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Creating acellular vascularized constructs from animal and plant tissue is one of the well‐known strategies for scaffold assembly. Decellularization takes an important position among these strategies. The most common method is chemical decellularization. This approach employs high concentrations of detergents, primarily Triton X‐100, sodium dodecyl sulfate (SDS), and sodium hypochlorite (SH). In this work, novel techniques for decellularizing spinach were developed using detergents frequently utilized in laboratories. Spinach leaves were decellularized using Tween‐20, SDS, and SH at low concentrations to generate an acellular plant matrix for tissue engineering. We measured the quantities of DNA and protein, as well as the decellularization using hematoxylin and eosin (H&E) staining. The biocompatibility and capacity of the biostructures to stimulate fibroblast wound healing were assessed using MTT and the Scratch assay. The antibacterial activity of the scaffolds was also tested against a gram‐positive bacterium, Staphilococcus aureus, which is a common pathogen associated with wound healing. The best shape, evident vascularization, and good biocompatibility were seen in the Tween‐20 decellularized samples at 1% concentration at 21°C and 37°C through the enhancement of cell proliferation and wound healing. In terms of antibacterial activity, all scaffold samples had a significant effect on Staphilococcus aureus, where the number of bacterial colonies in all six scaffold groups became zero after 4 h of treatment. The scaffolds also showed a 100% kill rate on Staphilococcus aureus, which could avoid wound infection during the repair process, and that can be suggested as a scaffold for tissue engineering applications and an important constituent for pharmacological activities. [ABSTRACT FROM AUTHOR]

Previous studies showed that the bladder extracellular matrix (B‐ECM) could increase the differentiation efficiency of mesenchymal cells into smooth muscle cells (SMC). This study investigates the potential of human amniotic membrane‐derived hydrogel (HAM‐hydrogel) as an alternative to xenogeneic B‐ECM for the myogenic differentiation of the rabbit adipose tissue‐derived MSC (AD‐MSC). Decellularized human amniotic membrane (HAM) and sheep urinary bladder (SUB) were utilized to create pre‐gel solutions for hydrogel formation. Rabbit AD‐MSCs were cultured on SUB‐hydrogel or HAM‐hydrogel‐coated plates supplemented with differentiation media containing myogenic growth factors (PDGF‐BB and TGF‐β1). An uncoated plate served as the control. After 2 weeks, real‐time qPCR, immunocytochemistry, flow cytometry, and western blot were employed to assess the expression of SMC‐specific markers (MHC and α‐SMA) at both protein and mRNA levels. Our decellularization protocol efficiently removed cell nuclei from the bladder and amniotic tissues, preserving key ECM components (collagen, mucopolysaccharides, and elastin) within the hydrogels. Compared to the control, the hydrogel‐coated groups exhibited significantly upregulated expression of SMC markers (p ≤.05). These findings suggest HAM‐hydrogel as a promising xenogeneic‐free alternative for bladder tissue engineering, potentially overcoming limitations associated with ethical concerns and contamination risks of xenogeneic materials. [ABSTRACT FROM AUTHOR]

Heart valve replacement surgeries are performed on patients suffering from abnormal heart valve function. In these operations, the problematic tissue is replaced with mechanical valves or with bioprosthetics that are being developed. The thrombotic effect of mechanical valves, reflecting the need for lifelong use of anticoagulation drugs, and the short‐lived nature of biological valves make these two types of valves problematic. In addition, they cannot adapt to the somatic growth of young patients. Although decellularized scaffolds have shown some promise, a successful translation has so far evaded. Although decellularized porcine xenografts have been extensively studied in the literature, they have several disadvantages, such as a propensity for calcification in the implant model, a risk of porcine endogenous retrovirus (PERV) infection, and a high xenoantigen density. As seen in clinical data, it is clear that there are biocompatibility problems in almost all studies. However, since decellularized sheep heart valves have not been tried in the clinic, a large data pool could not be established. This review compares and contrasts decellularized porcine and sheep xenografts for heart valve tissue engineering. It reveals that decellularized sheep heart valves can be an alternative to pigs in terms of biocompatibility. In addition, it highlights the potential advantages of bioinks derived from the decellularized extracellular matrix in 3D bioprinting technology, emphasizing that they can be a new alternative for the application. We also outline the future prospects of using sheep xenografts for heart valve tissue engineering. [ABSTRACT FROM AUTHOR]

The gross morphological examination of native caprine heart valves revealed distinctive structural characteristics of the caprine's cardiac anatomy. Four primary orifices were identified, each protected by thin, valve‐like structures. Atrioventricular orifices featured tricuspid and bicuspid valves, while the aorta and pulmonary arteries were guarded by semilunar valves. Within the atrioventricular apparatus, distinct features were observed including the tricuspid valve's three leaflets and the bicuspid valve's anterior and posterior leaflets. Ultrasonography provided insights into valve thickness and chordae tendineae lengths. Morphometric studies compared leaflets/cusps within individual native valves, showcasing significant variations in dimensions. Comparative analysis between native and decellularized valves highlighted the effects of decellularization on leaflet thickness and chordae tendineae lengths. Decellularized valves exhibited reduced dimensions compared to native valves, indicating successful removal of cellular components. While some dimensions remained unchanged post‐decellularization, significant reductions were observed in leaflet thicknesses and chordae tendineae lengths. Notably, semilunar valve cusps displayed varying responses to decellularization, with significant reductions in cusp lengths observed in the aortic valve, while the pulmonary valve exhibited more subtle changes. These findings underscore the importance of understanding structural alterations in heart valves post‐decellularization, providing valuable insights for tissue engineering applications and regenerative medicine. [ABSTRACT FROM AUTHOR]

The degeneration of intervertebral disc (IVD) is a disease of the entire joint between two vertebrae in the spine caused by loss of extracellular matrix (ECM) integrity, to date with no cure. The various regenerative approaches proposed so far have led to very limited successes. An emerging opportunity arises from the use of decellularized ECM as a scaffolding material that, directly or in combination with other materials, has greatly facilitated the advancement of tissue engineering. Here we focused on the decellularized matrix obtained from human umbilical cord Wharton's jelly (DWJ) which retains several structural and bioactive molecules very similar to those of the IVD ECM. However, being a viscous gel, DWJ has limited ability to retain ordered structural features when considered as architecture scaffold. To overcome this limitation, we produced DWJ‐based multifunctional hydrogels, in the form of 3D millicylinders containing different percentages of alginate, a seaweed‐derived polysaccharide, and gelatin, denatured collagen, which may impart mechanical integrity to the biologically active DWJ. The developed protocol, based on a freezing step, leads to the consolidation of the entire polymeric dispersion mixture, followed by an ionic gelation step and a freeze‐drying process. Finally, a porous, stable, easily storable, and suitable matrix for ex vivo experiments was obtained. The properties of the millicylinders (Wharton's jelly millicylinders [WJMs]) were then tested in culture of degenerated IVD cells isolated from disc tissues of patients undergoing surgical discectomy. We found that WJMs with the highest percentage of DWJ were effective in supporting cell migration, restoration of the IVD phenotype (increased expression of Collagen type 2, aggrecan, Sox9 and FOXO3a), anti‐inflammatory action, and stem cell activity of resident progenitor/notochordal cells (increased number of CD24 positive cells). We are confident that the DWJ‐based formulations proposed here can provide adequate stimuli to the cells present in the degenerated IVD to restart the anabolic machinery. [ABSTRACT FROM AUTHOR]

