Preservation Of Extracellular Matrix Research Articles

Engineering of a bilayer antibacterial wound dressing from bovine pericardium and electrospun chitosan/PVA/antibiotics for infectious skin wounds management: An in vitro and in vivo study

Tissue deterioration and post-injury infections are the primary cause of skin diseases. Tissue engineering has developed various synthetic and natural polymers to generate bioactive scaffolds that can closely replicate the natural extracellular matrix (ECM). Decellularized tissues have emerged as a potential solution for reconstructing cutaneous lesions due to their ability to preserve the intricate protein structure and provide essential functional domains for cellular differentiation. In this study, we selected bovine pericardium and subjected it to diverse decellularization methods to optimize ECM preservation. Polyvinyl alcohol (PVA)/chitosan (CS) infused with two clinically important antibiotics (colistin and meropenem) was directly electrospun onto the decellularized bovine pericardium (DBPS) to endow the dual-layer scaffold (DBPS-Abs) an antibacterial property. Both DBPS-Abs and DBPS demonstrated a consistent 3D microstructure with interlinked pore networks, minimal degradation, and robust mechanical stability. The DBPS-Abs group exhibited a potent antibacterial effect against standard and clinical strains of Escherichia coli. Moreover, implanting the constructs into full-thickness skin wounds in mice confirmed enhanced wound regeneration in cases treated with DBPS-Abs compared to other groups, observed over a 7- and 21-day post-implantation period. These findings highlight DBPS-Abs as a superior antibacterial wound dressing, requiring further clinical evaluations.

Identification of a Regenerative Protocol for Recellularizing Human Auricular Cartilage Scaffolds.

Utilizing biological scaffolds for cartilage tissue engineering is a promising tool for improving auricular reconstruction. Decellularized auricular scaffolds provide a means of regenerating cartilage for in vivo implantation, but identifying the ideal regenerative mix remains challenging. Human cadaver auricular cartilage was decellularized and recellularized with either auricular chondrocytes alone, auricular chondrocytes with adipose-derived stem cells, or both cells with platelet-rich plasma. Confirmation of decellularization and recellularization was done by hematoxylin and eosin staining. Extracellular matrix preservation and production were determined by Masson's trichrome, Alcian blue, and Verhoeff-van Gieson staining. Collagen II assessments were made using immunohistochemistry. Decellularization of cadaver auricular cartilage was confirmed by the absence of cells, reduction in glycosaminoglycans, and the preservation of collagen and elastin. Recellularization was more efficient when chondrocytes were seeded with adipose-derived stem cells, which was enhanced by adding platelet-rich plasma. Coculture with platelet-rich plasma yielded better total collagen (56% increase) and glycosaminoglycan (47% increase) induction. Moreover, when platelet-rich plasma was added, collagen II induction was significantly increased (42%; P < 0.05). We identified a regenerative protocol that included auricular chondrocytes, adipose-derived stem cells, and platelet-rich plasma, which stimulated chondrogenesis on decellularized auricular cartilage. This finding provides a model to explore cartilage formation and the potential for improving auricular and cartilage-based reconstruction.

New recellularizable cardio-matrix scaffold as a base for alternative in vitro models for the cardiotoxicity risk prediction

