommendations for the clinicians are provided. The preclinical and clinical evidence indicates that HA accelerates the wound healing process through inflammatory mechanisms and enhances blood clot stability when applied to the root surface. It also influences the expression of both mineralized tissue markers and cementoblast-specific genes, suggesting a potential role in cementum regeneration. HA strongly promotes osteoprogenitor growth while maintaining stemness, potentially regulating the balance between self-renewal and differentiation during bone regeneration. Additionally, HA enhances periodontal ligament (PDL) cell adhesion and proliferation. It has been shown to improve the proliferative and migratory abilities of cells while inducing the expression of collagen type III alpha 1 (COL3A1) and TGFβ-3 genes, which are characteristic of scarless fetal wound healing. Certain HA formulations upregulate the expression of genes encoding platelet-derived growth factor B (PDGFB), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF), all of which play crucial roles in the healing process. Histologic evidence from animal studies suggests that HA may promote periodontal regeneration when applied both non-surgically and surgically-particularly in intrabony defects, gingival recessions, and, to some extent, in furcation defects. The data from clinical studies revealed that HA leads to statistically significant and clinically relevant improvements of probing depths and clinical attachment levels when used in conjunction with nonsurgical periodontal therapy and surgical therapy in intrabony and recession defects. The available data from preclinical and clinical studies provide robust evidence on the effects of HA to enhance periodontal wound healing and regeneration, and on the improved clinical outcomes when HA is used in conjunction with nonsurgical periodontal therapy and regenerative surgery in intrabony and recession defects.

In maternal-fetal medicine, fetal surgery has become a game-changer, providing life-saving treatments for congenital defects that were previously thought to be incurable before to birth. The range of fetal interventions has increased because to developments in prenatal diagnostics, imaging modalities, and surgical procedures, which have improved long-term results and survival rates. Spina bifida, congenital diaphragmatic hernia, twin-to-twin transfusion syndrome, and sacrococcygeal teratoma are among the disorders that have been corrected thanks to open fetal surgery, fetoscopic treatments, and minimally invasive approaches. The dangers of preterm labor, complications for both the mother and the fetus, and the technical difficulties of in utero procedures are some of the obstacles that still exist in spite of these developments.Further influencing the development of this discipline are ethical issues pertaining to the autonomy of the mother and fetus, the possibility of fetal death, and the financial strain on healthcare systems. Enhancing minimally invasive procedures to lower maternal risks, developing biomaterials for fetal wound healing, and incorporating gene and stem cell therapy to treat genetic and developmental abnormalities in utero are the main goals of future perspectives in fetal surgery. Developments in regenerative medicine and artificial womb technology also have the potential to completely transform fetal interventions. Fetal surgery has the potential to provide even safer and more effective treatment choices with further study and technology advancement, revolutionizing newborn care and expanding the scope of perinatal medicine.

Background:The developmental abnormality spina bifida is hallmarked by missing tissues (e.g. skin) and exposure of the spinal cord to the amniotic fluid, which can negatively impact neurological development. Surgical closure of the skin in utero limits neurological damage, but in large defects this results in scarring and contractures. Stimulating skin regeneration in utero would greatly benefit treatment outcome. Previously, we demonstrated that a porous type I collagen (COL) scaffold, functionalized with heparin (HEP), fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) (COL-HEP/GF) improved pre- and postnatal skin regeneration in a fetal sheep full thickness wound model. In this study we uncover the early events associated with enhanced skin regeneration.Methods:We investigated the gene expression profiles of healing fetal skin wounds two weeks after implantation of the COL(-HEP/GF) scaffolds. Using laser dissection and microarrays, differentially expressed genes (DEG) were identified in the epidermis and dermis between untreated wounds, COL-treated wounds and wounds treated with COL-HEP/GF. Biological processes were identified using gene enrichment analysis and DEG were clustered using protein–protein-interaction networks.Results:COL-HEP/GF influences various interesting biological processes involved in wound healing. Although the changes were modest, using protein–protein-interaction networks we identified a variety of clustered genes that indicate COL-HEP/GF induces a tight but subtle control over cell signaling and extracellular matrix organization.Conclusion:These data offer a novel perspective on the key processes involved in (fetal) wound healing, where a targeted and early interference during wound healing can result in long-term enhanced effects on skin regeneration.

