Corneal Regeneration Research Articles

Corneal Treatment, Repair, and Regeneration: Exosomes at Rescue

Corneal Treatment, Repair, and Regeneration: Exosomes at Rescue

Progress in Transdifferentiation of Autologous Alternative Cell Sources into Corneal Epithelial Cells.

Corneal limbal epithelial stem cells (LESCs) play a crucial role in corneal epithelium regeneration. Severe damage to these cells can result in limbal stem cell deficiency (LSCD), characterized by repeated corneal conjunctivalization, leading to corneal turbidity and scar formation. Restoring functional LESCs and their ecological location are essential for treating LSCD. The goal of this review is to provide researchers and clinicians with key insights into LESCs biology and to conclude the current cell-based therapies advancement in LSCD treatments. Therapeutic cell resources mainly include mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), skin keratinocyte stem cells (SKCs), and oral mucosal epithelial cells (OMECs).

Long-term nerve regeneration in diabetic keratopathy mediated by a novel NGF delivery system.

Diabetic keratopathy (DK) is a common chronic metabolic disorder that causes ocular surface complications. Among various therapeutic approaches, local delivery of nerve growth factor (NGF) remains the most effective treatment for DK. However, achieving a sustained therapeutic effect with NGF and the frequent drug delivery burden remain challenging during clinical practice. Here, we developed a novel adeno-associated virus (AAV)-based NGF delivery system that achieved one-year-long-lasting effects by a single injection. We refined the corneal stromal injection technique, resulting in reduced corneal edema and improved AAV distribution homogeneity. AAV serotype AAV.rh10 exhibited high tropism and specificity to corneal nerves. A dose of 2×109 vector genomes (vg) was determined to achieve efficient Ngf gene expression without inducing corneal immune responses. Moreover, NGF protein was highly expressed in trigeminal ganglion (TG) through a retrograde transport mechanism, indicating the capacity for repairing corneal nerve damage both at the root and corneal nerve endings. In a mouse DK model, a single injection of AAV-Ngf into the corneal stroma led to marked corneal nerve regeneration for over 5 months. Together, we provide a novel therapeutic paradigm for long-term effective treatment of DK and this therapeutic approach is superior to current DK therapies.

A unique and biocompatible corneal collagen crosslinking in vivo

Corneal crosslinking (CXL) is a widely applied technique to halt the progression of ectatic diseases through increasing the thickness and mechanical stiffness of the cornea. This study investigated the biocompatibility and efficiency of a novel CXL procedure using ruthenium and blue light in rat corneas and evaluated parameters important for clinical application. To perform the CXL procedure, the corneal epithelium of rats was removed under anaesthesia, followed by the application of a solution containing ruthenium and sodium persulfate (SPS). The corneas were then exposed to blue light at 430 nm at 3 mW/cm2 for 5 min. Rat corneas were examined and evaluated for corneal opacity, corneal and limbal neovascularization, and corneal epithelial regeneration on days 0, 1, 3, 6, 8, and 14. On day 28, the corneas were isolated for subsequent tissue follow-up and analysis. CXL with ruthenium and blue light showed rapid epithelial healing, with 100% regeneration of the corneal epithelium and no corneal opacity on day 6. The ruthenium group also exhibited significantly reduced corneal (p < 0.01) and limbal neovascularization (p < 0.001). Histological analysis revealed no signs of cellular damage or apoptosis, which further confirms the biocompatibility and nontoxicity of our method. Confocal and scanning electron microscopy (SEM) images confirmed high density of collagen fibrils, indicating efficient crosslinking and enhanced structural integrity. This study is unique that demonstrates in vivo safety, biocompatibility, and functionality of ruthenium and blue light CXL. This approach can prevent toxicity caused by UV-A light and can be an immediate alternative compared to the existing crosslinking procedures that have side effects and clinical risks for the patients.