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): SID 227944/22 DOR 2331157/23 Background Cardiotoxicity is the occurrence of cardiac dysfunction induced by pathophysiological stimuli as a result of toxic effects. A considerable number of drugs, including chemotherapics, antibiotics antidepressants but also antiarrhythmics, could cause an alteration of cardiac physiology, and then cardiac damage. These include disturbances in ventricular repolarization and QT interval, arrhythmias, bradycardia, tachycardia, decreases in left ventricular ejection fraction, and congestive heart failure. Precinical evaluation of drug cardiotoxicity has used animal models, which tend to be expensive, have lo throughput, and have limitations as not ever faithfully reflect the human pathophysiology, while current two-dimensional cellular models revealed to be not sufficient and specific. Purpose The aim of this study is to develop a myocardial tissue-like scaffold, in order to realize an efficient and easy-replicable in vitro model for drug cardiotoxicity prediction. This must be able to mimic the morphological, cellular, and electrophysiological complexity of the intact adult human myocardium. Material and Methods Starting from fresh porcine hearts, cardiac specimens were isolated from left ventricles using a scalpel, sectioned with a vibratome and punched with a biopsy puncher obtaining myocardial patches. Thus, samples were decellularized through a novel serial decellularization treatment based on osmotic shock and detergents. The decellularization procedure exploited a new automated, dynamic perfusion device for a standardized and optimized removal of cardiac cells and non-ECM proteins. These samples were analysed for decellularization effectiveness by DNA quantification, histology and immunofluorescence staining to evaluate cell removal and extracellular matrix integrity. Moreover, they were tested for cytocompatibility using human mesenchymal stem cells and cardiac progenitors, also derived from induced pluripotent stem cells. Cellular adhesion, behaviour, vitality, proliferation, and possible apoptotic effects were tested, too. Results In order to advance a robust bioelectronics model, an automated platform was developed to generate a three-dimensional, bioengineered replica of human myocardial tissue. With respect to the common decellularization protocol characterized by mild agitation, results based on the automated, dynamic treatments have shown a superior ability to obtain acellular, biocompatible scaffolds in terms of cell elements’ removal and extracellular matrix preservation, as well as time- and cost-effectiveness. Indeed, these decellularized scaffolds allow cell penetration, adhesion and survival. Conclusions Further experiments will be focused on evaluating functional bioengineered myocardial tissues with electrophysiological and molecular analysis to evaluate this new model's throughput capacity. Finally, the validation of these bioelectronic platforms will be performed with drugs with known cardiotoxic effects.

Evaluation of Different Decellularization Protocols for Obtaining and Characterizing Canine Cardiac Extracellular Matrix.

Cardiovascular diseases are considered the leading cause of mortality globally; even with low mortality in dogs, such diseases are described in the same way in companion animals and humans. This study aimed to devise an effective decellularization protocol for the canine myocardium through the association of physical, chemical, and enzymatic methods, assessing resultant alterations in the myocardial extracellular matrix to obtain a suitable scaffold. Two canine hearts were collected; the samples were sectioned into ±1 cm2 fragments, washed in distilled water and 1× PBS solution, and followed by treatment under four distinct decellularization protocols. Sodium Dodecyl Sulfate (SDS) 1% 7 days + Triton X-100 1% for 48 h (Protocol I); Sodium Dodecyl Sulfate (SDS) 1% 5 days + Triton X-100 1% for 48 h (Protocol II); Trypsin 0.05% for 1 h at 36 °C + freezing -80 °C overnight + Sodium Dodecyl Sulfate (SDS) 1% for 3 days, Triton-X-100 for 48 h hours (Protocol III); 0.05% trypsin for 1 h at 36 °C + freezing at -80 °C overnight + 1% Sodium Dodecyl Sulfate (SDS) for 2 days + 1% Triton-X-100 for 24 h (Protocol IV). After analysis, Protocols I and II showed the removal of cellular content and preservation of extracellular matrix (ECM) contents, unlike Protocols III and IV, which retracted the ECM and removed essential elements of the matrix. In theory, although Protocols I and II have similar results, Protocol II stands out for the preservation of the architecture and components of the extracellular matrix, along with reduced exposure time to reagents, making it the recommended protocol for the development of a canine myocardial scaffold.

Histological evaluation of decellularization of freeze dried and chemically treated indigenously prepared bovine pericardium membrane.

