The physical method disrupts the cell membrane, induces cell lysis, and facilitates the transport of cellular debris (16–19). Commonly employed physical methods include repeated cycles of freezing and thawing, ultrasonic vibration, supercritical carbon dioxide, high hydrostatic pressure treatment, mechanical compression, and electroporation. In recent years, numerous studies have attempted to utilize physical techniques in facilitating decellularization and demonstrated its feasibility.

The freeze-thaw method can be optimized by increasing the temperature differential or adjusting the number of cycles, thereby enhancing its efficiency. Multiple freeze-thaw cycles may be employed throughout the decellularization process without compromising matrix protein preservation (20). It is important to note that complete removal of nuclear material should not be reported, as the formation of ice crystals during freezing and thawing disrupts cell membranes in tissues or organs (21). While this process effectively preserves biochemical composition and mechanical properties, insufficient removal of genetic material may lead to potential immunological rejection. To disrupt cell membranes, high hydrostatic pressure exceeding 600 MPa is applied; however, it should be acknowledged that such high pressure can induce protein deformation, as evidenced by observed collagen and elastic fiber deformations in decellularized blood vessels. Consequently, this results in a reduction of approximately 50% in the tensile strength of fibers compared to their original tissue state (22).

Supercritical carbon dioxide exhibits low viscosity and high transport properties, allowing for minimal damage to tissue mechanical properties when passed through at a controlled speed similar to the critical point. Various studies have demonstrated its ability to induce non-deformability in tissue fibers (23). However, its poor solubility for polymers and polar substances is a drawback that can be partially addressed with entraining agents such as ethanol, though this may introduce new impurities (24).

Nonthermal irreversible electroporation, typically achieved through the application of microsecond electrical pulses, disrupts the transmembrane electrical potential and induces micropore formation in the plasma membrane. Ultimately, this leads to cell death by perturbing its steady-state electrical balance while largely preserving the three-dimensional structure of tissues and organs. A novel porcine acellular dermal matrix (PADM) prepared by Xia et al. (25), utilizing laser micropore technology, was demonstrated to be both safe and effective in animal transplantation.

Chemical decellularization methods commonly employ a variety of detergents, including surfactants, acids, bases, hypertonic and hypotonic solutions, and chelating agents. Surfactants can be classified based on their charge as ionic, nonionic or zwitterionic; all of which have the ability to disrupt cell membranes and degrade DNA. Hogg et al. (26) utilized chemical treatment to remove the epidermis, followed by immersion in a hypotonic buffer solution for cell dissolution and subsequent addition of nuclease buffer to eliminate residual nucleic acid substances. This approach achieved rapid and efficient preparation of ADM. Draguňova et al. (27) also developed an effective method for preparing decellularized matrices using only a few chemicals with minimal procedures. Bera et al. (28) employed a method based on hypotonic/hypertonic saline solution to decellularize goat skin before formulating an ADM bio-ink and 3D bioprinting it. Ultimately, this resulted in an ADM with exceptional mechanical properties and cell adhesion.

Organisms (enzymes) selectively cleave the arginine carboxyl side of cell adhesion proteins, resulting in detachment and lysis of cells from the adjacent matrix. Commonly employed enzymes include trypsin, collagenase, nuclease, thermophilic protease, and dispase (29, 30). Excessive utilization of enzymes may lead to the degradation of natural matrix components, including collagen, elastin, and glycosaminoglycans (31). Following enzymatic decellularization, thorough rinsing of the tissue is essential to eliminate or neutralize any residual enzyme components and cellular debris (32). Therefore, it is recommended to use trypsin for short-term periods to prevent damage to the matrix components. It should be noted that trypsin does not exhibit cytotoxic effects on bioengineered materials which are crucial for in vitro cell culture.

As each method possesses its own set of advantages and disadvantages, a combination of methodologies can be employed to explore a more efficacious acellular process and prepare an ideal ADM with enhanced decellularization efficiency. While ionic SDS has demonstrated commendable efficacy in eluting cells and removing cellular components, it also leads to the degradation of the extracellular matrix due to the elution of other substances. Triton X-100, a nonionic eluent, exhibits inefficacy against the eluted cells while causing minimal damage to the extracellular matrix. Although enzymatic hydrolysis can selectively eliminate DNA and other cellular components, its sole effect on elution is suboptimal and may result in certain damages to blood vessels and ultrastructure. Therefore, combining physical, chemical, and enzymatic methods can yield superior outcomes in terms of decellularization.