Molecular and Cellular Mechanisms of the Therapeutic Effect of Mesenchymal Stem Cells and Extracellular Vesicles in Corneal Regeneration

The cornea is a vital component of the visual system, and its integrity is crucial for optimal vision. Damage to the cornea resulting from trauma, infection, or disease can lead to blindness. Corneal regeneration using mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) offers a promising alternative to corneal transplantation. MSCs are multipotent stromal cells that can differentiate into various cell types, including corneal cells. They can also secrete a variety of anti-inflammatory cytokines and several growth factors, promoting wound healing and tissue reconstruction. This review summarizes the current understanding of the molecular and cellular mechanisms by which MSCs and MSC-EVs contribute to corneal regeneration. It discusses the potential of MSCs and MSC-EV for treating various corneal diseases, including corneal epithelial defects, dry eye disease, and keratoconus. The review also highlights finalized human clinical trials investigating the safety and efficacy of MSC-based therapy in corneal regeneration. The therapeutic potential of MSCs and MSC-EVs for corneal regeneration is promising; however, further research is needed to optimize their clinical application.

Impact of Neurostimulation, Immunomodulation, Topical Medication Application, and Surgical Reconstruction on Corneal Nerve Function and Regeneration.

The corneal epithelium, supplied by thousands of nerve endings, plays a substantial role in absorbing and distributing nutrients along the ocular surface. Many studies have explored the influence of various modalities in regulating tear production to manage corneal disorders and dry eye disease. These findings have highlighted the advantages of enhancing corneal nerve function and regeneration through neurostimulation, neural signaling, immunomodulation, topical medication application, and surgical reconstruction. The purpose of this narrative review article was to provide an overview of the current state of knowledge on this topic based on a PubMed database literature search for relevant animal and human studies investigating the modification of the trigeminal pathway to restore corneal nerve function and improve overall ocular health. Further investigation into this area of research is important to help guide new therapeutic targets for the prevention and development of treatments of corneal degeneration.

Bio-printing method as a novel approach to obtain a fibrin scaffold settled by limbal epithelial cells for corneal regeneration

Treatment of Limbal Stem Cell Deficiency (LSCD), based on autologous transplantation of the patient’s stem cells, is one of the few medical stem cell therapies approved by the European Medicines Agency (EMA). It relies on isolating and culturing in vivo Limbal Epithelial Stem Cells (LESC) and then populating them on the fibrin substrate, creating a scaffold for corneal epithelial regeneration. Such a solution is then implanted into the patient’s eye. The epithelial cell culture process is specific, and its results strongly depend on the initial cell seeding density. Achieving control of the density and repeatability of the process is a desirable aim and can contribute to the success of the therapy. The study aimed to test bioprinting as a potential technique to increase the control over LESCs seeding on a scaffold and improve process reproducibility. Cells were applied to 0.5 mm thick, flat, transparent fibrin substrates using extrusion bioprinting; the control was the traditional manual application of cells using a pipette. The use of 3D printer enabled uniform coverage of the scaffold surface, and LESCs density in printed lines was close to the targeted value. Moreover, printed cells had higher cell viability than those seeded traditionally (91.1 ± 8.2% vs 82.6 ± 12.8%). The growth rate of the epithelium was higher in bioprinted samples. In both methods, the epithelium had favorable phenotypic features (p63 + and CK14 +). 3D printing constitutes a promising approach in LSCD therapy. It provides favorable conditions for LESCs growth and process reproducibility. Its application may lead to reduced cell requirements, thereby to using fewer cells on lower passages, which will contribute to preserving LESCs proliferative potential.

Fabrication of GelMA - Agarose Based 3D Bioprinted Photocurable Hydrogel with In Vitro Cytocompatibility and Cells Mirroring Natural Keratocytes for Corneal Stromal Regeneration.

The complex anatomy of the cornea and the subsequent keratocyte-fibroblast transition have always made corneal stromal regeneration difficult. Recently, 3D printing has received considerable attentionin terms of fabrication of scaffoldswith precise dimension and pattern. In the current work, 3D printable polymer hydrogels made of GelMA/agarose are formulated and its rheological properties are evaluated. Despite the variation in agarose content, both the hydrogels exhibited G'>G'' modulus. A prototype for 3D stromal model is created using Solid Works software, mimicking the anatomy of an adult cornea. The fabrication of 3D-printed hydrogels is performed using pneumatic extrusion. The FTIR analysis speculated that the hydrogel is well crosslinked and established strong hydrogen bonding with each other, thus contributing to improved thermal and structural stability. The MTT analysis revealed a higher rate of cell proliferation on the hydrogels. The optical analysis carried out on the 14th day of incubation revealed that the hydrogels exhibit transparency matching with natural corneal stromal tissue. Specific protein marker expression confirmed the keratocyte phenotype and showed that the cells do not undergo terminal differentiation into stromal fibroblasts. The findings of this work point to the potential of GelMA/A hydrogels as a novel biomaterial for corneal stromal tissue engineering.

