Comparative study on mechanical properties and biomineralization of hooks in the diaspores of three epizoochorous plant species.

Most of the plants using epizoochory show adaptations to this diaspore dispersal strategy by having the diaspores covered by barbs, hooks, spines or viscid outgrowths, which allow diaspores to easily attach to an animal surface. Many previous studies have been mainly focused on the dispersal distances and efficiency, or effectiveness of diverse attachment structures depending on their size, anatomy, and morphology. However, the knowledge about the mechanical properties of these structures remains rather poor. In this study, we use a combination of scanning electron microscopy, energy dispersive X-ray element analysis and nanoindentation, to examine the microstructure, biomineralization and mechanical properties of single hooks in Arctium minus, Cynoglossum officinale and Galium aparine. Both the biomineralization and mechanical properties of the hooks strongly differ in examined plant species; mechanical properties depend on the biomineralization pattern, such as the accumulation of silicon and calcium. Elastic modulus and hardness decrease in the series C. officinaleG. aparineA. minus. Anisotropic mechanical properties are found between the radial and longitudinal directions in each single hook. By characterizing the mechanical properties and biomineralization of plant hooks, this paper contributes to the understanding of attachment biomechanics related to seed dispersal. STATEMENT OF SIGNIFICANCE: The dispersal of seeds is essential for plant survival. Many of the plants that use the outside surface of animals to transport the seeds show adaptations to this dispersal strategy by having the seeds covered with hooks. Although these hooks have various sizes, morphologies and anatomical structures, all of them provide mechanical interlocking to animal surfaces. To reduce the risk of interlocking failure, the hooks are usually reinforced by mineralization. However, the relationship between mineralization, mechanical properties and specialized function of plant hooks has been largely overlooked. Here we perform a characterization study on the hooks of three plant species. Our results deepen the current understanding of the mineralization-material-function relationship in specialized hooks of plant seeds.

Sexual dimorphism in the structural colours of the wings of the black soldier fly (BSF) Hermetia illucens (Diptera: Stratiomyidae).

The black soldier fly (BSF) Hermetia illucens (Diptera: Stratiomyidae) plays a significant role at the larval stage in the circular economy due to its ability to convert organic waste into valuable products for energy, food, feed, and agricultural applications. Many data are available on larval development and biomass generation, but basic research on this species is lacking and little is known about adult biology, in particular about the cues involved in sexual recognition. In the present study, using various instruments (stereomicroscope, scanning and transmission electron microscope, hyperspectral camera and spectrophotometer), wing ultrastructure of both sexes was analysed, reflectance and transmission spectra of the wings were measured and behavioural bioassays were carried out to measure male response to specific visual stimuli. The collected data showed the existence of sexual dimorphism in the wings of H. illucens due to iridescent structural colouration generated by a multilayer of melanin located in the dorsal lamina of the central part of the wing. Wing sexual dimorphism is particularly evident regarding the strong emission of blue light of female wings. Blue colour induces in males a strong motivation to mate. The obtained results can help to improve and optimize the breeding techniques of BSF.

Nanoscale mesh acts as anti-adhesive surface against particulate contamination in eyes of whiteflies.

In many insects the surface of the eye is nanostructured by arrays of protuberances termed ommatidial gratings which provide the cuticle with anti-reflective, anti-wetting and self-cleaning properties. The hypothesised anti-contamination role of the gratings against dust and pollen results from theoretical predictions on grating geometry and experiments on synthetic replicas of ommatidia surfaces but has not yet been proven in an animal. Whiteflies are biological test beds for anti-contamination surfaces because they deliberately distribute wax particles extruded from abdominal plates over their entire bodies. The numerous particles protect the animal against water evaporation and radiation, but may severely impair vision. Using scanning electron microscopy (SEM) and CryoSEM, we here show that the cornea of whiteflies exhibits ~ 220 nm wide mesh-like structures forming hexagonal gratings with thin ~ 40 nm connecting walls. Quantitative measurements of wax particles on the eye show that the nanostructures reduce particle contamination by more than ~ 96% compared to other areas of the cuticle. Altogether, our study is the first description of a predicted optimized grating geometry for anti-contamination in an arthropod. The findings serve as evidence of the high effectiveness of nanostructured surfaces for reducing contact area and thus adhesion forces between biological surfaces and contaminating particles.

Numerical model of the locomotion of oscillating 'robots' with frictional anisotropy on differently-structured surfaces.

