Bionic artificial penile Tunica albuginea

•A method for preparing hydrogels with parallel wavy fibers was developed•ATA with a bionic double-layer structure was prepared•ATA has bionic mechanical properties and biocompatibility and can repair TA injury•This approach could provide a common platform for future tissue analog preparations A mammalian tunica albuginea (TA) is characterized by a double-layer orthogonal structure composed of stacked parallel wavy collagen fibrils. A TA mimic fiber with directional crimping was synthesized in this study by stretching an isotropic polyvinyl alcohol gel followed by covalent cross-linking to permanently fix the parallel aligned curved structures. Similar to the sequential response of the crimped structure in natural TA, the curved fibers first straighten to absorb strain and then stretch to induce a soft-to-firm transition. The proposed method paves the way for building a variety of weight-bearing organizations. Through the imitation of the structure, performance matching can be achieved. The mammalian tunica albuginea (TA) is characterized by a double-layer orthogonal structure composed of stacked parallel wavy collagen fibers. During an erection, these fibers undergo sequential straightening and stretching to mediate the soft-to-firm transition. Inspired by the delicate strain-adaptive structure of natural TA, we propose an artificial TA (ATA) composed of a strain-stiffening hydrogel consisting of aligned yet crimped fibers. This delicate structure is produced via the stretching of an isotropic polyvinyl alcohol gel followed by covalent cross-linking. ATA possesses several key mechanical features of natural TA, including rapid strain stiffening over small intervals of deformation, excellent fatigue resistance to sustain cyclic bursts, and high toughness to withstand pointwise acupuncture during suturing. Furthermore, ATA displays the capability to repair injuries and restore normal erectile function of the TA-damaged penile tissue in a pig model. Our study demonstrates that ATA has great promise for penile injury repair. The mammalian tunica albuginea (TA) is characterized by a double-layer orthogonal structure composed of stacked parallel wavy collagen fibers. During an erection, these fibers undergo sequential straightening and stretching to mediate the soft-to-firm transition. Inspired by the delicate strain-adaptive structure of natural TA, we propose an artificial TA (ATA) composed of a strain-stiffening hydrogel consisting of aligned yet crimped fibers. This delicate structure is produced via the stretching of an isotropic polyvinyl alcohol gel followed by covalent cross-linking. ATA possesses several key mechanical features of natural TA, including rapid strain stiffening over small intervals of deformation, excellent fatigue resistance to sustain cyclic bursts, and high toughness to withstand pointwise acupuncture during suturing. Furthermore, ATA displays the capability to repair injuries and restore normal erectile function of the TA-damaged penile tissue in a pig model. Our study demonstrates that ATA has great promise for penile injury repair. The tunica albuginea (TA) is a tough and elastic tissue covering the corpus cavernosum of the penis. It is a double-layered connective tissue consisting of an outer longitudinal layer and an inner circular layer.1Park H.J. Tissue-engineered approaches for penile reconstruction.Organ Tissue Eng. 2020; : 1-37https://doi.org/10.1007/978-3-030-18512-1_14-1Crossref Google Scholar The main components of TA are collagen fibers that are curly and parallel, as well as elastic fibers embedded within them. In penile erection, the orthogonal fiber structure of TA plays a vital role as a hydrostatic skeleton.2Kelly D.A. Penises as variable-volume hydrostatic skeletons.Ann. N. Y. Acad. Sci. 2007; 1101: 453-463https://doi.org/10.1196/annals.1389.014Crossref Scopus (17) Google Scholar Figure 1A illustrates that, before an erection, the fibers of the TA are crimped, which allows the penis to bend and twist with great flexibility. With penis erection, the fibers of TA are straightened, providing a strong ability to resist deformation. The TA can be damaged by Peyronie’s disease, injury, and/or aging, resulting in problems such as penile bending, painful erection, and difficult sexual intercourse, which routinely requires surgical treatment.3Andrew T.W. Kanapathy M. Murugesan L. Muneer A. Kalaskar D. Atala A. Towards clinical application of tissue engineering for erectile penile