Mitochondrial 'Birth-Death' coordinator: An intelligent hydrogen nanogenerator to enhance intervertebral disc regeneration.

Currently, mitochondrial dysfunction caused by oxidative stress is a growing concern in degenerative diseases, notably intervertebral disc degeneration (IVDD). Dysregulation of the balance of mitochondrial quality control (MQC) has been considered the key contributor, while it's still challenging to effectively harmonize different MQC components in a simple and biologically safe way. Hydrogen gas (H2) is a promising mitochondrial therapeutic molecule due to its bio-reductivity and diffusibility across cellular membranes, yet its relationship with MQC regulation remains unknown. Herein, we propose a mitochondrial 'Birth-Death' coordinator achieved by an intelligent hydrogen nanogenerator (Fe@HP-OD), which can sustainably release H2 in response to the unique microenvironment in degenerated IVDs. Both in vitro and in vivo results prove alleviation of cellular oxidative stress and restoration of nucleus pulposus cells function, thereby facilitating successful IVD regeneration. Significantly, this study for the first time proposes the mitochondrial 'Birth-Death' coordination mechanism: 1) attenuation of overactivated mitochondrial 'Death' process (UPRmt and unselective mitophagy); and 2) activation of Adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway for mitochondrial 'Birth-Death' balance (mitochondrial biogenesis and controlled mitophagy). These pioneering findings can fill in the gaps in molecular mechanisms for H2 regulation on MQC homeostasis, and pave the way for future strategies towards restoring equilibrium of MQC system against degenerative diseases.

Lens Regeneration: The Application of iSyTE and In Silico Approaches to Evaluate Gene Expression in Lens Organoids.

Several protocols have been established for the generation of lens organoids from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and other cells with regenerative potential in humans or various animal models. It is important to examine how well the regenerated lens organoids reflect lens biology, in terms of its development, homeostasis, and aging. Toward this goal, the iSyTE database (integrated Systems Tool for Eye gene discovery; https://research.bioinformatics.udel.edu/iSyTE/ ), a bioinformatics resource tool that contains meta-analyzed gene expression data in wild-type lens across different embryonic, postnatal, and adult stages, can serve as a resource for comparative analysis. This article outlines the approaches toward effective use of iSyTE to gain insights into normal gene expression in the mouse lens, enriched expression in the lens, and differential gene expression in select mouse gene-perturbation cataract/lens defects models, which in turn can be used to evaluate expression of key lens-relevant genes in lens organoids by transcriptomics (e.g., RNA-sequencing (RNA-seq), microarrays, etc.) or other downstream methods (e.g., RT-qPCR, etc.).

Experimental Framework for Assessing Mouse Retinal Regeneration Through Single-Cell RNA-Sequencing.

Retinal degenerative diseases including age-related macular degeneration and glaucoma are estimated to currently affect more than 14 million people in the United States, with an increased prevalence of retinal degenerations in aged individuals. An expanding aged population who are living longer forecasts an increased prevalence and economic burden of visual impairments. Improvements to visual health and treatment paradigms for progressive retinal degenerations slow vision loss. However, current treatments fail to remedy the root cause of visual impairments caused by retinal degenerations-loss of retinal neurons. Stimulation of retinal regeneration from endogenous cellular sources presents an exciting treatment avenue for replacement of lost retinal cells. In multiple species including zebrafish and Xenopus, Müller glial cells maintain a highly efficient regenerative ability to reconstitute lost cells throughout the organism's lifespan, highlighting potential therapeutic avenues for stimulation of retinal regeneration in humans. Here, we describe how the application of single-cell RNA-sequencing (scRNA-seq) has enhanced our understanding of Müller glial cell-derived retinal regeneration, including the characterization of gene regulatory networks that facilitate/inhibit regenerative responses. Additionally, we provide a validated experimental framework for cellular preparation of mouse retinal cells as input into scRNA-seq experiments, including insights into experimental design and analyses of resulting data.

Trabecular Meshwork Regeneration for Glaucoma Treatment Using Stem Cell-Derived Trophic Factors.

