Decellularization Strategies Research Articles

Assessment of decellularization strategy and biocompatibility testing of full-thickness abdominal wall to produce a tissue-engineered graft.

Restoration of the abdominal wall defects due to herniation or other complications represents a challenging task of the reconstructive surgery. Synthetic grafts or crosslinked animal-derived grafts, are utilized, followed by significant adverse reactions. This study aimed to evaluate primarily the production of a decellularized abdominal wall scaffold and secondly its biocompatibility upon transplantation in an animal model. Full-thickness abdominal wall samples were harvested from Wistar Rats and then decellularized utilizing a three-cycle process. To evaluate the decellularization efficacy, histological, biochemical and biomechanical analyses were performed. The biocompatibility assessment involved the implantation of the produced scaffolds to Sprague Dawley rats. The grafts remained for a total period of 4 weeks, followed by immunohistochemistry for the detection of CD11b+, CD4+ and CD8+ cells. Histological, biochemical and biomechanical results, indicated the production of compatible acellular full-thickness abdominal wall samples. After 4 weeks of implantation, a minor presence of immunity cells was observed. The data of this study indicated the successful production of a full-thickness abdominal wall scaffold. Biologically derived full-thickness abdominal wall scaffolds may have greater potential in restoration of the abdominal wall defects, bringing them one step closer to their clinical utility.

Comparative Analysis of Decellularization Methods for the Production of Decellularized Umbilical Cord Matrix.

The importance of decellularized extracellular matrix (dECM) as a natural biomaterial in tissue engineering and regenerative medicine is rapidly growing. The core objective of the decellularization process is to eliminate cellular components while maximizing the preservation of the ECM's primary structure and components. Establishing a rapid, effective, and minimally destructive decellularization technique is essential for obtaining high-quality dECM to construct regenerative organs. This study focused on human umbilical cord tissue, designing different reagent combinations for decellularization protocols while maintaining a consistent processing time. The impact of these protocols on the decellularization efficiency of human umbilical cord tissue was evaluated. The results suggested that the composite decellularization strategy utilizing trypsin/EDTA + Triton X-100 + sodium deoxycholate was the optimal approach in this study for preparing decellularized human umbilical cord dECM. After 5 h of decellularization treatment, most cellular components were eliminated, confirmed through dsDNA quantitative detection, hematoxylin and eosin (HE) staining, and DAPI staining. Meanwhile, Masson staining, periodic acid-silver methenamine (PASM) staining, periodic acid-Schiff (PAS) staining, and immunofluorescent tissue section staining results revealed that the decellularized scaffold retained extracellular matrix components, including collagen and glycosaminoglycans (GAGs). Compared to native umbilical cord tissue, electron microscopy results demonstrated that the microstructure of the extracellular matrix was well preserved after decellularization. Furthermore, Fourier-transform infrared spectroscopy (FTIR) findings indicated that the decellularization process successfully retained the main functional group structures of extracellular matrix (ECM) components. The quantitative analysis of collagen, elastin, and GAG content validated the advantages of this decellularization process in preserving and purifying ECM components. Additionally, it was confirmed that this decellularized matrix exhibited no cytotoxicity in vitro. This study achieved short-term decellularization preparation for umbilical cord tissue through a combined decellularization strategy.

Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney.

The extracellular matrix (ECM) is composed of collagens, ECM glycoproteins, and proteoglycans (also named core matrisome proteins) that are critical for tissue structure and function, and matrisome-associated proteins that balance the production and degradation of the ECM proteins. The identification and quantification of core matrisome proteins using mass spectrometry is often hindered by their low abundance and their propensity to form macromolecular insoluble structures. In this study, we aimed to investigate the added value of decellularization in identifying and quantifying core matrisome proteins in mouse kidney. The decellularization strategy combined freeze-thaw cycles and sodium dodecyl sulphate treatment. We found that decellularization preserved 95% of the core matrisome proteins detected in non-decellularized kidney and revealed few additional ones. Decellularization also led to an average of 59 times enrichment of 96% of the core matrisome proteins as the result of the successful removal of cellular and matrisome-associated proteins. However, the enrichment varied greatly among core matrisome proteins, resulting in a misrepresentation of the native ECM composition in decellularized kidney. This should be brought to the attention of the matrisome research community, as it highlights the need for caution when interpreting proteomic data obtained from a decellularized organ.