Decellularization is regarded as a xenogenic antigen-reduction technique because it effectively eliminates all cellular and nuclear components while mitigating any negative impact on the composition, biological functionality, and structural integrity of the remaining extracellular matrix. This study aimed to histologically evaluate native, freeze dried and chemically decellularized bovine pericardium membrane. Also, this study focused on preservation of extracellular matrix after decellularization. Bovine pericardium membrane was decellularized by freeze thaw cycle followed by freeze drying and 1% sodium dodecyl sulphate. Unprocessed pericardium was used as control. The effectiveness of Decellularization was assessed based on the reduction of histologically visible nuclei. Decellularization by freeze thaw cycle followed by freeze drying resulted in 17.84% reduction in nuclei content and decellularization by sodium dodecyl sulphate results in 92% reduction in nuclei content compare to control group. Picrosirius red staining for freeze dried group displayed loosely organised, thin collagen bundles that exhibit reddish-yellow birefringence and sodium dodecyl sulfate group revealed dense collagen bundles that are parallelly organised and compact, exhibiting reddish-yellow birefringence and showed good structural integrity. These results suggested that the sodium do decyl sulfate showed optimal decellularization results with better extracellular matrix preservation. It may be a suitable protocol for producing a suitable scaffold for periodontal tissue regeneration.

Physicochemical Decellularization of Bovine Pericardium: Effects on DNA Elimination, Extracellular Matrix Preservation, and Biocompatibility

Physicochemical Decellularization of Bovine Pericardium: Effects on DNA Elimination, Extracellular Matrix Preservation, and Biocompatibility

An update on a technical method of cartilage decellularization: (a physical-based protocol)

Introduction: Despite advances in orthopedic surgery, the lack of effective conventional treatment for cartilage defects has led to research in cartilage tissue engineering. One of the interesting topics is the use of decellularized extracellular matrix (ECM) as a suitable natural scaffold that supports the growth and function of cells cultured in it. A concern with decellularization protocols, especially those with high detergent concentrations, is the disruption of native ECM, which has deleterious effects on subsequent scaffold recellularization. Therefore, this study focused on optimizing cartilage decellularization by physical methods without the use of ionic detergents. Methods: The bovine tracheal cartilage fragments were decellularized by a combination of 8 cycles of freeze-thaw and ultrasound techniques. Then, the tissues were immersed and shaken in 0.25% trypsin for 24 hours. Efficient cell removal and preservation of ECM were confirmed by histological and cytocompatibility assessments. The in-vivo studies were performed to evaluate the biocompatibility and bioactivity of the scaffold. Results: The histological assessments indicated the appropriate cytocompatibility and the fibroblast cell culture study demonstrated that cells were able to proliferate and migrate on the decellularized cartilage. In-vivo evaluation also showed a reduced adverse immune response, including leukocyte infiltration into the ECM. Conclusions: These results suggest that a cartilage scaffold created using a physical decellularization protocol that efficiently removes cells while preserving the native ECM can be a suitable scaffold for cartilage reconstruction. The main advantage of this protocol is the absence of potentially toxic chemicals in the tissues.

Reconstruction of the human nipple-areolar complex: a tissue engineering approach.

Introduction: Nipple-areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization-recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast. Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7days, while human keratinocytes were seeded on the scaffold epidermal side for 10days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis. Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10days of culture. Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization

As the only reliable treatment option for end-stage liver diseases, conventional liver transplantation confronts major supply limitations. Accordingly, the decellularization of discarded livers to produce bioscaffolds that support recellularization with progenitor/stem cells has emerged as a promising translational medicine approach. The success of this approach will substantially be determined by the extent of extracellular matrix (ECM) preservation during the decellularization process. Here, we assumed that the matrix metalloproteinase (MMP) inhibition could reduce the ECM damage during the whole liver decellularization of an animal model using a perfusion-based system. We demonstrated that the application of doxycycline as an MMP inhibitor led to significantly higher preservation of collagen, glycosaminoglycans, and hepatic growth factor (HGF) contents, as well as mechanical and structural features, including tensile strength, fiber integrity, and porosity. Notably, produced bioscaffolds were biocompatible and efficiently supported cell viability and proliferation in vitro. We also indicated that produced bioscaffolds efficiently supported HepG2 cell function upon seeding onto liver ECM discs using albumin and urea assay. Additionally, MMP inhibitor pretreated decellularized livers were more durable in contact with collagenase digestion compared to control bioscaffolds in vitro. Using zymography, we confirmed the underlying mechanism that results in these promising effects is through the inhibition of MMP2 and MMP9. Overall, we demonstrated a novel method based on MMP inhibition to ameliorate the ECM structure and composition preservation during liver decellularization as a critical step in fabricating transplantable bioengineered livers.

