A sustainable approach to derive sheep corneal scaffolds from stored slaughterhouse waste

Abstract

Aim: The escalating demand for corneal transplants significantly surpasses the available supply. To bridge this gap, we concentrated on ethical and sustainable corneal grafting sources. Our objective was to create viable corneal scaffolds from preserved slaughterhouse waste.

Materials & methods: Corneas were extracted and decellularized from eyeballs that had been refrigerated for several days. These scaffolds underwent evaluation through DNA quantification, histological analysis, surface tension measurement, light propagation testing, and tensile strength assessment.

Results: Both the native and acellular corneas (with ~90% DNA removed using a cost-effective and environmentally friendly surfactant) maintained essential optical and biomechanical properties for potential clinical use.

Conclusion: Our method of repurposing slaughterhouse waste, stored at 4°C for several days, to develop corneal scaffolds offers a sustainable and economical alternative xenograft model.

Plain language summary

Article highlights.

• Innovative use of slaughterhouse waste: this study highlights the potential to repurpose slaughterhouse waste, refrigerated for several days among waste typically destined for incineration, to generate viable corneal scaffolds. This approach offers a sustainable solution to the global shortage of donor corneas for transplantation.

• Effective decellularization technique for sustainable tissue engineering: this technique utilizes a cost-effective and environmentally friendly zwitterionic surfactant to remove approximately 90% of DNA from the corneas, maintaining essential biomechanical and optical properties suitable for clinical use.

• Preservation and testing: corneas are preserved at 4°C and subjected to various evaluations, including DNA quantification, histology, surface tension measurement, light propagation testing , and tensile strength assessment to ensure their suitability for transplantation.

• Environmental and scientific benefits: the approach not only reduces global waste by repurposing slaughterhouse byproducts but also enhances resource utilization, demonstrating a significant reduction in the use of animals for research and increased availability of tissues for scientific study.

• Potential for clinical application: the study confirms that the decellularized corneal scaffolds retain critical properties necessary for clinically relevant models, suggesting their potential as an alternative to traditional corneal transplants and offering a new avenue for addressing corneal blindness.

• Challenges and limitations: the study acknowledges limitations, such as potential variations in scaffold properties due to the decellularization process and storage conditions, emphasizing the need for ongoing optimization and validation of the techniques used.

1. Introduction

Every year, it is estimated that approximately 36 million individuals worldwide will experience blindness, with corneal blindness being one of the top five causes [1,2]. Corneal blindness is a group of eye disorders that alters this tissue's transparency, causing scarring and vision loss. This form of vision loss can result from various conditions, including infectious, nutritional, hereditary, and degenerative diseases, as well as physical injuries/accidents [3,4]. The available treatment options for corneal blindness vary based on the severity and underlying cause of the condition. These options range from conservative measures such as glasses or contact lenses to highly invasive surgical interventions like laser photorefractive keratectomy and corneal transplantation (keratoplasty) [2,5]. Keratoplasty involves replacing the opaque cornea with a clear donor cornea to restore vision, reduce pain, and enhance the esthetic appearance of a damaged or diseased ocular surface [6,7]. However, this procedure has limitations and may lead to various complications after surgery, such as restenosis, aneurysms, infections, persistent tingling or numbness, wound reopening, and prolonged pain [8].

The scarcity of donor corneas remains a pressing global issue, with over 13 million people awaiting transplantation [6,9]. This shortage fuels the demand for corneal transplants. Consequently, tissue engineering, utilizing techniques like biofabrication and decellularization, offers a promising alternative to meet the growing demand for such transplants. Decellularization involves removing cellular components from donor corneas while leaving the acellular/decellularized extracellular matrix (ECM) intact to produce a nonimmunogenic scaffold [8,10]. This process encompasses a range of approaches – biological, chemical, and physical methods, which are used individually or in combination, depending on distinct features of tissues, including structure, components, size, and thickness [7,8]. Furthermore, this approach can facilitate the creation of bovine-, canine-, caprine-, feline-, ovine- and porcine-derived corneal scaffolds [11,12], compatible with human tissues, potentially providing an abundant and viable source for transplantation.