Fabrication and characterization of an electrospun silk fibroin/gelatin transparent graft material for corneal epithelial regeneration

The damaged corneal epithelial causing loss of vision can be restored by epithelial tissue regeneration through tissue engineering approach that provides an engineered corneal substitute with appropriate properties and nanofibrous architecture. This works reports a polymeric matrix that was derived from silk fibroin (SF) and gelatin (GE) natural polymer blend with optimized composition resembling the structure and function of the native epithelial tissue. SF/GE blend spinning solutions prepared with different volume ratios of SF/GE: 70/30, 50/50, and 30/70 were used to fabricate electrospun mats. The fabricated two-dimensional mats had nanofibrous porous architecture having interconnected pores with pore size range of 175–267 nm suitable for corneal epithelial regeneration and 891.2–558 nm fiber diameter range as revealed by scanning electron microscopic image analysis. The performed Fourier transform infrared spectra confirmed the presence of individual polymers in the scaffolds and the scaffolds are hydrophilic in nature. Among the scaffolds, SF/GE 70:30 and SF/GE 50:50 composition possess desired porosity range of 87–88%. The tensile strength was slightly decreased with increase in GE content and the strength obtained was in the range 2.74–2.26 Mpa. An enhanced transparency was obtained with increased GE concentration, the transparency range of 73–82% observed with SF/GE:50/50 matches with the transparency of natural corneal epithelium. The biocompatibility of the scaffolds was confirmed by cell attachment, cytotoxicity and ROS evaluation by culturing rabbit corneal fibroblast cells (SIRC) on the scaffolds. Among the scaffolds, SF/GE: 70/30 exhibited the highest cell viability, but with less transparency ranging 55–71%. Therefore, considering all the properties, SF/GE with 50:50 ratio scaffold may be a suitable substrate in the field of corneal tissue engineering.

TRPV1-targeted ion-responsive hydrogel against pyroptosis of dry eye disease

Dry eye disease (DED) is a complex ocular surface abnormality characterized by tear film hyperosmolarity, inflammation, and damage. Left untreated, DED can lead to a self-perpetuating vicious cycle of worsening symptoms. Existing treatments for DED face challenges related to limited drug delivery to the ocular surface and potential side effects from nonspecific drug targeting. In an effort to address these issues, we developed a novel ion-responsive in situ gelling system targeting the transient receptor potential vanilloid 1 (TRPV1), based on co-assembly of the hydrogelator KFQ12 with the functional peptide V1-Cal. The KFQ12/V1-Cal (KVC) aqueous solution, when dripped and mixed with the ionic tear film on the ocular surface, rapidly transformed into a honeycomb-like hydrogel. This hydrogel exhibited prolonged retention on the ocular surface, serving as a stable scaffold for corneal epithelium regeneration and providing protection against external pathogens while maintaining gas exchange. In a murine model of DED, the KVC hydrogel demonstrated superior therapeutic efficacy due to its sustained release characteristics and specific inhibition of the TRPV1 channel activated by hyperosmotic or desiccating stress. By performing transcriptome sequencing and experimental validation both in vivo and in vitro, we confirmed the mechanism of the TRPV1-Ca2+-Pyroptosis axis in the pathogenesis of DED. Our research provides novel perspectives on the understanding and treatment of DED, and introduces a potential ocular surface drug delivery system characterized by superior biosafety, prolonged retention, and improved therapeutic outcomes.

Harnessing Corneal Stromal Regeneration for Vision Restoration: A Comprehensive Review of the Emerging Treatment Techniques for Keratoconus.