Abstract P2133: Preservation &amp; Vascularization Of Cardiac Extracellular Matrix After Acute Myocardial Infarction

Following myocardial infarction (MI), the upregulated matrix metalloproteinases (MMPs) especially MMP-2/9 lead to the degradation of cardiac extracellular matrix (ECM), leading to impaired cardiac function. Therefore, preservation of cardiac ECM by decreasing MMP-2/9 acitivity will increase cardiac function. Meanwhile, vascularization is critical for cardiac ECM as otherwise its structure and composition change over time. In this work, a non-toxic peptide CTTHWGFTLC (CTT) that specifically inhibits MMP-2/9, and a pro-angiogenic basic fibroblast growth factor (bFGF) were used to preserve and vascularize the cardiac ECM. A fast gelling, degradable, and reactive oxygen species (ROS)-scavenging hydrogel was developed to deliver CTT and bFGF to the infarcted hearts. We evaluated the efficacy of the delivery system in preserving and vascularizing cardiac ECM, and improving cardiac function. The combination of CTT and bFGF synergistically promoted tube formation of HUVECs compared to the treatment with CTT or bFGF (**p&lt;0.01). Interestingly, both CTT and bFGF inhibited myofibroblast differentiation of cardiac fibroblasts. The combination of CTT and bFGF further significantly decreased the differentiation. After 28 days of implantation of the CTT/bFGF/Gel, the wall thickness was significantly increased compared to the CTT/Gel, bFGF/Gel, Gel only and MI. The released CTT and bFGF preserved cardiac ECM, decreased cardiac fibrosis, and stimulated angiogenesis in the infarcted area, leading to a significant increase of cardiac function.

Effect of Pressure Conditions in Uterine Decellularization Using Hydrostatic Pressure on Structural Protein Preservation.

Uterine regeneration using decellularization scaffolds provides a novel treatment for uterine factor infertility. Decellularized scaffolds require maximal removal of cellular components and minimal damage to the extracellular matrix (ECM). Among many decellularization methods, the hydrostatic pressure (HP) method stands out due to its low cytotoxicity and superior ECM preservation compared to the traditional detergent methods. Conventionally, 980 MPa was utilized in HP decellularization, including the first successful implementation of uterine decellularization previously reported by our team. However, structural protein denaturation caused by exceeding pressure led to a limited regeneration outcome in our previous research. This factor urged the study on the effects of pressure conditions in HP methods on decellularized scaffolds. The authors, therefore, fabricated a decellularized uterine scaffold at varying pressure conditions and evaluated the scaffold qualities from the perspective of cell removal and ECM preservation. The results show that by using lower decellularization pressure conditions of 250 MPa, uterine tissue can be decellularized with more preserved structural protein and mechanical properties, which is considered to be promising for decellularized uterine scaffold fabrication applications.

Development of a novel regenerative therapy for malignant bone tumors using an autograft containing tumor inactivated by high hydrostatic pressurization (HHP).