In engineering materials, surface anisotropy is known in certain textured patterns that appear during the manufacturing process. In biology, there are numerous examples of mechanical systems which combine anisotropic surfaces with the motion, elicited due to some actuation using muscles or stimuli-responsive materials, such as highly ordered cellulose fiber arrays of plant seeds. The systems supplemented by the muscles are rather fast actuators, because of the relatively high speed of muscle contraction, whereas the latter ones are very slow, because they generate actuation depending on the daily changes in the environmental air humidity. If the substrate has ordered surface profile, one can expect certain statistical order of potential trajectories (depending on the order of the spatial distribution of the surface asperities). If not, the expected trajectories can be statistically rather random. The same presumably holds true for the artificial miniature robots that use actuation in combination with frictional anisotropy. In order to prove this hypothesis, we developed numerical model helping us to study abovementioned cases of locomotion in 2D space on an uneven terrain. We show that at extremely long times, these systems tends to behave according to the rules of ballistic diffusion. Physically, it means that their motion tends to be associated with the "channels" of the patterned substrate. Such a motion is more or less the same as it should be in the uniform space. Such asymptotic behavior is specific for the motion in model regular potential and would be impossible on more realistic (and complex) fractal reliefs. However, one can expect that in any kind of the potential with certain symmetry (hexagonal or rhombic, for example), where it is still possible to find the ways, the motion along fixed direction during long (or even almost infinite) time intervals is possible.

Material composition and mechanical properties of the venom-injecting forcipules in centipedes.

BACKGROUND: Centipedes are terrestrial and predatory arthropods that possess an evolutionary transformed pair of appendages used for venom injection-the forcipules. Many arthropods incorporate reinforcing elements into the cuticle of their piercing or biting structures to enhance hardness, elasticity or resistance to wear and structural failure. Given their frequent exposure to high mechanical stress, we hypothesise that the cuticle of the centipede forcipule might be mechanically reinforced. With a combination of imaging, analytical techniques and mechanical testing, we explore the centipede forcipule in detail to shed light on its morphology and performance. Additionally, we compare these data to characteristics of the locomotory leg to infer evolutionary processes. RESULTS: We examined sclerotization patterns using confocal laser-scanning microscopy based on autofluorescence properties of the cuticle (forcipule and leg) and elemental composition by energy-dispersive X-ray spectroscopy in representative species from all five centipede lineages. These experiments revealed gradually increasing sclerotization towards the forcipular tarsungulum and a stronger sclerotization of joints in taxa with condensed podomeres. Depending on the species, calcium, zinc or chlorine are present with a higher concentration towards the distal tarsungulum. Interestingly, these characteristics are more or less mirrored in the locomotory leg's pretarsal claw in Epimorpha. To understand how incorporated elements affect mechanical properties, we tested resistance to structural failure, hardness (H) and Young's modulus (E) in two representative species, one with high zinc and one with high calcium content. Both species, however, exhibit similar properties and no differences in mechanical stress the forcipule can withstand. CONCLUSIONS: Our study reveals similarities in the material composition and properties of the forcipules in centipedes. The forcipules transformed from an elongated leg-like appearance into rigid piercing structures. Our data supports their serial homology to the locomotory leg and that the forcipule's tarsungulum is a fusion of tarsus and pretarsal claw. Calcium or zinc incorporation leads to comparable mechanical properties like in piercing structures of chelicerates and insects, but the elemental incorporation does not increase H and E in centipedes, suggesting that centipedes followed their own pathways in the evolutionary transformation of piercing tools.

Interleg coordination in free-walking bug Erthesina fullo (Hemiptera: Pentatomidae).

Insects can adapt their walking patterns to complex and varied environments and retain the ability to walk even after significant changes in their physical attributes, such as amputation. Although the interleg coordination of intact insects has been widely described in previous studies, the adaptive walking patterns in free-walking insects with amputation of 1 or more legs are still unclear. The pentatomid bug Erthesina fullo exhibits a tripod gait, when walking freely on horizontal substrates, like many other insects. In this study, amputations were performed on this species to investigate changes in interleg coordination. The walking parameters were analyzed, such as the locations of touchdown and liftoff, cycle period, walking speed, and head displacement of intact and amputated insects. The results show that E. fullo displays adaptive interleg coordination in response to amputations. With 1 amputated leg, bugs changed to a 3-unit gait, whereas with 2 amputated legs they employed a wave gait. These data are helpful in exploring the motion mode control in walking insects and provide the theoretical basis for the gait control strategy of robots, when leg failure occurs.

