Decellularization Procedure Research Articles

In Vitro Spermatogenesis on Human Decellularized Testicular Matrix Plates Following Exosome Treatment in a Dynamic Culture System.

Testicular tissue engineering for in vitro spermatogenesis aims to restore fertility, focusing on challenges like efficiency, ethical concerns, and the need for a deeper biological understanding. The use of decellularized scaffolds led to better cell seeding and differentiation, and exosomes led to enhanced spermatogenesis. Also, the dynamic culture systems are being explored to replicate in vivo conditions more accurately. In this study, we aimed to utilize a perfusion mini-bioreactor for the dynamic culture of mouse spermatogonial stem cells on decellularized testicular matrix plates supplemented with exosomes. Our goal was to assess the progression of the spermatogenesis process through histological, immunohistochemical, and molecular analyses over four weeks. Human testicular tissues were decellularized using 1% sodium dodecyl sulfate and were then fabricated into thin plates using a cryostat. Sertoli and spermatogonial stem cells were isolated from neonate mouse testis and seeded onto the decellularized testicular matrix plates. A mini-perfusion bioreactor was employed to create dynamic culture conditions. Also, MSCs-derived exosomes were introduced to the culture medium, alone or in combination with a spermatogenic medium containing numerous chemical factors. The histological, IHC, and molecular analyses were performed at the end of the experiment. Our decellularization procedure successfully preserved the ECM components, while eliminating native cells. The isolated cells expressed PLZF and VIMENTIN markers, confirming the presence of SSCs and Sertoli cells. The seeded scaffolds exhibited proper homing, viability, proliferation, and differentiation of the cells towards in vitro spermatogenesis. Also, exosome treatment is capable of enhancing the spermatogenic potential of SSCs. Our findings indicate that the dynamic culture system significantly promoted the proliferation and differentiation of SSCs into mature spermatozoa. The use of exosomes further enhanced these effects, as evidenced by improved cellular viability, reduced apoptosis, and advanced spermatogenesis to the elongated spermatid stage. The combined treatment of exosomes and spermatogenic medium showed a synergistic effect, yielding superior outcomes in terms of sperm cell maturity and functionality. This study underscores the potential of combining decellularized testicular matrices with exosome therapy in a dynamic culture set up to advance the field of reproductive biology and fertility restoration.

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.

Retinoic acid-releasing scaffold based on chitosan hydrogel and testis decellular plates

Introduction: The use of releasing scaffolds is promising for testes tissue engineering. Chitosan (CS) is a natural biopolymer extensively used as a delivery system. The decellularized testis provides a structure resembling natural extracellular matrix (ECM). All-trans retinoic acid (atRA) is an important factor for spermatogonia differentiation, meiosis completion, and mature sperm release. In this study, thermosensitive CS/βGP hydrogel was served as a novel atRA-releasing support for testis decellular plates (TDPs). Methods: The CS/βGP hydrogel was evaluated for gelation time, morphology, wettability, cytocompatibility, and atRA-releasing behavior. Mouse testes were treated with 1% SDS and evaluated for decellularization efficacy through morphological assessments, DNA content assays, and DAPI staining. TDPs were obtained from the decellularized testes and placed on an atRA-releasing CS/βGP hydrogel support. Results: The CS/βGP hydrogels were prepared with different formulations. It was found that increasing the βGP concentration significantly decreased the gelation time. The addition of atRA did not considerably affect the hydrophilicity of hydrogel. The in vitro release studies showed a sustained atRA release behavior, although an initial low burst release was recorded. Also, increasing the amount of atRA led to a decrease in the rate of drug release. The decellularization procedure successfully removed cells while preserving the ECM. The atRA-releasing CS-TDP scaffold was found to be non-toxic with good biocompatibility. Conclusion: Results showed that the novel atRA-releasing CS-TDP scaffold can sustainably deliver atRA to the culture system and create a cytocompatible environment for testicular cells. Therefore, this scaffold may be useful in developing new tissue engineering approaches for various types of male infertility diseases.

