Bioengineered corneal tissue for minimally invasive vision ...

Bioengineered Corneal Tissue for Minimally Invasive Vision Correction

Creating Collagen-Based Corneal Substitutes

Our team developed a collagen-based corneal stromal substitute named BPCDX, adhering to stringent good manufacturing practices (GMP). Produced in a Class 5 clean-room facility at LinkoCare Life Sciences AB, Sweden, BPCDX manufacturing emphasizes aseptic conditions throughout, even though a final sterilization process is also implemented.

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The production procedure begins with medical-grade, purified freeze-dried type I porcine dermal atelocollagen from SE Eng Company, South Korea. Dissolved in PBS at room temperature, it forms a 5% collagen solution, subjected to controlled vacuum evaporation. Incorporating crosslinkers (EDCM, NHS, and riboflavin) at 1% (w/v) ratios, the solution is mixed thoroughly and poured into custom molds to achieve final device thicknesses using 280 μm and 440 μm spacers. After curing at room temperature, molds are removed by immersion in PBS. A second photochemical crosslinking process using UVA light fortifies the mechanical strength and resistance of BPCDX. Compared to previous single-crosslinked human-collagen implants, BPCDX utilizes ultra-pure porcine dermal collagen for enhanced reproducibility in mechanical properties. The novel vacuum evaporation technique allows higher collagen content, while a higher ratio of chemical crosslinkers (1:1) provides improved crosslinking and durability.

BPCDX Packaging Process

Once manufactured, BPCDX is rinsed with sterile PBS to remove any residual reactions and stored in sterile PBS within a sealed blister-packed container. Containers are labeled per ISO 15223-1:2012 standards, offering protection during transportation and storage.

Ensuring Sterility of BPCDX

Additional safety is ensured by a final sterilization step using pulsed UVC irradiation, avoiding conventional methods unsuitable for hydrogel devices. Sterility validation follows ISO 14937:2016 and ISO 11137 standards, confirming that UVC exposure does not compromise BPCDX properties or packaging. Internal and external sterility tests are conducted to ensure compliance.

Optical Transparency

BPCDX light transmission is measured using a UV-Vis Spectrophotometer, compared with native human cornea data. Measurements confirmed BPCDX’s high optical transparency across multiple samples.

Mechanical Properties

Using an Instron Automated Materials Testing System, we tested BPCDX specimens for tensile strength, elasticity, stiffness, and toughness. The data, statistically analyzed from 22 samples per test, illustrated BPCDX’s enhanced mechanical properties.

Electron Microscopy Characterization

BPCDX morphology, studied via scanning electron microscopy (SEM), revealed structural nuances compared to native porcine corneas. This micrographic analysis was prepared by freezing, lyophilizing, and gold-coating specimens before examination.

Enzymatic Degradation Test

Tested against collagenase Type I, BPCDX exhibited superior resistance compared to donor human cornea and single-crosslinked versions. This test measured residual mass over time with multiple samples, indicating BPCDX's durability.

In Vitro Biocompatibility

Human corneal epithelial cells cultivated on pre-cut BPCDX demonstrated excellent biocompatibility. Cells sustained nutrient medium change every two days, confirming BPCDX's compatibility for biological purposes.

Biological Evaluation Compliance

Conducted per ISO 10993-1:2018 standards, biocompatibility tests included genotoxicity, cytotoxicity, hemolysis, and ocular irritation evaluations. Results validated BPCDX for safe medical use.

Endotoxin Testing

Ensuring BPCDX's safety, endotoxin testing adhered to ISO 11979-08 guidelines, confirming compliance with acceptable limits for ophthalmic devices.

Shelf-Life and Stability

Shelf-life stability testing involved both accelerated and real-time studies. BPCDX samples showed consistent performance, ensuring long-term reliability for global distribution.

In Vivo Rat Model Implantation

Subcutaneous implantation in rats over an eight-week period demonstrated biocompatibility post-surgery. Ethical approval permitted assessment of tissue surrounding the implant, indicating successful integration and stability.

Minimally Invasive Implantation in Minipigs

For keratoconus evaluations, BPCDX was tested using femtosecond laser surgery on minipigs. The model aimed to recreate thin corneal stroma and assess the device's integration, with observation of healing over extended periods.

Post-Surgery Assessments and Imaging

Six months post-surgery, comprehensive examinations including optical coherence tomography and confocal microscopy validated BPCDX's sustained performance and healing efficacy in treated minipigs.

Histology and Immunohistochemistry

Post-euthanasia tissue analysis via histology and immunohistochemistry revealed that BPCDX integrated smoothly without adverse cellular reactions, validating its biological compatibility.

Ethics and Human Application

Initial human trials in Iran and India, targeting advanced keratoconus, received ethical approvals. The exploratory trial aimed to assess the surgical method’s feasibility and revealed promising safety and efficacy comparable to standard corneal transplants, leading to approvals for more comprehensive randomized controlled trials within the EU.

Pilot Study in LMICs

Conducted in Iran and India, pilot studies aimed to establish guidelines for BPCDX usage. Varying clinical parameters confirmed the treatment's feasibility, supporting the framework for future controlled trials.

Subject Recruitment and Study Endpoints

Subjects were selected based on clinical history and study criteria through informed consent. The primary endpoint was to ensure BPCDX’s safety and stability over six months. Secondary endpoints assessed longer-term safety and efficacy at 12 months, with 24-month results now being reported.

Selection Criteria

Inclusion criteria required advanced keratoconus without corneal scars, suitable corneal thickness, and signed informed consent. Exclusion criteria focused on prior corneal surgeries, infections, systemic conditions, and other incompatible medical circumstances, ensuring the selection of suited candidates.

Clinical Surgical Method

The FLISK method involved femtosecond laser-assisted mid-stromal pocket creation for intrastromal BPCDX implantation, ensuring minimal invasiveness and optimal integration.

Post-Operative Care and Follow-Up

Both Iranian and Indian patients received tailored post-operative care regimes, emphasizing artificial tears, antibiotics, and corticosteroids to enhance healing and minimize complications. Regular follow-up ensured tracking of recovery and implant performance.

Statistical Analysis of Outcomes

Using SPSS software, statistical tests validated BPCDX’s efficacy. Significant improvements were reported in corneal thickness, visual acuity, and keratometry measurements over time, reflecting successful clinical outcomes.

Reporting Guidelines

Further detailed information about the research design is provided in the Nature Research Reporting Summary linked to the original article.

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