Comparison of three techniques for decellularization of porcine bone-tendon-bone grafts

ABSTRACT Anterior cruciate ligament injuries are frequent afflictions related to sports or physical trauma. Autograft reconstruction strategies cause secondary injury to the patient. One alternative, supported by clinical evidence, is porcine xenografts. For clinical use, xenografts must be conditioned to avoid immune rejection. The most widely accepted procedure is tissue decellularization. We analyzed three decellularization strategies: the application of the anionic detergent sodium dodecyl sulfate (SDS), sonication, and freezing and thawing cycles. The treated tissues were evaluated histologically using H&E, Masson’s trichrome, Verhoeff-van Gieson staining, and DAPI for fluorescent staining of nuclei. Finally, collagen fiber preservation was evaluated by quantifying this protein by colorimetry. The most efficient decellularization techniques were sonication and SDS. Collagen fibers were preserved in all experimental conditions.

Construction of Integral Decellularized Cartilage Using a Novel Hydrostatic Pressure Bioreactor.

The decellularized extracellular matrix (ECM) of cartilage is a widely used natural bioscaffold for constructing tissue-engineered cartilage due to its good biocompatibility and regeneration properties. However, current decellularization methods for accessing decellularized cartilaginous tissues require multiple steps and a relatively long duration to produce decellularized cartilage. In addition, most decellularization strategies lead to damage of the microstructure and loss of functional components of the cartilaginous matrix. In this study, a novel decellularization strategy based on a hydrostatic pressure (HP) bioreactor was introduced, which aimed to improve the efficiency of producing integral decellularized cartilage pieces by combining physical and chemical decellularization methods in a perfusing manner. Two types of cartilaginous tissues, auricular cartilage (AC) and nucleus pulposus (NP) fibrocartilage, were selected for comparison of the effects of ordinary, positive, and negative HP-based decellularization according to the cell clearance ratio, microstructural changes, ECM components, and mechanical properties. The results indicated that applying positive HP improved the efficiency of producing decellularized AC, but no significant differences in decellularization efficiency were found between the ordinary and negative HP-treated groups. However, compared with the ordinary HP treatment, the application of the positive or negative HP did not affect the efficiency of decellularized NP productions. Moreover, neither positive nor negative HP influenced the preservation of the microstructure and components of the AC matrix. However, applying negative HP disarranged the fibril distribution of the NP matrix and reduced glycosaminoglycans and collagen type II contents, two essential ECM components. In addition, the positive HP was beneficial for maintaining the mechanical properties of decellularized cartilage. The recellularization experiments also verified the good biocompatibility of the decellularized cartilage produced by the present bioreactor-based decellularization method under positive HP. Overall, applying positive HP-based decellularization resulted in a superior effect on the production of close-to-natural scaffolds for cartilage tissue engineering. Impact statement In this study, we successfully constructed a novel hydrostatic pressure (HP) bioreactor and used this equipment to produce decellularized cartilage by combining physical and chemical decellularization methods in a perfusing manner. We found that positive HP-based decellularization could improve the production efficiency of integral decellularized cartilage pieces and promote the maintenance of matrix components and mechanical properties. This new decellularization strategy exhibited a superior effect in the production of close-to-natural scaffolds and positively impacts cartilage tissue engineering.

Distinct HAND2/HAND2-AS1 Expression Levels May Fine-Tune Mesenchymal and Epithelial Cell Plasticity of Human Mesenchymal Stem Cells.