Construction of dental pulp decellularized matrix by cyclic lavation combined with mechanical stirring and its proteomic analysis

The decellularized matrix has a great potential for tissue remodeling and regeneration; however, decellularization could induce host immune rejection due to incomplete cell removal or detergent residues, thereby posing significant challenges for its clinical application. Therefore, the selection of an appropriate detergent concentration, further optimization of tissue decellularization technique, increased of biosafety in decellularized tissues, and reduction of tissue damage during the decellularization procedures are pivotal issues that need to be investigated. In this study, we tested several conditions and determined that 0.1% Sodium dodecyl sulfate and three decellularization cycles were the optimal conditions for decellularization of pulp tissue. Decellularization efficiency was calculated and the preparation protocol for dental pulp decellularization matrix (DPDM) was further optimized. To characterize the optimized DPDM, the microstructure, odontogenesis-related protein and fiber content were evaluated. Our results showed that the properties of optimized DPDM were superior to those of the non-optimized matrix. We also performed the 4D-Label-free quantitative proteomic analysis of DPDM and demonstrated the preservation of proteins from the natural pulp. This study provides a optimized protocol for the potential application of DPDM in pulp regeneration.

Liquid nitrogen improves the decellularization effectiveness of whole-ovary

BACKGROUND: Ovarian tissue cryopreservation for fertility preservation carries a risk of malignant cell re-seeding. Artificial ovary is a promising method to solve such a problem. However, ovary decellularization protocols are limited. Hence, further studies are necessary to get better ovarian decellularization techniques for the construction of artificial ovary scaffolds. OBJECTIVE: To establish an innovative decellularization technique for whole porcine ovaries by integrating liquid nitrogen with chemical agents to reduce the contact time between the scaffolds and chemical reagents. MATERIALS AND METHODS: Porcine ovaries were randomly assigned to three groups: novel decellularized group, conventional decellularized group and fresh group. The ovaries in the novel decellularized group underwent three cycles of freezing by liquid nitrogen and thawing at temperatures around 37??C before decellularization. The efficiency of the decellularization procedure was assessed through histological staining and DNA content analysis. The maintenance of ovarian decellularized extracellular matrix(ODECM) constituents was determined by analyzing the content of matrix proteins. Additionally, we evaluated the biocompatibility of the decellularized extracellular matrix(dECM) by observing the growth of granulosa cells on the ODECM scaffold in vitro.RESULTS: Hematoxylin and eosin staining, DAPI staining and DNA quantification techniques collectively confirm the success of the novel decellularization methods in removing cellular and nuclear components from ovarian tissue. Moreover, quantitative assessments of ODECM contents revealed that the novel decellularization technique preserved more collagen and glycosaminoglycan compared to the conventional decellularized group (P<0.05). Additionally, the novel decellularized scaffold exhibited a significantly higher number of granulosa cells than the conventional scaffold during in vitro co-culture (P<0.05). CONCLUSION: The novel decellularized method demonstrated high efficacy in eliminating DNA and cellular structures while effectively preserving the extracellular matrix. As a result, the novel decellularized method holds significant promise as a viable technique for ovarian decellularization in forthcoming studies.

The essential role of aligned architecture in decellularized tendon matrix mediated stem cell tenogenic differentiation and tendon repair

Decellularized tendon matrix (DTM) has shown great potential for inducing tenogenic differentiation and facilitating tendon repair. In which, the retained native tendon-derived matrix in DTM is considered as the main factor for these effects. However, whether preservation of the highly aligned architecture of DTM is also essential for tenogenic differentiation and tendon repair has not been well investigated yet. In this study, we prepared aligned DTM (A-DTM), in which the native aligned architecture was preserved and random DTM (R-DTM), in which the native architecture was disrupted. In vitro study indicated that after decellularization procedures, both A- and R-DTM retained most of the tendon extracellular matrix (ECM). In addition, A-DTM exhibited an inherent aligned architecture, whilst, R-DTM showed a random arranged surface topography. Both DTM materials exhibited good biocompatibility for tendon stem/progenitor cells (TSPCs) and adipose stem cells (ASCs). They were able to adhere and proliferate on the DTM materials and maintain high viability. Interestingly, A-DTM promoted longitudinally oriented growth pattern of TSPCs and ASCs, whereas, cells grown on the R-DTM showed a random spreading. Furthermore, both DTM materials induced tenogenic differentiation of TSPCs and ASCs without the addition of any exotic growth factors. However, A-DTM demonstrated a stronger pro-tendon differentiation compared to R-DTM. In vivo study demonstrated that A-DTM combined with TSPCs or ASCs significantly promoted functional repair of injured Achilles tendon while reducing fibrovascular scar and heterotopic ossification (HO) formation. Taken together, these results indicated that aligned architecture of DTM is essential for stem cell tenogenic differentiation and tendon repair. Thus, for the application of DTM, it is very important to preserve its native aligned architecture. In another scenario, when DTM was used to prepared a composite biomaterial, a tendon-mimic topography should be re-constructed.

