Cerium oxide-enhanced decellularized bovine pericardial biological scaffold

Abstract

The present invention relates to a decellularized bovine pericardium enhanced with cerium oxide nanoparticles, suitable for use in reconstructive surgeries, particularly cardiac surgeries. This invention addresses the limitations of existing commercial products, including weak mechanical properties and low biocompatibility. By optimizing the decellularization process and incorporating cerium oxide nanoparticles, the resulting material exhibits enhanced mechanical strength and improved biocompatibility.

Description

Title of Invention: Cerium Oxide-Enhanced Decellularized Bovine Pericardial Biological ScaffoldTechnical Field

[0001] The present invention relates generally to the fields of tissue engineering, regenerative medicine, and biomedical materials. More specifically, the invention is directed to a biologically derived scaffold obtained from decellularized bovine pericardial tissue, reinforced with cerium oxide nanoparticles to enhance mechanical strength, biocompatibility, and functional performance. The disclosed scaffold is suitable for use in medical and surgical applications.Background Art

[0002] Biological scaffolds derived from pericardial tissues are commonly used in tissue engineering and regenerative medicine. These scaffolds are employed in the production of various bioprosthetic devices, including vascular grafts, cardiac abdominal wall repair patches, vaginal reconstruction materials, and heart valve substitutes. Bovine pericardium has been widely used in cardiovascular and reconstructive surgeries due to its favorable properties. These include its structural similarity to native tissues, sufficient mechanical strength, biocompatibility, and its ability to support cellular infiltration and angiogenesis.

[0003] For clinical use, pericardial tissues typically undergo decellularization processes to remove cellular antigens and residual genetic material while preserving the integrity and architecture of the extracellular matrix. Various chemical, enzymatic, and physical methods have been developed for tissue decellularization; however, the selection of an optimal protocol depends on tissue type and often requires extensive optimization. Despite successful cell removal, decellularization procedures commonly result in alterations of biological and mechanical properties, including reduced mechanical strength and elasticity.

[0004] In cardiovascular tissue engineering applications, scaffolds are generally required to withstand mechanical stresses in the range of approximately 0.5 MPa while maintaining sufficient elasticity without permanent deformation under cyclic loading conditions. However, currently available decellularized bovine pericardial products may exhibit limitations, such as reduced mechanical strength, decreased resistance to cyclic strain following implantation, incomplete removal of residual DNA, delayed cellular attachment and proliferation, and long-term tissue degeneration associated with calcification and deterioration of the extracellular matrix.

[0005] Conventional decellularization methods typically use multiple chemical agents, enzymes, and prolonged processing times. This can drive up manufacturing costs and potentially compromise tissue integrity and biocompatibility due to leftover chemical residues. One significant challenge with decellularization is the mechanical weakening of tissues; while the process aims to reduce residual DNA content while preserving the extracellular matrix structure, it can also diminish mechanical resistance.

[0006] There is still a need for improved biological scaffolds derived from bovine pericardium, which demonstrate efficient removal of cellular components, enhanced mechanical properties, greater biocompatibility, and reduced processing time and costs.Summary of Invention

[0007] The present invention provides an improved biological scaffold derived from decellularized bovine pericardium for applications in tissue engineering and regenerative medicine. Conventional decellularization methods for pericardial tissue often require multiple chemical agents and enzymes, long processing times, and complex procedures, which can compromise mechanical strength, reduce biocompatibility, increase residual DNA, and result in calcification after implantation.

[0008] To address these limitations, the invention utilizes a simplified decellularization process employing a single chemical agent, sodium deoxycholate (SD), and a single enzyme, DNAse I, thereby reducing processing time and minimizing damage to the extracellular matrix. Furthermore, the scaffold is enhanced with cerium oxide nanoparticles to support cell adhesion, proliferation, and migration.

[0009] The decellularized and enhanced scaffold maintains the three-dimensional architecture of the extracellular matrix, achieving improved tensile strength and elasticity while maximizing DNA removal. Culturing mesenchymal stem cells on the scaffold further enhances mechanical resistance, demonstrating excellent biocompatibility and suggesting that, upon implantation, the scaffold can withstand hemodynamic forces in cardiovascular applications and support functional tissue regeneration.

[0010] The invention provides a cost-effective, robust, and biologically compatible pericardial scaffold suitable for cardiovascular repair, soft tissue reconstruction, and regenerative medicine applications.Brief Description of Drawings

[0011] The drawing Figures depict one or more implementations in accordance with the present teachings, by way of example only, not by way of limitation. In the Figures, reference numerals refer to the same or similar elements.

[0012] [Fig.l] illustrates a process flow chart for the preparation and production of the decellularized bovine pericardial scaffold according to an embodiment of the present invention.