regeneration.Nat. Rev. Urol. 2019; 16: 734-744https://doi.org/10.1038/s41585-019-0246-7Crossref Scopus (5) Google Scholar In most cases, the simple removal of fibrous plaque causes the penis to shorten.4Goldstein I. Hartzell R. Shabsigh R. The impact of Peyronie's disease on the patient: gaps in our current understanding.J. Sex Marital Ther. 2016; 42: 178-190https://doi.org/10.1080/0092623X.2014.985351Crossref PubMed Scopus (10) Google Scholar,5Milenkovic U. Albersen M. Castiglione F. The mechanisms and potential of stem cell therapy for penile fibrosis.Nat. Rev. Urol. 2019; 16: 79-97https://doi.org/10.1038/s41585-018-0109-7Crossref PubMed Scopus (25) Google Scholar The implantation of a patch graft after the removal of fibrous plaques may decrease penis shortening (Figure 1B).6Colombo F. Franceschelli A. Gentile G. Droghetti M. Fiorillo A. Palmisano F. The evolution in the surgical management of Peyronie’s disease.Urologia. 2021; 88: 79-89https://doi.org/10.1177/03915603211005326Crossref Google Scholar Currently, reported TA patches primarily consist of autologous tissues and extracellular matrix (ECM).3Andrew T.W. Kanapathy M. Murugesan L. Muneer A. Kalaskar D. Atala A. Towards clinical application of tissue engineering for erectile penile regeneration.Nat. Rev. Urol. 2019; 16: 734-744https://doi.org/10.1038/s41585-019-0246-7Crossref Scopus (5) Google Scholar,7Kadioglu A. Sanli O. Akman T. Ersay A. Guven S. Mammadov F. Graft materials in Peyronie's disease surgery: a comprehensive review.J. Sex. Med. 2007; 4: 581-595https://doi.org/10.1111/j.1743-6109.2007.00461.xAbstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar Some researchers recommend using autologous sheaths, small intestinal submucosa,8Valente P. Gomes C. Tomada N. 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The evolution in the surgical management of Peyronie’s disease.Urologia. 2021; 88: 79-89https://doi.org/10.1177/03915603211005326Crossref Google Scholar Furthermore, their microstructures are quite different from those of the natural TA, increasing the difficulty of maintaining the mechanical and morphological requirements of the TA; these structures do not achieve the restoration of normal erectile function.12García-Gómez B. González-Padilla D.A. Alonso-Isa M. Medina-Polo J. Romero-Otero J. Plication techniques in Peyronie’s disease: new developments.Int. J. Impot. Res. 2020; 32: 30-36https://doi.org/10.1038/s41443-019-0204-1Crossref PubMed Scopus (8) Google Scholar Hydrogels have many advantages over ECM derived from natural tissues. They can be synthesized from various raw materials with different properties, possess a definite composition, and have tunable mechanical properties.13Matsuda T. Kawakami R. Namba R. Nakajima T. Gong J.P. 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In practical applications, this mechanical mismatch may impair hydrogel effectiveness. In addition to strain-stiffening properties, the designed TA patches should be biocompatible, suture resistant, and blood compatible. We chose a sodium trimetaphosphate (STMP)-cross-linked polyvinyl alcohol (PVA) hydrogel to prepare an artificial TA (ATA) that meets these requirements. STMP-cross-linked PVA hydrogels have excellent mechanical properties and provide the possibility of suturing.26Shin C.S. Cabrera F.J. Lee R. Kim J. Ammassam Veettil R. Zaheer M. Adumbumkulath A. Mhatre K. Ajayan P.M. Curley S.A. et al.3D-Bioprinted inflammation modulating polymer scaffolds for soft tissue repair.Adv. Mater. 2021; 33: 2003778https://doi.org/10.1002/adma.202003778Crossref Scopus (9) Google Scholar,27Wu S. Hua M. Alsaid Y. Du Y. Ma Y. Zhao Y. Lo C.Y. Wang C. Wu D. Yao B. et al.Poly (vinyl alcohol) hydrogels with broad-range tunable mechanical properties via the hofmeister effect.Adv. Mater. 2021; 33: 2007829https://doi.org/10.1002/adma.202007829Crossref Scopus (107) Google Scholar At the same time, they are blood safe and could remain stable in vivo for a long time.28Stock U.A. Schenke-Layland K. Performance of decellularized xenogeneic tissue in heart valve replacement.Biomaterials. 