We previously developed several successful decellularization strategies that yielded porcine cardiac extracellular matrices (pcECMs) exhibiting tissue-specific bioactivity and bioinductive capacity when cultured with various pluripotent and multipotent stem cells. Here, we study the tissue-specific effects of the pcECM on seeded human mesenchymal stem cell (hMSC) phenotypes using reverse transcribed quantitative polymerase chain reaction (RT-qPCR) arrays for cardiovascular related gene expression. We further corroborated interesting findings at the protein level (flow cytometry and immunological stains) as well as bioinformatically using several mRNA sequencing and protein databases of normal and pathologic adult and embryonic (organogenesis stage) tissue expression. We discovered that upon the seeding of hMSCs on the pcECM, they displayed a partial mesenchymal-to-epithelial transition (MET) toward endothelial phenotypes (CD31+) and morphologies, which were preceded by an early spike (~Day 3 onward after seeding) in HAND2 expression at both the mRNA and protein levels compared to that in plate controls. The CRISPR-Cas9 knockout (KO) of HAND2 and its associated antisense long non-coding RNA (HAND2-AS1) regulatory region resulted in proliferation arrest, hypertrophy, and senescent-like morphology. Bioinformatic analyses revealed that HAND2 and HAND2-AS1 are highly correlated in expression and are expressed in many different tissue types albeit at distinct yet tightly regulated expression levels. Deviation (downregulation or upregulation) from these basal tissue expression levels is associated with a long list of pathologies. We thus suggest that HAND2 expression levels may possibly fine-tune hMSCs' plasticity through affecting senescence and mesenchymal-to-epithelial transition states, through yet unknown mechanisms. Targeting this pathway may open up a promising new therapeutic approach for a wide range of diseases, including cancer, degenerative disorders, and aging. Nevertheless, further investigation is required to validate these findings and better understand the molecular players involved, potential inducers and inhibitors of this pathway, and eventually potential therapeutic applications.

A proposed model of xeno-keratoplasty using 3D printing and decellularization.

Corneal opacity is a leading cause of vision impairment and suffering worldwide. Transplantation can effectively restore vision and reduce chronic discomfort. However, there is a considerable shortage of viable corneal graft tissues. Tissue engineering may address this issue by advancing xeno-keratoplasty as a viable alternative to conventional keratoplasty. In particular, livestock decellularization strategies offer the potential to generate bioartificial ocular prosthetics in sufficient supply to match existing and projected needs. To this end, we have examined the best practices and characterizations that have supported the current state-of-the-art driving preclinical and clinical applications. Identifying the challenges that delimit activities to supplement the donor corneal pool derived from acellular scaffolds allowed us to hypothesize a model for keratoprosthesis applications derived from livestock combining 3D printing and decellularization.

Impact of various detergent-based immersion and perfusion decellularization strategies on the novel caprine pancreas derived extracellular matrix scaffold.

Limited availability of the organs donors has facilitated the establishment of xenogeneic organ sources for transplantation. Numerous studies have decellularized several organs and assessed their implantability in order to provide such organs. Among all the decellularized organs studies for xenotransplantation, the pancreas has garnered very limited amount of research. The presently offered alternatives for pancreas transplantation are unable to liberate patients from donor dependence. The rat and mice pancreas are not of an accurate size for transplantation but can only be used for in-vitro studies mimicking in-vivo immune response in humans, while the porcine pancreas can cause zoonotic diseases as it carries porcine endogenous retrovirus (PERV- A/B/C). Therefore, we propose caprine pancreas as a substitute for these organs, which not only reduces donor dependence but also poses no risk of zoonosis. Upon decellularization the extracellular matrix (ECM) of different tissues responds differently to the detergents used for decellularization at physical and physiological level; this necessitates a comprehensive analysis of each tissue independently. This study investigates the impact of decellularization by ionic (SDS and SDC), non-ionic (Triton X-100 and Tween-20), and zwitterionic detergents (CHAPS). All these five detergents have been used to decellularize caprine pancreas via immersion (ID) and perfusion (PD) set-up. In this study, an extensive comparison of these two configurations (ID and PD) with regard to each detergent has been conducted. The final obtained scaffold with each set-up has been evaluated for the left-over cytosolic content, ECM components like sGAG, collagen, and fibronectin were estimated via Prussian blue and Immunohistochemical staining respectively, and finally for the tensile strength and antimicrobial activity. All the detergents performed consistently superior in PD than in ID. Conclusively, PD with SDS, SDC, and TX-100 successfully decellularizes caprine pancreatic tissue while retaining ECM architecture and mechanical properties. This research demonstrates the viability of caprine pancreatic tissue as a substitute scaffold for porcine organs and provides optimal decellularization protocol for this xenogeneic tissue. This research aims to establish a foundation for further investigations into potential regenerative strategies using this ECM in combination with other factors.

