Bioengineering

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15 pages, 5601 KB

Open AccessArticle

Putative Self-Organizing Human Corneal Organoids Recapitulate Human Corneal Architecture and Cellular Diversity

by Timothy A. Blenkinsop and Anne Z. Eriksen

Bioengineering 2026, 13(5), 518; https://doi.org/10.3390/bioengineering13050518 - 29 Apr 2026

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Abstract

Background: Corneal organoids derived from pluripotent stem cells have emerged as powerful tools for studying corneal development, disease modeling, and regenerative medicine applications. While previous protocols have successfully generated corneal tissue structures, there remains a need for three-dimensional models that recapitulate the complex [...] Read more.

Background: Corneal organoids derived from pluripotent stem cells have emerged as powerful tools for studying corneal development, disease modeling, and regenerative medicine applications. While previous protocols have successfully generated corneal tissue structures, there remains a need for three-dimensional models that recapitulate the complex cellular architecture and diversity of native human cornea. Methods: We developed a modified spontaneous three-dimensional corneal organoid model using human embryonic stem cells (hESCs) through an adapted Self-formed Ectoderm Autonomous Multi-zone (SEAM) protocol. hESCs were cultured as spheroids in ultra-low-binding plates under normoxic conditions and differentiated over 7–8 weeks. Organoids were characterized using immunofluorescence staining for corneal-specific markers and single-cell RNA sequencing to assess cellular composition and gene expression patterns. Results: Approximately 20% of organoids developed transparent regions characteristic of corneal tissue by day 30 of differentiation. Immunofluorescence analysis revealed spatially organized expression of corneal markers, including ZO-1 and E-cadherin in the outermost epithelial layers, P63α-positive putative limbal stem cells at the epithelial–stromal interface, vimentin-positive stromal cells in the interior, and laminin-1 deposition that suggests Bowman’s membrane formation. The organoids expressed cornea-specific keratins (K3, K12, and K15) and the master regulator PAX6 in appropriate cellular compartments. Single-cell RNA sequencing identified 18 distinct cell clusters, including three corneal epithelium subclusters with differential expression of MUC16, KRT12, and ΔNp63α, two stromal populations with distinct inflammatory profiles, and a corneal endothelium cluster. Transcriptomic analysis confirmed expression of key corneal genes, including AQP3, CDH1, multiple keratins, mucins, and extracellular matrix components (HAS2, CD34, CD44, COL8A1, and KERA). Conclusions: This three-dimensional spheroid-based putative corneal organoid model successfully recapitulates the multilayered architecture and cellular diversity of human cornea, including stratified epithelium, putative limbal stem cells, stroma, and endothelium in spatially appropriate arrangements. The model demonstrates molecular signatures consistent with native corneal tissue and provides a valuable platform for studying corneal development, disease mechanisms, and potential therapeutic applications. Future optimization to improve organoid formation efficiency and functional maturation will enhance the utility of this system for both basic research and translational medicine. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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13 pages, 3139 KB

Open AccessArticle

Cytocompatibility of PMMA and Titanium Boston Keratoprosthesis Backplates with Human Corneal Fibroblasts

by Antonio Esquivel Herrera, Liangju Kuang, Mark Krauthammer, Michael Bednar, Eleftherios I. Paschalis and Thomas H. Dohlman

Bioengineering 2026, 13(5), 517; https://doi.org/10.3390/bioengineering13050517 - 29 Apr 2026

Viewed by 528

Abstract

This study evaluates how titanium and polymethyl methacrylate (PMMA) Boston Keratoprosthesis backplate substrates influence human corneal fibroblast proliferation, cytotoxicity, morphology, activation phenotype, and mechanotransductive signaling. Human corneal fibroblasts were cultured on titanium and PMMA, with tissue culture plastic or glass as controls. Proliferation [...] Read more.