2006; 27: 1-2https://doi.org/10.1016/j.biomaterials.2005.05.100Crossref PubMed Scopus (51) Google Scholar However, reports on the design of their microstructure are still very rare. The double-layer crimped fiber structure in the TA is the key to its erectile function. Therefore, the bionic preparation of oriented crimped fiber structures is the main challenge of an ATA. Here, we remolded isotropic PVA hydrogels into double-layered elastic patches composed of parallel curved fibers. Similar to the sequential response of the crimped structure in natural TA, the curved fibers first straighten to absorb strain and then stretch to induce a soft-to-firm transition. Using STMP cross-linking, a bionic ATA is obtained by linking two anisotropic PVA hydrogels together. The ATA displays strain-stiffening properties similar to natural TA, and its burst pressure is much higher than the intra-cavernous pressure during an erection. In addition, the superior suture performance of the ATA makes it an attractive option for clinical use. Furthermore, we examined the effect of the two-layer arrangement of the ATA on penis hardness. Then, the biocompatibility, antithrombotic properties, and long-term stability of ATA were suggested in vitro and in vivo. Finally, the ATA was used in a Bama pig TA injury model. The erection of the penis returned to normal after suturing the ATA at the injured part, and the long-term prognosis was satisfactory. PVA is a strong, physiologically safe hydrogel that has been widely used for tissue repair and regeneration.29Liu J. Lin S. Liu X. Qin Z. Yang Y. Zang J. Zhao X. Fatigue-resistant adhesion of hydrogels.Nat. 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Hierarchical multiscale hydrogels with identical compositions yet disparate properties via tunable phase separation.Adv. Funct. Mater. 2022; 32: 2110277https://doi.org/10.1002/adfm.202110277Crossref Scopus (3) Google Scholar,34Pan J. Gao L. Sun W. Wang S. Shi X. Length effects of short alkyl side chains on phase-separated structure and dynamics of Hydrophobic Association Hydrogels.Macromolecules. 2021; 54: 5962-5973https://doi.org/10.1021/acs.macromol.1c00471Crossref Scopus (9) Google Scholar However, the strong hydrophobic effect of these PVA hydrogels is not favorable for their long-term application in vivo. We first stretched the PVA cryogel to produce the orientation and subsequently fixed this structure using the biosafe cross-linker STMP to construct the biomimetic convoluted fiber structure. Figure 2A illustrates the preparation of the anisotropic PVA patch (PVA hydrogel containing curled oriented fibers). Isotropic PVA hydrogels were prepared by freezing and thawing PVA aqueous solutions for at least three cycles. We added low-molecular-weight PVA (87%–90% hydrolyzed, Mw 30,000–70,000) to a high molecular weight PVA (99% hydrolyzed, Mw 146,000–186,000) solution to obtain isotropic PVA hydrogels with a homogeneous porous network (Figure S1). The isotropic PVA was stretched to λ = 3 and soaked in 15 wt % STMP solution for 24 h to obtain oriented hydrogels. To trigger covalent cross-linking, the pH of the STMP solution was adjusted to approximately 12. Fourier transform infrared (FTIR) spectra indicate the formation of stable cross-links (Figure 2C), and the obtained hydrogels are called stretched PVA. The stretched PVA was dried for 24 h to create microcrystalline regions and increase the hydrogel’s toughness and stability. Finally, the dried patches were immersed in a phosphate-buffered saline (PBS) solution to rehydrate to obtain an anisotropic PVA patch. FTIR spectroscopy confirmed the phosphate cross-linking between adjacent PVA chains on the molecular scale (Figure 2C). All spectra were very similar and showed typical PVA bands. The spectra of isotropic PVA were almost indistinguishable from those of pure PVA, indicating that no new chemical cross-links formed during the freeze-thaw cycles and STMP immersion treatment. In the spectrum of anisotropic PVA, the bands at 1,284 cm−1 (O=P–O stretching) and 1,021 cm–1 (P–O bending) are usually used to confirm polymer cross-linking by STMP.35Leone G. Consumi M. Pepi S. Pardini A. Bonechi C. Tamasi G. Donati A. Rossi C. Magnani A. Poly-vinyl alcohol (PVA) crosslinked by trisodium trimetaphosphate (STMP) and sodium hexametaphosphate (SHMP): effect of molecular weight, pH and phosphorylating agent on length of spacing arms, crosslinking density and water interaction.J. Mol. 