The regeneration of osteochondral lesions by tissue engineering techniques is challenging due to the lack of physicochemical characteristics and dual‐lineage (osteogenesis and chondrogenesis). A scaffold with better mechanical properties and dual lineage capability is required for the regeneration of osteochondral defects. In this study, a hydrogel prepared from decellularized human umbilical cord tissue was developed and evaluated for osteochondral regeneration. Mesenchymal stem cells (MSCs) isolated from the umbilical cord were seeded with hydrogel for 28 days, and cell‐hydrogel composites were cultured in basal and osteogenic media. Alizarin red staining, quantitative polymerase chain reaction, and immunofluorescent staining were used to confirm that the hydrogel was biocompatible and capable of inducing osteogenic differentiation in umbilical cord‐derived MSCs. The findings demonstrate that human MSCs differentiated into an osteogenic lineage following 28 days of cultivation in basal and osteoinductive media. The expression was higher in the cell‐hydrogel composites cultured in osteoinductive media, as evidenced by increased levels of messenger RNA and protein expression of osteogenic markers as compared to basal media cultured cell‐hydrogel composites. Additionally, calcium deposits were also observed, which provide additional evidence of osteogenic differentiation. The findings demonstrate that the hydrogel is biocompatible with MSCs and possesses osteoinductive capability in vitro. It may be potentially useful for osteochondral regeneration. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) is a dynamic and complex network of proteins and molecules that surrounds cells and tissues in the nervous system and orchestrates a myriad of biological functions. This review carefully examines the diverse interactions between cells and the ECM, as well as the transformative chemical and physical changes that the ECM undergoes during neural development, aging, and disease. These transformations play a pivotal role in shaping tissue morphogenesis and neural activity, thereby influencing the functionality of the central nervous system (CNS). In our comprehensive review, we describe the diverse behaviors of the CNS ECM in different physiological and pathological scenarios and explore the unique properties that make ECM‐based strategies attractive for CNS repair and regeneration. Addressing the challenges of scalability, variability, and integration with host tissues, we review how advanced natural, synthetic, and combinatorial matrix approaches enhance biocompatibility, mechanical properties, and functional recovery. Overall, this review highlights the potential of decellularized ECM as a powerful tool for CNS modeling and regenerative purposes and sets the stage for future research in this exciting field. This article is categorized under:Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and ReplacementTherapeutic Approaches and Drug Discovery > Nanomedicine for Neurological DiseaseImplantable Materials and Surgical Technologies > Nanomaterials and Implants [ABSTRACT FROM AUTHOR]

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three‐dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1‐positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two‐dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35‐day mark, the percentage of Prm1‐positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio‐scaffold for enhancing mouse in vitro spermatogenesis. [ABSTRACT FROM AUTHOR]

Background: Epothilone B (EpoB) is a microtubule‐stabilizing agent with neuroprotective properties. Objectives: This study examines the regenerative properties of ANA supplemented with EpoB on a sciatic nerve deficit in male Wistar rats. Methods: For this purpose, the 10 mm nerve gap was filled with acellular nerve allografts (ANAs) containing EpoB at 0.1, 1, and 10 nM concentrations. The sensorimotor recovery was evaluated up to 16 weeks after the operation. Real‐time PCR, histomorphometry analysis, and electrophysiological evaluation were also used to evaluate the process of nerve regeneration. Results: ANA/EpoB (0.1 nM) significantly improved sensorimotor recovery in rats compared to ANA, ANA/EpoB (1 nM), and ANA/EpoB (10 nM) groups. This led to reduced muscle atrophy, improved sciatic functional index, and thermal paw withdrawal reflex latency, indicating nerve regeneration and target organ reinnervation. The electrophysiological and histomorphometry findings also confirmed the ANA/EpoB regenerative properties (0.1 nM). EpoB only enhanced ANA regenerative properties at 0.1 nM, with no therapeutic effects at higher concentrations. Conclusion: Totally, we concluded that ANA loaded with 0.1 nM EpoB can effectively reconstruct the transected sciatic nerve in rats, likely by enhancing axonal sprouting and extension. [ABSTRACT FROM AUTHOR]

Focal chondral defects (FCDs) significantly impede quality of life for patients and impose severe economic costs on society. One of the most promising treatment options—autologous matrix‐induced chondrogenesis (AMIC)—could benefit from a scaffold that contains both of the primary cartilage matrix components—sulfated glycosaminoglycans (sGAGs) and collagen type II. Here, 17 different protocols were evaluated to determine the most optimum strategy for decellularizing (decelling) the bovine nucleus pulposus (bNP) to yield a natural biomaterial with a cartilaginous constituency. The resulting scaffold was then characterized with respect to its biochemistry, biomechanics and cytocompatibility. Results indicated that the optimal decell protocol involved pre‐crosslinking the tissue prior to undergoing decell with trypsin and Triton X‐100. The residual DNA content of the scaffold was found to be 32.64 ± 9.26 ng/mg dry wt. of tissue with sGAG and hydroxyproline (HYP) contents of 72.53 ± 16.43. and 78.38 ± 8.46 μg/mg dry wt. respectively. The dynamic viscoelastic properties were found to be preserved (complex modulus: 17.92–16.62 kPa across a range of frequencies) while the equilibrium properties were found to have significantly decreased (aggregate modulus: 11.51 ± 9.19 kPa) compared to the non‐decelled fresh bNP tissue. Furthermore, the construct was also found to be cytocompatible with bone marrow stem cells (BMSCs). While it was not permissive of cellular infiltration, the BMSCs were still found to have lined the laser drilled channels in the scaffold. Taken together, the biomaterial developed herein could be a valuable addition to the AMIC family of scaffolds or serve as an off‐the‐shelf standalone option for cartilage repair. [ABSTRACT FROM AUTHOR]

Solubilizing extracellular matrix (ECM) materials and transforming them into hydrogels has expanded their potential applications both in vitro and in vivo. In this study, hydrogels are prepared by decellularization of human placental tissue using detergent and enzymes and by the subsequent creation of a homogenized acellular placental tissue powder (P‐ECM). A perfusion‐based decellularization approach is employed using detergent and enzymes. The P‐ECM with and without gamma irradiation is then utilized to prepare P‐ECM hydrogels. Physical and biological evaluations are conducted to assess the suitability of the P‐ECM hydrogels for biocompatibility. The decellularized tissue has significantly reduced cellular content and retains the major ECM proteins. Increasing the concentration of P‐ECM leads to improved mechanical properties of the P‐ECM hydrogels. The biocompatibility of the P‐ECM hydrogel is demonstrated through cell proliferation and viability assays. Notably, gamma‐sterilized P‐ECM does not support the formation of a stable hydrogel. Nonetheless, the use of HCl during the digestion process effectively decreases spore growth and bacterial bioburden. The study demonstrates that P‐ECM hydrogels exhibit physical and biological attributes conducive to soft tissue reconstruction. These hydrogels establish a favorable microenvironment for cell growth and the need for investigating innovative sterilization methods. [ABSTRACT FROM AUTHOR]

Porcine small intestinal submucosa, despite its successful use as a scaffold in regenerative medicine, has innate biomechanical heterogeneity. In this study, we hypothesized that human small intestinal submucosa could be a viable alternative bio‐scaffold. For the first time, we characterize submucosal extraction from human small intestine and examine appropriate decellularization methods. In total, 16 human small intestinal submucosal samples were obtained and decellularized using three reported methods of porcine decellularization: Abraham, Badylak, and Luo. For each method, four specimens were decellularized. The remaining four specimens were designated as non‐decellularized. We measured the amount of residual DNA and growth factors in decellularized human intestinal samples. Additionally, decellularized human small intestinal submucosa was co‐cultured with mouse bone marrow‐derived mesenchymal stem cells to examine mesenchymal stem cell survival and proliferation. The reference value for the amount of residual DNA deemed appropriate in decellularized tissue was established as 50 ng/mg of extracellular matrix dry weight or less. Abraham's method most successfully met this criterion. Measurement of residual growth factors revealed low levels observed in samples decellularized using the Abraham and Badylak methods. Co‐culture of each small intestinal submucosal sample with mouse bone marrow‐derived mesenchymal stem cells confirmed viable cell survival and proliferation in samples derived using protocols by Abraham and Badylak. Abraham's method most successfully met the criteria for efficient tissue decellularization and cell viability and proliferation. Thus, we consider this method most suitable for decellularization of human small intestinal submucosa. [ABSTRACT FROM AUTHOR]