Glaucoma is one of the leading causes of irreversible blindness. Stem cell therapy has shown promise in the treatment of primary open-angle glaucoma in animal models. Stem cell-free therapy using stem cell-derived trophic factors might be in demand in patients with high-risk conditions or religious restrictions. In this chapter, we describe methods for trabecular meshwork stem cell (TMSC) cultivation, secretome harvesting, and protein isolation, as well as assays to ensure the health of TMSC post-secretome harvesting and for secretome periocular injection into mice for therapeutic purposes.

Isolation and Characterization of Extracellular Vesicles to Activate Retina Regeneration.

Mammals do not possess the ability to spontaneously repair or regenerate damaged retinal tissue. In contrast to teleost fish which are capable of retina regeneration through the action of Müller glia, mammals undergo a process of reactive gliosis and scarring that inhibits replacement of lost neurons. Thus, it is important to discover novel methods for stimulating mammalian Müller glia to dedifferentiate and produce progenitor cells that can replace lost retinal neurons. Inducing an endogenous regenerative pathway mediated by Müller glia would provide an attractive alternative to stem cell injections or gene therapy approaches. Extracellular vesicles (EVs) are now recognized to serve as a novel form of cell-cell communication through the transfer of cargo from donor to recipient cells or by the activation of signaling cascades in recipient cells. EVs have been shown to promote proliferation and regeneration raising the possibility that delivery of EVs could be a viable treatment for visual disorders. Here, we provide protocols to isolate EVs for use in retina regeneration experiments.

Automated In Vivo Phenotypic Screening Platform for Identifying Factors that Affect Cell Regeneration Kinetics.

Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.

Controlled Release of Molecules to Enhance Cell Survival and Regeneration.

Controlled release or controlled drug delivery comprises the set of techniques and approaches to improve bioavailability through improved safety and/or efficacy using a carrier material for the molecule of interest. The predictability and tunability of these carriers make them ideal for protection, localization, and sustained presentation of a wide range of therapeutics, including growth factors implicated in cell survival and regeneration. Here we provide a method for encapsulating epidermal growth factor in a degradable polymer matrix for delivery to the cornea. Additional notes are included to demonstrate the wide-ranging capabilities of such methods for other materials, therapeutic agents, and sites of action within the eye.

Efficiently directing differentiation and homing of mesenchymal stem cells to boost cartilage repair in osteoarthritis via a nanoparticle and peptide dual-engineering strategy.

Mesenchymal stem cells (MSCs) are expected to be useful therapeutics in osteoarthritis (OA), the most common joint disorder characterized by cartilage degradation. However, evidence is limited with regard to cartilage repair in clinical trials because of the uncontrolled differentiation and weak cartilage-targeting ability of MSCs after injection. To overcome these drawbacks, here we synthesized CuO@MSN nanoparticles (NPs) to deliver Sox9 plasmid DNA (favoring chondrogenesis) and recombinant protein Bmp7 (inhibiting hypertrophy). After taking up CuO@MSN/Sox9/Bmp7 (CSB NPs), the expressions of chondrogenic markers were enhanced while hypertrophic markers were decreased in response to these CSB-engineered MSCs. Moreover, a cartilage-targeted peptide (designated as peptide W) was conjugated onto the surface of MSCs via a click chemistry reaction, thereby prolonging the residence time of MSCs in both the knee joint cavity of mice and human-derived cartilage. In a surgery-induced OA mouse model, the NP and peptide dual-modified W-CSB-MSCs showed an enhancing therapeutic effect on cartilage repair in knee joints compared with other engineered MSCs after intra-articular injection. Most importantly, W-CSB-MSCs accelerated cartilage regeneration in damaged cartilage explants derived from OA patients. Thus, this new peptide and NPs dual engineering strategy shows potential for clinical applications to boost cartilage repair in OA using MSC therapy.

Multilayered hydrogel scaffold construct with native tissue matched elastic modulus: A regenerative microenvironment for urethral scar-free healing.