This study evaluates how titanium and polymethyl methacrylate (PMMA) Boston Keratoprosthesis backplate substrates influence human corneal fibroblast proliferation, cytotoxicity, morphology, activation phenotype, and mechanotransductive signaling. Human corneal fibroblasts were cultured on titanium and PMMA, with tissue culture plastic or glass as controls. Proliferation was assessed over 7 days using metabolic assays, and cytotoxicity was measured by lactate dehydrogenase release. Cell morphology and surface coverage were examined by scanning electron microscopy, while immunofluorescence quantified fibroblast-specific protein 1 (FSP-1) and α-smooth muscle actin (α-SMA). Gene expression of α-SMA, collagen I, FSP-1, and focal adhesion kinase (FAK) was analyzed by quantitative PCR. Cells cultured on both substrates maintained stable viability with modest increases in estimated cell numbers and comparable proliferation curves, indicating preserved metabolic activity without growth suppression. Cytotoxicity remained low and similar between groups. SEM demonstrated broader and more continuous cell spreading on titanium, whereas cells on PMMA were more sparsely distributed. Immunofluorescence showed higher FSP-1 expression on titanium and increased α-SMA on PMMA. Gene expression analysis revealed higher FAK transcripts on PMMA, with no significant differences in α-SMA, FSP-1, or collagen I. These results confirm the cytocompatibility of both titanium and PMMA backplates with human corneal fibroblasts and support their use with the Boston Keratoprosthesis. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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23 pages, 325 KB

Open AccessArticle

Changes in Ocular Biomechanics During Adolescence and Its Relationship with Lifestyle and Myopic Progression: The Oporto Myopia Study

by Pedro M. L. Baptista, Gabriel Santos, João H. Marques, André Ferreira, Beatriz Vieira, Paulo Sousa, Ricardo Parreira, Renato Ambrósio, Jr., Pedro M. A. M. Menéres and João N. M. Beirão

Bioengineering 2026, 13(3), 367; https://doi.org/10.3390/bioengineering13030367 - 20 Mar 2026

Viewed by 665

Abstract

The relationship between lifestyle, ocular biomechanical behavior, and myopia is not well established in the literature. The present study aims to describe changes in ocular biomechanics during adolescence and to explore their relationship with lifestyle factors and myopic progression. Prospective cohort study including [...] Read more.

The relationship between lifestyle, ocular biomechanical behavior, and myopia is not well established in the literature. The present study aims to describe changes in ocular biomechanics during adolescence and to explore their relationship with lifestyle factors and myopic progression. Prospective cohort study including 63 adolescents (126 eyes) with a mean age of 14.1 ± 2.6 years old examined twice over a 30 ± 0.9-month period. The data from biomechanics, biometry, corneal tomography, and lifestyle was addressed. The relationships between biomechanical changes, biometric and refractive variation, and lifestyle variables were analyzed using parametric and non-parametric statistics with a significance level of p < 0.05. A biomechanical stiffening trend was found. Axial elongation was 0.12 ± 0.17 mm, and refractive shift was −0.32 ± 0.87 D. The history of allergies was associated with greater axial growth (p = 0.032) and smaller increase in stress–strain-index (SSI) (p = 0.01). Myopization was higher in eyes with ocular surface symptoms (p = 0.049) and those with reported eye-rubbing habits (p = 0.04), with a lower gain in stiffness (p < 0.05). Outdoor activities were associated with higher gain in corneo-scleral stiffness (p < 0.05). Reduced myopization correlated directly with the increase in the SSI (p < 0.05) and inversely with the Integrated Radius (p < 0.05). Greater increases in axial length (AL), vitreous cavity length (VCL), and the ratio between VCL and AL (R_VCL/AL) correlated negatively with the increase in the SSI (p < 0.05). The increase in the R_VCL/AL correlated positively with the time spent on digital devices and negatively with the amount of outdoor activity (p < 0.05). Biomechanics may represent the physiological bridge between the environmental exposure and myopization, as lower gain in corneo-scleral stiffness was consistently associated with greater axial elongation and refractive myopization, with outdoor activity appearing to be protective. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

12 pages, 3017 KB

Open AccessArticle

A Novel, Low-Cost, 3D-Printed Motorized Injector for Retinal Sheet Transplantation

by Jerald Lim, Francis Ung, Samir Malhotra, Jacob C. Diaz, Austen Hamilton, Clare Chen, William C. Tang, Magdalene J. Seiler and Andrew W. Browne

Bioengineering 2026, 13(2), 188; https://doi.org/10.3390/bioengineering13020188 - 6 Feb 2026

Viewed by 899

Abstract

Retinal transplantation offers promise for restoring vision in advanced retinal degeneration. However, manual delivery of retinal sheets is often hindered by imprecise placement and collateral tissue damage resulting from instrument instability. We introduce a novel, 3D-printed, motorized retinal sheet injector designed to enhance [...] Read more.