There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long‐segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T‐cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration. [ABSTRACT FROM AUTHOR]

Fish skin grafting as a new skin substitute is currently being used in clinical applications. Acceleration of the wound healing, lack of disease transmission, and low cost of the production process can introduce fish skin as a potential alternative to other grafts. An appropriate decellularization process allows the design of 3D acellular scaffolds for skin regeneration without damaging the morphology and extracellular matrix content. Therefore, the role of decellularization processes is very important to maintain the properties of fish skin. In this review article, recent studies on various decellularization processes as well as biological, physical, and mechanical properties of fish skin and its applications with therapeutic effects in wound healing were investigated. [ABSTRACT FROM AUTHOR]

Achilles tendon, which connects the calf muscles to heel, is the strongest tendon in the body. Despite its strength, it is more prone to injury due to its limited blood supply. Tendon‐related injuries are more common in sportspersons, people with labor‐intensive work and the aged community. The currently available treatment mode is surgery which is expensive with chances of re‐injury. Present study made an attempt to fabricate a tissue‐engineered tendon product using decellularized tendon (DT) seeded with stem cells and bioactive components of Tinospora cordifolia extract (TCE). The bare DT tissue scaffold/substitute may also serve as a drug delivery platform for growth factors and cells with a new approach to promote tissue regeneration in clinical applications. DT construct showed good regenerative potential and easily promoted new tissue formation. Decellularization of the tendon was carried out by chemical method using tri (n‐butyl) phosphate (TnBP). DT was physicochemically characterized by contact angle measurement, thermal gravimetric analysis (TGA), and mechanical testing. Rabbit adipose derived mesenchymal stem cells (RADMSCs) were isolated and phenotypically characterized by flow cytometry analysis, tri lineage differentiation, and so forth. Further, stem cell seeded DT scaffolds were prepared and found to be non‐toxic by cytotoxicity, cell adhesion by scanning electron microscope (SEM) analysis, cell viability by live dead assays, and so forth. The findings of this study yield valid proof for the employability of cell‐seeded DT construct as a natural scaffold in repairing injured tendons—the toughest chords of the skeleton. This is a cost effective method for the replacement of injured/damaged tendons for athletes, people in labor‐intensive occupations, the elderly population, and so forth—a boon towards the repair of the tendon in damage/injury. [ABSTRACT FROM AUTHOR]

Background: Nitric oxide is a chemical agent produced by endothelial cells in a healthy blood vessel, inhibiting the overgrowth of vascular smooth muscle cells and regulating vessel tone. Liposomes are biocompatible and biodegradable drug carriers with a similar structure to cell bilayer phospholipid membrane that can be used as useful nitric oxide carriers in vascular grafts. Method: Using a custom‐designed apparatus, the sheep carotid arteries were decellularized while still maintaining important components of the vascular extracellular matrix (ECM), allowing them to be used as small‐diameter vascular grafts. A chemical signal of sodium nitrite was applied to control smooth muscle cells' behavior under static and dynamic cell culture conditions. The thin film hydration approach was used to create nano‐liposomes, which were then used as sodium nitrite carriers to control the drug release rate and enhance the amount of drug loaded into the liposomes. Results: The ratio of 80:20:2 for DPPC: Cholesterol: PEG was determined as the optimum formulation of the liposome structure with high drug encapsulation efficiency (98%) and optimum drug release rate (the drug release rate was 40%, 65%, and 83% after 24, 48, and 72 h, respectively). MTT assay results showed an improvement in endothelial cell proliferation in the presence of nano‐liposomal sodium nitrite (LNS) at the concentration of 0.5 μg/mL. Using a suitable concentration of liposomal sodium nitrite (0.5 μg/mL) put onto the constructed scaffold resulted in the controllable development of smooth muscle cells in the experiment. The culture of smooth muscle cells in a pulsatile perfusion bioreactor indicated that in the presence of synthesized liposomal sodium nitrite, the overgrowth of smooth muscle cells was inhibited in dynamic cell culture conditions. The mechanical properties of ECM graft were measured, and a multi‐scale model with an accuracy of 83% was proposed to predict mechanical properties successfully. Conclusion: The liposomal drug‐loaded small‐diameter vascular graft can prevent the overgrowth of SMCs and the formation of intimal hyperplasia in the graft. Aside from that, the effect of LNS on endothelial has the potential to stimulate endothelial cell proliferation and re‐endothelialization. [ABSTRACT FROM AUTHOR]

Peripheral nerve regeneration after injury is still a clinical problem. The application of autologous nerve grafting, the gold standard treatment, is greatly restricted. Acellular nerve allografts (ANAs) are considered promising alternatives, but they are difficult to achieve satisfactory therapeutic outcomes, which may be attributed to their compact inherent ultrastructure and substantial loss of extracellular matrix (ECM) components. Regarding these deficiencies, this study developed an optimized multichannel ANA by a modified decellularization method. These innovative ANAs were demonstrated to retain more ECM bioactive molecules and regenerative factors, with effective elimination of cellular antigens. The presence of microchannels with larger pore size allowed ANAs to gain higher porosity and better swelling performance, which improves their internal ultrastructure. Their mechanical properties were more similar to those of native nerves. Moreover, the optimized ANAs exhibited good biocompatibility and possessed significant advantages in supporting the proliferation and migration of Schwann cells in vitro. The in vivo results further confirmed their superior capacity to promote axon regrowth and myelination as well as restore innervation of target muscles, leading to better functional recovery than the conventional ANAs. Overall, this study demonstrates that the optimized multichannel ANAs have great potential for clinical application and offer new insight into the further improvement of ANAs. [ABSTRACT FROM AUTHOR]

Background: Pig‐derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α‐Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. Methods: The N‐glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1‐KO and GGTA1/CMAH‐KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser‐induced fluorescence detection. Results: We identified biantennary and core‐fucosylated N‐glycans terminating with immunogenic α‐Gal‐ and α‐Gal‐/Neu5Gc‐epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH‐KO pigs, respectively. Levels of N‐glycans terminating with galactose bound in β(1‐4)‐linkage to N‐acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N‐glycans capped with Neu5Gc were increased in GGTA1‐KO pigs compared to WT, but were not detected in GGTA1/CMAH‐KO pigs. Similarly, the ganglioside Neu5Gc‐GM3 was found in WT and GGTA1‐KO but not in GGTA1/CMAH‐KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. Conclusion: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human‐like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic. [ABSTRACT FROM AUTHOR]

One of the approaches to restoring the structure of damaged cartilage tissue is an intra‐articular injection of tissue‐engineered medical products (TEMPs) consisting of biocompatible matrices loaded with cells. The most interesting are the absorbable matrices from decellularized tissues, provided that the cellular material is completely removed from them with the maximum possible preservation of the structure and composition of the natural extracellular matrix. The present study investigated the mechanical, biochemical, and biological properties of decellularized porcine cartilage microparticles (DCMps) obtained by techniques, differing only in physical treatments, such as freeze–thaw cycling (Protocol 1), supercritical carbon dioxide fluid (Protocol 2) and ultrasound (Protocol 3). Full tissue decellularization was achieved, as confirmed by the histological analysis and DNA quantification, though all the resultant DCMps had reduced glycosaminoglycans (GAGs) and collagen. The elastic modulus of all DCMp samples was also significantly reduced. Most notably, DCMps prepared with Protocol 3 significantly outperformed other samples in viability and the chondroinduction of the human adipose‐derived stem cells (hADSCs), with a higher GAG production per DNA content. A positive ECM staining for type II collagen was also detected only in cartilage‐like structures based on ultrasound‐treated DCMps. The biocompatibility of a xenogenic DCMps obtained with Protocol 3 has been confirmed for a 6‐month implantation in the thigh muscle tissue of mature rats (n = 18). Overall, the results showed that the porcine cartilage microparticles decellularized by a combination of detergents, ultrasound and DNase could be a promising source of scaffolds for TEMPs for cartilage reconstruction. [ABSTRACT FROM AUTHOR]