ADM removes cellular elements that have potential immunogenicity while retaining the original extracellular matrix as a supportive framework (46). This three-dimensional structure comprises biomaterials such as collagen, elastin, and proteoglycan that serve as an ideal substrate for epithelial cell growth, fibroblast proliferation, and neovascularization post-transplantation (47). ADM can be regarded as a collagen-based scaffold with preserved three-dimensional structure obtained through physical-chemical-biological methods involving elimination of epidermal cells and other constituents from natural skin tissue. Consequently, ADM possesses slight variations in its physicochemical characteristics compared to native tissues; particularly exhibiting inferior mechanical properties along with reduced resistance against enzymatic degradation (48).

The xenogeneic ADM undergoes a process where immunogenic cells and skin appendages are removed, resulting in the presence of mainly collagen, laminin, hyaluronic acid, elastin, keratan sulfate, fibronectin, and a small amount of cell-associated proteins along with abundant type I collagen (49, 50). Being a xenogeneic protein, the telopeptide of this however, numerous experiments have demonstrated that the immunogenicity of collagen is relatively weak. The primary constituent found in ADM is collagen I which exhibits excellent histocompatibility (51). It should be noted that xenogeneic ADMs elicit a more severe inflammatory response compared to allografts possibly due to the presence of basement membrane constituents such as collagen IV and laminin in the superficial dermis.

In practical applications, ADM often requires properties similar to native tissue in order to meet the specific requirements. Therefore, for better application performance, a certain degree of cross-linking modification is advantageous in further reducing the immune response triggered by the material. Commonly used modification methods include physical and chemical approaches, with thermal modification, radiation irradiation, and drilling being the primary physical methods. Chemical methods involve crosslinking modifications using glutaraldehyde, epoxides, carbodiimide, nanomaterials, dialdehyde polysaccharides and others. Previous studies have demonstrated that cross-linking can effectively decrease immunogenicity by inducing covalent bond formation between amino acid residues in ADM and collagen molecules which can mask tissue antigenicity and reduce immune responses (52).

Chen et al. (53) employed chemical cross-linking and the synergistic binding of ADM and chitosan under freezing conditions to fabricate a composite scaffold with dual physical and chemical, thereby significantly enhancing the survival rate of autologous rat skin grafts. Feng et al. (54) synthesized glutaraldehyde-modified heparin with cross-linked active aldehyde groups, which was subsequently cross-linked with porcine ADM and chemically modified to enhance its anticoagulant performance. The results demonstrated that the modified ADM exhibited superior thermal stability and biocompatibility, particularly in terms of its enhanced anticoagulant and anti-platelet adhesion properties, leading to a reduced incidence of coagulation, thrombocytopenia, and bleeding. Improved properties can be achieved through the utilization of newly developed nanoengineered ECM scaffold technologies (55, 56). The complete elimination of immunogenicity in ADM cannot be guaranteed solely by removing cellular components alone. The rate at which residual cellular components are removed from dermis can be enhanced by increasing temperature differentials and altering freeze-thaw cycles (57).

The development of ADM aims to harness the properties of native ECM and facilitate tissue regeneration in practical clinical settings. ADM exhibits properties such as toughness, elasticity, water retention, and robust mechanical force buffering (58). It can serve as a dermal scaffold, facilitating the rapid migration and infiltration of various host cells including fibroblasts, myofibroblasts, lymphocytes, macrophages, granulocytes, mast cells and more (47, 58,59). Following inflammatory cell infiltration, there increased levels of collagen and elastin production leading to the formation of new connective tissue and blood vessels (61). ADM demonstrates excellent biological strength and effectively supports fixation while reducing tension during cell proliferation (58).