Wax Protrusions on Anti-Adhesive Plant Surfaces and Their Interactions with Insect Adhesive Pads: A Mechanical Interpretation.

Insect attachment devices enhance adhesion to complex-geometry substrates by increasing the real contact area. In nature, insects mainly interact with plant surfaces that are often covered by 3D wax structures. Here, we describe, discuss, and give a mechanical interpretation of plant waxes and the possible fracture mechanisms of these wax structures during their interactions with the adhesive pads of insects. It is argued that these plant surface microstructures significantly influence insect adhesion through reducing the contact area and contaminating the insect pads.

Voronoi diagrams and Delaunay triangulation for modelling animal territorial behaviour.

We explore the use of movable automata in numerical modelling of male competition for territory. We used territorial dragonflies as our biological inspiration for the model, assuming two types of competing males: (a) faster and larger males that adopt a face-off strategy and repulse other males; (b) slower and smaller males that adopt a non-aggressive strategy. The faster and larger males have higher noise intensity, leading to faster motion and longer conservation of motion direction. The velocity distributions resemble the Maxwell distributions of velocity, expected in Brownian dynamics, with two probable velocities and distribution widths for the two animal subpopulations. The fast animals' trajectories move between visually fixed density folds of the slower animal subpopulation. A correlation is found between individual velocity and individual area distribution, with smaller animals concentrated in a region of small velocities and areas. Attraction between animals results in a modification of the system behaviour, with larger animals spending more time being surrounded by smaller animals and being slowed down by their interaction with the surroundings. Overall, the study provides insights into the dynamics of animal competition for territory and the impact of attraction between animals.

Interaction of a non-axisymmetric artificial single spatula with rough surfaces.

Hair-like attachment structures are frequently used by animals to create stable contact with rough surfaces. Previous studies focused primarily on axisymmetric biomimetic models of artificial spatulas, such as those with a mushroom-shaped and cylinder-shaped geometry, in order to simulate the so-called gecko effect. Here, two geometric prototypes of artificial adhesive structures with non-axisymmetric properties were designed. The investigation of the prototype's interactions with rough surfaces was carried out using the finite element software ABAQUS. Under increasing vertical displacement, the effect of asperity size on the contact pressure evolution of the spatula was investigated. It has been demonstrated that the contact behaviour is greatly affected by the flexibility of the spatula, which is caused by its variable thickness. The thinner spatula shows a higher nominal contact area and attaches more strongly to various rough surfaces. Although a thicker spatula is more susceptible to the 'leverage' phenomenon, which occurs when excessively applied displacements prematurely reduce the nominal contact area, it obtains the ability to regulate attachment during unidirectional loading. Two non-axisymmetric prototypes provide different design concepts for the artificial adhesives. It is hoped that this study will provide fresh viewpoints and innovations that contribute to the development of biologically inspired adhesives.

From fibres to adhesives: evolution of spider capture threads from web anchors by radical changes in silk gland function.

Spider webs that serve as snares are one of the most fascinating and abundant type of animal architectures. In many cases they include an adhesive coating of silk lines-so-called viscid silk-for prey capture. The evolutionary switch from silk secretions forming solid fibres to soft aqueous adhesives remains an open question in the understanding of spider silk evolution. Here we functionally and chemically characterized the secretions of two types of silk glands and their behavioural use in the cellar spider, Pholcus phalangioides. Both being derived from the same ancestral gland type that produces fibres with a solidifying glue coat, the two types produce respectively a quickly solidifying glue applied in thread anchorages and prey wraps, or a permanently tacky glue deployed in snares. We found that the latter is characterized by a high concentration of organic salts and reduced spidroin content, showing up a possible pathway for the evolution of viscid properties by hygroscopic-salt-mediated hydration of solidifying adhesives. Understanding the underlying molecular basis for such radical switches in material properties not only helps to better understand the evolutionary origins and versatility of ecologically impactful spider web architectures, but also informs the bioengineering of spider silk-based products with tailored properties.

The Greasy Pole Syndrome in Alliaria petiolata (Brassicaceae): The Pubescence and Wax Coverage on Stems Reduce Invasion by Lasius niger Ants.