Background: Bioengineered transplantable heart‐lung scaffolds could be potentially lifesaving in a large number of congenital and acquired cardiothoracic disorders including terminal heart‐lung disease. Methods: We decellularized heart‐lung organ‐blocks from rats (n = 10) by coronary and tracheal perfusion with ionic detergents in a modified Langendorff circuit. Results: In the present project, we were able to achieve complete decellularization of the heart‐lung organ‐block. Decellularized heart‐lung organ‐blocks lacked intracellular components but maintained structure of the cellular walls with collagen and elastic fibers. Conclusions: We present a novel model of combined perfusion and decellularization of heart‐lung organ‐blocks. This model is the first step on the pathway to creating bioengineered transplantable heart‐lung scaffolds. We believe that further development of this technology could provide a life‐saving conduit, significantly reducing the risks of heart‐lung failure surgery and improving postoperative quality of life. [ABSTRACT FROM AUTHOR]

Bone marrow mesenchymal stem cell sheet‐derived spheroids (BMSCs spheroids) have been widely studied as native bioactive scaffolds. However, the abundant cells in BMSCs spheroids cause immunogenicity and make them difficult to store. This paper aimed to construct a new bioactive scaffold called 3D‐decellularized extracellular matrix spheroids (ECM spheroids) via decellularization of BMSCs spheroids to enhance bone formation. Hematoxylin and eosin staining (HE), nuclear and cytoskeletal fluorescence, immunofluorescence (IF), and scanning electron microscopy (SEM) were utilized to detect the characteristics and components of ECM spheroids. Furthermore, the biological properties of migration, adhesion, and recellularization of cells in ECM spheroids were assessed in vitro, and bone formation was evaluated in rat calvarial defects. The results showed that both the nuclei and cytoskeleton in ECM spheroids were greatly altered and one of the major components of FN was intact. The migration, adhesion, and recellularization potential were improved in vitro. Meanwhile, ECM spheroids promoted osteogenesis in rat skull defects after 3 months (p <.01). In conclusion, ECM spheroids were successfully prepared and proven to promote cell migration, adhesion, and proliferation. Bone formation in vivo was also accelerated. We believe that ECM spheroids can be used as bioactive and biocompatible 3D scaffolds in the future. [ABSTRACT FROM AUTHOR]

Background: Healing the full‐thickness skin wounds has remained a challenge. One of the most frequently used grafts for skin regeneration is xenogeneic acellular dermal matrices (ADMs), including bovine ADMs. This study investigated the effect of the source animal age, enzymatic versus non‐enzymatic decellularization protocols, and gamma irradiation versus ethylene oxide (EO) sterilization on the scaffold. Methods: ADMs were prepared using the dermises of fetal bovine or calf skins. All groups were decellularized through chemical and mechanical methods, unless T‐FADM samples, in which an enzymatic step was added to the decellularization protocol. All groups were sterilized with ethylene oxide (EO), except G‐FADM which was sterilized using gamma irradiation. The scaffolds were characterized through scanning electron microscopy, differential scanning calorimetry, tensile test, MTT assay, DNA quantification, and real‐time PCR. The performance of the ADMs in wound treatment was also evaluated macroscopically and histologically. Results: All ADMs were effectively decellularized. In comparison to FADM (EO‐sterilized fetal ADM), morphological, and mechanical properties of G‐FADM, T‐FADM, and CADM (EOsterilized calf ADM) were changed to different extents. In addition, the CADM and G‐FADM were thermally more stable than the FADM and T‐FADM. Although all ADMs were noncytotoxic, the wounds of the FADM, T‐FADM, and G‐FADM groups were contracted to almost 30.0% of the original area on day 7, significantly faster than the CADM (17.5% ± 1.7) and control (12.2% ± 1.59) groups. However, by day 21, all ADMs were mostly closed except for the untreated group (60.1 ± 1.8). Conclusion: Altogether, fetal source and EO‐sterilized samples performed better than calf source and gamma‐sterilized samples unless in some mechanical properties. There was no added value in using enzymatic treatment during the decellularization process. Our results suggest that the age, decellularization, and sterilization methods of animal source should be selected based on the clinical requirements. [ABSTRACT FROM AUTHOR]

The decellularization of plant tissues can be one of the design options of scaffolds in tissue engineering. Chemical detergents such as Triton X‐100 and sodium dodecyl sulfate (SDS) in different concentrations were used to decellularize olive leaves as an acellular plant matrix for tissue engineering. The samples were investigated by different analyses such as Hematoxylin and Eosin staining, SEM, tensile strength, swelling, water vapor transmission, and toxicity. The results of staining and toxicity tests showed that the Triton X‐100 decellularized samples at a concentration of 0.1% had the best morphology and the lowest toxicity. Mechanical results showed that the elasticity modulus of acellular samples was significantly reduced compared to normal leaf samples. While swelling rate and water vapor transmission in acellular samples compared to the control sample doubled and tripled, respectively. In general, acellular olive leaf can be suggested as a scaffold for tissue engineering applications. [ABSTRACT FROM AUTHOR]

Background: Tympanic membrane perforation (TMP) is a common disease in otology, and few acellular techniques have been reported for repairing this condition. Decellularized extracellular matrix (ECM) scaffolds have been used in organ reconstruction. Objective: This study on tissue engineering aimed to develop a tympanic membrane (TM) scaffold prepared using detergent immersion and bone marrow mesenchymal stem cells (BMSCs) as repair materials to reconstruct the TM. Results: General structure was observed that the decellularized TM scaffold with BMSCs retained the original intact anatomical ECM structure, with no cell residue, as observed using scanning electron microscopy (SEM), and exhibited low immunogenicity. Therefore, we seeded the decellularized TM scaffold with BMSCs for recellularization. Histology and eosin staining, SEM and immunofluorescence in vivo showed that the recellularized TM patch had a natural ultrastructure and was suitable for the migration and proliferation of BMSCs. The auditory brainstem response (ABR) evaluated after recellularized TM patch repair was slightly higher than that of the normal TM, but the difference was not significant. Conclusion: The synthetic ECM scaffold provides temporary physical support for the three‐dimensional growth of cells during the tissue developmental stage. The scaffold stimulates cells to secrete their own ECM required for tissue regeneration. The recellularized TM patch shows potential as a natural, ultrastructure biological material for TM reconstruction. [ABSTRACT FROM AUTHOR]

Transplantation of hematopoietic stem cells (HSCs)—rare cells that can self‐renew and differentiate into cells of all hematopoietic lineages—is an important treatment option for patients with hematologic diseases. Key to the regenerative potential of HSCs is their natural in vivo microenvironment—the bone marrow niche—but in vitro engineering approaches to recapitulate the biochemical and biophysical properties of the bone marrow niche are still insufficiently explored. Herein, modular macroporous cryogels, mimicking the natural 3D architecture of trabecular bone, are seeded with human bone marrow‐derived mesenchymal stromal cells and decellularized to generate tissue‐specific extracellular matrix (ECM)‐decorated scaffolds. To recapitulate the physiological concentration of biomolecules in bone marrow, the cell culture medium is supplemented with macromolecular crowders that modulate the composition, ultrastructure, and mechanical properties of the cell‐deposited ECM. The ECM‐coated cryogel scaffolds generated under crowding conditions exhibit physicochemical properties similar to those of the endosteal niche of the bone marrow. The ECM‐functionalized cryogels presented could be used in the future for the ex vivo expansion of HSCs for transplantation and may also aid in the development of more realistic hematological disease models. [ABSTRACT FROM AUTHOR]