Retinal transplantation offers promise for restoring vision in advanced retinal degeneration. However, manual delivery of retinal sheets is often hindered by imprecise placement and collateral tissue damage resulting from instrument instability. We introduce a novel, 3D-printed, motorized retinal sheet injector designed to enhance placement accuracy and minimize tissue injury. The motorized injector features an Arduino-controlled foot pedal with three discrete actuator positions (“Min”, “Mid”, “Max”). When compared via frame-by-frame motion analysis, the motorized system reduced tip variance by approximately threefold over manual methods. In addition, in in vitro gelatin trials, the motorized injector achieved significantly higher placement accuracy versus the manual injector, which suffered from occasional complete misplacements. The novel motorized retinal sheet injector markedly improves stability and placement accuracy relative to manual methods, potentially reducing complications associated with subretinal delivery. Safer subretinal delivery can pave the way for innovative research and advanced treatment for retinal disease. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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12 pages, 1427 KB

Open AccessArticle

Comparative Analysis of Two Measurement Modalities for Ex Vivo Analysis of Corneal Stiffness in Porcine Corneas

by Sophia A. Reifeltshammer, Hannah Seferovic, Malavika H. Nambiar, Philippe Büchler, Theo G. Seiler, Jascha Wendelstein, Matthias Bolz, Peter Hinterdorfer, Yoo Jin Oh and Isaak Fischinger

Bioengineering 2025, 12(12), 1308; https://doi.org/10.3390/bioengineering12121308 - 28 Nov 2025

Viewed by 779

Abstract

Uniaxial tensile testing and atomic force microscopy (AFM) nanoindentation experiments are two valuable methods used to quantify changes in stiffness after corneal crosslinking (CXL). Here, we apply these methods by characterizing corneal stiffness ex vivo before and after CXL. Sixty-two fresh porcine corneas [...] Read more.

Uniaxial tensile testing and atomic force microscopy (AFM) nanoindentation experiments are two valuable methods used to quantify changes in stiffness after corneal crosslinking (CXL). Here, we apply these methods by characterizing corneal stiffness ex vivo before and after CXL. Sixty-two fresh porcine corneas were divided into three groups: an untreated control group, a CXL3 group treated with the Dresden protocol, and a CXL9 group treated with the accelerated protocol. Biomechanical testing was then performed using either uniaxial tensile testing or AFM nanoindentation. Uniaxial tensile testing revealed a significant increase in corneal stiffness for the CXL3 group compared to the control group (p < 0.05). At 10% strain, the CXL3 and CXL9 groups exhibited increases in stiffness of 96% and 48%, respectively, compared to the control group. In contrast, AFM analysis revealed no significant differences in stiffness, showing 28% and 16% increases in the CXL3 and CXL9 groups, respectively, compared to the control group. The results suggest that uniaxial tensile testing provides a robust, sample-averaged measure of global stiffening. Interestingly, AFM nanoindentation enables mapping of localized biomechanical changes with high spatial resolution but is less sensitive to overall biomechanical changes induced by CXL. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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24 pages, 2761 KB

Open AccessArticle

An Explainable AI Framework for Corneal Imaging Interpretation and Refractive Surgery Decision Support

by Mini Han Wang

Bioengineering 2025, 12(11), 1174; https://doi.org/10.3390/bioengineering12111174 - 28 Oct 2025

Cited by 3 | Viewed by 2055

Abstract

This study introduces an explainable neuro-symbolic and large language model (LLM)-driven framework for intelligent interpretation of corneal topography and precision surgical decision support. In a prospective cohort of 20 eyes, comprehensive IOLMaster 700 reports were analyzed through a four-stage pipeline: (1) automated extraction [...] Read more.