Biliary atresia (BA) is a cholangiopathy affecting the extrahepatic bile duct (EHBD) of newborns. The etiology and pathophysiology of BA are not fully understood; however, multiple causes of damage and obstruction of the neonatal EHBD have been identified. Initial damage to the EHBD likely occurs before birth. We discuss how different developmental stages in utero and birth itself could influence the susceptibility of the fetal EHBD to damage and a damaging wound-healing response. We propose that a damage-repair response of the fetal and neonatal EHBD involving redox stress and a program of fetal wound healing could-regardless of the cause of the initial damage-lead to either obstruction and BA or repair of the duct and recovery. This overarching concept should guide future research targeted toward identification of factors that contribute to recovery as opposed to progression of injury and fibrosis. Viewing BA through the lens of an in utero damage-repair response could open up new avenues for research and suggests exciting new therapeutic targets.

The potential of human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hucMSC-EVs) in wound healing is promising, yet a comprehensive understanding of how fibroblasts and keratinocytes respond to this treatment remains limited. This study utilizes single-cell RNA sequencing (scRNA-seq) to investigate the impact of hucMSC-EVs on the cutaneous wound microenvironment in mice. Through rigorous single-cell analyses, we unveil the emergence of hucMSC-EV-induced hematopoietic fibroblasts and MMP13+ fibroblasts. Notably, MMP13+ fibroblasts exhibit fetal-like expressions of MMP13, MMP9, and HAS1, accompanied by heightened migrasome activity. Activation of MMP13+ fibroblasts is orchestrated by a distinctive PIEZO1-calcium-HIF1α-VEGF-MMP13 pathway, validated through murine models and dermal fibroblast assays. Organotypic culture assays further affirm that these activated fibroblasts induce keratinocyte migration via MMP13-LRP1 interactions. This study significantly contributes to our understanding of fibroblast heterogeneities as well as intercellular interactions in wound healing and identifies hucMSC-EV-induced hematopoietic fibroblasts as potential targets for reprogramming. The therapeutic targets presented by these fibroblasts offer exciting prospects for advancing wound healing strategies.

Unlike adult mammalian wounds, early embryonic mouse skin wounds completely regenerate and heal without scars. Analysis of the underlying molecular mechanism will provide insights into scarless wound healing. Twist2 is an important regulator of hair follicle formation and biological patterning; however, it is unclear whether it plays a role in skin or skin appendage regeneration. Here, we aimed to elucidate Twist2 expression and its role in fetal wound healing. ICR mouse fetuses were surgically wounded on embryonic day 13 (E13), E15, and E17, and Twist2 expression in tissue samples from these fetuses was evaluated via in situ hybridization, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction. Twist2 expression was upregulated in the dermis of E13 wound margins but downregulated in E15 and E17 wounds. Twist2 knockdown on E13 left visible marks at the wound site, inhibited regeneration, and resulted in defective follicle formation. Twist2-knockdown dermal fibroblasts lacked the ability to undifferentiate. Furthermore, Twist2 hetero knockout mice (Twist + /-) formed visible scars, even on E13, when all skin structures should regenerate. Thus, Twist2 expression correlated with skin texture formation and hair follicle defects in late mouse embryos. These findings may help develop a therapeutic strategy to reduce scarring and promote hair follicle regeneration.

Antifibrotic properties of hyaluronic acid crosslinked polyisocyanide hydrogels

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia

Chronic non-healing wounds are a significant healthcare challenge. Various biomaterials have been developed to treat chronic wounds but there are still opportunities for improvement of biomaterial therapeutics. This review discusses how fetal wound healing could be used as inspiration to develop pro-healing materials. Compared to adults, fetuses have enhanced wound healing outcomes and healing without scarring. Scarless fetal wound healing is associated with various key differences in several growth factors, cytokines, extracellular matrix components, and coagulation parameters. Mimicking the fetal wound healing environment through bioinspired materials could create improved therapeutics to treat chronic wounds. This review addresses the key differences between adult and fetal wound healing that allow for enhanced scarless fetal healing and discusses how these differences can be used to develop pro-healing materials.