The in vitro reconstruction of the extracellular matrix (ECM) is required in tissue engineering and regenerative medicine because the ECM can regulate cell functions in vivo. For ECM reconstruction, a decellularization technique is used. ECM reconstructed by decellularization (dECM) is prepared from tissues/organs and cultured cells. Although decellularization methods have been optimized for tissue‐/organ‐derived dECM, the methods for cultured cell‐derived dECM have not yet been optimized. Here, two physical (osmotic shocks) and five chemical decellularization methods are compared. The decellularization efficacies were changed according to the decellularization methods used. Among them, only the Triton X‐100 and Tween 20 treatments could not decellularize completely. Additionally, when the efficacies were compared among different types of cells (monolayered cells with/without strong cell adhesion, multilayered cells), the efficacies were decreased for multilayered cells or cells with strong cell adhesion. Retained ECM contents tended to be greater in the dECM prepared by osmotic shocks than in those prepared by chemical methods. The contents impacted cell adhesion, shapes, growth and intracellular signal activation on the dECM. The comparison would be helpful for the optimization of decellularization methods for cultured cells, and it could also provide new insights into developing milder decellularization methods for tissues and organs. [ABSTRACT FROM AUTHOR]

Regenerative bio‐scaffolds, widely used for clinical tissue reconstruction and tissue repairs, are functionally diversified and structurally complex decellularized tissue materials (e.g., extracellular matrix, ECM). ECM is naturally cross‐linked and can be further selectively cross‐linked upon processing. Identification, quantification and bioinformatics functional comparison of all ECM proteins are challenging for regenerative bio‐scaffolds. In this study, we have applied proteomic profiling with a two‐step sequential trypsinization method, and identified and quantified 300–400 constituent proteins in three commercially available regenerative bio‐scaffolds (BioDesign Surgisis, ReGen tissue matrix, and ThormalGEN mesh). These proteins were classified into four categories and 14 subcategories based on their mainly biological function. The main components of regenerative bio‐scaffolds were highly abundant ECM structural proteins, and the minor parts of bio‐scaffolds were lowly abundant, less cross‐linked, functionally more diversified proteins, especially extracellular fluid proteins that were easily solubilized by trypsin. The comparative analysis has revealed large differences in the number, type, abundance and function of identified proteins, as well as the extent of decellularization and cross‐linking among regenerative bio‐scaffolds. So, the proteomic profiling with a two‐step sequential trypsinization method could not only provide the molecular basis to better understand the degradation process of regenerative bio‐scaffolds in vivo and different clinical outcomes among various regenerative bio‐scaffolds, facilitate the exploration of the response mechanisms in the host's early clinical stages of ECM‐induced tissue regeneration that is still poorly understood, but also can be used for optimization of the decellularization and cross‐linking process, product characterization and rational design of new ECM products. [ABSTRACT FROM AUTHOR]

Purpose: Meniscus tears are one of the most frequent orthopedic knee injuries, which are currently often treated performing meniscectomy. Clinical concerns comprise progressive degeneration of the meniscus tissue, a change in knee biomechanics, and an early onset of osteoarthritis. To overcome these problems, meniscal transplant surgery can be performed. However, adequate meniscal replacements remain to be a great challenge. In this research, we propose the use of a decellularized and sterilized human meniscus allograft as meniscal replacement. Methods: Human menisci were subjected to a decellularization protocol combined with sterilization using supercritical carbon dioxide (scCO2). The decellularization efficiency of human meniscus tissue was evaluated via DNA quantification and Hematoxylin & Eosin (H&E) and DAPI staining. The mechanical properties of native, decellularized, and decellularized + sterilized meniscus tissue were evaluated, and its composition was determined via collagen and glycosaminoglycan (GAG) quantification, and a collagen and GAG stain. Additionally, cytocompatibility was determined in vitro. Results: Human menisci were decellularized to DNA levels of ~ 20 ng/mg of tissue dry weight. The mechanical properties and composition of human meniscus were not significantly affected by decellularization and sterilization. Histologically, the decellularized and sterilized meniscus tissue had maintained its collagen and glycosaminoglycan structure and distribution. Besides, the processed tissues were not cytotoxic to seeded human dermal fibroblasts in vitro. Conclusions: Human meniscus tissue was successfully decellularized, while maintaining biomechanical, structural, and compositional properties, without signs of in vitro cytotoxicity. The ease at which human meniscus tissue can be efficiently decellularized, while maintaining its native properties, paves the way towards clinical use. [ABSTRACT FROM AUTHOR]

Human‐induced pluripotent stem cells (hiPSCs) hold great promise in the fields of regenerative therapy and personalized in vitro tissue models as they provide an almost endless autologous source of virtually any cell type. Their potential benefits can be highly enhanced through polymeric cell microencapsulation, which offers the cells structural support and immune protection from the host tissue. However, in contrast to biologically‐inert polymers, decellularized extracellular matrix (dECM)‐based microencapsulation also provides the cells a physiologically mimetic bioactive microenvironment while providing reproducibility and scalability. Here we describe the entrapment of hiPSCs in a porcine pancreatic ECM (pECM)‐based microencapsulation platform and address its key aspects. To promote high levels of cell survival and growth, several encapsulation parameters were optimized, including cell dissociation level, the process layout, and Rho‐associated kinase (ROCK) inhibitor addition. This has allowed extensive proliferation of the cells for at least 21 days in vitro. Furthermore, the encapsulation of embryoid bodies (EBs) was assessed to address the entrapment of hiPSCs at various stages of differentiation, and the culture conditions that promote lasting encapsulated EB survival were investigated. Finally, to study the in vivo safety of dECM‐encapsulated hiPSC‐derived products, completely undifferentiated pECM‐encapsulated hiPSCs were implanted in mice. The cells displayed a slower proliferation rate in vivo and showed no signs of cell escape from the microcapsules. Altogether, the results of this study point to the potential of dECM‐based microencapsulation to provide an efficient polymeric platform for transplantation of hiPSC‐derived products and the development of iPSC‐based 3D tissue models. [ABSTRACT FROM AUTHOR]

Decellularized meniscus extracellular matrix (dmECM)‐based biological scaffolds in the forms of sponge, hydrogel, nanofiber, and composite have gained increasing interest in meniscus tissue engineering and regeneration. A common shortcoming of those scaffolds is insufficient mechanical strength and poor elasticity. Herein, we report a practicable protocol for milder meniscus decellularization to prepare elastic, porous dmECM scaffolds. Porcine meniscus was pulverized by cyclic freeze–thaw grinding and then treated with DNase to obtain fine dmECM particles. Individual dmECM particles were condensed to bulk preparation by centrifuge, followed by lyophilization to form blocks, and finally crosslinked by dehydrothermal treatment to obtain porous dmECM scaffolds. Our results show that the freeze–thaw grinding method was effective in removing cellular DNA with good retention of meniscus‐derived bioactive components. The dmECM scaffold had porous structure with interconnected mesopores and good mechanical properties. Primary articular chondrocytes proliferated robustly and maintained chondrogenic characteristics and produce abundant collagen on dmECM scaffolds. Evaluation of biocompatibility in a rat model shows that the dmECM scaffold elicited minor foreign body reactions, indicating effective antigen removal from dmECM. This study provides an alternative for preparing dmECM and fabricating porous scaffolds for meniscus repair and regeneration. [ABSTRACT FROM AUTHOR]

In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non‐ionic (0.1% v/v triton X‐100, t‐octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation‐driven set‐up. After 48 h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold‐like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72 h on normal insulin‐producing cells (MIN‐6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non‐toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source. [ABSTRACT FROM AUTHOR]