Decellularized extracellular matrix particle-based biomaterials for cartilage repair applications

Cartilage Decellularized ExtraCellular Matrix (dECM) materials have shown promising cartilage regeneration capacity due to their chondrogenic bioactivity. However, the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes impair the biomimicry of these materials. The current study aims to fabricate biomimetic materials containing decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair. For this, we established a novel two-fraction decellularization strategy for the preparation of reconstituted dECM (rdECM) particles by mixing the two-fraction components, as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM (bdECM) particles. Hyaluronic acid-tyramine (THA) hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test, swelling and stability evaluation, and compression test. The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of decellularization. The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties. bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70% and high retention of glycosaminoglycans and collagen after long-term incubation, while rdECM particle-embedded THA hydrogels showed unsatisfactory stability as self-standing biomaterials. Compared to pure THA hydrogels, the addition of bdECM particles significantly enhanced the compression moduli. In summary, our decellularization methods are successful in the retention of functional and intact cartilage components with high yield. Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing. Hydrogels with cartilage bdECM particles possess the functional structure and the natural composition of cartilage ECM, long-term stability, and enhanced mechanical properties, and are promising biomaterials for cartilage repair.

Bioactive ECM-Loaded SIS Generated by the Optimized Decellularization Process Exhibits Skin Wound Healing Ability in Type I Diabetic Rats.

Patients with diabetes have 15-25% chance for developing diabetic ulcers as a severe complication and formidable challenge for clinicians. Conventional treatment for diabetic ulcers is to surgically remove the necrotic skin, clean the wound, and cover it with skin flaps. However, skin flap often has a limited efficacy, and its acquisition requires a second surgery, which may bring additional risk for the patient. Skin tissue engineering has brought a new solution for diabetic ulcers. Herein, we have developed a bioactive patch through a compound culture and the optimized decellularization strategy. The patch was prepared from porcine small intestinal submucosa (SIS) and modified by an extracellular matrix (ECM) derived from urine-derived stem cells (USCs), which have low immunogenicity while retaining cytokines for angiogenesis and tissue regeneration. The protocol included the optimization of the decellularization time and the establishment of the methods. Furthermore, the in vitro mechanism of wound healing ability of the patch was investigated, and its feasibility for skin wound healing was assessed through an antishrinkage full-thickness skin defect model in type I diabetic rats. As shown, the patch displayed comparable effectiveness to the USCs-loaded SIS. Our findings suggested that this optimized decellularization protocol may provide a strategy for cell-loaded scaffolds that require the removal of cellular material while retaining sufficient bioactive components in the ECM for further applications.

Detergent-Free Decellularization Preserves the Mechanical and Biological Integrity of Murine Tendon.

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. In this study, we adapted and established the advantages of a detergent-free decellularization method that relies on latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Our method maintained the collagen molecular structure (i.e., minimal extent of denaturation), while adequately removing cells and preserving bulk mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/"provisional"), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact statement In this study, we present a decellularization method for tendon that does not rely on any detergent or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of gross fiber architecture, collagen molecular structure, and extracellular matrix (ECM)-associated biological cues that are essential for studying physiological cell-ECM interactions. Finally, we demonstrated the applicability of this method for healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

Exhale and Decell: Environmentally Sustainable Sterilization and Decellularization of Cartilage Grafts via Supercritical Carbon Dioxide