Xenotransplantation, the clinical cross-species grafting of animal organs and tissues, offers a promising solution to the critical shortage of viable human organs. Corneal xenografts, in particular, present several advantages over traditional allografts, including the potential for greater availability of donor tissues, reduced risk of graft rejection, and decreased reliance on human donors [13,14]. This approach not only addresses the challenge of corneal shortages for transplantation but also facilitates the development of advanced in vitro models, reducing the reliance on animal models. Annually, slaughterhouses generate around 150 million tons of organic waste, containing valuable substrates and bioactive molecules that can be harnessed for regenerative medicine [9,15,16]. Therefore, it is imperative to explore sustainable methods to maximize the utilization of these byproducts that can be repurposed for transplantation models [17].

Corneal xenotransplantation utilizes animals that share anatomical and physiological similarities to humans in an attempt to improve our clinical applications. Notably, despite the sheep’s eye being slightly larger, it shares significant structural and biomechanical similarities to the human eye, presenting advantages for surgical techniques and ocular research. Additionally, sheep corneas are marginally thicker than human corneas, making them viable candidates for crosslinking studies employing the same surface irradiance as humans [18]. Furthermore, these livestock, typically raised for food, benefit from a short gestation period and prolific reproduction rates. Like advances in vascular tissue engineering [19], repurposing byproducts, such as eyes, from slaughterhouse waste offers a pathway to produce personalized transplantable grafts. This approach not only boosts organ availability but does so in a manner that is both sustainable and cost effective [20–22].

Previous studies have underscored the difficulties in developing viable corneal substitutes, highlighting the potential of corneas sourced from agrifood waste to mitigate the scarcity of corneal tissues for transplantation. Such materials offer a unique alternative to generate sustainable and ethical xenograft models and support circular economic practices [8]. In this study, our objective was to create viable native and acellular corneal scaffolds from slaughterhouse waste stored at 4°C for several days. This approach offers multiple scientific and environmental benefits, including global waste reduction, enhanced resource utilization, cost efficiency, a reduction in the number of animals used for research, and improving the availability of tissues and organs to study and develop transplantation models. It also leverages current storage mechanisms at the abattoir that can potentially reduce its reliance on incineration and landfilling.

2. Materials & methods

2.1. Study design

Ovine eyeballs from the prominent Arabian sheep breeds (Najdi, Awassi, and Orb) were obtained from a local slaughterhouse in Abu Dhabi. These eyes, which were discarded during meat processing, were carefully excised from the ocular globe and subsequently stored in a refrigerator with other discarded tissues set for incineration. Following the acquisition of the eyes, decellularization was performed to remove cellular components while maintaining the ECM. Once decellularized, DNA quantifications, histology, light propagation, surface tension and biomechanical tests were conducted on the native and decellularized samples. These experiments were performed in accordance with the ARRIVE criteria and the Automated Slaughterhouse of the Municipality of Abu Dhabi City and the Animal Research Oversight Committee at Khalifa University of Science and Technology (Abu Dhabi, UAE), protocol #H22-036. Further experimental details are presented in the subsequent sections.

2.2. Corneal extraction & decellularization

30 cadaveric eyes were excised from their ocular globes and stored at a local slaughterhouse, where they were refrigerated for several days among waste typically destined for incineration. The eyes were then kept in a cooler for transportation from the slaughterhouse to the research facility for decellularization and experimentation. Subsequently, they underwent thorough washing in isotonic saline several times and were refrigerated in a sealed container filled with saline containing antibiotics to minimize the risk of contamination and create a suitable physical environment for preservation [23]. The corneas were carefully extracted using iris surgical scissors preserving a 5 mm scleral ring while ensuring the limbus was intact.

For decellularization, we used a zwitterionic biosurfactant solution (Ecover, Malle, and Belgium) that consisted of 15–30% nonionic surfactants, 5–15% anionic surfactants, ethanol, sodium citrate, glycerine, trisodium ethylenediamine disuccinate, polypropylene terephthalate and citric acid. While high concentrations of the detergent can lead to more efficient removal of cellular components and debris, they may also damage the ECM and tissue integrity [18]. Therefore, for this experiment, we opted to use lower concentrations of the detergent. The total volume of the surfactant solution prepared for immersion, distributed across all bottles, each containing 75 ml, was 1 l. The isolated eyes were immersed in these bottles containing either 1 or 4% of the surfactant solution [24]. The durations of decellularization were set for 2, 4, and 7 days. Each cornea was placed into a different container based on its detergent concentration and the decellularization period at room temperature throughout the process. The corneas were divided into five groups, as outlined in Table 1.