Decellularization and Their Significance for Tissue Regeneration in the Era of 3D Bioprinting.

Three-dimensional bioprinting is an emerging technology that has high potential application in tissue engineering and regenerative medicine. Increasing advancement and improvement in the decellularization process have led to an increase in the demand for using a decellularized extracellular matrix (dECM) to fabricate tissue engineered products. Decellularization is the process of retaining the extracellular matrix (ECM) while the cellular components are completely removed to harvest the ECM for the regeneration of various tissues and across different sources. Post decellularization of tissues and organs, they act as natural biomaterials to provide the biochemical and structural support to establish cell communication. Selection of an effective method for decellularization is crucial, and various factors like tissue density, geometric organization, and ECM composition affect the regenerative potential which has an impact on the end product. The dECM is a versatile material which is added as an important ingredient to formulate the bioink component for constructing tissue and organs for various significant studies. Bioink consisting of dECM from various sources is used to generate tissue-specific bioink that is unique and to mimic different biometric microenvironments. At present, there are many different techniques applied for decellularization, and the process is not standardized and regulated due to broad application. This review aims to provide an overview of different decellularization procedures, and we also emphasize the different dECM-derived bioinks present in the current global market and the major clinical outcomes. We have also highlighted an overview of benefits and limitations of different decellularization methods and various characteristic validations of decellularization and dECM-derived bioinks.

Developing Porcine Acellular Dermal Matrix by Using Sodium Dodecyl Sulfate and Biomechanical Property Testing

ABSTRACT Introduction: An alternative for supporting wound closure is acellular dermal matrix (ADM), which serves as a scaffold. Humans and porcine possess a similar biochemical makeup. Using sodium dodecyl sulfate (SDS), a decellularization technique was developed and its biomechanical properties were assessed. Methods: This work uses a pig dermis layer for an in vitro experimental investigation with a posttest-only control group. Using SDS 0.5% for 14 days, the decellularization procedure compares the biomechanical properties and cellular components of the ADM with control. The Mann–Whitney U-test for data with a nonnormal distribution or the t-test for continuous variables with a normal distribution was used for the study. Results: Histological analysis revealed that none of the cells were detected in four fields of analysis in the treatment group; however, 48.00 ± 4.86 cells were observed in the control group (P < 0.001); the collagen organization in the control group appeared to be identical. The variables elastic modulus (MPa) (136.78 vs. 129.19; P = 0.556), thickness (mm) (3.27 vs. 3.15; P = 0.397), and width (mm) (8.50 vs. 8.56; P = 0.40) did not differ statistically. The following data showed significant differences between the treatment group and the control group: break strain (%) (108.46 vs. 67.48; P < 0.001) and tensile strength stress (MPa) (19.916 vs. 22.1; P = 0.030). Conclusions: SDS decellularization is an efficient method for creating an ADM. Although the break strain was considerably lower, the treatment group’s tensile strength was higher. Elastic modulus changes were not observed.

Using a Xenogeneic Acellular Dermal Matrix Membrane to Enhance the Reparability of Bone Marrow Mesenchymal Stem Cells for Cartilage Injury.

Due to its avascular organization and low mitotic ability, articular cartilage possesses limited intrinsic regenerative capabilities. The aim of this study is to achieve one-step cartilage repair in situ via combining bone marrow stem cells (BMSCs) with a xenogeneic Acellular dermal matrix (ADM) membrane. The ADM membranes were harvested from Sprague-Dawley (SD) rats through standard decellularization procedures. The characterization of the scaffolds was measured, including the morphology and physical properties of the ADM membrane. The in vitro experiments included the cell distribution, chondrogenic matrix quantification, and viability evaluation of the scaffolds. Adult male New Zealand white rabbits were used for the in vivo evaluation. Isolated microfracture was performed in the control (MF group) in the left knee and the tested ADM group was included as an experimental group when an ADM scaffold was implanted through matching with the defect after microfracture in the right knee. At 6, 12, and 24 weeks post-surgery, the rabbits were sacrificed for further research. The ADM could adsorb water and had excellent porosity. The bone marrow stem cells (BMSCs) grew well when seeded on the ADM scaffold, demonstrating a characteristic spindle-shaped morphology. The ADM group exhibited an excellent proliferative capacity as well as the cartilaginous matrix and collagen production of the BMSCs. In the rabbit model, the ADM group showed earlier filling, more hyaline-like neo-tissue formation, and better interfacial integration between the defects and normal cartilage compared with the microfracture (MF) group at 6, 12, and 24 weeks post-surgery. In addition, neither intra-articular inflammation nor a rejection reaction was observed after the implantation of the ADM scaffold. This study provides a promising biomaterial-based strategy for cartilage repair and is worth further investigation in large animal models.