Secondly, ADM harbors a multitude of signaling factors, such as vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), basic fibroblast growth factor (BFGF), and others (62). These growth factors have demonstrated the ability to retain their biological activity post-sterilization and during extended storage, while also promoting cell adhesion, proliferation, differentiation, and tissue formation (59). Additionally, ADM exhibits low antigenicity and excellent histocompatibility, creating an advantageous environment for the growth and proliferation of seed cells (63, 64). Following implantation in the body, ADM gradually undergoes degradation and is subsequently replaced by new tissue. Compared to other biological materials, ADM provides ample space for normal cell growth, making it a suitable candidate for use as a biological injection material (65–67). ADM can be considered as an “off-the-shelf” natural biomaterial that effectively addresses the issue of donor site insufficiency within the host itself (68). In summary, ADM scaffolds possess a complex composition consisting of diverse molecules that play pivotal roles in the process of tissue regeneration.

In the initial stage, Lin et al. (78) successfully achieved the reconstruction of 16 male urethral diseases by suturing ADM into a tubular structure, which included 13 complicated urethral strictures and one urethral hypospadias. The average length of urethral replacement was 4.7 cm, ranging from 2 to 10 cm. Postoperative urethrography demonstrated excellent integration of ADM with the surrounding tissue, while urethroscope examination revealed complete epithelialization of the graft urethra. No postoperative infections or rejections were observed; however, three cases experienced postoperative urethral strictures that were effectively managed through dilatation or incision. ADM is tentatively considered an ideal substitute for the urethra. On the other hand, Liu et al. (79) implanted sutured allogeneic ac dermal matrix into a tubular structure to repair 5.0 cm long urethral defects in 15 dogs. After a follow-up period of 24 weeks, no infections or anastomotic stenosis occurred, and there was no histological evidence of rejection with significant infiltration of inflammatory cells at any time point during evaluation. However, it should be noted that achieving normal structural approximation in the central area of ADM after transplantation requires an extended duration. Yang et al. (80) surgically treated five patients with anterior urethral strictures ranging from 5 to 9 cm in length by excising the affected segment and replacing it with tubular ADM. Three patients achieved successful voiding after catheter removal, while the remaining two underwent intermittent urethral dilation for a period of 2 months. All five patients demonstrated satisfactory voiding function 3 months post-operation, as confirmed by urethrography which revealed excellent continuity of the reconstructed urethra. ADM as a substitute for autologous tissue is a safe, effective, invasive, and cost-efficient method to avoid the trauma associated with harvesting autologous tissue in previous procedures. However, long-term outcomes regarding urethral stenosis remain uncertain.

Tang et al. (81) conducted a review of 49 patients with urethral strictures ranging from 1.5 to 3.8 cm in length who underwent urethroplasty with ADM and were followed up for 12 months. Cystoscopy revealed satisfactory coverage of the urethral epithelial mucosa in 11 cases. Infection occurred in two cases within the postoperative period of 2–4 weeks, while one case developed a urethral fistula at 5 months and seven cases experienced non-infective urethral strictures between 6 and 10 months after surgery. In this study, one out of two patients who developed postoperative infection had a stricture length of 3.0 cm, while the patient with a urethral fistula also presented with a stricture length close to 3.0 cm. Therefore, the use of ADM without seeded cells in an Onlay method for urethral reconstruction may have limited therapeutic efficacy for strictures longer than 3.0 cm. Additionally, the health status of the urethral bed should also be taken into consideration (82). El-Kassaby et al. (83) reported that acellular matrix would be suitable for use in surgical treatment of early strictures with an apparently healthy urethral bed and minimal spongiofibrosis. Primary ADM should not be performed in patients with preoperative infection complicated by urethral stones due to long-term stone irritation and poor local mucosal conditions, which increase the risk of infection or poor healing outcomes (81). In general, ADM represents a novel approach for material selection in urethral repair and reconstruction that offers benefits such as straightforward implementation, convenience, minimal invasiveness, and no additional sampling.

The most direct tissue engineering strategy for urethral reconstruction involves the utilization of natural or synthetic cell-free scaffolds, which are subsequently infiltrated with host cells and eventually degraded to form new tissue. The successful implantation of cell-free grafts relies on a healthy urethral bed, sufficient vascular supply, and the absence of spongiform fibrosis; otherwise, there is a risk of graft atrophy, inadequate tissue regeneration, and fibrosis. Considering that urethral stricture is characterized by ischemic cavernous fibrosis as a pathological process, it may impact the quality of the newly constructed urethra. Therefore, this simplified procedure can only be considered as an option for patients with short to moderate urethral defects. Clinical data demonstrates a high failure rate in treating urethral strictures longer than 4.0 cm.