Table 1.

A description of control (native) and decellularized sample groups.

Group	Number of corneas per group	Concentration (%)	Days	Decellularization condition
1	5	0	0	Native
2	5	1	2	1% 2D
3	5	1	4	1% 4D
4	5	4	2	4% 2D
5	5	4	4	4% 4D
6	5	4	7	4% 7D

The samples were set up on Ohaus Analog Heavy Duty Shaker (Thermo Fischer Scientific, MA, USA) and agitated at 300 rpm for their assigned decellularization periods (2, 4, or 7 days). Following decellularization, the corneas were subjected to an extensive 3-day washing, where they were washed, immersed in deionized water, and agitated more at 300 rpm to remove residual traces of the detergent from the tissues. After the 3rd day of washing, the corneas were immersed in glycerol and subjected to continuous agitation for 16 h to reverse opacity, as shown in Figure 1.

Figure 1.

Visual comparisons of the degrees of corneal opacity reversal achieved using glycerol for different groups; native, 1% 2D, 1% 4D, 4% 2D, 4% 4D and 4% 7D.

2.3. DNA quantifications & histology assessments

Briefly, to assess innate and residual DNA contents in native and acellular scaffolds, respectively, we utilized approximately 20 mg of tissue from which we extracted the DNA using a QIAamp DNA Mini Kit #51306 (Qiagen, MD, USA) and assessed DNA quantities with a nanodrop spectrophotometer (Thermo Fisher Scientific). In comparison, for histological analyses, first, we fixed both native and decellularized corneas in 10% formalin and dehydrated the samples via a series of graded ethanol incubations (70, 80, 95, and 100%). Second, xylene clearing and paraffin infiltration were performed. Last, we collected approximately 3.5 μm sections, which were stained with hematoxylin and eosin and examined under a CX43 microscope (Olympus Corporation, Tokyo, Japan). Further experimental details can be found in the literature [9,25].

2.4. Surface tension test

In this test, we evaluated the extent of surfactant removal post-decellularization. The test was conducted using a BP100 Bubble Pressure Tensiometer (Kruss Scientific, NC, USA [9]. The corneas were subjected to a 3-day washing protocol, with washes performed three times a day at 3 h intervals. The elutes washouts collected from each washing cycle were utilized to assess the surface tension and gauge the presence of residual detergent in the acellular scaffolds.

For this test, we used deionized water, 1, 4, and 100% detergent concentrations as control groups. We started by measuring the surface tensions of five samples for each control group. Then, we measured the surface tension of 1% 2D, 1% 4D, 4% 2D, 4% 4D, and 4% 7D decellularized solutions and the elutes gathered from the 3 days of washing. The data was then collected and plotted using KRÜSS lab desktop software 3.2.2.3068 and Microsoft Excel. The data collected from the washouts were compared with data from the control groups and the final decellularization solution. The Kruskal–Wallis test, a nonparametric test for comparing two or more independent groups, was performed on the data obtained from the software. This test is used when the data does not meet the assumptions required for parametric tests, such as normal distribution or equal variances.

2.5. Light propagation

Our methods provide a concise overview of the procedures extensively detailed in the literature. Specifically, transmission spectra across the visible spectrum for native and decellularized corneal scaffolds were acquired using a UV-Vis spectrophotometer (USB 4000+, Ocean Optics, FL, USA) connected to a Zeiss Axioscope [9,25] (Zeiss Group, Oberkochen, Germany). Tissue measurements were averaged and compared with a glass slide reference, which provides roughly 100% transmission, to evaluate the degree of visible light propagation that can be achieved after reversing corneal opacity.

2.6. Tensile strength

The corneas were sectioned into 1x1 cm samples (Figure 2) to ensure uniformity in testing. Although the eye is based on a spherical coordinate system, known as the ophthalmic coordinate system [26], our experimental approach for measuring tensile strength was based on a linear uniaxial model. This test was conducted using a CellScale machine [27], which offers precise actuator adjustments. It is also equipped with a high-resolution camera that enables it to capture images of samples simultaneously during testing. The data were collected using Lab Joy software and Microsoft Excel, with each sample subjected to a force of 5000 mN for 60 s. For each sample, we created a stress–strain curve and calculated the Young’s Modulus (YM) to determine the elastic properties of the scaffolds. We then gathered data on the elastic modulus, yield strength, yield strain, tensile strength, ductility, resilience, and toughness, as well as their mean and standard deviation, to compare values among the samples. To assess the significant differences between our control group (native corneas) and the decellularized scaffolds, we employed MATLAB R2020b to conduct the Kruskal–Wallis test, and a p-value of less than 0.05 was considered statistically significant for all evaluations.