PURPOSE: Current decellularization techniques have important limitations, including obligatory use of harsh ionized substances or detergents that deplete bioactive cytokines and compromise the physical integrity of the resulting extracellular matrix (ECM) scaffold. Furthermore, the detergents used are increasingly recognized as harmful to the environment and are poised to be restricted from commercial use. Supercritical carbon dioxide (scCO2) is an appealing alternative, applying the principles behind supercritical fluid extraction and sterilization to the removal of immunogenic cellular components. We endeavor to evaluate the efficacy of scCO2 in both sterilization and decellularization of ovine costal cartilage xenograft and human costal cartilage allograft to develop a highly efficacious, environmentally sustainable, and biocompatible alternative to currently available decellularization strategies. METHODS AND MATERIALS: Xenograft preparation: Racks of lamb were purchased from a local butcher. Floating ribs were minced into ∼8 mm3 cubes or zested into flakes (1 mm × 1 mm). Allograft harvest: Human costal cartilage was obtained from patients undergoing microsurgical breast reconstruction necessitating partial rib resection and processed via mincing or zested as above. All samples were subjected to: 1) sterilization; 2) sterilization and decellularization; or 3) sterilization, decellularization, and a pretreatment wash. Sterilization: Samples were placed in a NovaSterilis ™ Nova2200 unit and subject to standard NovaSterilis ™ sterilization parameters. At least 3 samples with biologic indicators were placed within the packaging and processed with each run. Decellularization: Samples were subject to the standard parameters of the Nova2200 system as previously described by NovaSterilis ™ with an ethanol to scCO2 volume ratio of 1:3.3. Wash: Samples were serially soaked in saline and agitated with exposure to 16 mL 3% H2O2 and a 30-minute scCO2 run at 35° C and 1,436 psi. Histology: After treatment, H&E, DAPI, and safranin-O staining were performed. DNA Quantification: DNA content was quantified in unprocessed and decellularized graft samples using the DNeasy Blood & Tissue kit (Qiagen Inc.). RESULTS: Sterilization conditions were sufficient for a 6 log10 sterilization of B. atrophaeus. H&E staining revealed preservation of tissue architecture after both sterilization and decellularization in both allograft and xenograft cartilage. DAPI staining demonstrated depletion of nuclei in the decellularized zested samples, but persistence of visible nuclei in the minced samples. Safranin-O staining revealed immunogenic GAG depletion after decellularization. DNA content in minced and zested samples was 192.2 ng DNA/mg tissue and 321.6 ng DNA/mg tissue, respectively, while DNA content in sterilized and decellularized minced and zested samples were 24.8 ng DNA/mg tissue and 11.6 ng DNA/mg tissue, respectively (industry standard requirement for decellularization <50 ng DNA/mg tissue); after sterilization, decellularization, and the chemical wash, minced and zested samples contained 17.6 ng DNA/mg tissue and 4 ng DNA/mg tissue respectively. CONCLUSION: These preliminary data suggest that scCO2 sufficiently sterilizes and decellularizes cartilage derived from both human and animal sources, thereby supporting scCO2 as an efficacious, commercially appealing, and ecologically responsible alternative to current decellularization strategies. Biomechanical testing on treated samples, as well as the evaluation of scCO2 as a decellularization agent for human nasal septal cartilage are ongoing.

Smooth muscle matrix bioink promotes myogenic differentiation of encapsulated adipose-derived stem cells.

Hydrogels derived from decellularized extracellular matrices (dECM) can mimic the biochemical composition of the native tissue. They can also act as a template to culture reseeded cells in vitro. However, detergent-based decellularization methods are known to alter the biochemical compositions, thereby compromising the bioactive potential of dECM. This study proposes a facile detergent-free method to achieve dECM from smooth muscle tissue. We have used the muscle layer of caprine esophageal tissue and decellularized using hypo and hyper-molar sodium chloride solutions alternatingly. Then, a hydrogel was prepared from this decellularized smooth muscle matrix (dSMM) and characterized thoroughly. A comparative analysis of the dSMM prepared with our protocol with the existing detergent-based protocol suggests successful and comparable decellularization with minimal residual DNA content. Interestingly, an 8.78-fold increase in sulfated glycosaminoglycans content and 1.62-fold increased collagen content indicated higher retention of ECM constituents with NaCl-based decellularization strategy. Moreover, the dSMM gel induces differentiation of the encapsulated adipose-derived mesenchymal stem cells toward smooth muscle cells (SMCs) as observed by their expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, the hallmarks of SMCs. Finally, we optimized the process parameter for productive bioprinting with this dSMM bioink and fabricated 3D muscle constructs. Our results suggest that dSMM has the potential to be used as a bioink to engineer personalized esophageal tissues.