To reduce negative effects of floral visitation by ants, which do not serve as reliable cross-pollinators, some plants have developed a non-floral, stem-based defense mechanism called greasy pole syndrome. In the present study, we examined the effects of two surface features (trichomes and three-dimensional epicuticular wax coverage) on stems of Alliaria petiolata plants on visiting frequencies, travelled distances, and running velocities of Lasius niger ants. The experiments were performed with stem samples prepared from different (apical and basal) stem portions showing different surface morphologies (smooth control, covered by wax and trichomes + wax, respectively). The control, mechanically wiped stem samples lacking any surface features were significantly more often visited by ants, where they travelled significantly longer distances and moved with significantly higher velocities, compared to the intact stems. The apical and basal stem portions showed no significant differences in the measured parameters. Based on data obtained, we conclude about the main contribution of the wax to the greasy pole function of the A. petiolata stem via reduction of ant adhesion to the wax-bearing stem surface, whereas trichomes presumably serve as the first barrier for ants approaching usually from the ground level and protect the fragile wax coverage from an excessive deterioration.

Structure, mechanical and adhesive properties of the cellulosic mucilage in Ocimum basilicum seeds.

Plant seeds and fruits, like those of Ocimum basilicum, develop a mucilaginous envelope rich in pectins and cellulosic fibers upon hydration. This envelope promotes adhesion for attachment to soils and other substrates for dispersal and protection of the seed for a safe germination. Initially at hydration, the mucilage envelope demonstrates low adhesion and friction, but shows increasing adhesive and frictional properties during dehydration. However, the mechanisms underlying the cellulose fiber arrangement and the mechanical properties, especially the elasticity modulus of the mucilage envelope at different hydration conditions are not fully known. In this study, which is based on scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and light microscopy, the structure of the seed coat and arrangement of the cellulose fibers of basil seeds were characterized. Moreover, we performed pull-off force measurements to estimate adhesive properties and JKR-tests to estimate E-modulus of the mucilage at different hydration levels. Microscopy results demonstrate that cellulose fibers are split at their free ends into smaller fibrils, which might enhance the adhesive properties of the mucilage. Adhesive forces in contact increased during dehydration and reached maximum of 33 mN shortly before complete dehydration. The E-modulus of the mucilage changed from 1.4 KPa in water to up to 2.1 MPa in the mucilage at the maximum of its adhesion performance. Obtained results showed hydrogel-like mechanical properties during dehydration and cellulose fiber structures similar to the nanofibrous systems in other organisms with strong adhesive properties. STATEMENT OF SIGNIFICANCE: This paper reveals the hierarchical cellulose fiber structure in Ocimum basilicum's mucilaginous seed coat, suggesting increased fiber splitting towards the end, potentially enhancing adhesion contact areas. Mechanical tests explore elasticity modulus and adhesion force during various hydration stages, crucial as these properties evolve with mucilage desiccation. A rare focus on mucilaginous seed coat mechanical properties, particularly cellulose-reinforced fibers, provides insight into the hydrogel-like mucilage of plant seeds. Adhesion forces peak just before complete desiccation and then decline rapidly. As mucilage water content decreases, the E-modulus rises, displaying hydrogel-like properties during early dehydration stages with higher water content. This study might bring the focus to plant seeds as inspiration for biodegradable glues and applications for hydrogel research.

The effect of age on the attachment ability of stick insects (Phasmatodea).

Many insect species have found their way into ageing research as small and easy-to-keep model organisms. A major sign of ageing is the loss of locomotory functions due to neuronal disorders or tissue wear. Soft and pliable attachment pads on the tarsi of insects adapt to the substrate texture to maximize their real contact area and, thereby, generate attachment during locomotion. In the majority of stick insects, adhesive microstructures covering those pads support attachment. Stick insects do not molt again after reaching the imaginal stage; hence, the cuticle of their pads is subject to continuous ageing. This study aims to quantify how attachment ability changes with age in the stick insect Sungaya aeta Hennemann, 2023 and elucidate the age effects on the material and microstructure of the attachment apparatus. Attachment performance (adhesion and friction forces) on substrates with different roughnesses was compared between two different age groups, and the change of attachment performance was monitored extending over a larger time frame. Ageing effects on the morphology of the attachment pads and the autofluorescence of the cuticle were documented using light, scanning electron, and confocal laser scanning microscopy. The results show that both adhesion and friction forces decline with age. Deflation of the pads, scarring of the cuticle, and alteration of the autofluorescence, likely indicating stiffening of the cuticle, were observed to accumulate over time. This would reduce the attachment ability of the insect, as pads lose their pliant properties and cannot properly maintain sufficient contact area with the substrate.

Performance of biological food processing interfaces: Perspectives on the science of mollusc radula.