Figure 2.

Corneal extraction and section preparation for biomechanical analyses. (A) Corneal extraction. (B) Measurement of corneas to be sectioned into 1x1 cm.

3. Results

3.1. DNA quantifications & histology assessments

The decellularization process effectively removed around 90% of the innate DNA. The conditions that met the desired threshold-removal of at least 90% of innate DNA [28,29] were samples decellularized with 1% detergent for 4 days, 4% for 2 days, and 4% detergent for 4 days. The residual DNA contents are presented in Table 2 and Figure 3. Similarly, brightfield micrographs, presented in Figure 4, outline the effective removal of cellular components from the scaffolds, as well as the different effects the protocols had on components in the ECM.

Table 2.

A description of control (native) and decellularized sample groups.

Type of scaffold	DNA (ng)	% residual DNA
Native	8540.00	0
1% 2D	1521.33	82
1% 4D	877.33	90
4% 2D	653.33	92
4% 4D	620.67	93
4% 7D	1054.67	88

Figure 3.

A comparison of residual contents of acellular scaffolds compared with innate DNA contents in native scaffolds.

Figure 4.

A comparison of the microarchitecture of native and acellular scaffolds. (A) Native cornea with its intact various layers (epithelium, Bowman's layer, and stroma). (B) Decellularized cornea generated from 1% detergent treatment for two days. (C) Decellularized cornea generated from 1% detergent treatment for four days. (D) Decellularized cornea generated from 4% detergent treatment for two days. (E) Decellularized cornea generated from 4% detergent treatment for four days. (F) Decellularized cornea generated from 4% detergent treatment for seven days.

3.2. Surface tension measurements

The surface tension of deionized water averaged 72.0 mN/m. In contrast, the surface tension of the zwitterionic detergent without being diluted was found to be 33.6 mN/m. The surface tension measurements for 1% and 4% concentrated detergent solutions yielded an average value of 34 and 32.8 mN/m, respectively. These results suggest that the addition of the detergent reduces the surface tension of water. When the decellularized corneas were washed, the surface tensions of the washouts became progressively closer to that of water with each wash. The average surface tension of 1% 2D, 1% 4D, 4% 2D, 4% 4D, and 4% 7D decellularized corneas were found to be 33.6, 66.3, 32.2, 71.6, and 72.3 mN/m, respectively. The comparison between the surface tension of the decellularized solution before washing and the surface tension of the washouts is shown in Figures 5 and 6. The Kruskal-Wallis test was conducted to assess the statistical significance of the increasing trend in surface tension observed in the washing process. The p-values obtained from these tests demonstrated a significant decrease from 0.34 on the first day to 0.002 on the last day of the washing process.

Figure 5.

Surface tension measurements of water, different concentrations of detergent, and experimental groups with different decellularization periods.

Figure 6.

An evaluation of surface tensions of the elutes for all experimental groups. (A) Surface tension measurements of 1% 2D, 1% 4D, 4% 2D, 4% 4D, and 4% 7D decellularized solutions before washing and after washing. (B) Kruskal–Wallis test for the surface tension measurements of 1% 2D, 1% 4D, 4% 2D, 4% 4D, and 4% 7D decellularized corneas’ decellularized solution and washouts.

3.3. Optical transparency & light propagation

We compared the restoration of transparency in five different groups of corneas, which varied based on the concentration of detergent used and the duration of decellularization: 1% for 2 days (1% 2D), 1% for 4 days (1% 4D), 4% for 2 days (4% 2D), 4% for 4 days (4% 4D), and 4% for 7 days (4% 7D), to assess the degree of transparency restoration. The results showed varying degrees of transparency restoration among the groups, with the 4% 7D group achieving the highest level of transparency. This significant restoration of transparency, observed exclusively in the 4% 7D group, is consistent with our previous studies that utilized samples processed immediately after harvesting [9].