Impact of Three Different Processing Techniques on the Strength and Structure of Juvenile Ovine Pulmonary Homografts.

Homografts are routinely stored by cryopreservation; however, donor cells and remnants contribute to immunogenicity. Although decellularization strategies can address immunogenicity, additional fixation might be required to maintain strength. This study investigated the effect of cryopreservation, decellularization, and decellularization with additional glutaraldhyde fixation on the strength and structure of ovine pulmonary homografts harvested 48 h post-mortem. Cells and cellular remnants were present for the cryopreserved group, while the decellularized groups were acellular. The decellularized group had large interfibrillar spaces in the extracellular matrix with uniform collagen distribution, while the additional fixation led to the collagen network becoming dense and compacted. The collagen of the cryopreserved group was collapsed and appeared disrupted and fractured. There were no significant differences in strength and elasticity between the groups. Compared to cryopreservation, decellularization without fixation can be considered an alternative processing technique to maintain a well-organized collagen matrix and tissue strength of homografts.

90. Sustainable and Efficacious Sterilization and Decellularization of Xenograft Cartilage Using Supercritical Carbon Dioxide

Purpose: Current decellularization techniques have important limitations, including obligatory use of harsh ionized substances or detergents that compromise the physical integrity of the resulting extracellular matrix (ECM) scaffold and, consequently, its biological performance. Furthermore, the detergents used are increasingly recognized to be harmful to the environment and may soon restricted from commercial use. Supercritical CO2 (scCO2) is an appealing alternative, applying the principles behind supercritical fluid extraction and sterilization to the removal of cellular components. In this work, we evaluate the efficacy of scCO2 in both sterilization and decellularization of cartilage xenograft in order to develop a highly efficacious, environmentally conscious, and biocompatible alternative to currently available technologies. Methods:Xenograft preparation: Racks of lamb were purchased from a local butcher. Floating ribs were minced into ∼8 mm3 cubes or zested into flakes (1 mm × 1 mm). Samples were subjected to: 1) sterilization; 2) sterilization and decellularization; or 3) sterilization, decellularization, and a pretreatment wash. Sterilization: Samples were placed in a NovaSterilis™ Nova2200 unit and subject to standard NovaSterilis™ sterilization parameters, including temperature (35 °C), NovaKill solution (16 mL), scCO2 dwell time (90 min.), and pressure (>1,200 psi). All samples were plated to allow for enumeration of bioburden reduction. At least 3 samples with biologic indicators were placed within the packaging and processed with each run. Decellularization: Samples were subject to the standard parameters of the Nova2200 system as previously described by NovaSterilis™ with an ethanol to scCO2 volume ratio of 1:3.3. Wash: Samples were serially soaked in saline and agitated with exposure to 16 mL 3% H2O2 and a 30-minute scCO2 run at 35 °C and 1,436 psi. Histology: After treatment, hematoxylin and eosin (H&E), DAPI, and safranin-O staining were performed. DNA Quantification: DNA content was quantified in raw and decellularized graft samples using the DNeasy Blood & Tissue kit (Qiagen Inc.) with a DNA content <50 ng DNA/mg tissue considered decellularized as per the literature standard. Results: No microbial growth was detected after 14 days incubation. H&E staining revealed preservation of tissue architecture after both sterilization and decellularization. DAPI staining demonstrated depletion of nuclei in the decellularized zested samples, but persistence of visible nuclei in the minced samples. Safranin-O staining revealed GAG depletion after decellularization. DNA content in raw minced and zested samples was 192.2 ng DNA/mg tissue and 321.6 ng DNA/mg tissue, respectively, while DNA content in sterilized and decellularized minced and zested samples were 24.8 ng DNA/mg tissue and 11.6 ng DNA/mg tissue, respectively (industry standard requirement for decellularization <50 ng DNA/mg tissue); after sterilization, decellularization, and the chemical wash, minced and zested samples contained 17.6 ng DNA/mg tissue and 4 ng DNA/mg tissue respectively. Conclusions: These preliminary data suggest that scCO2 sufficiently sterilizes and decellularizes ovine xenograft, thereby supporting scCO2 as an efficacious, commercially appealing, and ecologically responsible alternative to current decellularization strategies.