Keratoconus is a progressive corneal disorder characterized by thinning and conical protrusion, leading to visual impairment that often necessitates advanced treatment strategies. Traditional management options, including corrective lenses, corneal cross-linking (CXL), and surgical interventions such as corneal transplants and intracorneal ring segments (ICRS), address symptoms but have limitations, especially in progressive or advanced cases. Recent advancements in corneal stromal regeneration offer promising alternatives for enhancing vision restoration and halting disease progression. This review explores emerging techniques focused on corneal stromal regeneration, emphasizing cell-based therapies, tissue engineering, and gene therapy. Cell-based approaches, including corneal stromal stem cells and adipose-derived stem cells, are promising to promote tissue repair and functional recovery. Tissue engineering techniques, such as developing synthetic and biological scaffolds and 3D bioprinting, are being investigated for their ability to create viable corneal grafts and implants. Additionally, gene therapy and molecular strategies, including gene editing technologies and the application of growth factors, are advancing the potential for targeted treatment and regenerative medicine. Despite these advancements, challenges remain, including technical limitations, safety concerns, and ethical considerations. This review aims to provide a comprehensive overview of these innovative approaches, highlighting their current status, clinical outcomes, and future directions in keratoconus management.

Transcriptomic landscape of quiescent and proliferating human corneal stromal fibroblasts

This study analyzed the transcriptional changes in primary human corneal stromal fibroblasts (hCSFs) grown under quiescent (serum-free) and proliferating (serum-supplemented) culture conditions to identify genes, pathways, and protein‒protein interaction networks influencing corneal repair and regeneration. Primary hCSFs were isolated from donor human corneas and maintained in serum-free or serum-laden conditions. RNA was extracted from confluent cultures using Qiagen kit and subjected to RNA sequencing (RNAseq) analysis. Differential gene expression (DGE) and pathway enrichment analyses were conducted using DESeq2 and Gene Set Enrichment Analysis (GSEA), respectively. Protein‒protein interaction (PPI) networks were created exploiting the STRING database and analyzed with Cytoscape and the cytoHubba plugin. RNA-seq revealed 5,181 genes that were significantly differentially expressed/changed among the 18,812 annotated genes (p value ˂0.05). A cutoff value of a log2-fold change of ±1.5 or greater was used to identify 674 significantly upregulated and 771 downregulated genes between quiescent and proliferating hCSFs. Pathway enrichment analysis revealed significant changes in genes linked to cell cycle regulation, inflammatory, and oxidative stress response pathways, such as E2F Targets, G2M Checkpoint, and MYC Targets, TNFA signaling via NF-kB, and oxidative phosphorylation. Protein-protein interaction network analysis highlighted critical hub genes. The FGF22, CD34, ASPN, DPT, LUM, FGF10, PDGFRB, ECM2, DCN, VEGFD, OMD, OGN, ANGPT1, CDH5, and PRELP were upregulated, whereas genes linked to cell cycle regulation and mitotic progression, such as BUB1, TTK, KIF23, KIF11, BUB1B, DLGAP5, NUSAP1, CCNA2, CCNB1, BIRC5, CDK1, KIF20A, AURKB, KIF2C, and CDCA8, were downregulated. The RNA sequences and gene count files have been submitted to the Gene Expression Omnibus (accession # GSE260476). Our study provides a comprehensive information on the transcriptional and molecular changes in hCSFs under quiescent and proliferative conditions and highlights key pathways and hub genes.

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.

Preparation and characterization of peptide-modified core-shell fibrous substrates with UV-blocking properties for corneal regeneration applications

Effective UV protection is a key aspect of substrates directly exposed to UV radiation. Therefore, in the present study, fibrous substrates of core–shell morphology (PCL-core, PVP-shell) containing peptides based on tryptophan, tyrosine and cysteine (W6, YYC2 and YYC3) were prepared. Spectrophotometric studies showed UV absorption by peptides containing tyrosine and tryptophan in the UVB (up to 80%) and UVA (up to 40%) ranges. Cysteine, in turn, contributed to high antioxidant properties, confirmed by DPPH assay. The presence of peptides contributed to a nonwoven fabric characterized by the ability to absorb UV radiation and prevent the occurrence of oxidative stress (caused by the presence of free radicals). In turn, the increase in the surface zeta potential of the nonwoven after UV irradiation and higher thermal stability (demonstrated by DSC studies) indicated the crosslinking of the PVP layer under UVR, which further contributes to the increased protection of the nonwoven against its effects. In summary, obtained nonwoven exhibited functional similarity to the native cornea, providing a potential solution for enhancing corneal tissue engineering and regenerative medicine applications.