The utilization of acellular fetal skin offers potential advantages compared to other acellular matrices. Sobhani et al. (84) employed an early gestational age fetal ADM scaffold for repairing hypospadias in a rabbit model. The decellularized fetal skin demonstrated favorable angiogenesis and re-epithelialization, resulting in reduced postoperative complications and shortened operation time, thereby establishing a solid foundation for treating complex hypospadias. The composition of the fetal dermis plays a critical role in scar-free wound healing and reducing complications associated with fetal dermal grafting (85). There is a significant disparity between the extracellular matrix of adult skin and fetal skin, primarily due to type Ⅰ collagen being the primary component. However, compared to adult skin, fetal skin exhibits higher levels of type Ⅲ collagen, type Ⅰ collagen, and cutin (86), which may explain its suitability as a “ready-made” material for tissue engineering urethra. Furthermore, recent studies have shown that utilizing ADM grafts in the second stage repair of hypospadias for ventral side elongation effectively corrects ventral curvature without increasing the risk of urethroplasty complications while also providing aesthetic benefits (87).

In order to enhance the efficacy of ADM materials, scholars have compared various preparation methods. Morgante et al. (88) conducted a study on porcine urethral repair using ADM and compared two types of acellular matrices: full-thickness porcine bladder matrix (PABM) and commercially derived cross-linked porcine dermal matrix (Permacol™). Anatomical and immunohistochemical evaluations were performed 3 months after the operation. The PABM graft showed complete fusion, while the Permacol™ graft remained palpable. Immunohistochemical analysis demonstrated a non-inflammatory remodeling response to both biomaterials. PABM implants displayed extensive infiltration of interstitial cells and neovascularization with significantly higher cell density than Permacol™. We believe that the poor growth of cells in Permacol™ may be attributed to variations in decellularization methods employed by different research groups, resulting in differential effects on cell removal, extracellular matrix composition, and subsequent tissue remodeling. It is evident that natural acellular matrices hold great promise as biomaterials for reconstructive and regenerative surgery; however, their physical and biological properties can potentially be influenced by chemical or radiation exposure. To fully realize the clinical potential of ADMs, it is crucial to conduct comprehensive evaluations of their safety and efficacy both in vitro and in vivo while also establishing standardized protocols for their utilization in clinical practice (Table 3).

Cell seeding with acellular dermal matrix is more suitable for tubular implants (92). The biological scaffolds are seeded with different types of cells, which can effectively promote cell expansion by releasing growth factors, cytokines, etc., thereby improving the mechanical properties of the grafts (93). Studies have demonstrated that in animal models, cell-inoculated matrix have been compared with uninoculated matrix, and the results are better with the former, which provides a robust empirical foundation for clinical research (94). The utilization of cell-based grafts has exhibited a remarkable 5.7-fold increase in long-term success rates compared to unseeded grafts (95). Importantly, incorporating cells within grafts has been shown to significantly reduce the incidence of stenosis, fistula formation, and infection. This can be attributed to the potential promotion of vascularization and urothelial barrier formation by cellular components, effectively mitigating local inflammation and fibrosis resulting from urine leakage. Autologous stem cells hold immense promise in urethral tissue engineering, with several scholars exploring the feasibility of combining ADM with stem cells. Fu et al. (96) employed bone marrow mesenchymal stem cells seeded on ADM, subsequently repairing rabbit urethral injuries. Histological examination using HE staining revealed that over time, the reconstructed area in the experimental group exhibited a transition from a single layer of urethral epithelial cells to multiple layers, closely resembling the structure of normal urethral tissue at 12 weeks post-operation. In contrast, the control group without the use of bone marrow mesenchymal stem cells displayed inferior urethral regeneration. Urethrography demonstrated an absence of urinary fistulas and urethral strictures or other complications in the experimental group, while such complications were observed in the control group. These findings indicate that combining bone marrow mesenchymal stem cells with ADM yields superior outcomes for repairing urethral injuries compared to using ADM alone. Therefore, ADM provides support for cell adhesion, growth, and proliferation, facilitating material exchange and signal transduction pathway formation. Both cellular-matrix interaction and tissue engineered graft-host tissue environment interaction are crucial factors for successful outcomes in urethral reconstruction. However, the mechanism by which seed cells participate in the regeneration process after binding to ADM remains unclear. Although preclinical animal studies tend to suggest that cell-bound ADM is more effective with fewer complications, these findings have not been substantiated by a limited number of clinical studies.