Decellularization has been applied to many tissues and organs to obtain biomaterials for applications in tissue engineering. In this study, decellularization and characterization of chicken skin was performed to provide comprehensive information and in‐depth details on this material as a potential tissue scaffold. Application of Triton X‐100 and sodium dodecyl sulfate (SDS) on tissues at different time intervals as two decellularization protocols were compared according to various aspects, such as removal of cellular components, DNA quantification, protection of extracellular matrix (ECM), mechanical properties, and cytocompatibility, to find the optimum technique during preparation of decellularized scaffolds. The results showed that treatment with SDS revealed better results when compared with Triton X‐100 regarding the preservation of tissue structure and morphology, although there was no difference in the efficiency of decellularization. In general, the tissues decellularized with SDS demonstrated higher levels of cytocompatibility and better mechanical properties in comparison with samples treated with Triton X‐100. In conclusion, this study revealed that decellularized chicken skin is a cheap, abundant, and biocompatible material that supports cell attachment, growth, and proliferation. Therefore, it could be used as a proper candidate to prepare scaffolds for further studies on tissue engineering, especially for skin tissue engineering. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) is a principal contributor to stroke and coronary heart disease in humans characterized by chronic low‐grade inflammation. The extracellular matrix (ECM) plays critical roles in regulating the function of arteries. However, the effect of changes in ECM on AS development is rarely studied. In this context, we intend to study the effect of oxidizing agent peroxynitrite (ONOO−)‐mediated oxidization of ECM proteins on the biological behaviors of vascular smooth muscle cells (SMCs) and the development of AS. AS mouse models were established, and mouse coronary artery smooth muscle cells (MCASMCs) were cultured in vitro to derive ECM (SMC‐ECM), which was obtained by deoxycholate (DOC)‐based decellularization. Further, MCASMCs were subjected to the determination of ECM oxidative damage and ECM protein structure. Finally, roles of ONOO−‐mediated oxidization of ECM in SMC adhesion and migration and in AS development were explored through Transwell assay, transcriptome sequencing, and gene enrichment analysis. High concentration of ONOO− was found in the serum of AS mice, and ONOO− could stimulate the development of AS. SMC‐ECM with intact structure can be obtained in vitro by DOC treatment. Functionally, ONOO−‐mediated oxidization destroyed the three‐dimensional structure of SMC‐ECM proteins, affected SMC adhesion and migration and promoted the absorption efficiency of lipids while reducing the efflux of cholesterol. In addition, the expression of inflammation‐ and oxidative stress‐related genes was significantly increased in ECM subjected to ONOO−‐mediated oxidization, thereby contributing to AS progression. ONOO−‐mediated oxidative modification of ECM aggravates AS by affecting the biological behavior of SMCs. [ABSTRACT FROM AUTHOR]

Background: To prevent ovarian hyperstimulation syndrome, in vitro maturation (IVM) allows the oocytes for infertility treatment without hormone therapy. Although many oocytes matured during IVM, some deficiencies in the culture conditions lead to inhibition of the growth and development of the cumulus cells and the oocyte nuclear and cytoplasmic maturation. Objectives: The challenge of improving the oocyte culture conditions prompted us to use greater omentum (GOM), full of growth factors and proteins, as a rich supplement to the base culture medium. Methods: Cumulus‐oocyte complexes were recovered from rabbits and divided into 3D and 2D conditions cultured for 12 and 24 h. In 3D cultures, the oocytes embedded in alginate containing FBS decellularized GOM. Corresponding supplements were also added in 2D conditions—maturation of the oocytes evaluated by Aceto‐Orcein, TEM, and RT‐PCR for MAP2K1 and Cdk2. Results: DNA quantification, Hoechst, and H&E staining confirmed cell depletion from GOM, and SEM showed the preservation of ultra‐architecture after decellularization. Histochemical staining methods showed appropriate extracellular matrix preservation. ELISA assessment showed retention of VEGF content. MTT assessment indicated decellularized GOM was non‐toxic. Both Aceto‐Orcein assessment and ultra‐structure study of the oocytes showed that supplementation of 2D or 3D cultures with decellularized omentum promoted oocyte maturation. Expression of MAP2K1 and Cdk2 also increased in the presence of GOM. Conclusions: GOM supplementation has a beneficial impact on oocyte maturation, probably due to the presence of growth factors and proteins. [ABSTRACT FROM AUTHOR]

Background: Hypopharynx reconstruction after hypopharyngectomy is still a great challenge. Perfusion decellularization is for extracellular matrix (ECM) scaffolding and had been used in organ reconstruction. Our study aimed to prepare an acellular, natural, three‐dimensional biological hypopharynx with vascular pedicle scaffold as the substitute materials to reconstruct hypopharynx. Result: Scanning electron microscope and histology staining showed that the decellularized hypopharynx with vascular pedicle scaffold retained intact native anatomical ECM structure. Myoblasts were observed on the recellularized scaffolds with bone marrow mesenchymal stem cells induced by 5‐azacytidine implanted in the rabbit greater omentum by immunohistochemical analysis. Conclusion: The decellularized hypopharynx with vascular pedicle scaffold prepared by detergent perfusion in our study has a potential to be an alternative material to pharynx reconstruction. [ABSTRACT FROM AUTHOR]

Background: Decellularized extracellular matrixs (dECMs) derived from organs and tissues have emerged as a promising tool, as they encompass the characteristics of an ideal tissue scaffold: complex composition, vascular networks and unique tissue‐specific architecture. Consequently, their use has propagated throughout tissue engineering and regenerative medicine. dECM can be easily obtained from various tissues/organs by appropriate decellularization protocolsand is entitled to provide necessary cues to cells homing. Methods: In this review, we describe the decellularization and sterilization methods that are commonly used in recent research, the effects of these methods upon biologic scaffold material are discussed. Also, we summarize the recent developments of recellularization and vascularization techniques in regeneration medicine. Additionally, dECM preservation methods is mentioned, which provides the basis for the establishment of organ bank. Results: Biomedical applications and the status of current research developments relating to dECM biomaterials are outlined, including transplantation in vivo, disease models and drug screening, organoid, 3D bioprinting, tissue reconstruction and rehabilitation and cell transplantation and culture. Finally, critical challenges and future developing technologies are discussed. Conclusions: With the development of tissue engineering and regenerative medicine, dECM will have broader applications in the field of biomedicine in the near future. [ABSTRACT FROM AUTHOR]

Background: We have previously applied in vivo tissue‐engineered vascular grafts constructed in patients' subcutaneous spaces. However, since the formation of these vascular grafts depends on host health, their application is challenging in patients with suppressed regenerative ability. Therefore, the allogeneic implantation of grafts from healthy donors needs to be evaluated. This study aimed to fabricate allogeneic cardiovascular grafts in animals. Materials and methods: Silicone rod molds were implanted into subcutaneous pouches in dogs; the implants, along with surrounding connective tissues, were harvested after four weeks. Tubular connective tissues were decellularized and stored before they were cut open, trimmed to elliptical sheets, and implanted into the common carotid arteries of another dog as vascular patches (n = 6); these were resected and histologically evaluated at 1, 2, and 4 weeks after implantation. Results: No aneurysmal changes were observed by echocardiography. Histologically, we observed neointima formation on the luminal graft surface and graft wall cell infiltration. At 2 and 4 weeks after implantation, α‐SMA‐positive cells were observed in the neointima and graft wall. At 4 weeks after implantation, the endothelial lining was observed at the grafts' luminal surfaces. Conclusion: Our data suggest that decellularized connective tissue membranes can be prepared and stored for later use as allogeneic cardiovascular grafts. [ABSTRACT FROM AUTHOR]