Injured adult tendons heal fibrotically and possess high re-injury rates, whereas fetal tendons appear to heal scarlessly. However, knowledge of fetal tendon wound healing is limited due in part to the need for an accessible animal model. Here, we developed and characterized an in vivo and ex vivo chick embryo tendon model to study fetal tendon healing. In both models, injury sites filled rapidly with cells and extracellular matrix during healing, with wound closure occurring faster in vivo. Tendons injured at an earlier embryonic stage improved mechanical properties to levels similar to non-injured controls, whereas tendons injured at a later embryonic stage did not. Expression levels of tendon phenotype markers, collagens, collagen crosslinking regulators, matrix metalloproteinases, and pro-inflammatory mediators exhibited embryonic stage-dependent trends during healing. Apoptosis occurred during healing, but ex vivo tendons exhibited higher levels of apoptosis than tendons in vivo. Future studies will use these in vivo and ex vivo chick embryo tendon injury models to elucidate mechanisms of stage-specific fetal tendon healing to inform the development of therapeutic approaches to regeneratively heal adult tendons.

Endogenous Interleukin-10 Contributes to Wound Healing and Regulates Tissue Repair

Understanding the complex processes of fetal wound healing and skin regeneration can help to improve fetal surgery. As part of the integumentary system, the skin protects the newborn organism against environmental factors and serves various functions. Glucocorticoids can enter the fetal circulatory system by either elevated maternal stress perception or through therapeutic administration and are known to affect adult skin composition and wound regeneration. In the present study, we aimed at investigating the effects of local glucocorticoid administration on the process of embryonic wound healing. We performed in-ovo bead implantation of dexamethasone beads into skin incisional wounds of avian embryos and observed the local effects of the glucocorticoid on the process of skin regeneration through histology, immunohistochemistry and in-situ hybridization, using vimentin, fibronectin, E-cadherin, Dermo-1 and phospho-Histone H3 as investigational markers. Local glucocorticoid administration decelerated the healing of the skin incisional wounds by impairing mesenchymal contraction and re-epithelialization resulting in morphological changes, such as increased epithelialization and disorganized matrix formation. The results contribute to a better understanding of scarless embryonic wound healing and how glucocorticoids might interfere with the underlying molecular processes, possibly indicating that glucocorticoid therapies in prenatal clinical practice should be carefully evaluated.

Multiple transitions occur in the healing ability of the skin during embryonic development in mice. Embryos up to embryonic day 13 (E13) regenerate completely without a scar after full-thickness wounding. Then, up to E16, dermal structures can be formed, including skin appendages such as hair follicles. However, after E17, wound healing becomes incomplete, and scar formation is triggered. Lhx2 regulates the switch between maintenance and activation of hair follicle stem cells, which are involved in wound healing. Therefore, we investigated the role of Lhx2 in fetal wound healing. Embryos of ICR mice were surgically wounded at E13, E15, and E17, and the expression of Lhx2 along with mitotic (Ki67 and p63) and epidermal differentiation (keratin-10 and loricrin) markers was analyzed. The effect of Lhx2 knockdown on wound healing was observed. Lhx2 expression was not noticed in E13 due to the absence of folliculogenesis but was evident in the epidermal basal layer of E15 and E17 and at the base of E17 wounds, along with Ki67 and p63 expression. Furthermore, Lhx2 knockdown in E15 markedly prolonged wound healing and promoted clear scar formation. Therefore, Lhx2 expression is involved in cell division associated with wound healing and may contribute to scar formation in late embryos.