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Throughout the preparation process, X-ray diffraction profiles showed an increasing intensity ratio between the crystalline peak (2θ = 19.9°) and amorphous peaks (2θ = 28° and 41°) in anisotropic PVA patches, suggesting increasing crystallinity (Figure S2A). The chemical bonds, hydrogen bonds, and crystalline regions between PVA molecular chains form multiple cross-linked networks, which gives the anisotropic PVA patch excellent mechanical properties. The changes in the microstructure of the hydrogel during preparation were studied using laser scanning confocal microscopy (Figure 2B). In the isotropic PVA hydrogel, the polymer chains showed a loose and irregular orientation, and there were many obvious gaps. In the stretched PVA hydrogel, the polymer chains were highly oriented, and a large number of parallel fibers were arranged in the stretching direction. After drying and rehydration, the anisotropic PVA patch contained oriented curly fibers with a decreased overall degree of orientation, but the fibers were generally still aligned in the stretch direction. At the same time, the water content of hydrogels in different stages was studied. With the emergence of new cross-linking sites, the water content decreased in each stage (Figure S2B). We further investigated the orientation of anisotropic PVA patches from the micron scale to the nanoscale. The scanning electron microscopy (SEM) images of lyophilized anisotropic PVA patches revealed parallel curved fibers (Figure 2D), which were significantly different from the random entangled fibers of isotropic PVA (Figure S2C). It should be noted that ice crystal growth during freeze drying squeezes the polymer chain, which may cause differences from the actual state of anisotropic PVA patches. The nanostructures of isotropic PVA and the anisotropic PVA patch were studied using small angle neutron scattering (SANS), and the two-dimensional patterns of their absolute scattering intensities are shown in Figure 2E. Unlike the circular pattern of isotropic PVA, the SANS pattern of the anisotropic PVA patch exhibits a distinct elliptical pattern, indicating that its structure is oriented along the stretching direction. Anisotropic materials usually have high strength in one direction and significantly lower strength in the other direction. During penile erection, TA is subjected to both axial and radial stretching, and a single-layer anisotropic PVA patch has difficulty meeting the demand of a TA patch. This problem can be solved through precise mimicry of the natural TA double-layer structure. Two anisotropic PVA patches were rotated to 90° crossed and adhered according to the direction of stretching orientation, and an ATA with a double-layer orthogonal structure was constructed. To obtain ATA, two pieces of anisotropic PVA patch need to be laminated firmly without compromising the tensile strength. As abundant residual hydroxyl groups still existed in the anisotropic PVA patch, we used a PVA/STMP solution as a binder to laminate the anisotropic PVA patches. The adhesive mechanism of orthogonal ATA is proposed in Figure 3A . The confocal and SEM images clearly showed the two-tier microstructure of the oriented ATA in flabby and tensed states (Figures 3B and 3C). Additionally, Figures S3 and S4 show photos of two pieces of anisotropic PVA patch adhesion, which are dyed red and blue. The adhered ATA (the adhesion area is approximately 2.5 cm2) withstood mechanical loads of 2.08 kg without delamination, suggesting tough adhesion. Indeed, the adhesion energy of the ATA was approximately equal to the fracture energy of the pristine anisotropic PVA patch (Figure S5). We prepared three groups of samples with the same thickness to verify the performance of the double-layer bionic structure: isotropic PVA, anisotropic PVA, and ATA. The uniaxial tensile curves and bursting pressures of these samples are shown in Figures 3D, 3E, and S5. Compared with TA tissues from pigs and baboons, the ATA has very similar breaking strength and elongation at break, indicating its potential as a TA patch. As shown in Figure S5C, both ATA and anisotropic PVA exhibited typical J-shaped stress-strain curves when stretched along a direction parallel to the fiber orientation, demonstrating their strain-stiffening properties. When the deformation was small, the response of the material to stretching was mainly a conformational transition of the bending fibers when the modulus was low. As stretching proceeded, the bending-dominated response to stretching of the fibers in the hydrogel transitioned to a stretching-dominated response as the fibers gradually straightened. The stress-strain curves of isotropic PVA showed typical J-shaped curves, but the critical stretch point at the onset of strain stiffening (λ = 2.5) was much larger than that of anisotropic PVA and ATA (λ = 1.2–1.5), indicating that aligned fibers produce strain-stiffening effects faster.37Kier W.M. The diversity