Mesenchymal stem cell therapy in veterinary ophthalmology: clinical evidence and prospects.

Mesenchymal stem cell (MSC) therapy presents a promising strategy for treating various ocular conditions in veterinary medicine. This review explores the therapeutic potential of MSCs in managing corneal ulcers, immune-mediated keratitis, chronic superficial keratitis, keratoconjunctivitis sicca, retinal degeneration, and ocular burns in feline, equine, and canine patients. Studies have demonstrated the immunomodulatory and regenerative properties of MSCs, highlighting their ability to mitigate inflammation and promote tissue regeneration. Experimental studies have shown the potential of MSC therapy in reducing corneal opacity and vascularization, indicating significant therapeutic advantages. Delivery methods play a crucial role in optimizing the therapeutic efficacy of MSCs in ocular diseases. Various delivery methods, such as intravitreal injection, subconjunctival injection, topical administration, and scaffold-mediated delivery, are being explored to optimize MSC delivery to the target ocular tissues. Clinical trials have shown significant improvements in clinical signs following MSC therapy, underscoring its efficacy in treating ocular diseases. Additionally, tissue engineering approaches incorporating MSCs, growth factors, and scaffolds offer innovative strategies for corneal regeneration and tissue repair. Despite challenges such as standardization of protocols and long-term safety assessment, ongoing research endeavours seek to unlock the full therapeutic potential of MSC therapy in ocular diseases. Future prospects in MSC therapy involve exploring scaffold and hydrogel-based approaches and cell-free therapies leveraging the bioactive molecules released by MSCs. Continued research and development efforts are essential to unlock the full therapeutic potential of MSCs and realize their transformative impact on ocular diseases in veterinary patients.

Hepatocyte Growth Factor Modulates Corneal Endothelial Wound Healing In Vitro.

In this study, we assessed the impact of hepatocyte growth factor (HGF) on corneal endothelial cells (CECs), finding that HGF concentrations of 100-250 ng/mL significantly increased CEC proliferation by 30%, migration by 32% and improved survival under oxidative stress by 28% compared to untreated controls (p < 0.05). The primary objective was to identify non-fibrotic pharmacological strategies to enhance corneal endothelial regeneration, addressing a critical need in conditions like Fuchs' endothelial dystrophy (FED), where donor tissue is scarce. To confirm the endothelial nature of the cultured CECs, Na+/K+-ATPase immunohistochemistry was performed. Proliferation rates were determined through BrdU incorporation assays, while cell migration was assessed via scratch assays. Cell viability was evaluated under normal and oxidative stress conditions using WST-1 assays. To ensure that HGF treatment did not trigger epithelial-mesenchymal transition, which could lead to undesirable fibrotic changes, α-SMA staining was conducted. These comprehensive methodologies provided robust data on the effects of HGF, confirming its potential as a therapeutic agent for corneal endothelial repair without inducing harmful EMT, as indicated by the absence of α-SMA expression. These findings suggest that HGF holds therapeutic promise for enhancing corneal endothelial repair, warranting further investigation in in vivo models to confirm its clinical applicability.

Innovative Bioscaffolds in Stem Cell and Regenerative Therapies for Corneal Pathologies.

Corneal diseases, which can result in substantial visual impairment and loss of vision, are an important worldwide health issue. The aim of this review was to investigate the novel application of bioscaffolds in stem cell and regenerative treatments for the treatment of corneal disorders. The current literature reports that organic and artificial substances create bioscaffolds that imitate the inherent structure of the cornea, facilitating the attachment, growth, and specialization of stem cells. Sophisticated methods such as electrospinning, 3D bioprinting, and surface modification have been reported to enhance the characteristics of the scaffold. These bioscaffolds have been shown to greatly improve the survival of stem cells and facilitate the regrowth of corneal tissue in both laboratory and live animal experiments. In addition, the incorporation of growth factors and bioactive compounds within the scaffolds can promote a favorable milieu for corneal regeneration. To summarize, the advancement of these groundbreaking bioscaffolds presents a hopeful treatment strategy for the regeneration of the cornea, which has the potential to enhance the results for individuals suffering from corneal disorders. This study highlights the possibility of utilizing the fields of biomaterials science and stem cell treatment to tackle medical demands that have not yet been satisfied in the field of ophthalmology.