Background: Decellularized tissues based on well‐conserved extracellular matrices (ECMs) are a common area of research in tissue engineering. Although several decellularization protocols have been suggested for several types of tissues, studies on the optic nerve have been limited. Methods: We report decellularization protocol with different detergent for the preparation of acellular optic nerve and tissues were examined. DNA, glycosaminoglycan (GAG), and collagen content of the groups were evaluated with biochemical analyses and examined with histological staining. Mechanical properties, chemical components as well as cytotoxic properties of tissues were compared. Results: According to the results, it was determined that TX‐100 (Triton X‐100) was insufficient in decellularization when used alone. In addition, it was noticed that 85% of GAG content was preserved by using TX‐100 and TX‐100‐SD (sodium deoxycholate), while this ratio was calculated as 30% for SDS. In contrast, the effect of the decellularization protocols on ECM structure of the tissues was evaluated by scanning and transmission electron microscopy (SEM and TEM) and determined their mechanical properties. Cytotoxicity analyses were exhibited minimum 95% cell viability for all groups, suggesting that there are no cytotoxic properties of the methods on L929 mouse fibroblast cells. Conclusions: The combination of TX‐100‐SD and TX‐100‐SDS (sodium dodecyl sulfate) were was determined as the most effective methods to the literature for optic nerve decellularization. [ABSTRACT FROM AUTHOR]

Engineered replacement materials have tremendous potential for vascular applications where over 400,000 damaged and diseased blood vessels are replaced annually in the United States alone. Unlike large diameter blood vessels, which are effectively replaced by synthetic materials, prosthetic small‐diameter vessels are prone to early failure, restenosis, and reintervention surgery. We investigated the differential response of varying 0%–6% sodium dodecyl sulfate and sodium deoxycholate anionic detergent concentrations after 24 and 72 h in the presence of DNase using biochemical, histological, and biaxial mechanical analyses to optimize the decellularization process for xenogeneic vascular tissue sources, specifically the porcine internal thoracic artery (ITA). Detergent concentrations greater than 1% were successful at removing cytoplasmic and cell surface proteins but not DNA content after 24 h. A progressive increase in porosity and decrease in glycosaminoglycan (GAG) content was observed with detergent concentration. Augmented porosity was likely due to the removal of both cells and GAGs and could influence recellularization strategies. The treatment duration on the other hand, significantly improved decellularization by reducing DNA content to trace amounts after 72 h. Prolonged treatment times reduced laminin content and influenced the vessel's mechanical behavior in terms of altered circumferential stress and stretch while further increasing porosity. Collectively, DNase with 1% detergent for 72 h provided an effective and efficient decellularization strategy to be employed in the preparation of porcine ITAs as bypass graft scaffolding materials with minor biomechanical and histological penalties. [ABSTRACT FROM AUTHOR]

Due to its availability and minimal invasive harvesting human adipose tissue‐derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell‐derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively toward their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose‐derived stem cells, toward their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared with cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM‐based biomaterial for their specific application and the interpretation of obtained results. [ABSTRACT FROM AUTHOR]

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4′,6‐diamidino‐2‐phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood‐derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation. [ABSTRACT FROM AUTHOR]

Damage to the nervous system can result in loss of sensory and motor function, paralysis, or even death. To facilitate neural regeneration and functional recovery, researchers have employed biomaterials strategies to address both peripheral and central nervous system injuries. Injectable hydrogels that recapitulate native nerve extracellular matrix are especially promising for neural tissue engineering because they offer more flexibility for minimally invasive applications and provide a growth‐permissive substrate for neural cell types. Here, we explore the development of injectable hydrogels derived from decellularized rat peripheral nerves (referred to as "injectable peripheral nerve [iPN] hydrogels"), which are processed using a newly developed sodium deoxycholate and DNase (SDD) decellularization method. We assess the gelation kinetics, mechanical properties, cell bioactivity, and drug release kinetics of the iPN hydrogels. The iPN hydrogels thermally gel when exposed to 37°C in under 20 min and have mechanical properties similar to neural tissue. The hydrogels demonstrate in vitro biocompatibility through support of Schwann cell viability and metabolic activity. Additionally, iPN hydrogels promote greater astrocyte spreading compared to collagen I hydrogels. Finally, the iPN is a promising delivery vehicle of drug‐loaded microparticles for a combinatorial approach to neural injury therapies. [ABSTRACT FROM AUTHOR]

Stirred bioreactor systems integrating microcarriers represent a promising approach for therapeutic cell manufacturing. While a variety of microcarriers are commercially available, current options do not integrate the tissue‐specific composition of the extracellular matrix (ECM), which can play critical roles in directing cell function. The current study sought to generate microcarriers comprised exclusively of ECM from multiple tissue sources. More specifically, porcine decellularized dermis, porcine decellularized myocardium, and human decellularized adipose tissue were digested with α‐amylase to obtain ECM suspensions that could be electrosprayed into liquid nitrogen to generate 3D microcarriers that were stable over a range of ECM concentrations without the need for chemical crosslinking or other additives. Characterization studies confirmed that all three microcarrier types had similar soft and compliant mechanical properties and were of a similar size range, but that their composition varied depending on the native tissue source. In vivo testing in immunocompetent mice revealed that the microcarriers integrated into the host tissues, supporting the infiltration of host cells including macrophages and endothelial cells at 2 weeks post‐implantation. In vitro cell culture studies validated that the novel microcarriers supported the attachment of tissue‐specific stromal cell populations under dynamic culture conditions within spinner flasks, with a significant increase in live cell numbers observed over 1 week on the dermal‐ and adipose‐derived microcarriers. Overall, the findings demonstrate the versatility of the electrospraying methods and support the further development of the microcarriers as cell culture and delivery platforms. [ABSTRACT FROM AUTHOR]

The limited availability of human donor organs suitable for transplantation has resulted in ever‐increasing patient waiting lists globally. Xenotransplantation is considered a potential option, but is yet to reach clinical practice. Although remarkable progress has been made in overcoming immunological rejection, issues with functionality are still to be resolved. Bioengineering approaches have been used to create cardiac tissues with optimized functions. The use of decellularized xenogeneic cardiac tissues seeded with donor‐derived cardiac cells may prove to be a viable strategy as supporting structures of the native tissue such as vasculature can be utilized. Here we used sequential perfusion to decellularize adult rat hearts. The acellular scaffolds were reseeded with human endothelial cells, human fibroblasts, human mesenchymal stem cells, and cardiac cells derived from human‐induced pluripotent stem cells. The ability of the resultant recellularized rat scaffolds to activate human naïve neutrophils in vitro was investigated to measure xenogeneic recognition. Our results demonstrate that in contrast to cadaveric xenogeneic hearts, acellular and recellularized xenogeneic scaffolds did not activate human naïve neutrophils and suggest that decellularization removes the xenogeneic antigens that lead to human naïve neutrophil activation thus allowing human cells to populate the now "allogenized" xenogeneic scaffolds. [ABSTRACT FROM AUTHOR]

Introduction/Aims: While the peripheral nervous system has the inherent ability to recover following injury, results are often unsatisfactory, resulting in permanent functional deficits and disability. Therefore, methods that enhance regeneration are of significant interest. The present study investigates an injectable nerve‐tissue‐specific hydrogel as a biomaterial for nerve regeneration in a rat nerve crush model. Methods: Nerve‐specific hydrogels were injected into the subepineurial space in both uninjured and crushed sciatic nerves of rats to assess safety and efficacy, respectively. The animals were followed longitudinally for 12 wk using sciatic functional index and kinematic measures. At 12 wk, electrophysiologic examination was also performed, followed by nerve and muscle histologic assessment. Results: When the hydrogel was injected into an uninjured nerve, no differences in sciatic functional index, kinematic function, or axon counts were observed. A slight reduction in muscle fiber diameter was observed in the hydrogel‐injected animals, but overall muscle area and kinematic function were not affected. Hydrogel injection following nerve crush injury resulted in multiple modest improvements in sciatic functional index and kinematic function with an earlier return to normal function observed in the hydrogel treated animals as compared to untreated controls. While no improvements in supramaximal compound motor action potential were observed in hydrogel treated animals, increased axon counts were observed on histologic assessment. Discussion: These improvements in functional and histologic outcomes in a rapidly and fully recovering model suggest that injection of a nerve‐specific hydrogel is safe and has the potential to improve outcomes following nerve injury. [ABSTRACT FROM AUTHOR]