Effect of different decellularization protocols on reendothelialization with human cells for a perfused renal bioscaffold of the rat

BackgroundScaffolds for tissue engineering can be received by whole organ decellularization while maintaining the site-specific extracellular matrix and the vascular tree. One among other decellularization techniques is the perfusion-based method using specific agents e.g. SDS for the elimination of cellular components. While SDS can disrupt the composition of the extracellular matrix and impair the adherence and growth of site-specific cells there are indications that xenogeneic cell types may benefit from protein denaturation by using higher detergent concentrations. The aim of this work is to investigate the effect of two different SDS-concentrations (i.e. 0.66% and 3%) on the ability of human endothelial cells to adhere and proliferate in an acellular rat kidney scaffold.Material and methodsAcellular rat kidney scaffold was obtained by perfusion-based decellularization through the renal artery using a standardized protocol including SDS at concentrations of 0.66% or 3%. Subsequently cell seeding was performed with human immortalized endothelial cells EA.hy 926 via the renal artery. Recellularized kidneys were harvested after five days of pressure-controlled dynamic culture followed sectioning, histochemical and immunohistochemical staining as well as semiquantitative analysis.ResultsEfficacy of decellularization was verified by absence of cellular components as well as preservation of ultrastructure and adhesive proteins of the extracellular matrix. In semiquantitative analysis of recellularization, cell count after five days of dynamic culture more than doubled when using the gentle decellularization protocol with a concentration of SDS at 0.66% compared to 3%. Detectable cells maintained their endothelial phenotype and presented proliferative behavior while only a negligible fraction underwent apoptosis.ConclusionRecellularization of acellular kidney scaffold with endothelial cells EA.hy 926 seeded through the renal artery benefits from gentle decellularization procedure. Because of that, decellularization with a SDS concentration at 0.66% should be preferred in further studies and coculture experiments.

Extracellular matrix-derived scaffolds in constructing artificial ovaries for ovarian failure: a systematic methodological review.

What is the current state-of-the-art methodology assessing decellularized extracellular matrix (dECM)-based artificial ovaries for treating ovarian failure? Preclinical studies have demonstrated that decellularized scaffolds support the growth of ovarian somatic cells and follicles both in vitro and in vivo. Artificial ovaries are a promising approach for rescuing ovarian function. Decellularization has been applied in bioengineering female reproductive tract tissues. However, decellularization targeting the ovary lacks a comprehensive and in-depth understanding. PubMed, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials were searched from inception until 20 October 2022 to systematically review all studies in which artificial ovaries were constructed using decellularized extracellular matrix scaffolds. The review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol. Two authors selected studies independently based on the eligibility criteria. Studies were included if decellularized scaffolds, regardless of their species origin, were seeded with ovarian cells or follicles. Review articles and meeting papers were removed from the search results, as were articles without decellularized scaffolds or recellularization or decellularization protocols, or control groups or ovarian cells. The search returned a total of 754 publications, and 12 papers were eligible for final analysis. The papers were published between 2015 and 2022 and were most frequently reported as coming from Iran. Detailed information on the decellularization procedure, evaluation method, and preclinical study design was extracted. In particular, we concentrated on the type and duration of detergent reagent, DNA and extracellular matrix detection methods, and the main findings on ovarian function. Decellularized tissues derived from humans and experimental animals were reported. Scaffolds loaded with ovarian cells have produced estrogen and progesterone, though with high variability, and have supported the growth of various follicles. Serious complications have not been reported. A meta-analysis could not be performed. Therefore, only data pooling was conducted. Additionally, the quality of some studies was limited mainly due to incomplete description of methods, which impeded specific data extraction and quality analysis. Several studies that used dECM scaffolds were performed or authored by the same research group with a few modifications, which might have biased our evaluation. Overall, the decellularization-based artificial ovary is a promising but experimental choice for substituting insufficient ovaries. A generic and comparable standard should be established for the decellularization protocols, quality implementation, and cytotoxicity controls. Currently, decellularized materials are far from being clinically applicable to artificial ovaries. This study was funded by the National Natural Science Foundation of China (Nos. 82001498 and 81701438). The authors have no conflicts of interest to declare. This systematic review is registered with the International Prospective Register of Systematic Reviews (PROSPERO, ID CRD42022338449).