Wnt proteins secrete glycoproteins that are involved in various cellular processes to maintain homeostasis during development and adulthood. However, the expression and role of Wnt in wound healing have not been fully documented. Our previous studies have shown that, in an early-stage mouse fetus, no scarring occurred after cutaneous wounding, and complete regeneration was achieved. In this study, the expression and localization of Wnt proteins in a mouse fetal-wound-healing model and their associations with scar formation were analyzed. Wnt-related molecules were detected by in-situ hybridization, immunostaining, and real-time polymerase chain reaction. The results showed altered expression of Wnt-related molecules during the wound-healing process. Moreover, scar formation was suppressed by Wnt inhibitors, suggesting that Wnt signaling may be involved in wound healing and scar formation. Thus, regulation of Wnt signaling may be a possible mechanism to control scar formation.

Adult mammalian wounds leave visible scars, whereas skin wounds in developing mouse fetuses are scarless until a certain point in development when complete regeneration occurs, including the structure of the dermis and skin appendages. Analysis of the molecular mechanisms at this transition will provide clues for achieving scarless wound healing. The fibroblast growth factor (FGF) family is a key regulator of inflammation and fibrosis during wound healing. We aimed to determine the expression and role of FGF family members in fetal wound healing. ICR mouse fetuses were surgically wounded at embryonic day 13 (E13), E15, and E17. Expression of FGF family members and FGF receptor (FGFR) in tissue samples from these fetuses was evaluated using in situ hybridization and reverse transcription-quantitative polymerase chain reaction. Fgfr1 was downregulated in E15 and E17 wounds, and its ligand Fgf7 was upregulated in E13 and downregulated in E15 and E17. Recombinant FGF7 administration in E15 wounds suppressed fibrosis and promoted epithelialization at the wound site. Therefore, the expression level of Fgf7 may correlate with scar formation in late mouse embryos, and external administration of FGF7 may represent a therapeutic option to suppress fibrosis and reduce scarring.

In early pregnancy, fetal skin wounds can heal quickly and undergo a transition period from scarless healing to scar formation. The aim of the present study was to identify potential biomarkers associated with scarless repair of cleft lips, in order to determine the intrinsic factors leading to scar formation in embryonic tissue. A stable model of cleft lip was established using microsurgery by constructing a wedge-shaped cleft lip-like defect in fetal rats at gestational age (GA) 16.5 and GA18.5. The GA16.5 and GA18.5 groups were used to model scarless healing and scar formation, respectively. The fetuses were returned to the uterus following surgery, then removed 72 h after the procedure. Macroscopic observation of the cleft defect and histological examination were carried out. Reverse transcription-quantitative (RT-q) PCR and parallel reaction monitoring (PRM) were used to detect mRNA and protein expression levels, respectively. The upper-left lip completely healed 72 h after surgery in the GA16.5 group of fetal rats. However, this was not the case in the GA18.5 group. Histological examination indicated new follicles visible under the epidermis of the scarless group (GA16.5). Scarring was visible on the upper-left cleft lip wound of the fetal rats in the GA18.5 group. The expression of some growth and pro-inflammatory factors, including TNF-α, were also different between two groups. Label-free quantification was used to identified differentially expressed proteins and five differentially expressed proteins (Smad4, Fabp5, S100a4, S100a8 and S100a9) were identified. The relative expression of these molecules at the mRNA and protein levels were measured using RT-qPCR and PRM. These molecules may represent potential biomarkers for the scarless repair of fetal rat cleft lip wounds.

Neural tube (NT) closure is a complex developmental process that takes place in the early stages of embryogenesis and that is a key step in neurulation. In mammals, the process by which the neural plate generates the NT requires organized cell movements and tissue folding, and it terminates with the fusion of the apposed ends of the neural folds. Here we describe how almost identical cellular and molecular machinery is used to fuse the spinal neural folds as that involved in the repair of epithelial injury in the same area of the embryo. For both natural and wound activated closure of caudal neural tissue, hyaluronic acid and platelet-derived growth factor signaling appear to be crucial for the final fusion step. There seems to be no general wound healing machinery for all tissues but rather, a tissue-specific epithelial fusion machinery that embryos activate when necessary after abnormal epithelial opening.