The Mollusca comprises a diverse range of organisms, with the class Gastropoda alone boasting approximately 80 000 extant species. Their adaptability across various habitats is facilitated by the evolution of the radula, a key structure for food acquisition. The radula's composition and mechanical properties, including its chitinous membrane, teeth, and supporting structures, enable efficient food gathering and processing. Through adaptive tooth morphology and composition, an interplay between radular components is facilitated, which results in collective effects to withstand forces encountered during feeding and reduce structural failure, with the broad range of variations reflecting ecological niches. Furthermore, teeth consist of composite materials with sometimes high contents of iron, calcium, or silicon to reduce wear. During interaction with the food, the radula performs complex three-dimensional motions, challenging to document. Here, we provide a review on the morphology, the mechanical properties, the composition, and various other parameters that contribute to radular performance. Due to, e.g., the smallness of these structures, there are, however, limitations to radular research. However, numerical simulations and physical models tested on substrates offer avenues for further understanding radular function and performance during feeding. These studies not only advance our knowledge of molluscan biology and ecology but also provide inspirations for biomimetic design and further advances in materials engineering.

Parasitoid attachment ability and the host surface wettability.

Climbing animals such as geckos and arthropods developed astonishing adhesive mechanisms which are fundamental for their survival and represent valuable models for biomimetic purposes. A firm adhesion to the host surface, in order to successfully lay eggs is necessary for the reproduction of most parasitoid insects. In the present study, we performed a comparative investigation on the attachment ability of four parasitoid species (the egg parasitoid Anastatus bifasciatus (Eupelmidae), the aphid parasitoid Aphidius ervi (Braconidae), the fly pupal ectoparasitoid Muscidifurax raptorellus (Pteromalidae) and the pupal parasitoid of Drosophila Trichopria drosophilae (Diapriidae)) with hosts characterized by a surface having different wettability properties. The friction force measurements were performed on smooth artificial (glass) surfaces showing different contact angles of water. We found that attachment systems of parasitoid insects are tuned to match the wettability of the host surface. Sexual dimorphism in the attachment ability of some tested species has been also observed. The obtained results are probably related to different microstructure and chemical composition of the host surfaces and to different chemical composition of the parasitoid adhesive fluid. The data here presented can be interpreted as an adaptation, especially in the female, to the physicochemical properties of the host surface and contribute to shed light on the coevolutionary processes of parasitoid insects and their hosts.

Comparative analysis of the ultrastructure and adhesive secretion pathways of different smooth attachment pads of the stick insect Medauroidea extradentata (Phasmatodea).

The mechanism by which insects achieve attachment and locomotion across diverse substrates has long intrigued scientists, prompting extensive research on the functional morphology of attachment pads. In stick insects, attachment and locomotion are facilitated by two distinct types of smooth cuticular attachment pads: the primary adhesion force-generating arolium and the friction force-generating euplantulae. They are both supported by an adhesive secretion delivered into the interspace between the attachment pads and the substrate. In this study, we analysed and compared internal morphology, material composition and ultrastructure, as well as the transportation pathways in both adhesive organs in the stick insect Medauroidea extradentata using scanning electron microscopy, micro-computed tomography, light microscopy, and confocal laser scanning microscopy. Our observations revealed structural differences between both attachment pads, reflecting their distinct functionality. Furthermore, our results delineate a potential pathway for adhesive secretions, originating from exocrine epidermal cells and traversing various layers before reaching the surface. Within the attachment pad, the fluid may influence the viscoelastic properties of the pad and control the attachment/detachment process. Understanding the material composition of attachment pads and the distribution process of the adhesive secretion can potentially aid in the development of more effective artificial attachment systems.

The puncture mechanics: an example from the bed bug Cimex lectularius showing traumatic insemination using the paramere.

Cimicidae are well-known for traumatic insemination, and males pierce females with their parameres and transfer sperm through them. The shape of parameres is relatively stable in the family, but in some genera, the paramere is elongated, appearing less resistant against lateral deflection. To understand the mechanical limitations of the paramere, we studied its penetration mechanics of the common bed bug, Cimex lectularius. We examined the post-abdominal morphology, paramere geometry and material properties and conducted breaking stress experiments on the paramere under wet and dry conditions. Mechanical property gradients are present with the paramere tip as the stiffest region and the base as the most flexible one. These mechanical properties relate to the presence of Ca, Zn and Si. The basal wing-shaped structure is flexible, enabling it to interlock with the anal region during mating. The paramere is slightly twisted; the tip region is circular in cross-section, and the geometry of the rest is rather complex. In the mechanical tests, wet parameres mainly buckled, while dried parameres broke off. The level of structural failures depended on directions from which the compression forces were applied. Structural, material and mechanical strengthening mechanisms preventing the paramere from mechanical failure are discussed.