Characterization of three washing/decellularization procedures for the production of bioactive human micronized neural tissue (hMINT).

We developed a novel, injectable and decellularized human peripheral nerve-based scaffold, named Micronized Human Neural Tissue (hMINT), designed to be used as a supportive matrix for stem cell transplantation in the context of spinal cord injury (SCI). Human donated sciatic nerves were micronized at liquid nitrogen temperature prior to decellularization using 3 different procedures of various harshness. hMINT were characterized in terms of particle size, DNA, sulfated glycosaminoglycans (sGAG) and growth factors content. To test the biocompatibility and bioactivity of the various preparations, we used a type of mesenchymal stromal cells (MSCs), termed MIAMI cells, which were placed in contact with hMINT to monitor cell attachment by confocal microscopy and gene expression by RT-qPCR in vitro. The content of DNA, sGAG and growth factors left in the product after processing was highly dependent on the decellularization procedure used. We demonstrated that hMINT are biocompatible and promoted the attachment and long-term survival of MIAMI cells in vitro. Finally, combination with hMINT increased MIAMI cells mRNA expression of pro-survival and anti-inflammatory factors. Importantly, the strongest bioactivity on MIAMI cells was observed with the hMINT decellularized using the mildest decellularization procedure, therefore emphasizing the importance of achieving an adequate decellularization without losing the hMINT's bioactivity. The capacity of hMINT/stem cells to facilitate protection of injured neural tissue, promote axon re-growth and improve functional recovery will be tested in an animal model of SCI and other neurodegenerative disorders in the future.

Hemocompatibility tuning of an innovative glutaraldehyde-free preparation strategy using riboflavin/UV crosslinking and electron irradiation of bovine pericardium for cardiac substitutes

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.

Strategies for improving the 3D printability of decellularized extracellular matrix bioink.

3D bioprinting is a revolutionary technology capable of replicating native tissue and organ microenvironments by precisely placing cells into 3D structures using bioinks. However, acquiring the ideal bioink to manufacture biomimetic constructs is challenging. A natural extracellular matrix (ECM) is an organ-specific material that provides physical, chemical, biological, and mechanical cues that are hard to mimic using a small number of components. Organ-derived decellularized ECM (dECM) bioink is revolutionary and has optimal biomimetic properties. However, dECM is always "non-printable" owing to its poor mechanical properties. Recent studies have focused on strategies to improve the 3D printability of dECM bioink. In this review, we highlight the decellularization methods and procedures used to produce these bioinks, effective methods to improve their printability, and recent advances in tissue regeneration using dECM-based bioinks. Finally, we discuss the challenges associated with manufacturing dECM bioinks and their potential large-scale applications.

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.

Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep.

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve.

Preparation and Characterization of Extracellular Matrix Hydrogels Derived from Acellular Cartilage Tissue.

Decellularized matrices can effectively reduce severe immune rejection with their cells and eliminated nucleic acid material and provide specific environments for tissue repair or tissue regeneration. In this study, we prepared acellular cartilage matrix (ACM) powder through the decellularization method and developed ACM hydrogels by physical, chemical, and enzymatic digestion methods. The results demonstrated that the small size group of ACM hydrogels exhibited better gel conditions when the concentration of ACM hydrogels was 30 and 20 mg/mL in 1N HCl through parameter adjustment. The data also confirmed that the ACM hydrogels retained the main components of cartilage: 61.18% of glycosaminoglycan (GAG) and 78.29% of collagen, with 99.61% of its DNA removed compared to samples without the decellularization procedure (set as 100%). Through turbidimetric gelation kinetics, hydrogel rheological property analysis, and hydrogel tissue physical property testing, this study also revealed that increasing hydrogel concentration is helpful for gelation. Besides, the ex vivo test confirmed that a higher concentration of ACM hydrogels had good adhesive properties and could fill in cartilage defects adequately. This study offers useful information for developing and manufacturing ACM hydrogels to serve as potential alternative scaffolds for future cartilage defect treatment.

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

A Preliminary Study of Constructing the Tissue-Engineered Corpus Cavernosum With Autologous Adipose Stem Cells In Vivo.