Surgical resection of malignant bone tumors leads to significant defects in the normal surrounding tissues that should be reconstructed to avoid amputation. Our research aimed to inactivate osteosarcoma (OS)-affected bone to obtain autologous bone grafts for bone defect reconstruction using a novel therapy called high hydrostatic pressurization (HHP) therapy. The key points are complete tumor death and preservation of the non-denatured native extracellular matrix (ECM) and bone tissue by HHP. Previously, we found that HHP at 200MPa for 10min can completely inactivate cells in normal skin and skin tumors, including malignant melanoma and squamous cell carcinoma while maintaining their original biochemical properties and biological components. Based on our previous research, this study used HHP at 200MPa for 10min to eradicate OS. We prepared an OS cell line (LM8), pressurized it at 200MPa for 10min, and confirmed its inactivation through morphological observation, WST-8 assay, and live/dead assay. We then injected OS cells with or without HHP into the bone marrow of the murine tibia, after which we implanted tumor tissues with or without HHP into the anterior surface of the tibia. After HHP, OS cells did not proliferate and were assessed using a live/dead assay. The pressurized cells and tumors did not grow after implantation. The pressurized bone was well prepared as tumor-free autologous bone tissues, resulting in the complete eradication of OS. This straightforward and short-pressing treatment was proven to process the tumor-affected bone to make a transplantable and tumor-free autologous bone substitute.

Decellularized vascularized bone grafts: A preliminary in vitro porcine model for bioengineered transplantable bone shafts.

Introduction: Durable reconstruction of critical size bone defects is still a surgical challenge despite the availability of numerous autologous and substitute bone options. In this paper, we have investigated the possibility of creating a living bone allograft, using the perfusion/decellularization/recellularization (PDR) technique, which was applied to an original model of vascularized porcine bone graft. Materials and Methods: 11 porcine bone forelimbs, including radius and ulna, were harvested along with their vasculature including the interosseous artery and then decellularized using a sequential detergent perfusion protocol. Cellular clearance, vasculature, extracellular matrix (ECM), and preservation of biomechanical properties were evaluated. The cytocompatibility and in vitro osteoinductive potential of acellular extracellular matrix were studied by static seeding of NIH-3T3 cells and porcine adipose mesenchymal stem cells (pAMSC), respectively. Results: The vascularized bone grafts were successfully decellularized, with an excellent preservation of the 3D morphology and ECM microarchitecture. Measurements of DNA and ECM components revealed complete cellular clearance and preservation of ECM's major proteins. Bone mineral density (BMD) acquisitions revealed a slight, yet non-significant, decrease after decellularization, while biomechanical testing was unmodified. Cone beam computed tomography (CBCT) acquisitions after vascular injection of barium sulphate confirmed the preservation of the vascular network throughout the whole graft. The non-toxicity of the scaffold was proven by the very low amount of residual sodium dodecyl sulfate (SDS) in the ECM and confirmed by the high live/dead ratio of fibroblasts seeded on periosteum and bone ECM-grafts after 3, 7, and 16days of culture. Moreover, cell proliferation tests showed a significant multiplication of seeded cell populations at the same endpoints. Lastly, the differentiation study using pAMSC confirmed the ECM graft's potential to promote osteogenic differentiation. An osteoid-like deposition occurred when pAMSC were cultured on bone ECM in both proliferative and osteogenic differentiation media. Conclusion: Fully decellularized bone grafts can be obtained by perfusion decellularization, thereby preserving ECM architecture and their vascular network, while promoting cell growth and differentiation. These vascularized decellularized bone shaft allografts thus present a true potential for future in vivo reimplantation. Therefore, they may offer new perspectives for repairing large bone defects and for bone tissue engineering.

Effects of Different Perfusing Routes through The Portal Vein, Hepatic Vein, and Biliary Duct on Whole Rat Liver Decellularization.

Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through three anatomical routes. In this experimental study, rat liver decellularization was performed through biliary duct (BD), portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs). ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines. Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD route produced an opaque scaffold with incomplete decellularization. H and E staining confirmed these results. Maximum DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay showed the biocompatibility of the decellularized scaffold. The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.