The unsuitable deformation stimulus, harsh urine environment, and lack of a regenerative microenvironment (RME) prevent scaffold-based urethral repair and ultimately lead to irreversible urethral scarring. The researchers clarify the optimal elastic modulus of the urethral scaffolds for urethral repair and design a multilayered PVA hydrogel scaffold for urethral scar-free healing. The inner layer of the scaffold has self-healing properties, which ensures that the wound effectively resists harsh urine erosion, even when subjected to sutures. In addition, the scaffold's outer layer has an extracellular matrix-like structure that synergizes with adipose-derived stem cells to create a favorable RME. In vivo experiments confirm successful urethral scar-free healing using the PVA multilayered hydrogel scaffold. Further mechanistic study shows that the PVA multilayer hydrogel effectively resists the urine-induced inflammatory response and accelerates the transition of urethral wound healing to the proliferative phase by regulating macrophage polarization, thus providing favorable conditions for urethral scar-free healing. This study provides mechanical criteria for the fabrication of urethral tissue-engineered scaffolds, as well as important insights into their design.

ß-Cell Regeneration Is Driven by Pancreatic Plasticity.

The pancreas has been considered a non-regenerative organ. ß cells lost in diabetes are not replaced due to the inability of the pancreas to regenerate. However, ample evidence generated in the last few decades using murine models has demonstrated that the pancreas has a remarkable plasticity wherein differentiated cells can change cell fate toward a ß-like cell phenotype. Although this process is observed after using rather artificial stimuli and the conversion efficiency is very limited, these findings have shed some light on novel pathways for ß-cell regeneration. In this chapter, we will summarize the different cellular interconversion processes described to date, the experimental details and molecular regulation of such interconversions, and the genomic technologies that have allowed the identification of potential new ways to generate ß cells.

Phyto-safe in vitro regeneration and harnessing antimicrobial-resistant endophytes as bioinoculants for enhanced growth and secondary metabolites yield in Nilgirianthus ciliatus.

Nilgirianthus ciliatus, extensively exploited for its pharmacological properties, is now classified as vulnerable. In vitro micropropagation offers a sustainable approach for ecological conservation and rational utilization of this biodiversity resource. This study aimed to reduce endophytes during in vitro propagation and isolating antimicrobial-resistant endophytes from N. ciliatus by employing various concentrations and exposure times of Plant Preservative Mixture (PPM). Optimal results were observed when nodal explants treated with 0.3% PPM for 8 h, followed by inoculation in Murashige and Skoog (MS) medium supplemented with 3 mg/L 6-benzylaminopurine (BAP) and 0.3% PPM. This protocol achieved 82% shoot regeneration with minimal endophytic contamination, suggesting that the duration of explant exposure to PPM significantly influences endophyte reduction. Two antimicrobial-resistant endophytes were isolated and identified as Bacillus cereus and Acinetobacter pittii through 16S rDNA sequencing. These endophytes exhibited plant growth-promoting characteristics, including amylolytic, proteolytic, lipolytic activities, indole-3-acetic acid production, phosphate solubilization, and stress tolerance. In vivo application of these endophytes as bioinoculants to N. ciliatus not only improved growth parameters but also significantly increased the levels of pharmacologically important compounds, squalene, and stigmasterol, as confirmed by High-performance thin-layer chromatography (HPTLC). This study demonstrates that PPM is a promising alternative for sustainable micropropagation of N. ciliatus. Furthermore, it highlights the potential of antimicrobial-resistant endophytes as bioinoculants to improve growth and medicinal value, offering a sustainable solution for conservation and large-scale cultivation of this species.

The role of erythropoietin-loaded hydrogel versus adipose derived stem cell secretome in the regeneration of tongue defects.