A comprehensive review on natural macromolecular biopolymers for biomedical applications: Recent advancements, current challenges, and future outlooks

Versatile material properties coupled with high degree of biocompatibility and biodegradability has made biopolymers as potential candidates for diverse applications in the biomedical field. Natural biopolymers derived from various plant, animal and microbial sources with different biochemical compositions are extensively used in biomaterial industry with or without further medication to their native form. Biopolymeric biomaterials have been employed in a wide range of biomedical applications like tissue engineering, drug delivery, bone regeneration, wound dressings and cardiovascular surgery. Carbohydrate based biopolymers and protein based biopolymers are extensively used for several applications in the biomedical field including cartilage regeneration, periodontal tissue regeneration, bone regeneration, corneal regeneration, drug delivery and wound healing. This review work presents a comprehensive outlook on the applications of various biopolymers in biomedical field. The work elaborates the biochemistry of these polymers with special focus on their crucial properties in the biomedical industry. Further a detailed description on the most recent application of various biopolymers in the biomedical filed is presented in this review. This work further summarizes the current challenges and future prospects in the use of biopolymers in biomedical field.

The landscape of clinical trials in corneal regeneration: A systematic review of tissue engineering approaches in corneal disease.

The limited availability of a healthy donor cornea and the incidence of allograft failure led researchers to seek other corneal substitutes via tissue engineering. Exploring the trend of clinical trials of the cornea with the vision of tissue engineering provides an opportunity to reveal future potential corneal substitutes. The results of this clinical trial are beneficial for future study designs to overcome the limitations of current therapeutic approaches. In this study, registered clinical trials of bio-based approaches were reviewed for corneal regeneration on March 22, 2024. Among the 3955 registered trials for the cornea, 392 trials were included in this study, which categorized in three main bio-based scaffolds, stem cells, and bioactive macromolecules. In addition to the acellular cornea and human amniotic membrane, several bio-based materials have been introduced as corneal substrates such as collagen, fibrin, and agarose. However, some synthetic materials have been introduced in recent studies to improve the desired properties of bio-based scaffolds for corneal substitutes. Nevertheless, new insights into corneal regenerative medicine have recently emerged from cell sheets with autologous and allogeneic cell sources. In addition, the future perspective of corneal regeneration is described through a literature review of recent experimental models.

Assessment of corneal nerve regeneration after axotomy in a compartmentalized microfluidic chip model with automated 3D high resolution live-imaging.

Damage to the corneal nerves can result in discomfort and chronic pain, profoundly impacting the quality of life of patients. Development of novel in vitro method is crucial to better understand corneal nerve regeneration and to find new treatments for the patients. Existing in vitro models often overlook the physiology of primary sensory neurons, for which the soma is separated from the nerve endings. To overcome this limitation, our novel model combines a compartmentalized microfluidic culture of trigeminal ganglion neurons from adult mice with live-imaging and automated 3D image analysis offering robust way to assess axonal regrowth after axotomy. Physical axotomy performed by a two-second aspiration led to a reproducible 70% axonal loss and altered the phenotype of the neurons, increasing the number of substance P-positive neurons 72 h post-axotomy. To validate our new model, we investigated axonal regeneration after exposure to pharmacological compounds. We selected various targets known to enhance or inhibit axonal regrowth and analyzed their basal expression in trigeminal ganglion cells by scRNAseq. NGF/GDNF, insulin, and Dooku-1 (Piezo1 antagonist) enhanced regrowth by 81, 74 and 157%, respectively, while Yoda-1 (Piezo1 agonist) had no effect. Furthermore, SARM1-IN-2 (Sarm1 inhibitor) inhibited axonal regrowth, leading to only 6% regrowth after 72 h of exposure (versus 34% regrowth without any compound). Combining compartmentalized trigeminal neuronal culture with advanced imaging and analysis allowed a thorough evaluation of the extent of the axotomy and subsequent axonal regrowth. This innovative approach holds great promise for advancing our understanding of corneal nerve injuries and regeneration and ultimately improving the quality of life for patients suffering from sensory abnormalities, and related conditions.