Currently, there is a limited number of clinical reports available on the construction of urethra using cell-loaded ADM grafts. Liu et al. (79) sutured allogeneic acellular dermal matrix into a tube to repair two patients with urethral defects measuring 12.0 cm and 12.7 cm in length, respectively. Urethral mucosa homogenate and bladder mucosa homogenate were applied on the inner surface of the ADM cavity, respectively. No anastomotic stenosis was observed. In the clinical application of long segment urethral reconstruction using ADM, the repair process relies on host cell regeneration at both ends, resulting in time-consuming procedures. However, by utilizing homogenization seeding of host urothelium and multicentric growth of epithelium based on nutrients infiltrated into the ADM, it is possible to effectively shorten the repair time. Fossum et al. (97) conducted a study in which autologous urethral epithelial cells were cultured in vitro and subsequently transplanted onto ADM for surgical treatment of 6 patients with severe hypospadias, aged between 14 and 44 months. All patients underwent a two-stage surgical approach. Initially, urothelial cells were obtained through bladder lavage and then seeded onto ADM. In the second operation, an ADM scaffold containing urothelial cells was implanted to construct a new urethra. During the follow-up period of 3.5–5 years, one patient experienced partial stenosis without receiving any specific treatment, one patient developed proximal anastomotic obstruction, and two patients developed urinary fistula requiring surgical correction. Urethroscopy performed on all patients revealed widening of the newly constructed urethra, while biopsy results from three patients indicated that the inner mucosa consisted of urothelial cells. The authors Bhargava et al. (82) performed surgical treatment on five patients with urethral strictures ranging from 4 to 11 cm buccal mucosa keratinocytes and fibroblasts on ADM. Buccal mucosa biopsies were obtained from each patient for cell collection. These grafts were utilized for two-stage procedures (n = 3) and urethroplasty (n = 2). Results revealed that two patients experienced complications of urethral fibrosis and constriction, resulting in complete or partial removal of the graft. Three other patients required internal fixation after a follow-up period of 33 months. The application of tissue engineering techniques for implanting expanded cells on ADM can effectively enhance urethral tissue regeneration and accelerate lesion healing, making it particularly suitable for patients with long and complex urethral strictures or defects. However, the optimal conditions for cell differentiation and maturation in cell-loaded ADM remain unclear, and there are stringent requirements for in vitro cell culture, especially when considering human treatment. Once a tissue-engineered urethra is successfully constructed in vitro, timely scheduling of the implantation procedure is crucial to prevent graft failure. Additionally, it should be noted that using cell-inoculated matrices would result in higher overall costs compared to using cell-free matrices.

Another aspect that has received limited attention in the design of tissue engineered urethral scaffolds is the prevention of urinary fistulas. Recent research indicates that the utilization of ADM derived from bovine skin as a coverage material for proximal hypospadias repair can effectively decrease the occurrence of urinary fistula formation (89). Wu et al. (91) investigated the use of ADM in hypospadias repair and demonstrated a significant reduction in urinary fistula complications without an increased risk of infection and urethral stricture. Given the detrimental effects of urine on the cellular components of tissue-engineered urethra, it is crucial for the scaffold to possess sufficient impermeability as an isolation barrier. Furthermore, appropriately modified ADM materials can effectively decrease the incidence of associated complications and other adverse reactions.

Wang et al. (90) utilized bovine ADM loaded with collagen binding vascular endothelial growth factor (CBD-VEGF) for the repair of canine urethral injury. A few months later, the group with urethral injury was compared to the groups receiving ADM implantation and CBD-VEGF modified ADM implantation, respectively. The findings revealed that while the control group experienced urethral stricture and diverticulum, one case in the ADM group developed urinary fistula. However, no associated complications or adverse reactions were observed in the CBD-VEGF modified ADM implantation group. Importantly, ADM effectively prevented both urethral stricture and diverticulum when compared to the control group, demonstrating its efficacy in averting these conditions. Notably, combining ADM with growth factors promotes functional vascular system formation, thereby maintaining extracellular matrix metabolism balance and facilitating urethra remodeling without inducing severe inflammation or scar hyperplasia (Table 4).