of hydrostatic skeletons.J. Exp. Biol. 2012; 215: 1247-1257https://doi.org/10.1242/jeb.056549Crossref PubMed Scopus (127) Google Scholar Anisotropic PVA has a low strength in the vertical direction, resulting in its low burst pressure (<20 kPa). The ATA shows rapid strain stiffening within λ < 1.5, which is very similar to natural TA. In addition, 1-mm-thick ATA has a higher burst pressure (71.5 kPa), which is much higher than the intra-cavernosal pressure (5.1–31.5 kPa) of a human erection,15An G. Guo F. Liu X. Wang Z. Zhu Y. Fan Y. Xuan C. Li Y. Wu H. Shi X. et al.Functional reconstruction of injured corpus cavernosa using 3D-printed hydrogel scaffolds seeded with HIF-1α-expressing stem cells.Nat. Commun. 2020; 11: 2687https://doi.org/10.1038/s41467-020-16192-xCrossref PubMed Scopus (25) Google Scholar indicating that it can be used safely. Based on these results, the ATA achieved excellent performance through structural bionic design, such as rapid strain stiffening, high burst pressure, and superb suture performance. The aligned fibers conferred tear resistance to the ATA. Figure 3F shows that a notched anisotropic PVA patch withstood cyclic stretching under a prescribed stretch of λ = 2 parallel to the aligned fibers, suggesting a remarkable crack-pinning ability. By providing such tear resistance, the ATA can function in the presence of small cracks and can be stitched to the injured part. Figure 3G shows the tensile stress-strain curves after suturing in different groups. Multiple notches were introduced during suturing, while the thinner sutures had a minimal area of force. The mechanical properties of all groups were significantly decreased after suturing. According to statistics, when an adult male has an erection, the elongation of the penis is approximately 120%–190%.38Ponchietti R. Mondaini N. Bonafè M. Di Loro F. Biscioni S. Masieri L. Penile length and circumference: a study on 3, 300 young Italian males.Eur. Urol. 2001; 39: 183-186https://doi.org/10.1159/000052434Crossref PubMed Scopus (110) Google Scholar,39Chen X.B. Li R.X. Yang H.N. Dai J.C. A comprehensive, prospective study of penile dimensions in Chinese men of multiple ethnicities.Int. J. Impot. Res. 2014; 26: 172-176https://doi.org/10.1038/ijir.2014.9Crossref PubMed Scopus (18) Google Scholar Because of the sparse fiber network structure, the elongation and strength at break of isotropic PVA are low, preventing it from easily meeting the requirements of clinical applications. For anisotropic PVA, its elongation at break reaches 210% when it is stretched parallel to the orientation direction, but when stretched perpendicular to the orientation direction, similar to isotropic PVA, it still does not meet the application scenario of bi-axial stretching. The ATA can achieve an elongation at a break of 190%, enabling it to match the percentage of penile elongation during erection in most men. Therefore, under physiological conditions, the ATA cannot be damaged easily. After 200 cycles of the tensile fatigue test with a deformation of λ = 1.5, the stitched pinhole showed no obvious damage, and the strength of the ATA did not significantly decrease according to the stress-strain curve, suggesting the durability of the ATA (Figure 3H). A mammalian penis is a typical hydrostatic skeleton. A hydrostatic skeleton is composed of a cavity filled with liquid (corpus spongiosum filled with blood, in the penis) and outer connective tissue fibers (TA in the penis). The connective tissue fiber arrangement plays a key role in controlling and limiting shape change. The cross-fiber spiral connective tissue array is the most common arrangement of connective tissue fibers in nature. With the elongation (the fiber angle, which is the angle relative to the long axis, decreases) and shortening (the fiber angle increases) of the pitch of the spiral, an overall change in length is realized.40Miranda A.F. Sampaio F.J.B. A geometric model of plaque incision and graft for P eyronie's disease with geometric analyses of different techniques.J. Sex. Med. 2014; 11: 1546-1553https://doi.org/10.1111/jsm.12462Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar The fiber structure of the TA is different and is arranged in an orthogonal array; it is composed of circular and longitudinal connective tissue fibers. During an erection, this structure allows the penis to resist external deformation.2Kelly D.A. Penises as variable-volume hydrostatic skeletons.Ann. N. Y. Acad. Sci. 2007; 1101: 453-463https://doi.org/10.1196/annals.1389.014Crossref Scopus (17) Google Scholar We evaluated whether ATA, like natural TA, gives the penis enough strength to resist bending when the ATA is vertically crossed, but not when it is spirally crossed. Based on the balloon model (replaces the penile corpus cavernosum and provides pressure) commonly used in Peyronie’s disease research,41Vatankhah-Varnosfaderani M. Keith A.N. Cong Y. Liang H. Rosenthal M. Sztucki M. Clair C. Magonov S. Ivanov D.A. Dobrynin A.V. et al.Chameleon-like elastomers with molecularly encoded strain-adaptive stiffening and coloration.Science. 