Preservation of Extracellular Matrix in Decellularized Human Auricular Cartilage for Recellularization

Preservation of Extracellular Matrix in Decellularized Human Auricular Cartilage for Recellularization

An efficient strategy to recellularization of a rat aorta scaffold: an optimized decellularization, detergent removal, and Apelin-13 immobilization

BackgroundTissue engineering of native vessels is an alternative approach for patients with vascular disease who lack sufficient saphenous vein or other suitable conduits for autologous vascular graft. Moreover, the harvest of vessels prolongs the surgical procedure and it may lead to the morbidity of donor site in elder patients: therefore, it seems that the use of tissue-engineered vessels would be an attractive and less invasive substitute for autologous vascular grafts. Apelin-13 plays a pivotal role in cell proliferation, survival, and attachment; therefore, covalent attachment of apelin-13 to the acellular scaffolds might be a favorable approach for improving recellularization efficacy.MethodsIn the present study, the decellularization process was performed using various detergents. Afterward, the efficacy of decellularization procedure was evaluated using multiple approaches including assessment of DNA, hydroxyproline, and GAG content as well as Masson’s trichrome and orcein staining used for collagen and elastin determination. Subsequently, the scaffold was bioconjugated with apelin-13 using the EDC-NHS linker and acellular scaffolds were recellularized using fibroblasts, endothelial cells, and smooth muscle cells. SEM images and characterization methods were also used to evaluate the effect of apelin-13 attachment to the acellular scaffold on tissue recellularization. We also developed a novel strategy to eliminate the remnant detergents from the scaffold and increase cell viability by incubating acellular scaffolds with Bio-Beads SM-2 resin. Testometric tensile testing machine was also used for the assessment of mechanical properties and uniaxial tensile strength of decellularized and recellularized vessels compared to that of native tissues.ResultsOur results proposed 16-h perfusion of 0.25% sodium dodecyl sulfate (SDS) + 0.5% Triton X-100 combination to the vessel as an optimal decellularization protocol in terms of cell elimination as well as extracellular matrix preservation. Furthermore, the results demonstrated considerable elevation of cell adhesion and proliferation in scaffolds bioconjugated with apelin-13. The immunohistochemical (IHC) staining of CD31, α-SMA, and vimentin markers suggested placement of seeded cells in the suitable sites and considerable elevation of cell attachment within the scaffolds bioconjugated with apelin-13 compared to the non-bioconjugated, and decellularized groups. Moreover, the quantitative analysis of IHC staining of CD31, α-SMA, and vimentin markers suggested considerable elevation in the number of endothelial, smooth muscle, and fibroblast cells in the recellularized scaffolds bioconjugated with apelin-13 group (1.4% ± 0.02, 6.66% ± 0.23, and 9.87% ± 0.13%, respectively) compared to the non-bioconjugated scaffolds (0.03% ± 0.01, 0.28% ± 0.01, and 1.2% ± 0.09%, respectively) and decellularized groups (0.03% ± 0.007, 0.05% ± 0.01, and 0.13% ±0.005%, respectively). Although the maximum strain to the rupture was reduced in tissues decellularized using 0.5% SDS and CHAPS compared to that of native ones (116% ± 6.79, 139.1% ± 3.24, and 164% ± 8.54%, respectively), ultimate stress was decreased in all decellularized and recellularized groups. Besides, our results indicated that cell viability on the 1st, 3rd, and 7th day was 100.79% ± 0.7, 100.34% ± 0.08, and 111.24% ± 1.7% for the decellularized rat aorta conjugated with apelin-13, which was incubated for 48-h with Bio-Beads SM-2, and 73.37% ± 7.99, 47.6% ± 11.69, and 27.3% ± 7.89% for decellularized rat aorta scaffolds conjugated with apelin-13 and washed 48-h by PBS, respectively. These findings reveal that the incubation of the scaffold with Bio-Beads SM-2 is a novel and promising approach for increasing cell viability and growth within the scaffold.ConclusionsIn conclusion, our results provide a platform in which xenograft vessels are decellularized properly in a short time, and the recellularization process is significantly improved after the bioconjugation of the acellular scaffold with apelin-13 in terms of cell adhesion and viability within the scaffold.Graphical

Three-dimensional in vitro maturation of rabbit oocytes enriched with sheep decellularized greater omentum.