Figure 7 presents the results of the light propagation assessment. These results reveal significant variations in light transmittance between decellularized and native tissues. Notably, native tissue showed relatively higher transmittance percentages across the visible spectrum, likely due to its preserved structural properties conducive to light transmission. Among the acellular tissues, transmittance levels were relatively similar despite the notable reductions in corneas decellularized for the longest period of 7 days. This variation may result from increased tissue density or microstructural alterations that scatter light more, thereby reducing transmittance. This highlights that the duration of decellularization can significantly affect the optical properties of the tissue.

Figure 7.

Visible light propagation through native and acellular scaffolds.

3.4. Tensile test

The mechanical testing was performed on samples from the four groups (4% 6D, 4% 7D, fresh native scaffolds, and native corneas immersed in saline). The aim of this analysis was to compare the mechanical properties of the corneas in this batch and determine if there were any significant differences among the groups. There were five samples in each group. For calculating the YM, average stress and strain values were used, resulting in YM values of 0.073, 0.015, 0.021, and 0.070 for the native, native in saline for 2 weeks, 4% 6D, and 4% 7D groups, respectively. Moreover, the data recorded from the CellScale machine can be seen in the stress-strain graph presented in Figure 8 below. We used the Kruskal-Wallis test to compare the mechanical properties of the corneas in this batch. The results from this statistical analysis, summarized in Table 3, show that the values have a p-value greater than 0.05.

Figure 8.

Scaffold biomechanical evaluations. (A) Box and Whisker plots for Young modulus of four different groups. (B) Kruskal–Wallis test results for tensile test.

Table 3.

Summary of Kruskal–Wallis test for tensile strength of sheep cornea.

	Elastic modulus (MPa)	Yield strength (MPa)	Yield strain	Tensile strength	Ductility (%EL)	Resilience (Pa)	Toughness $\left(\frac{J}{m^3}\right)$
p-value	0.7909	0.5439	0.99846	0.0947	0.1881	0.7924	0.2874
H value	1.04	2.14	0.15	6.38	4.79	1.04	3.77

4. Discussion

Our study presents a timely approach that offers multiple scientific and environmental benefits, including global waste reduction, enhanced resource utilization, cost efficiency, a reduction in the number of animals used for research, and an improving the availability of tissues and organs for the study and development of transplantation models. By focusing on stored slaughterhouse waste rather than freshly collected tissues, our study builds upon previous research and offers an additional dimension of using these tissues in transplantation model development. We employed decellularization using zwitterionic surfactant to remove cellular components while maintaining the structure of ECM to reduce the risk of immune rejection for use a template for personalized graft development. Once decellularized, the corneas were immersed in glycerol to restore their transparency [9]. The use of glycerol is a cost-effective and simple method for preserving corneal tissues intended for keratoplasties for an extended period of up to 5 years [30]. Glycerol also serves as a cryoprotectant, preventing cellular damage and maintaining the structural integrity of the cornea during storage [9,30,31]. It acts by reducing the formation of ice crystals, which can cause structural damage to the ECM of corneal tissue [31]. The effectiveness of glycerol preservation is evident from the noticeable improvement in transparency observed in the decellularized corneas. For this approach, we performed routine DNA, histological, and optical transmission tests. We also wanted to conduct a pilot study to examine biomechanical studies. For this, we chose to compare the biomechanical properties of native tissues to residual tissues (scaffolds) that we exposed to the most intense decellularization method: longest decellularization time and highest detergent concentration (4% 7D). The rationale for this is that this particular decellularization method would have caused the greatest alterations to tissue structure. The retention of transparency in decellularized corneas is a critical aspect of their suitability for various applications, particularly in the context of corneal transplantation and sustainable tissue engineering. The native cornea enables clear light penetration, the corneal curvature focuses the image rays on the retina, and eyesight is determined by the light refraction depending on the corneal curvature, thickness, and eye size [7,24]. In our study, the degree of retention of transparency in the corneas varies among the decellularized groups and the native cornea, after undergoing, a generally effective degree of decellularization. Alterations in corneal tissues can arise from experimental conditions of extraction and storage procedures. This opacity tends to increase during decellularization. In general, numerous layers causing scattering within biological tissues restrict the optical penetration depth, resulting in reduced transmission [9]. Despite these challenges, our findings demonstrate a promising degree of transparency retention in decellularized corneas, with transmittance levels closer to that of aged human corneal tissues in the range of 300–800 nm [25,32]. These findings are noteworthy given the use of discarded waste for the experiment and the ability to regain a substantial amount of transparency and transmittance.