2018; 359: 1509-1513https://doi.org/10.1126/science.aar5308Crossref Scopus (236) Google Scholar the simulated penis was constructed using a balloon and ATA with two different crossed structures (vertical or spiral). The simulated penis constructed using the ATA with different fiber arrangement structures is shown in Figure 4A . When weights were suspended at the end of the simulated penis to apply pressure, the spiral cross-over group showed significant bending, indicating that the structure of the spiral cross-over is not conducive to maintaining penile stiffness. The vertically crossed ATA group had no bending and showed good resistance to tension. This result is consistent with our expectation, indicating that the assembly mode of the double-layer TA has a significant impact on its performance. As shown in Figure 4B, two types of fiber orientations are simulated through the simplified finite element model. The elongated TA after erection is approximated as non-stretchable fibers with a high modulus and only bears tension without pressure. Because of the incompressibility of the liquid, the macro stretching of the penis must be accompanied by the reduction of the cross-section and vice versa. During compression, the diameter of the section must increase, while the circular fibers are stretched. The results show that the relative rotation mainly occurs in the helical structure, while the orthogonal structure does not change significantly. When subjected to a large bending force, the orthogonal fibers break and are destroyed (corresponding with the condition of penile fracture), while the spiral fibers bend smoothly. Furthermore, an isolated pig penis was used to experimentally repair TA injuries. The results and statistical analysis of the angle after erection are shown in Figure 4C. A normal penis can be completely straightened, and its included angle with the horizontal direction is approximately 20°. The penis cannot be erect normally when the TA is damaged, because it cannot maintain internal pressure. The direct suturing of TA injuries causes local stress concentration and bends the penis in one direction. After the ATA was sutured to the injured part, the penis resumed normal erection, and the angle of the penis after erection was similar to that of the normal group. Through the interview and investigation of clinicians, we set the normal range after erection as ±30°. As shown in Figure 4D, the penises of the normal group and the ATA group were within the normal range, and that of the direct suture group was beyond the normal range, which shows that our ATA has potential as a TA repair patch. The biocompatibility of the ATA was analyzed further to verify the possibility of its clinical application. Since the cells surrounding TA are mainly fibroblasts, rat skin fibroblasts were selected and cultured in the presence of an ATA for 5 days. The results of the Cell Counting Kit-8 (CCK-8) assay and live-dead staining showed no significant differences between the ATA group and the blank control group (Figure S6), proving that the ATA had no significant cytotoxicity. A large amount of blood in the corpus cavernosum means that the ATA will come into contact with blood after transplantation, so the hemocompatibility of the ATA is critical. Fresh rabbit blood was used to conduct the platelet adhesion assay. Isotropic PVA was chosen as the control group. As we expected, the ATA has a low platelet adhesion and coagulation effect (Figure S7), suggesting that it has a low thrombogenic tendency and avoids local tissue necrosis due to thrombosis. Using lactate dehydrogenase (LDH), adhesion to the ATA and isotropic PVA was quantitatively studied. The adhesion of the ATA to blood platelets was surprisingly low, possibly because of the STMP’s introduction of a large number of negative charges on its surface. The in vivo fibrotic effect of the ATA is very important for its future use as