This study introduces an explainable neuro-symbolic and large language model (LLM)-driven framework for intelligent interpretation of corneal topography and precision surgical decision support. In a prospective cohort of 20 eyes, comprehensive IOLMaster 700 reports were analyzed through a four-stage pipeline: (1) automated extraction of key parameters—including corneal curvature, pachymetry, and axial biometry; (2) mapping of these quantitative features onto a curated corneal disease and refractive-surgery knowledge graph; (3) Bayesian probabilistic inference to evaluate early keratoconus and surgical eligibility; and (4) explainable multi-model LLM reporting, employing DeepSeek and GPT-4.0, to generate bilingual physician- and patient-facing narratives. By transforming complex imaging data into transparent reasoning chains, the pipeline delivered case-level outputs within ~95 ± 12 s. When benchmarked against independent evaluations by two senior corneal specialists, the framework achieved 92 ± 4% sensitivity, 94 ± 5% specificity, 93 ± 4% accuracy, and an AUC of 0.95 ± 0.03 for early keratoconus detection, alongside an F1 score of 0.90 ± 0.04 for refractive surgery eligibility. The generated bilingual reports were rated ≥4.8/5 for logical clarity, clinical usefulness, and comprehensibility, with representative cases fully concordant with expert judgment. Comparative benchmarking against baseline CNN and ViT models demonstrated superior diagnostic accuracy (AUC = 0.95 ± 0.03 vs. 0.88 and 0.90, p < 0.05), confirming the added value of the neuro-symbolic reasoning layer. All analyses were executed on a workstation equipped with an NVIDIA RTX 4090 GPU and implemented in Python 3.10/PyTorch 2.2.1 for full reproducibility. By explicitly coupling symbolic medical knowledge with advanced language models and embedding explainable artificial intelligence (XAI) principles throughout data processing, reasoning, and reporting, this framework provides a transparent, rapid, and clinically actionable AI solution. The approach holds significant promise for improving early ectatic disease detection and supporting individualized refractive surgery planning in routine ophthalmic practice. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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14 pages, 2173 KB

Open AccessArticle

Different Expression of Vascularization and Inflammatory Regulators in Cells Derived from Oral Mucosa and Limbus

by Eleni Voukali, Joao Victor Cabral, Natalia Smorodinova, Vojtech Kolin, Magdalena Netukova, Tomáš Vacík and Katerina Jirsova

Bioengineering 2025, 12(7), 688; https://doi.org/10.3390/bioengineering12070688 - 24 Jun 2025

Cited by 1 | Viewed by 1121

Abstract

Bilateral limbal stem cell deficiency (LSCD) can be effectively treated with cultivated oral mucosa epithelial cell transplantation (COMET). However, COMET is associated with greater superficial neovascularization than limbal stem cell (LESC) transplantation, the gold standard for unilateral LSCD. To investigate the intrinsic molecular [...] Read more.

Bilateral limbal stem cell deficiency (LSCD) can be effectively treated with cultivated oral mucosa epithelial cell transplantation (COMET). However, COMET is associated with greater superficial neovascularization than limbal stem cell (LESC) transplantation, the gold standard for unilateral LSCD. To investigate the intrinsic molecular features of cells intended for grafting, we assessed the in vitro expression of genes involved in vascularization and inflammation using real-time quantitative PCR and multifactorial linear models. Oral mucosal epithelial cells (OMECs) and limbal epithelial cells (LECs) were cultured in either conventional (COM) or xenobiotic-free (XF) media on fibrin substrates. Gene expression profiling revealed distinct transcriptional signatures. The pro-angiogenic genes AGR2, ANGPTL2, CRYAB, EREG, JAM3, and S100A4 were significantly higher in LECs (adjusted p < 0.01), whereas FGF2 was higher in OMECs (adjusted p < 0.001). The anti-angiogenic genes TIMP3 and SERPINF1 were higher in LECs (adjusted p < 0.01), while COL18A1 was higher in OMECs (adjusted p < 0.01). OMECs also showed significantly greater expression of the immunoregulatory genes IL1B, IL6, TNF, CXCL10, and IL1RN (adjusted p < 0.01). Cultivation induced phenotypic changes in OMECs, with COM and XF media exerting comparable effects. These results highlight the contribution of inflammatory mediators to neovascularization following COMET. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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Review

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45 pages, 8058 KB

Open AccessEditor’s ChoiceReview

Bioengineered 3D Human Trabecular Meshwork Models for Outflow Physiology and Glaucoma Research

by Andrea Valarezo, Pujhitha Ramesh, Rong Du, Rohit Sharma, Evan Davis, Susan T. Sharfstein, John Danias, Yiqin Du and Yubing Xie

Bioengineering 2026, 13(3), 291; https://doi.org/10.3390/bioengineering13030291 - 28 Feb 2026

Viewed by 1018

Abstract

Primary open angle glaucoma (POAG) is one of the leading causes of irreversible blindness and is associated with dysfunction of the trabecular meshwork (TM), a three-dimensional (3D) structure that regulates aqueous humor outflow and, consequently, intraocular pressure (IOP). IOP is the only modifiable [...] Read more.