[0013] [Table.1] illustrates the chemical and enzymatic treatment conditions, including the type of materials, concentrations, and treatment durations used during the decellularization process.

[0014] [Fig.2] illustrates a comparison of residual DNA content among fresh native bovine pericardium, bovine pericardium decellularized according to the method of the present invention, and a commercially available decellularized bovine pericardium sample.

[0015] [Fig.3] illustrates Young’s modulus values and representative stress-strain curves of bovine pericardial tissues under different conditions.

[0016] [Fig.4] illustrates the percentage of resazurin reduction at days 3 and 7 following cell seeding on commercial decellularized bovine pericardium, decellularized bovine pericardium without cerium oxide, and cerium oxide-loaded decellularized bovine pericardium.Description of Embodiments

[0017] In the present invention, instead of using multiple chemical agents, physical methods, and various enzymatic treatments, only a single chemical agent, sodium deoxycholate (SD), and a single enzyme, DNAse-I, are employed. Furthermore, in order to enhance the biocompatibility of the decellularized tissue, in another embodiment, a second sample is prepared in which the scaffold is enhanced with cerium oxide nanoparticles. As a result of reduced manipulation, the decellularized bovine pericardial tissue obtained according to the present invention exhibits improved mechanical properties and enhanced biocompatibility.

[0018] The objective of tissue decellularization is the maximal removal of cellular components and genetic material in a manner that preserves the three-dimensional architecture and composition of the native extracellular matrix and consequently maintains the mechanical strength of the tissue to the greatest possible extent. It isrecognized that the presence of residual cells and DNA may contribute to the preservation of mechanical resistance.

[0019] Another challenge associated with the use of biological scaffolds in medical applications is biocompatibility, meaning that cells should be capable of adhering to the scaffold surface, proliferate, and maintaining their normal biological functions. Additionally, cells should be capable of migrating and proliferating both on the surface and within the scaffold structure.

[0020] Culturing adipose-derived mesenchymal cells on the scaffold according to the present invention resulted in a significant increase in the tensile mechanical strength of the decellularized bovine pericardium. This increase appears to be due to cell-mediated contraction, whereby the tensile strength of the cell-seeded scaffold approached that of native untreated tissue. This observation is important for two reasons. First, it indicates suitable biocompatibility of the decellularized tissue and effective removal of potentially harmful substances used during the decellularization process. Second, it demonstrates that, when the decellularized tissue is used as a patch, and host cells grow within it after implantation, the tensile strength of the patch may adequately withstand hemodynamic demands in organs such as the heart or large arteries.

[0021] Method of Preparation :In one embodiment, bovine pericardium is obtained from a slaughterhouse and transported to the laboratory in phosphate-buffered saline (PBS) containing 1-3% penicillin and streptomycin at about 4 °C. In the laboratory, blood vessels and adipose tissue are gently removed using scissors and forceps with minimal mechanical damage, followed by washing with cold PBS containing antibiotics.

[0022] Unlike previous bovine pericardium decellularization methods that generally utilize multiple chemical agents and physical and enzymatic treatments, including sodium dodecyl sulfate (SDS), Triton X-100, trypsin, and others, the present invention applies minimal manipulation using only sodium deoxycholate (SD) and DNAse-I, followed by extensive washing with PBS and deionized water. For decellularization, bovine pericardium is treated with approximately 1% SD solution for about 24 hours in a shaking incubator at approximately 37 °C. Subsequently, the tissue is exposed to DNAse-I enzyme at a concentration of approximately 10 units for about 12 hours with the aim of developing a biocompatible and bioactive scaffold suitable for cardiac tissue engineering applications (Table 1).

[0023] Between each step of decellularization, the pericardial tissue is washed with PBS. After completion of the decellularization process, extensive final washing with PBS and deionized water is performed for approximately three days, with replacement every 12 hours, to remove residual treatment agents and detergents. The resulting product at this stage is decellularized bovine pericardium, which may be used alone. In another embodiment, to further enhance the biocompatibility of the decellularized tissue, a second sample referred to as cerium oxide-reinforced decellularized bovine pericardium may be produced by immersing the decellularized tissue for approximately 24 hours in a solution containing approximately 8 mg / mL cerium oxide nanoparticles (Ce2Os) to achieve nanoparticle loading.