a long-term alternative material. A major cause of Peyronie’s disease is abnormal fibrosis after damage to the TA. If the ATA causes inflammation and induces fibrosis, plaques may form at the implantation site and result in Peyronie’s disease recurrence. A subcutaneous embedding experiment in Sprague-Dawley (SD) rats verified the biocompatibility of the ATA (Figure S8). At 30 and 60 days after subcutaneous implantation, no obvious fibrosis or abnormal inflammatory reaction was detected in the ATA group. Additionally, ATA group participants had less inflammatory infiltrate around the implanted area than isotropic PVA group participants, suggesting that the ATA does not easily cause an inflammatory response around the implanted area. The ATA’s ability to capture positively charged pro-inflammatory factors is attributed to its negatively charged phosphate groups.26Shin C.S. Cabrera F.J. Lee R. Kim J. Ammassam Veettil R. Zaheer M. Adumbumkulath A. Mhatre K. Ajayan P.M. Curley S.A. et al.3D-Bioprinted inflammation modulating polymer scaffolds for soft tissue repair.Adv. Mater. 2021; 33: 2003778https://doi.org/10.1002/adma.202003778Crossref Scopus (9) Google Scholar Based on the results above, the ATA has high biocompatibility and can be used as a long-term replacement material. Finally, using TA injury pig models, the effect of an ATA was verified in a TA repair experiment. The Bama miniature pig has no apparent penile spine and a penile size that is similar to that in humans. The operation process is shown in Figures 5A and S9. A square incision with a side length of approximately 8 mm was made on the penis of all experimental pigs to create a TA injury model. In the suture group, the injured part was directly sutured; in the ATA group, an ATA slightly larger than the wound was used as a patch and sutured to the injured part; the control group consisted of untreated normal pigs. The corpus spongiosum was injected with normal saline to erect the penis, and the shape of the penis after erection was observed (Figure 5B and Video S1). In the suture group, obvious bending was noted, and the erectile state resembled the pathological state of Peyronie’s disease. The ATA restored the normal erectile morphology to a level comparable with that of normal penile tissue after surgery, indicating that the ATA patch replaced the function of the natural TA during the erection procedure. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1MTM3NWQ4MDYzYzFkZjU0MDE3NGY1YWVjZGQxNWE2NyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjcyOTE4MzQyfQ.duBwyhhYVWq5Oqt63YUSEQNcHDot59wLS5uzM25qtX8oPhdjrZ1DBvbnxOnF7ajdtp9AAmZk9xarDZdMskD9ZYI_xOmNhk4A_1GUecYYlnuJrizenV3bfBzJn4WtcIBh0F22uI12QyT7KfgSP4qRaExn5ID4rkhaXRpKqVIYe90fGAPI3TAmGlNcUiITmIyGOxdtlV8AL5oF3Ez_5ZQsqCHhRmCd3TCYUPjHtcfXQzvprwyYL_6nRj1AYwqn0USiEuINOK2qqNM_DBErlIekfZ7NAw6ycSEu0kNTw2QqR1VeKcv67u5HxMSbYXzxEnE_Au0RnS4F5LH03w2pVjoKYg Download .mp4 (12.53 MB) Help with .mp4 files Video S1. Erectile effect of pig penis treated with different treatments All pigs were euthanized 1 month after the operation and sampled to study the repair effect. The results obtained after sectioning and staining are shown Figure 5C. In the suture group, there was evident abnormal fibrosis and hematoma, suggesting poor wound healing and the possibility of recurrence of Peyronie’s disease. Despite the inability of the ATA to restore the microstructure of natural tissue, the amount of fibrosis was comparable with that of normal tissue, and a normal erection was achieved after injection of normal saline (Figure S9). This indicates that the use of ATA had a positive impact on TA injuries. Almost all available TA patches are prepared from natural tissues, which may be attributed to the difficulty in replicating the structure of the double-layer crimped fiber of TA using artificial materials. Using a simple stretching-cross-linking procedure, we prepared an ATA with a bionic double-layer structure. The random reticular fibers generated using the freeze-thaw cycle and salting-out method have clear orientations after stretching and continue to retain this orientation after STMP cross-linking. The ATA exhibited a J-type stress-strain curve in the uni-axial tensile test, similar to pig TA. In vivo and in vitro experiments demonstrate that our ATA is biocompatible and has a positive effect on pig TA injuries. This ATA represents a novel approach to improving outcomes and decreasing acute and long-term complications associated with the repair of TA injuries.