Surface tension measurements were conducted in order to check the presence of surfactant after subjecting the decellularized corneas to an extensive 3-day washing process. This test aimed to quantify the amount of detergent remaining in the decellularized corneas after each step of washing. Studies have shown that detergents are known to reduce the surface tension of water significantly [33]. This has significant implications as the restoration of surface tension is critical for the proper functioning and integrity of corneas. It also suggests a reduced risk associated with the presence of surfactants [9]. We used the surface tension of deionized water (72 mN/m) as our baseline, against which the original surfactant agent, 4% detergent concentrations, and 4% 7D decellularized corneas elutes’ were compared. The observed trend in increasing surface tension in the washouts, as shown in Figure 5, indicates the effective removal of surfactant from the corneas during the washing process, gradually restoring the corneas’ surface tension closer to water, and consistent with our previous studies [8].

The cornea plays a major role in human vision by providing approximately two-thirds of the refractive power of the eye [34]. The meniscus shape of the cornea results from a mechanical equilibrium of the cornea with respect to intraocular pressure [34,35]. A change in mechanical homeostasis can alter the corneal shape and thereby affect visual acuity [36]. The data of our study demonstrated valuable mechanical properties of decellularized ovine corneas using uniaxial tensile testing. Tensile strength testing measures the ability to withstand forces applied in tension or stretching [35]. By subjecting corneal scaffolds to tensile forces, it is possible to evaluate their mechanical strength and determine if they can withstand the stresses experienced during transplantation and normal eye movements [35]. Additionally, this test assesses the structural integrity and durability of the scaffold in order to identify weak points and potential failure sites, such as areas prone to tearing or breaking. This crucial information is necessary for optimizing decellularization techniques and the manufacturing process of the scaffold.

Uniaxial tensile testing of the decellularized ovine cornea can provide valuable mechanical properties that characterize tissue behavior under tension [35]. The mechanical and viscoelastic characteristics of the cornea play an important role in maintaining its functionality, particularly since the cornea is a load-bearing tissue [9,25]. Besides, these variables are related to cell behavior through mechanotransduction, defined as the process by which cells perceive and respond to mechanical stimuli, leading to different molecular processes that regulate cellular behavior [18,37]. The mechanical properties of a tissue are related to the arrangement and integrity of the structural proteins of the ECM. In the cornea, these properties and optical transmission depend mostly on collagen I fibers [38], as well as the degree of hydration and glycosaminoglycan contents [39,40]. YM has been used as an indicator for the preservation of the mechanical properties of a tissue after decellularization, generally measured through tensile tests [18]. However, during decellularization, the stromal ECM might be disrupted, which in turn decreases the corneal strength [24]. This is evident from our data, where the decellularized samples had less tensile strength than the native. Nonetheless, the values for all the samples were relatively close, ranging between 0.3 and 0.7, which indicates that the ECM was maintained relatively intact after decellularization.

4.1. Limitations

It is important to stress that our research encompassed several significant limitations that necessitate acknowledgment within this manuscript. Our studies focused on assessing the physio-morphological properties of these scaffolds in a limited group. Nevertheless, our previous studies have shown that these detergent concentrations and agitative decellularization approach can effectively provide a threshold useful for scaffold preparation by removing residual ECM components or neo-epitopes that can activate the immune system, resulting in inflammatory reactions, tissue rejection or fibrotic responses [9,41]. Furthermore, all initial tissue extraction and storage processes were performed by abattoir personnel untrained in biological tissue handling, potentially leading to significant experimental variations.

4.2. Recommendations

To validate the effectiveness of decellularization, it is important to conduct a comprehensive evaluation to ensure the complete removal of cellular material. This can be achieved by quantifying the DNA content and residual cellular components using appropriate assays, such as DAPI staining or DNA quantification techniques.

When considering long-term storage of decellularized sheep corneas, it is essential to identify suitable storage conditions, such as freezing or cryopreservation, that will preserve the integrity and functionality of the tissues for an extended duration. It is essential to regularly monitor the long-term stability of the decellularized tissues and assess any potential alterations in the properties of the ECM.