BACKGROUND: Tongue defects have several etiologies and significantly affect the quality of life. This study was conducted to compare the regenerative potential of erythropoietin (EPO)-loaded hydrogel and adipose derived stem cell (ADSC) secretome on tongue dorsum defects focusing on the role of anti-inflammatory M2 macrophage phenotype. METHODS: Rats were subjected to induction of mechanical circular defects on the dorsal surface of the tongue, then divided into three groups; Group I (control): received 0.1 ml phosphate buffered saline, Group II (EPO): received 5000 U/kg EPO-hydrogel, and Group III (ADSC-Secretome): received 0.1 ml ADSC-Secretome. Treatments were injected circumferentially around wound margins after induction. Seven and fourteen days after treatment, specimens were obtained and processed for histological and immunohistochemical staining followed by the relevant histomorphometric and statistical analyses. RESULTS: Seven days after treatment, groups II and III presented defects with some epithelial regeneration at the lateral margins, while the center of the defect showed granulation tissue with much inflammatory cells. The base of the defects showed some muscle fibers and new blood vessels, however group III showed more enhanced neovascularization. Fourteen days after therapeutic intervention, group II defects were completely covered with epithelium showing a thin keratin layer with regular rete pegs interdigitating with the underlying connective tissue papillae, but tongue papillae were not restored. Group III expressed much better healing with developing filiform papillae. The connective tissue showed more vascularity and well-arranged muscle bundles. Both treated groups showed a significant decrease in defect depth and significant increase in anti-inflammatory macrophages compared to the control group at both time intervals, however there was no significant difference between the two treated groups. CONCLUSION: Both treatments showed promising and comparable results in the treatment of tongue defects reducing inflammation and restoring tongue histological architecture with significant upregulation of M2 macrophage.

The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration.

The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration; however, many questions remain about its exact role. Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration. We find in cryoinjured myd88-/- ventricles a significant reduction in neutrophil and macrophage numbers and the expansion of a collagen-rich endocardial population. Further analyses reveal compromised PI3K/AKT pathway activation in the myd88-/- endocardium and increased myofibroblasts and scarring. Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic and scarring phenotypes. Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88 signaling pathway, and using loss-of-function and gain-of-function tools, we show that it controls neutrophil recruitment. Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration.

PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens.

Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens.

1,25-Dihydroxyvitamin D Enhances the Regenerative Function of Lgr5+ Intestinal Stem Cells In Vitro and In Vivo.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestines without a cure. Current therapies suppress inflammation to prevent further intestinal damage. However, healing already damaged intestinal epithelia is still an unmet medical need. Under physiological conditions, Lgr5+ intestinal stem cells (ISCs) in the intestinal crypts replenish the epithelia every 3-5 days. Therefore, understanding the regulation of Lgr5+ ISCs is essential. Previous data suggest vitamin D signaling is essential to maintain normal Lgr5+ ISC function in vivo. Our recent data indicate that to execute its functions in the intestines optimally, 1,25(OH)2D requires high concentrations that, if present systemically, can cause hypercalcemia (i.e., blood calcium levels significantly higher than physiological levels), leading to severe consequences. Using 5-bromo-2'-deoxyuridine (BrdU) to label the actively proliferating ISCs, our previous data suggested that de novo synthesized locally high 1,25(OH)2D concentrations effectively enhanced the migration and differentiation of ISCs without causing hypercalcemia. However, although sparse in the crypts, other proliferating cells other than Lgr5+ ISCs could also be labeled with BrdU. This current study used high-purity Lgr5+ ISC lines and a mouse strain, in which Lgr5+ ISCs and their progeny could be specifically tracked, to investigate the effects of de novo synthesized locally high 1,25(OH)2D concentrations on Lgr5+ ISC function. Our data showed that 1,25(OH)2D at concentrations significantly higher than physiological levels augmented Lgr5+ ISC differentiation in vitro. In vivo, de novo synthesized locally high 1,25(OH)2D concentrations significantly elevated local 1α-hydroxylase expression, robustly suppressed experimental colitis, and promoted Lgr5+ ISC differentiation. For the first time, this study definitively demonstrated 1,25(OH)2D's role in Lgr5+ ISCs, underpinning 1,25(OH)2D's promise in IBD therapy.

Keratin 8/18a.1 Expression Influences Embryonic Neural Crest Cell Dynamics and Contributes to Postnatal Corneal Regeneration in Zebrafish.