IntroductionThe autologous skin flap is still the mainstream method for penile reconstruction, but it is very difficult to reconstruct a functional corpus cavernosum. Tissue engineering provides a new idea aiming to restore the damaged or absent corpus cavernosum.AimTo assess the feasibility of constructing the tissue-engineered corpus cavernosum with autologous adipose stem cells in a rabbit model.MethodsA total of 30 New Zealand male white rabbits. Among them, 20 rabbits were used to obtain the original corpus cavernosum which were used to prepare the acellular corporal scaffolds (ACSs). The others were used for acquiring autologous adipose stem cells (ADSCs) and constructing tissue-engineered corpus cavernosum in vivo.OutcomeACSs were obtained from rabbit penile tissues through an established decellularization procedure. Rabbit autologous ADSCs as seed cells were harvested and expanded. The ADSCs seeded and unseeded ACSs were implanted back into the intramuscular and subcutaneous site in vivo, and the tissue-engineered corpus cavernosum was harvested and analyzed with gross morphology, histological staining, and real-time PCR assay after 1, 3, and 6 months.ResultsACSs were successfully prepared. The cell non-cytotoxicity and integrity of micro-architecture of ACSs was confirmed in vitro. The cell-seeded scaffold in the intramuscular group was considered as the better strategy for constructing the tissue-engineered corpus cavernosum compared with the other groups. Some α-SMA and CD31 positive cells were detected and identified by immunofluorescent staining and real-time PCR assay in the tissue-engineered corpus cavernosum.Clinical TranslationThis study provides a new method for constructing the tissue-engineered corpus cavernosum.Strengths and LimitationsFirst, it is urgent to improve the transformation rate of the endothelial cells and smooth muscle cells from ADSCs. Second, the scaffold harvested in this study was not a complete matrix. Third, further study is needed to explore the potential mechanism of which scaffolds are more suitable for living in intramuscular rather than subcutaneous environment.ConclusionIn this study, we used the autologous ADSCs as seed cells, the acellular corpus cavernosum as scaffolds, and implanted the grafts back into the rabbit model to preliminarily construct the tissue-engineered corpus cavernosum. This study would provide help for further development in tissue-engineered corpus cavernosum.Cao Z, Liu L, Jiao H, et al. A Preliminary Study of Constructing the Tissue-Engineered Corpus Cavernosum With Autologous Adipose Stem Cells In Vivo. Sex Med 2022;10:100563.

Effects of decellularized extracellular matrix derived from Jagged1-treated human dental pulp stem cells on biological responses of stem cells isolated from apical papilla.

Objective: Indirect Jagged1 immobilization efficiently activates canonical Notch signaling in human dental pulp stem cells (hDPSCs). This study aimed to investigate the characteristics of the Jagged1-treated hDPSC-derived decellularized extracellular matrix (dECM) and its biological activity on odonto/osteogenic differentiation of stem cells isolated from apical papilla (SCAPs). Methods: Bioinformatic database of Jagged1-treated hDPSCs was analyzed using NetworkAnalyst. hDPSCs seeded on the Jagged1 immobilized surface were maintained with normal or osteogenic induction medium (OM) followed by decellularization procedure, dECM-N, or dECM-OM, respectively. SCAPs were reseeded on each dECM with either the normal medium or the OM. Cell viability was determined by MTT assay. Characteristics of dECMs and SCAPs were evaluated by SEM, EDX, immunofluorescent staining, and alcian blue staining. Mineralization was assessed by alizarin red S, Von Kossa, and alkaline phosphatase staining. Statistical significance was considered at p < 0.05. Results: RNA-seq database revealed upregulation of several genes involved in ECM organization, ECM–receptor interaction, and focal adhesion in Jagged1-treated hDPSCs. Immobilized Jagged1 increased the osteogenesis of the hDPSC culture with OM. dECMs showed fibrillar-like network structure and maintained major ECM proteins, fibronectin, type I-collagen, and glycosaminoglycans. A decrease in calcium and phosphate components was observed in dECMs after the decellularized process. Cell viability on dECMs did not alter by 7 days. Cell attachment and f-actin cytoskeletal organization of SCAPs proliferated on Jagged1-treated dECMs were comparable to those of the control dECMs. SCAPs exhibited significantly higher mineralization on dECM-N in OM and markedly enhanced on dECM-OM with normal medium or OM conditions. Conclusion: Jagged1-treated hDPSC-derived dECMs are biocompatible and increase odonto/osteogenic differentiation of SCAPs. The results suggested the potential of Jagged1 dECMs, which could be further developed into ECM scaffolds for application in regenerative medicine.