Alveolar ridge augmentation can be used to obtain appropriate alveolar ridge for dental implantation. A variety of bone graft materials including autogenous bone, allograft, xenograft, and alloplastic material are used in alveolar ridge augmentation. Autogenous tooth‐derived bone graft material has received much attention for the past few years, because the structure and physicochemical characteristics of tooth are similar to those of bones. Compared to autogenous tooth, allogenic tooth has the advantage of extensive resources. However, the problem of cell‐derived immunological rejection of allogenic tooth remains unresolved. In the present study, acellular tooth root (ATR) is obtained by an innovative combination procedure. The biocompatibility of ATR is assessed using cytotoxicity test, hemolysis test, intracutaneous reactivity test, and acute systemic toxicity test. Osseointegration is evaluated in vivo by implanting ATR into the rat tibia defect as an allograft material. The results show that the ATR has fine biocompatibility, and there is an osseointegration between ATR and bone bed at 8 weeks post operation. This study demonstrates that the ATR could be used in alveolar ridge augmentation as a kind of new tooth‐derived bone graft material. [ABSTRACT FROM AUTHOR]

Biomaterials derived from extracellular matrices (ECMs) were extensively used for skin tissue engineering and wound healing. ECM is a complex network of biomolecules (e.g., proteins), which provide organizational support to cells for growth. Thus, ECM could be an ideal biomaterial for fabricating the scaffold. However, oxidative stress and biofilm formation at the wound site remains a major challenge that could be neutralized using herbal ingredients (e.g., curcumin). In this study, ECM was extracted from the biowaste of the goat abattoir by using decellularization. The goat small intestine submucosa (G‐SIS) is decellularized to obtain the decellularized G‐SIS (DG‐SIS) and curcumin (in different concentrations) was incorporated in the DG‐SIS to fabricate curcumin‐embedded DG‐SIS scaffolds. Changes brought by increasing the concentrations of the curcumin in DG‐SIS were observed in various properties, including free radical scavenging and antibacterial properties. Results depicted that the scaffolds are porous, biodegradable, biocompatible, antibacterial, and hydrophilic and showed sustained release of curcumin. Besides, it showed free radicals scavenging property. The porosity and hydrophilicity of the scaffolds were decreased with an increase in the curcumin content. However, biodegradability, free radical scavenging, biocompatibility, and antibacterial properties of the scaffolds increased with an increase in the curcumin content. The DG‐SIS scaffold containing 1 wt % of curcumin may be a potential biomaterial for wound‐healing and skin tissue engineering. [ABSTRACT FROM AUTHOR]

Decellularization (DC) of biomaterials with bioreactors is widely used to produce scaffolds for tissue engineering. This study uses 3D printing to develop efficient but low‐cost DC bioreactors. Two bioreactors were developed to decellularize pericardial patches and vascular grafts. Flow profiles and pressure distribution inside the bioreactors were optimized by steady‐state computational fluid dynamics (CFD) analysis. Printing materials were evaluated by cytotoxicity assessment. Following evaluation, all parts of the bioreactors were 3D printed in a commercial fused deposition modeling printer. Samples of bovine pericardia and porcine aortae were decellularized using established protocols. An immersion and agitation setup was used as a control. With histological assessment, DNA quantification and biomechanical testing treatment effects were evaluated. CFD analysis of the pericardial bioreactor revealed even flow and pressure distribution in between all pericardia. The CFD analysis of the vessel bioreactor showed increased intraluminal flow rate and pressure compared to the vessel's outside. Cytotoxicity assessment of the used printing material revealed no adverse effect on the tissue. Complete DC was achieved for all samples using the 3D printed bioreactors while DAPI staining revealed residual cells in aortic vessels of the control group. Histological analysis showed no structural changes in the decellularized samples. Additionally, biomechanical properties exhibited no significant change compared to native samples. This study presents a novel approach to manufacturing highly efficient and low budget 3D printed bioreactors for the DC of biomaterials. When compared to standard protocols, the bioreactors offer a cost effective, fast, and reproducible approach, which vastly improves the DC results. [ABSTRACT FROM AUTHOR]

Scaffold materials suitable for the scale‐up and subsequent commercialization of tissue engineered products should ideally be cost effective and accessible. For the in vitro culture of certain adherent cells, synthetic fabrication techniques are often employed to produce micro‐ or nano‐patterned substrates to influence cell attachment, morphology, and alignment via the mechanism of contact guidance. Here we present a natural scaffold, in the form of decellularized amenity grass, which retains its natural striated topography and supports the attachment, proliferation, alignment and differentiation of murine C2C12 myoblasts, without the need for additional functionalization. This presents an inexpensive, sustainable scaffold material and structure for tissue engineering applications capable of influencing cell alignment, a desired property for the culture of skeletal muscle and other anisotropic tissues. [ABSTRACT FROM AUTHOR]

Decellularization approaches have been commonly used as alternative techniques to reconstruct tissues. However, due to the complex tissue compartmentation of the larynx, the decellularization process may not retain the characteristics necessary for the successful recreation of the larynx. The aim of this study was to assess the effect of the decellularization process on the framework of the human cadaveric larynx generally and the cricoarytenoid joint specifically. In this work, five freshly frozen human cadaveric larynges were decellularized utilizing a protocol that was previously demonstrated to be effective in decellularizing a porcine larynx. The decellularization protocol included: biological, chemical, and physical decellularization methods. Each specimen served as its own control to assess changes after decellularization. Studies and measurements included: histological, using Hematoxylin and Eosin (H&E) and Live/Dead™ stains; DNA quantification; micro‐computed tomography (μ‐CT) imaging; and biomechanical testing of the cricoarytenoid joints. The decellularization protocol took 12 days for each specimen. Microscopy of H&E stained samples demonstrated substantial removal of cells with preservation of the extracellular matrix that was more evident in cartilage than muscle specimens. Confocal microscope images of Live/Dead™ stained specimens also demonstrated almost complete removal of cells. Pre‐decellularization cartilage‐DNA quantity range was 27.0 to 336.8 ng/mg while post‐decellularization DNA quantity range was 0 to 30.4 ng/mg (p = 0.031). For muscles, pre‐decellularization DNA quantity range was 150.0 to 3,384.6 ng/mg, while post‐decellularization DNA quantity range was 0 to 45.5 ng/mg (p = 0.031). μ‐CT demonstrated preservation of the cartilaginous framework with a slight reduction of cricoarytenoid joint space. Furthermore, μ‐CT demonstrated no significant reduction in the Housefield unit (p = 0.25) and mineral density (p = 0.25) after decellularization. Biomechanical testing demonstrated a non‐significant reduction of forces required for anterior displacement of the arytenoid (mean reduction of forces, 0.1 ± 0.2 N, p = 0.16) and forces required for posterior displacement of the arytenoid (mean reduction of forces, 0.2 ± 0.3 N, p = 0.05). This study demonstrates effective decellularization of human larynges as evidenced by significant DNA depletion and preservation of extracellular matrix, which are outcomes that are required for a biological scaffold to regenerate a non‐immunogenic larynx. The decellularization process caused minimal weakness in the cricoarytenoid joints due to treatment with multiple detergents and enzymes in the decellularization protocol. [ABSTRACT FROM AUTHOR]