A complete understanding of neural crest cell mechanodynamics during ocular development will provide insight into postnatal neural crest cell contributions to ophthalmic abnormalities in adult tissues and inform regenerative strategies toward injury repair. Herein, single-cell RNA sequencing in zebrafish during early eye development revealed keratin intermediate filament genes krt8 and krt18a.1 as additional factors expressed during anterior segment development. In situ hybridization and immunofluorescence microscopy confirmed krt8 and krt18a.1 expression in the early neural plate border and migrating cranial neural crest cells. Morpholino oligonucleotide (MO)-mediated knockdown of K8 and K18a.1 markedly disrupted the migration of neural crest cell subpopulations and decreased neural crest cell marker gene expression in the craniofacial region and eye at 48 h postfertilization (hpf), resulting in severe phenotypic defects reminiscent of neurocristopathies. Interestingly, the expression of K18a.1, but not K8, is regulated by retinoic acid (RA) during early-stage development. Further, both keratin proteins were detected during postnatal corneal regeneration in adult zebrafish. Altogether, we demonstrated that both K8 and K18a.1 contribute to the early development and postnatal repair of neural crest cell-derived ocular tissues.

A novel injectable boron doped-mesoporous nano bioactive glass loaded-alginate composite hydrogel as a pulpotomy filling biomaterial for dentin regeneration.

BACKGROUND: Different materials have been used as wound dressings after vital pulp therapies. Some of them have limitations such as delayed setting, difficult administration, slight degree of cytotoxicity, crown discoloration and high cost. Therefore, to overcome these disadvantages, composite scaffolds have been used in regenerative dentistry. This study aims to construct and characterize the physicochemical behavior of a novel injectable alginate hydrogel loaded with different bioactive glass nanoparticles in various concentrations as a regenerative pulpotomy filling material. METHODS: Alginate hydrogels were prepared by dissolving alginate powder in alcoholic distilled water containing mesoporous bioactive glass nanoparticles (MBG NPs) or boron-doped MBG NPs (BMBG NPs) at 10 and 20 wt% concentrations. The mixture was stirred and incubated overnight in a water bath at 50 0 C to ensure complete solubility. A sterile dual-syringe system was used to mix the alginate solution with 20 wt% calcium chloride solution, forming the hydrogel upon extrusion. Then, constructed hydrogel specimens from all groups were characterized by FTIR, SEM, water uptake percentage (WA%), bioactivity and ion release, and cytotoxicity. Statistical analysis was done using One-Way ANOVA test for comparisons between groups, followed by multiple pairwise comparisons using Bonferroni adjusted significance level (p < 0.05). RESULTS: Alginate/BMBG loaded groups exhibited remarkable increase in porosity and pore size diameter [IIB1 (168), IIB2 (183) (µm)]. Similarly, WA% increased (~ 800%) which was statistically significant (p < 0.05). Alginate/BMBG loaded groups exhibited the strongest bioactive capability displaying prominent clusters of hydroxyapatite precipitates on hydrogel surfaces. Ca/P ratio of precipitates in IIA2 and IIB1 (1.6) were like Ca/P ratio for stoichiometric pure hydroxyapatite (1.67). MTT assay data revealed that the cell viability % of human gingival fibroblast cells have declined with increasing the concentration of both powders and hydrogel extracts in all groups after 24 and 48 h but still higher than the accepted cell viability % of (˃70%). CONCLUSIONS: The outstanding laboratory performance of the injectable alginate/BMBGNPs (20 wt%) composite hydrogel suggested it as promising candidate for pulpotomy filling material potentially enhancing dentin regeneration in clinical applications.

Spatio-temporal expression of Sox2+ progenitor cells regulates the regeneration of rat submandibular gland.