Additional tests can be conducted to evaluate the effectiveness of the decellularization protocol for sheep corneas. These tests include histological analysis to examine the tissues structure and composition, transmission electron microscopy to assess the preservation of ECM components, and DNA quantification assays to measure the removal of cellular DNA. Additional computational approaches can be employed to access the ECM structure [41–45] and the presence of residual nuclei [46–51].

By comparing the results of these tests with native corneas, a thorough evaluation of the decellularization protocol can be achieved. Such a comparison can provide valuable insights into tissue structure preservation, removal of cellular components, and the overall success of the decellularization process for sheep corneas.

5. Conclusion

The constraints surrounding the accessibility of deceased human donor corneas require the investigation of alternative methods and approaches, as corneal blindness is one of the top five causes of visual impairment. The utilization of slaughterhouse waste for the production of functional corneal scaffolds not only addresses transplantation challenges but also fosters sustainability and promotes a circular economy. As the field of sustainable tissue engineering continues to advance, the optimization of a decellularization protocol for sheep corneas holds significant potential. By preserving the natural environment of tissues, decellularization promotes cell proliferation and stimulates cell differentiation. We were able to create scaffolds that could regain a substantial amount of transparency and transmittance, as well as retain viable biomechanical/histological properties in natives and those obtained after effective decellularization. The DNA quantification results show that decellularization effectively removed 80–95% of the innate DNA. Additionally, the surface tension test confirmed the successful removal of the detergent from the decellularized scaffolds. Comparisons between the native corneas and the scaffolds resulting from 6 or 7 days of 4% detergent decellularization demonstrated similarity in mechanical properties using stored and unprocessed slaughterhouse waste. These findings suggest that the decellularization process can be optimized by reducing the duration to 6 days, thereby streamlining the experimental period.

The decellularization protocol for sheep corneas provides essential insights for tissue engineering and regenerative medicine. Our approach can be used to refine decellularization outcomes and provide novel insight into corneal xenograft data. The protocol effectively restores corneal transparency, offering potential applications in corneal transplantation and tissue engineering. Moreover, biomechanical testing confirms the satisfactory properties of the decellularized scaffolds, suggesting their suitability for tissue regeneration. In summary, this protocol shows promise for future research and its application to address corneal restoration and vision enhancement challenges. Additionally, our ability to generate scaffolds with such stored waste is a testament to the potential to devise a sustainable tissue engineering approach to derive sheep corneal scaffolds from discarded slaughterhouse waste.

Funding Statement

This study was supported by funds awarded to PR Corridon by Khalifa University of Science and Technology, Grant Numbers: FSU-2020-25 and RC2-2018-022 (HEIC), ESIG-2023-005 and the Center for Biotechnology, the College of Medicine and Health Sciences, the Abu Dhabi Automated Slaughterhouse and the Abu Dhabi Municipality.

Author contributions

X Wang, ZM Ali, MG Shibru, A Alnuaimi, FF Murtaza, A Zaid, R Khaled and PR Corridon collected the tissues and performed decellularization. X Wang, AE Salih, ZM Ali and MG Shibru performed the surface tension studies and PR Corridon interpreted the data. X Wang, A Alnuaimi, A Zaid, FF Murtaza, R Khaled and PR Corridon performed mechanical tests, and PR Corridon interpreted the data. X Wang and AE Salih performed transmittance tests, and H Butt and PR Corridon interpreted the data. S Daoud prepared histological sections, and X Wang and PR Corridon performed imaging and analyses. H Vurivi performed DNA measurements, and X Wang and PR Corridon interpreted the data. V Chan, IV Pantic, and J Paunovic obtained approval for the study. PR Corridon conceptualized and designed the study, obtained approval and funding for the study, coordinated sample and data acquisition/validation, and performed the statistical analyses with ZM Ali and MG Shibru. All authors contributed to the final draft of the manuscript.

Financial disclosure

This study was supported by funds awarded to PR Corridon by Khalifa University of Science and Technology, Grant Numbers: FSU-2020-25 and RC2-2018-022 (HEIC), ESIG-2023-005 and the Center for Biotechnology, the College of Medicine and Health Sciences, the Abu Dhabi Automated Slaughterhouse and the Abu Dhabi Municipality. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval (#H22036) and/or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.