IntroductionThere is a shortage of available organs for transplantation worldwide, with over 100,000 patients consistently on the organ waiting list in the United States alone1. While the number of patients on the waiting list has rapidly increased over the past 15 years, the number of available transplants has remained stagnant. As a result, researchers have actively pursued the advancement of organ and tissue engineering methodologies. This field, known as tissue engineering2, has shown great promise in potentially alleviating the organ shortage. As tissue engineering has progressed, there has been an emphasis on integrating cells, scaffolds, and scaffold biofunctionalizations into constructs that resemble native tissues.Bioinspired analogs to human tissue structures have provided exciting opportunities for the development and improvement of engineered tissues. A prime example of applying biomimetic strategies in tissue engineering is evident in the careful design and choice of biomaterials intended for use as scaffolds. Human tissues are composed of cells embedded in a complex mixture of proteins, carbohydrates, and glycoproteins called the extracellular matrix (ECM)3,4. ECM was originally thought to be a mere supporting framework for the cells in a tissue or organ, but more recent research has revealed that the ECM has important roles in directing function at cellular, tissue, and organ levels5,6. Not only does the composition of the ECM affect the overall tissue function, but so does its hierarchical structure7. For instance, blood vessels that are integral to tissue viability must be considered when designing scaffolds for engineered tissues and organs8. In fact, one of the main hurdles currently affecting the clinical translation of tissue engineered grafts is the density and lack of properly sized vasculature, especially microvascular networks. A reliable, patent vascular supply is critical as it allows for blood and nutrient delivery to cells within the tissue. The delivery of oxygenated blood is limited by the diffusion limit of oxygen in tissue, which requires blood to be transported within 100–200 microns of cells within the tissue9. Without adequate blood distribution, engineered tissues will die when grown to clinically relevant sizes10, thereby limiting the ability to engineer larger tissue sections and whole organs. Overcoming this limitation will help enable the field to solve the organ shortage problem on a feasible scale.Researchers have looked toward the native structure found in tissues and organs in the body and tried to harness those structures directly to create vascularized tissue. One such approach that has shown great promise is decellularization11. Decellularization is a technique where cells are washed away from a tissue or organ, leaving behind the ECM and the hierarchical structure of the tissue. This technique is commonly performed using different detergents, which work to disrupt the cell membrane thereby destroying the cells. These detergents can be perfused through the vasculature of the organ being decellularized which allows for retention of the hierarchical structure of the organ11,12. The ECM can then be studied13, used by itself as a therapeutic14, or engineered as a tissue scaffold15,16. Because the tissue or organ is devoid of cellular material, its usage of an implant should elicit minimal to no host immune rejection17.However, the process of removing cells from tissues and organs has its own set of limitations, mostly due to the variability and availability of suitable tissues and organs for this procedure. The donor’s natural ECM is affected by factors like the donor’s health status18 and lifestyle habits19,20, making it challenging to establish a consistent and controllable baseline for human tissue quality. Furthermore, the requirement for donor tissue for this process presents a paradoxical obstacle to the initial goal of reducing donor dependence. While using animal tissue offers a potential extension, it remains inadequate to meet the current demand. Along with availability concerns, animal tissues offer high risks of infection from multiple pathogens and viral agents21. Animal sources also contribute to ecological concerns such as biodiversity loss, freshwater depletion, and deforestation22. These limitations have led our group to investigate the use of plant tissues, particularly spinach leaves, as prevascularized scaffolds for tissue engineering23. Despite the apparent differences between plant and animal tissue, there are many similarities in tissue architecture. The branching pattern of leaf vasculature closely resembles the branching pattern of the human cardiovascular system24, and these structures can withstand large fluid pressure gradients ranging to upwards of 1 MPa25. Harvesting plant tissues is both sustainable and highly controllable using techniques such as Good Agricultural Practices (GAPs) or hydroponic growth. We were successful in the decellularization and subsequent recellularization of plant tissues with functional human cells24. Furthermore, we were able to measure the diameter of the lumens within the vasculature of leaf scaffolds and found them suitable for the transport of red blood cells. These initial results and the potential of leaf scaffolds led us to expand upon our initial investigation.Our previous plant decellularization method used a highly concentrated formulation of detergents, which had potential cytotoxic consequences. Decellularization can be performed with different detergents, acids and bases, hypotonic solutions, enzymes, and by physical means26. The most common methods include the use of detergents due to their effectiveness, ease of use, availability, and cost. Different detergents can be combined to maximize the process. For instance, sodium dodecyl sulfate (SDS) is an anionic detergent that is effective at removing nuclei from dense tissues27, whereas the nonionic surfactant, TritonX-100, is effective in dilapidation and removal of other detergents28. Because of these differences, SDS and TritonX-100 are often used serially when decellularizing tissue11,15. The timing of treatment and concentrations of the solution can affect the final decellularized tissue. Higher concentrations can reduce the time needed to decellularize, but there could be residual detergent leading to cytotoxic effects29,30. Likewise, lower detergent concentrations can lengthen decellularization but result in the tissue being exposed to cytotoxic agents for longer than necessary. Because of these concerns, we aimed to optimize a process to maximize the decellularization in the shortest amount of time with the minimum permissive concentration of detergents31,32,33. We have hypothesized that the removal of the waxy outer layer of the leaf, known as the cuticle, prior to decellularization would increase efficiency. The cuticle encases the leaf and works to protect it from invading pathogens and retention of water34. Furthermore, if these leaf scaffolds were employed in engineered tissues, their biocompatibility must be understood. Thereby, we aim to investigate the in vivo immunological response to decellularized leaf scaffolds. Furthermore, we previously functionalized the surfaces of decellularized leaves and stems with an arginine-glycine-aspartate-dopamine (RGD-dopamine) peptide35,36, allowing for large-scale cellular attachment and expansion over 50 days. Biofunctionalization of the leaves could offer potential immunomodulatory effects by attenuating the body’s response to the implanted leaf scaffold. Therefore, we investigated the biocompatibility and possible immunomodulatory effects of both non-functionalized and RGD-functionalized decellularized leaf scaffolds through subcutaneous implantation in a rat animal model.ResultsRemoval of leaf cuticle allows for modification of decellularization processThe modified decellularization protocol (Fig. 1B) decellularized the leaves in 5 days whereas the original process (Fig. 1A) required 9 days to complete. A tris-buffer wash was introduced post-decellularization to help change the pH of the leaf scaffold, which has previously been shown to be effective in releasing bound SDS from the tissue37.Fig. 1: Modification of the Decellularization Process.The (A) original decellularization process was (B) modified to shorten the process and lower the concentration of detergents.Full size imageThis process was verified through quantification measures of both DNA and protein. DNA analysis revealed that both the original and modified protocol produced fully decellularized leaves that had significantly (p