Application of Recurrence Plot Analysis to Examine Dynamics of Biological Molecules on the Example of Aggregation of Seed Mucilage Components.

The goal of the research is to describe the aggregation process inside the mucilage produced by plant seeds using molecular dynamics (MD) combined with time series algorithmic analysis based on the recurrence plots. The studied biological molecules model is seed mucilage composed of three main polysaccharides, i.e. pectins, hemicellulose, and cellulose. The modeling of biological molecules is based on the assumption that a classical-quantum passage underlies the aggregation process in the mucilage, resulting from non-covalent interactions, as they affect the macroscopic properties of the system. The applied recurrence plot approach is an important tool for time series analysis and data mining dedicated to analyzing time series data originating from complex, chaotic systems. In the current research, we demonstrated that advanced algorithmic analysis of seed mucilage data can reveal some features of the dynamics of the system, namely temperature-dependent regions with different dynamics of increments of a number of hydrogen bonds and regions of stable oscillation of increments of a number of hydrophobic-polar interactions. Henceforth, we pave the path for automatic data-mining methods for the analysis of biological molecules with the intermediate step of the application of recurrence plot analysis, as the generalization of recurrence plot applications to other (biological molecules) datasets is straightforward.

Tree frogs (Polypedates dennysi) landing on horizontal perches: the effects of perch diameter.

Secure landing is indispensable for both leaping animals and robotics. Tree frogs, renowned for their adhesive capabilities, can effectively jump across intricate 3D terrain and land safely. Compared with jumping, the mechanisms underlying their landing technique, particularly in arboreal environments, have remained largely unknown. In this study, we focused on the landing patterns of the tree frog Polypedates dennysi on horizontally placed perches, explicitly emphasizing the influence of perch diameters. Tree frogs demonstrated diverse landing postures, including the utilization of: (1) single front foot, (2) double front feet, (3) anterior bellies, (4) middle bellies, (5) posterior bellies, (6) single hind foot, or (5) double hind feet. Generally, tree frogs favoured bellies on slimmer targets but double front feet on large perches. Analysis of limb-trunk relationships revealed their adaptability to modify postures, including body positions and limb orientations, for successful landing. The variations in the initial landing postures affected the subsequent landing procedures and, consequently, the dynamics. As the initial contact position switched from front foot back to the hind foot, the stabilization time decreased at first, reaching a minimum in middle belly landings, and then increased again. The maximum vertical forces showed an inverse trend, whereas the maximum fore-aft forces continuously increased as the initial contact position switched. As the perch diameter increased, the time expended dropped, whereas the maximum impact force increased. These findings not only add to our understanding of frog landings but also highlight the necessity of considering perch diameters and landing styles when studying the biomechanics of arboreal locomotion.

Optimization of Enzymatic and Chemical Decellularization of Native Porcine Heart Valves for the Generation of Decellularized Xenografts.

One of the most important medical interventions for individuals with heart valvular disease is heart valve replacement, which is not without substantial challenges, particularly for pediatric patients. Due to their biological properties and biocompatibility, natural tissue-originated scaffolds derived from human or animal sources are one type of scaffold that is widely used in tissue engineering. However, they are known for their high potential for immunogenicity. Being free of cells and genetic material, decellularized xenografts, consequently, have low immunogenicity and, thus, are expected to be tolerated by the recipient's immune system. The scaffold ultrastructure and ECM composition can be affected by cell removal agents. Therefore, applying an appropriate method that preserves intact the structure of the ECM plays a critical role in the final result. So far, there has not been an effective decellularization technique that preserves the integrity of the heart valve's ultrastructure while securing the least amount of genetic material left. This study demonstrates a new protocol with untraceable cells and residual DNA, thereby maximally reducing any chance of immunogenicity. The mechanical and biochemical properties of the ECM resemble those of native heart valves. Results from this study strongly indicate that different critical factors, such as ionic detergent omission, the substitution of Triton X-100 with Tergitol, and using a lower concentration of trypsin and a higher concentration of DNase and RNase, play a significant role in maintaining intact the ultrastructure and function of the ECM.