Preliminary Study on the Antigen-Removal from Extracellular Matrix via Different Decellularization.

Due to the abundance of bioactive components, surficial decoration with cell-derived extracellular matrix (ECM) is a promising strategy to improve the biological functionality of the tissue engineering scaffolds. However, decellularization is necessary to remove antigenic components in the ECM that may trigger adverse immune response. Freeze-thaw (FT) cycles and treatment with Triton X-100/ammonium hydroxide (TN) are two commonly used decellularization methods for ECM, but their effects on both growth factor retention and antigen removal are still controversial. The objectives of this study are to compare the preservation of ECM texture and beneficial ingredients and the removal of cellular antigens by these two methods. First, the constructs combined bone marrow mesenchymal stem cell-derived ECM and poly(lactic-co-glycolic acid) (PLGA) membrane are prepared and decellularized using FT and TN treatments. Moreover, the effects of decellularization on the ultrastructure and the composition of ECM-decorated PLGA membrane are compared by scanning electron microscope observation and protein quantification. Furthermore, the ECM deposited on PLGA is stripped off and then implanted subcutaneously in rats, and the host macrophage and local lymphocyte responses were investigated. Finally, ECM-decorated porous PLGA scaffolds are implanted into rat calvarial defects, and the new bone formation is evaluated. Our results showed that both methods effectively removed DNA. TN treatment partially retained collagen, glycosaminoglycan, bone morphogenetic protein-2, and vascular endothelial growth factor, and better preserved structural integrity than FT treatment. ECM implants decellularized by both methods induced a mild host response after subcutaneous implantation. Although the total content of residual DNA in the two ECMs digested by the DNA enzyme seemed to be similar and very low, the interfaces between implanted materials and natural tissues in the TN group recruited lower numbers of CD68+ macrophages, CD68+CD86+ (M1) macrophages, and CD4+ T lymphocytes than that in FT group, implying that there exist other ECM antigens to influence immune response besides DNA. Furthermore, ECM-decorated scaffolds decellularized by TN treatment induced greater bone formation than that of bare scaffolds in vivo, demonstrating the effective retention of ECM bioactive components after decellularization. This study showed that TN treatment was a more effective and safer decellularization method than FT cycles. Impact statement Decellularization is a prerequisite for extracellular matrix (ECM) application, but there is still no standard for its selection. This study demonstrated that detergent treatment was more effective than freeze-thaw (FT) cycles in removing ECM antigens besides DNA, and the prepared ECM elicited a milder allogenic immune response, which ensured the safety of ECM. Moreover, detergent better preserved the ECM integrity than FT cycles, and effectively retained growth factors, and the decellularized ECM-decorated scaffolds significantly promoted bone repair, which ensured the effectiveness of ECM. This study provides the theoretical and experimental bases for the decellularization strategy of ECM-modified tissue engineering scaffolds.

Small-diameter artery decellularization: Effects of anionic detergent concentration and treatment duration on porcine internal thoracic arteries.

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 72h 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.