OBJECTIVE: Sox2 plays crucial roles in tissues homeostasis and regeneration. However, there are lack of a comprehensive examination of Sox2 expression and its functional role in submandibular gland regeneration. Therefore, we aimed to elucidate the impact of Sox2 on submandibular gland regeneration. MATERIALS AND METHODS: A Sprague-Dawley rat submandibular gland duct ligation/de-ligation regeneration model was conducted in this study. Sox2-shRNA vectors were retro-ductally administered into the submandibular gland to establish a stable Sox2 knockdown model. Conventional histopathological and molecular biological methods were used to investigate phenotypic changes. RESULTS: The submandibular gland normalized completely 28 days after ligature removal (following 7 days of duct ligation). AQP5 expression gradually increased after ligation removal until returning to normal levels. In submandibular gland regeneration, Sox2 re-expressed and co-expressed with AQP5+ acinar cells, and Sox2 expression peaked on day 14, recovered to normal on day 28, reproducing the developmental pattern. Sox2 knockdown hindered gland regeneration and induced irreversible fibrosis. The AQP5 expression was significantly lower than the contemporaneous solely ligated group, while the blue collagen deposition and the Vimentin expression increased prominently. The expression of CD68, IL-1ß, TNF-α and IL-17A increased significantly, and epithelial cells in the Sox2 knockdown group expressed higher levels of IL-17A. CONCLUSIONS: These findings highlight Sox2 as a crucial regulator of the acinar cell lineage. Sox2+ progenitor cells are pivotal for acinar cell maintenance, which is indispensable for submandibular gland regeneration. Collectively, our findings may help develop targeted interventions for enhancing tissue repair and preventing irreversible fibrosis in salivary gland disorders.

Tcap deficiency impedes striated muscle function and heart regeneration with elevated ROS and autophagy.

Telethonin/titin-cap (TCAP) encodes a Z-disc protein that plays important roles in sarcomere/T-tubule interactions, stretch-sensing and signaling. Mutations in TCAP are associated with muscular dystrophy and cardiomyopathy; however, the complete etiology and its roles in myocardial infarction and regeneration are not fully understood. Here, we generated tcap gene knockout zebrafish with CRISPR/Cas9 technology and observed muscular dystrophy-like phenotypes and abnormal mitochondria in skeletal muscles. The stretch-sensing ability was inhibited in tcap-/- mutants. Moreover, Tcap deficiency led to alterations in cardiac morphology and function as well as increases in reactive oxygen species (ROS) and mitophagy. In addition, the cardiac regeneration and cardiomyocyte proliferation ability of tcap-/- mutants were impaired, but these impairments could be rescued by supplementation with ROS scavengers or autophagy inhibitors. Overall, our study demonstrates the essential roles of Tcap in striated muscle function and heart regeneration. Additionally, elevations in ROS and autophagy may account for the phenotypes resulting from Tcap deficiency and could serve as novel therapeutic targets for muscular dystrophy and cardiomyopathy.

The dual role of glucocorticoid regeneration in inflammation at parturition.

Introduction: Fetal membrane inflammation is an integral event of parturition. However, excessive pro-inflammatory cytokines can impose threats to the fetus. Coincidentally, the fetal membranes express abundant 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which generates biologically active cortisol to promote labor through induction of prostaglandin synthesis. Given the well-recognized anti-inflammatory actions of glucocorticoids, we hypothesized that cortisol regenerated in the fetal membranes might be engaged in restraining fetus-hazardous pro-inflammatory cytokine production for the safety of the fetus, while reserving pro-labor effect on prostaglandin synthesis to ensure safe delivery of the fetus. Methods: The hypothesis was examined in human amnion tissue and cultured primary human amnion fibroblasts as well as a mouse model. Results: 11ß-HSD1 was significantly increased in the human amnion in infection-induced preterm birth. Studies in human amnion fibroblasts showed that lipopolysaccharide (LPS) induced 11ß-HSD1 expression synergistically with cortisol. Cortisol completely blocked NF-κB-mediated pro-inflammatory cytokine expression by LPS, but STAT3-mediated cyclooxygenase 2 expression, a crucial prostaglandin synthetic enzyme, remained. Further studies in pregnant mice showed that corticosterone did not delay LPS-induced preterm birth, but alleviated LPS-induced fetal organ damages, along with increased 11ß-HSD1, cyclooxygenase 2, and decreased pro-inflammatory cytokine in the fetal membranes. Discussion: There is a feed-forward cortisol regeneration in the fetal membranes in infection, and cortisol regenerated restrains pro-inflammatory cytokine expression, while reserves pro-labor effect on prostaglandin synthesis. This dual role of cortisol regeneration can prevent excessive pro-inflammatory cytokine production, while ensure in-time delivery for the safety of the fetus.