Primary open angle glaucoma (POAG) is one of the leading causes of irreversible blindness and is associated with dysfunction of the trabecular meshwork (TM), a three-dimensional (3D) structure that regulates aqueous humor outflow and, consequently, intraocular pressure (IOP). IOP is the only modifiable factor for glaucoma. Outflow facility is the inverse of aqueous humor outflow resistance caused by the presence of the TM and adjacent tissues, and reflects the TM’s central role in IOP control, representing the most physiologically relevant measure of human trabecular meshwork (HTM) function. Therefore, development of ex vivo systems to study outflow facility and IOP regulation is critical for advancing glaucoma research. We present a comprehensive review of bioengineering approaches to generation of 3D HTM models using synthetic, natural, and hybrid hydrogels, micro- and nanofabricated synthetic substrates or porous scaffolds, and microfluidic devices. These 3D HTM systems have been designed to capture key features such as topography, stiffness, and fluid flow in the conventional outflow pathway. In particular, we highlight HTM models that recapitulate IOP regulation and allow measurement of outflow facility, which directly reflect pressure-dependent outflow resistance in dynamic HTM physiology and glaucoma pathophysiology. By integrating these bioengineering approaches with emerging stem cell technologies, this review offers an evidence-based landscape overview and framework for designing next-generation 3D human-relevant TM models for outflow physiological studies and IOP-modulating drug discovery. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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21 pages, 1748 KB

Open AccessReview

Extracellular Vesicles in Ophthalmology: From Natural Nanocarriers to Engineered Therapeutics

by Christopher Flores, Fabiana Mastantuono, Lu Huang, Tina B. McKay, Grace M. Coyne, Brenna Hefley, Brenda Vasini, Dimitrios Karamichos and Menglu Yang

Bioengineering 2026, 13(3), 275; https://doi.org/10.3390/bioengineering13030275 - 27 Feb 2026

Viewed by 1256

Abstract

Extracellular vesicles (EVs) are increasingly recognized as programmable bioactive carriers in non-viral gene delivery and adaptable bioengineering platforms. Beyond their roles as natural nanocarriers in intercellular communication, EVs can promote ocular surface repair and retinal neuroprotection with potential for low immunogenicity and high [...] Read more.

Extracellular vesicles (EVs) are increasingly recognized as programmable bioactive carriers in non-viral gene delivery and adaptable bioengineering platforms. Beyond their roles as natural nanocarriers in intercellular communication, EVs can promote ocular surface repair and retinal neuroprotection with potential for low immunogenicity and high biocompatibility. Bioengineering now enables cargo encapsulation, surface targeting, and integration of EVs with biomaterial platforms to enhance tissue penetration, retention, and precision delivery. The emergence of induced pluripotent stem cell-derived EVs (iMSC-EVs) offers improved batch uniformity and potential for personalized therapy. However, progress hinges on resolving knowledge gaps in ocular EV biology, standardizing isolation and storage, scaling reproducible manufacturing, and executing focused clinical trials. We synthesize the current developments and outline how EVs are moving from biological mediators to engineered therapeutics to accelerate the translation of EV diagnostics and therapeutics for eye diseases. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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29 pages, 2092 KB

Open AccessReview

Bioengineering Strategies for Corneal Endothelial Cell Injection Therapy: Advances, Challenges, and Clinical Translation

by Yura Choi, Mi-Young Jung, Eunsun Han and Choul Yong Park

Bioengineering 2025, 12(11), 1162; https://doi.org/10.3390/bioengineering12111162 - 26 Oct 2025

Viewed by 3155

Abstract

Corneal endothelial dysfunction is a leading cause of vision impairment globally, traditionally managed through donor-dependent keratoplasty procedures. However, limitations in donor tissue availability, surgical complexity, and long-term graft survival have prompted the development of cell-based regenerative therapies. Among these, corneal endothelial cells (CECs) [...] Read more.

Corneal endothelial dysfunction is a leading cause of vision impairment globally, traditionally managed through donor-dependent keratoplasty procedures. However, limitations in donor tissue availability, surgical complexity, and long-term graft survival have prompted the development of cell-based regenerative therapies. Among these, corneal endothelial cells (CECs) injection therapy has emerged as a minimally invasive alternative, offering the potential to restore endothelial function. This review provides a comprehensive analysis of recent advances in bioengineering strategies for CECs therapy, including cell sourcing from donor tissue, pluripotent stem cells, and transdifferentiated somatic cells; optimization of culture conditions and substrates; and delivery protocols that enhance cell adhesion and survival. We further examine clinical trial outcomes and propose future directions for clinical translation. The convergence of cell biology, biomaterials engineering, and translational medicine positions CECs injection therapy as a transformative solution to corneal blindness. Full article

(This article belongs to the Special Issue Bioengineering and the Eye—3rd Edition)

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