Decellularization Strategies to Engineer Fibrocartilage Bioscaffolds: A Review

Fibrocartilage or known as meniscus tissues located in between the tibia and femur always subjected to extreme forces that can lead to injury especially for the sportsperson. The meniscal injury mean incidence in the general population is 66 per 100,000. The principal methods for the surgical management of fibrocartilage injury have been improvised from meniscectomy to meniscal repair and meniscal reconstruction that portrays different advantages and disadvantages in the short and long-term results. The inability to treat meniscus injury without osteoarthritis development in long-term results also motivates to find new treatment strategies. In this current era, the development of the multidisciplinary fields of tissue engineering and regenerative medicine provides new alternatives for the treatment approaches. This field involves the regeneration of the required tissue using scaffolds such as synthetic, natural, and biological scaffolds to restore the damaged one. Biological scaffolds are preferable because it tremendously mimics the native anatomical structure and has similar ratios and concentration of the proteins and growth factors that influence tissue repair and remodeling. The development of biological scaffolds with low immunogenic levels involves the decellularization process that eliminates all the cellular components while preserved the extracellular matrix (ECM) integrity and mechanics. In this review, the pros and cons of the recent decellularization strategies to engineer fibrocartilage scaffolds have been discussed. We believed that the ideal decellularization methods still need to be explored to develop suitable biological scaffolds that structurally and functionally mimic native tissue as a replacement for new tissue regeneration.

Optimizing Decellularization Strategies for the Efficient Production of Whole Rat Kidney Scaffolds.

Renal dysfunction remains a global issue, with chronic kidney disease being the 18th most leading cause of death, worldwide. The increased demands in kidney transplants, led the scientific society to seek alternative strategies, utilizing mostly the tissue engineering approaches. Unlike to perfusion decellularization of kidneys, we proposed alternative decellularization strategies to obtain acellular kidney scaffolds. The aim of this study was the evaluation of two different decellularization approaches for producing kidney bioscaffolds. Rat kidneys from Wistar rats, were submitted to decellularization, followed two different strategies. The decellularization solutions used in both approaches were the same and involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate and sodium dodecyl sulfate buffers for 12h each, followed by incubation in a serum medium. Both approaches involved 3 decellularization cycles. Histological analysis, biochemical and DNA quantification were performed. Cytotoxicity assay and repopulation of acellular kidneys were also applied. Histological, biochemical and DNA quantification confirmed that the 2nd approach had the best outcome regarding the kidney composition and cell elimination. Acellular kidneys from both approaches were successfully recellularized. Based on the above data, the production of kidney scaffolds with the proposed cost- effective decellularization approaches, was efficient.

Fabrication of an injectable acellular adipose matrix for soft tissue regeneration

Abstract The acellular adipose matrix (AAM) represents a promising source of biomaterial for applications in soft tissue regeneration. In this study, we aimed to prepare an injectable AAM to serve as a ready-to-use allograft. A fabrication procedure including harvesting, delipidating, and decellularizing was established. Accordingly, we proposed a mechanical disruption during delipidation and a final homogenization to produce the AAM powder in an injectable form. Our results demonstrated that mechanical disruption would enhance the efficiency of fat removal from the tissue and would significantly shorten the delipidation duration. A decellularization strategy composed of Tris–HCl and Sodium Dodecyl Sulfate was developed for adipose tissue. The evidence for a complete cellular remnant removal was shown through Hematoxylin and Eosin staining and DNA measurements. The AAM powder was prepared by mechanical disruption under reduced temperatures, which resulted in an AAM particle size in the range of 100 μm. In vitro cytotoxicity testing via extraction demonstrated that AAM had no toxicity on human adipose derived stem cells (hADSCs). Further experiments showed a positive support of the AAM extraction on the migration and proliferation of hADSCs, which indicated its role as a modulator for cell recruitment and proliferation during the tissue remodeling process. Subcutaneous injection of AAM presented a gradual degradation and integration with the surrounding tissues. In the following 8 week post-injection period, AAM supported the in vivo adipogenesis observed in the peripheral side along the implant. Overall, the results of this study demonstrated that the fabrication protocol for the acellular adipose matrix can be applied to injectable materials, which provide proper biocompatibility and potential applicability as off-the-shelf material for soft tissue regeneration.