BackgroundTo 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.ObjectivesThe 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.MethodsCumulus‐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.ResultsDNA 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.ConclusionsGOM supplementation has a beneficial impact on oocyte maturation, probably due to the presence of growth factors and proteins.

A novel method for providing scaffold: Decellularization of parathyroid capsule

This study aimed to generate a novel biomatrix from the decellularized human parathyroid capsule using different methods and to compare the efficiency of decellularization in the means of cell removal, structural integrity and extracellular matrix preservation. The parathyroid capsules, which were carefully dissected from the parathyroid tissue, were randomly divided into four groups and then decellularized using three different protocols: freeze-thaw only, sodium dodecyl sulphate and Triton X-100 treatments after freeze-thawing. Quantitative DNA analysis, agarose gel electrophoresis, sulphated glycosaminoglycan assay, histological analysis, immunohistochemistry and scanning electron microscopy were used to observe the efficiency of parathyroid capsule decellularization and preservation of extracellular matrix components. Considering all the results, it can be said that only freeze-thawing is not an effective method in parathyroid capsule decellularization. When the tissue was treated with a detergent agent in addition to freeze-thawing, the amount of DNA decreased by 90% while sulphated glycosaminoglycan amount maintained 50% compared to untreated tissue. Comparing the effects of the two detergents on the preservation of extracellular matrix such as collagen and sulphated glycosaminoglycan, it was seen that the integrity of tissues treated with Triton X-100 was preserved more than tissues treated with sodium dodecyl sulphate. It is concluded that Triton X-100 treatment with freeze-thawing is the most suitable and effective method for decellularizing the human parathyroid capsule. The biomatrix obtained with this method can be applied in the transplantation of parathyroid tissue and other endocrine tissue types in the body.

Interindividual heterogeneity affects the outcome of human cardiac tissue decellularization

The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold’s intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Decellularization of kidney tissue: comparison of sodium lauryl ether sulfate and sodium dodecyl sulfate for allotransplantation in rat.

An automatic decellularization device was developed to perfuse and decellularize male rats' kidneys using both sodium lauryl ether sulfate (SLES) and sodium dodecyl sulfate (SDS) and to compare their efficacy in kidney decellularization and post-transplantation angiogenesis. Kidneys were perfused with either 1% SDS solution for 4h or 1% SLES solution for 6h. The decellularized scaffolds were stained with hematoxylin and eosin, periodic acid Schiff, Masson's trichrome, and Alcian blue to determine cell removal and glycogen, collagen, and glycosaminoglycan contents, respectively. Moreover, scanning electron microscopy was performed to evaluate the cell removal and preservation of microarchitecture of both SDS and SLES scaffolds. Additionally, DNA quantification assay was applied for all groups in order to measure residual DNA in the scaffolds and normal kidney. In order to demonstrate biocompatibility of the decellularized scaffolds, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were seeded on the scaffolds. In addition, the allotransplantation was performed in back muscle and angiogenesis was evaluated. Complete cell removal in both SLES and SDS groups was observed in scanning electron microscopy and DNA quantification assays. Moreover, the extracellular matrix (ECM) architecture of rat kidney in the SLES group was significantly preserved better than the SDS group. The hUC-MSCs were successfully migrated from the cell culture plate surface into the SDS and SLES decellularized scaffolds. The formation of blood vessels was observed in the kidney in both SLES and SDS decellularized kidneys. The better preservation of ECM than SDS introduces SLES as the solvent of choice for kidney decellularization.