[0024] One of the major challenges associated with the use of this tissue is maintaining its mechanical properties over time and under physiological conditions. Mechanical strength of bovine pericardium is considered a key factor in the success of biological prostheses and heart valves. Decellularization processes generally result in a significant reduction in mechanical strength. In the present invention, a significant decrease in Young’s modulus of the decellularized tissue compared with fresh native tissue was observed (from approximately 9.62 MPa to approximately 6.57 MPa). However, the mechanical strength of the decellularized tissue produced according to the present invention (approximately 6.57 MPa) remained more than twice that of commercially available decellularized bovine pericardium (approximately 3.18 MPa). Addition of cerium oxide nanoparticles did not produce a significant change in mechanical strength (approximately 6.51 MPa); however, cell seeding on cerium oxide-loaded decellularized bovine pericardium increased the mechanical strength (approximately 8.78 MPa), approaching that of fresh bovine pericardium (approximately 9.62 MPa). This indicates that, upon implantation, cerium oxide-reinforced decellularized bovine pericardium may demonstrate substantially improved structural integrity compared with commercially available tissue.

[0025] Biocompatibility and cellular adhesion to bovine pericardium are critical aspects in cardiac surgery and tissue engineering, directly influencing treatment success and tissue regeneration. Resazurin assay results demonstrated that, over seven days of cell culture on commercially available decellularized bovine pericardium, no progressive cell growth was observed. However, cell adhesion and proliferation on the decellularized bovine pericardium produced according to the present invention increased progressively, and the cerium oxide-reinforced sample demonstrated markedly greater cellular growth,approximately twofold higher than the commercial sample at day 3 and approximately threefold higher at day 7. Since suitable biocompatibility may improve cellular function and facilitate tissue regeneration processes, the cerium oxide-reinforced decellularized bovine pericardium may integrate more rapidly in vivo compared with commercially available tissue following implantation.Residual DNA content in the decellularized bovine pericardium produced according to the present invention showed approximately 97.5% reduction (from approximately 22.86 pg / mL to approximately 0.57 pg / mL), indicating effective decellularization.

[0026] Decellularization processes generally lead to a reduction in the mechanical resistance of the tissue. In the present invention, although a reduction in Young’s modulus was observed compared to fresh tissue, the mechanical strength remained substantially higher than that of commercially available products, thereby preventing tearing during suturing procedures. Addition of cerium oxide nanoparticles followed by cell seeding further improved mechanical strength toward native tissue levels. Resazurin assay results further confirm enhanced cellular proliferation on cerium oxide-reinforced decellularized bovine pericardium compared with commercial samples (Figure 4), indicating that after implantation, the reinforced scaffold may integrate more rapidly within the host tissue compared with existing materials]

Claims

What is claimed is:

1. A biological scaffold comprising a decellularized bovine pericardium, wherein the scaffold is processed to remove cellular components while preserving the extracellular matrix architecture, and the scaffold is reinforced with cerium oxide nanoparticles.

2. The scaffold according to claim 1, wherein the scaffold retains a three-dimensional extracellular matrix structure and native biochemical composition after decellularization.

3. The scaffold according to claim 1, wherein the scaffold exhibits mechanical strength within a range of 6.57 to 8.87 MPa.

4. The scaffold according to claim 1 , wherein the residual DNA content is reduced by at least 97.5% relative to native tissue.

5. The scaffold according to claim 1, wherein the scaffold supports adhesion, migration, and proliferation of mesenchymal stem cells.

6. The scaffold according to claim 1 , wherein the scaffold is configured for use in cardiac or vascular tissue repair.

7. A method for decellularization of bovine pericardium comprising:a) Isolating the bovine pericardial tissue;b) Washing the tissue in phosphate-buffered saline (PBS) containing antibiotics at an approximate temperature of 4°C;c) Removing blood vessels and adipose tissue;d) Washing the tissue;e) Treating the tissue with a sodium deoxycholate (SD) solution;f) Treating the tissue with DNAse I enzyme to remove cellular genetic materials; g) Washing the tissue with aqueous solutions to remove residual chemical substances;

8. The method according to claim 7, wherein the chemical agent comprises sodium deoxy cholate (SD) and the enzyme comprises DNAse-I.

9. The method according to claim 7, wherein the sodium deoxycholate treatment is performed at 1 percent concentration for 24 hours at 37 °C.

10. A method for enhancing the biocompatibility and resistance to calcification of a biological scaffold after implantation, comprising immersing the dehydrated scaffold in a solution containing cerium oxide nanoparticles.

11. The method according to claim 10, wherein cerium oxide nanoparticles are incorporated into the scaffold by immersing the decellularized tissue in a nanoparticlecontaining solution.

12. The method according to claim 10, wherein the immersion is performed for 24 hours in a solution containing 8 mg / mL cerium oxide nanoparticles (Ce2Ch).

13. The method according to claim 10, wherein reinforcement with cerium oxide nanoparticles improves biocompatibility and resistance to calcification of the scaffold after implantation.

14. The method according to claim 10, wherein culture of adipose-derived mesenchymal cells on the scaffold improves the mechanical strength to approach that of native bovine pericardium.

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