Residual DNA Content Research Articles

Vascraft: new decellularized and re-endothelialized human vascular graft produced by tissue engineering for use in arterial bypass

Abstract Introduction Small-diameter vascular grafts are crucial for patients undergoing coronary artery bypass surgery. Inherent comorbidities and the absence of optimal autologous vessels have prompted the development of vascular grafts through tissue engineering. The objective of the study is to develop a human biological substitute, VasCraft, for use as a coronary bypass graft. Methods VasCraft is an inter-institutional project carried out in four phases (F1–F4) for the design, development, and production of the bioimplant.F1: Decellularization of human cadaveric saphenous veins. The optimal decellularization protocol was identified by evaluating extracellular matrix (ECM) preservation, residual DNA content, morphohistological structure, and cytotoxicity.F2: Reendothelialization using endothelial cells derived from Wharton’s jelly mesenchymal stem cells (EC-MSC, WJ) in 3D bioreactors.F3: Generation of an Advanced Therapy Medicinal Product (ATMP) under Good Manufacturing Practice (GMP) conditions.F4: Pre-clinical validation of the VasCraft ATMP in the femoro-femoral position in a porcine model. Graft implantation was monitored fluoroscopically, and structural and inflammatory assessments were performed at 30 days post-implantation. Results Eleven decellularization protocols were tested. All protocols achieved a DNA concentration of less than 50 ng/mg of dry tissue. Protocol 1 was selected due to its high preservation of the morphology and ECM of the decellularized vein and the low cytotoxicity of the decellularization reagents. Additionally, a 3D bioreactor was developed to facilitate the recellularization of grafts with EC-MSC, WJ under continuous flow conditions. Conclusion The VasCraft bioimplant aims to solve an unmet need in the development of artificial vascular grafts. Phases F1 and F2 are well underway, with plans to move into Phases F3 and F4 in the coming months.

The development of a nucleus pulposus-derived cartilage analog scaffold for chondral repair and regeneration.

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

Automated Decellularization of the Rodent Epigastric Free Flap: A Comparison of Sodium Dodecyl Sulfate-Based Protocols.

Free tissue transfer to cover complex wounds with exposed critical structures results in donor-site morbidity. Perfusion decellularization and recellularization of vascularized composite tissues is an active area of research to fabricate complex constructs without a donor site. Sodium dodecyl sulfate (SDS)-based protocols remain the predominant choice for decellularization despite the deleterious effects on tissue ultrastructure and capillary networks. We aimed to develop an automated decellularization process and compare different SDS perfusion times to optimize the protocol. A three-dimensional-printed closed-system bioreactor capable of continuously perfusing fluid through the vasculature was used for decellularization. The artery and vein of rat epigastric fasciocutaneous free flaps were cannulated and connected to the bioreactor. Protocols had varying durations of 1% SDS solution (3, 5, and 10 days) followed by 1 day of 1% Triton X-100 and 1 day of 1x phosphate-buffered saline. The residual DNA was quantified. Microarchitecture of the constructs was assessed with histology, and the vascular network was visualized for qualitative assessment. The structural integrity and the microarchitecture of the extracellular matrix was preserved in the 3- and 5-day SDS perfusion groups; however, the subcutaneous tissue of the 10-day protocol lost its structure. Collagen and elastin structures of the pedicle vessels were not compromised by the decellularization process. Five-day SDS exposure group had the least residual DNA content (p < 0.001). Across all protocols, skin consistently had twice as much residual DNA over the subcutaneous tissues. A compact and integrated bioreactor can automate decellularization of free flaps to bioengineer regenerative constructs for future use in reconstruction of complex defects. A decellularization protocol with 5 days of 1% SDS exposure was the most successful to keep the residual DNA content at a minimum while preserving the structural integrity of the tissues.

Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration.

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized “self-developing” esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.

Preparation and mechanical behavior of the acellular porcine common bile duct and its immunogenicity in vivo

The current clinical treatments for complications caused by hepatobiliary surgery still have some inevitable weaknesses. This study aimed to prepare the acellular porcine common bile duct (APCBD) for repairing biliary defects and damage. The porcine common bile duct was decellularized by the freeze-thaw method combined with nuclease treatment, and the efficacy of acellularization was confirmed by the DNA quantification and histological structure. The results showed that the residual DNA content was reduced from 854.67 ± 9.71 ng/mg to 5.43 ± 0.85 ng/mg, and the natural structure and shape of the bile duct were well preserved. The biomechanical properties such as the tensile strength, elastic modulus, and elongation-at-break of the APCBD in the transverse and longitudinal direction indicated that the APCBD meets the requirements of the biomechanical strength in replacement. In addition, the results of the immunotoxicity test showed there was no significant difference in the body weights, organ coefficient, hematology, and immune histology between the experimental groups (three subgroups) and the negative control group, which demonstrated the prepared APCBD had no obvious toxicity to the immune system in vivo and might be a suitable biomaterial for the bile duct repairing.

Decellularized Pancreatic Tail as Matrix for Pancreatic Islet Transplantation into the Greater Omentum in Rats.

Infusing pancreatic islets into the portal vein currently represents the preferred approach for islet transplantation, despite considerable loss of islet mass almost immediately after implantation. Therefore, approaches that obviate direct intravascular placement are urgently needed. A promising candidate for extrahepatic placement is the omentum. We aimed to develop an extracellular matrix skeleton from the native pancreas that could provide a microenvironment for islet survival in an omental flap. To that end, we compared different decellularization approaches, including perfusion through the pancreatic duct, gastric artery, portal vein, and a novel method through the splenic vein. Decellularized skeletons were compared for size, residual DNA content, protein composition, histology, electron microscopy, and MR imaging after repopulation with isolated islets. Compared to the other approaches, pancreatic perfusion via the splenic vein provided smaller extracellular matrix skeletons, which facilitated transplantation into the omentum, without compromising other requirements, such as the complete depletion of cellular components and the preservation of pancreatic extracellular proteins. Repeated MR imaging of iron-oxide-labeled pancreatic islets showed that islets maintained their position in vivo for 49 days. Advanced environmental scanning electron microscopy demonstrated that islets remained integrated with the pancreatic skeleton. This novel approach represents a proof-of-concept for long-term transplantation experiments.

Decellularization alters the unfavorable regenerative adverse microenvironment of the injured spinal cord to support neurite outgrowth.

BackgroundAcellular tissue has been transplanted into the injury site as an external microenvironment to intervene with imbalance microenvironment that occurs after spinal cord injury (SCI) and stimulating axonal regeneration, although the mechanism is unclear. Given decellularization is the key means to obtain acellular tissues, we speculated changes in the internal components of tissue caused by decellularization may be the key reason why acellular tissues affect remodeling of the microenvironment.MethodsComplete spinal cord crush in a mouse model was established, and the dynamic of extracellular matrix (ECM) expression and distribution during SCI was studied with immunohistochemistry (IHC). Normal spinal cord (NSC) and 14-day injury spinal cord (ISC) were obtained to prepare the decellularized NSC (DNSC) and decellularized ISC (DISC) through a well-designed decellularization method, and the decellularization effects were evaluated by residual DNA content determination, hematoxylin and eosin staining (H&E), and IHC. Rat dorsal root ganglia (DRG) were co-cultured with NSC, ISC, DNSC, and DISC to evaluate their effect on neurite outgrowth. Furthermore, the mechanisms by which decellularized tissue promotes axonal growth were explored with proteomics analysis of the protein components and function of 14-day ISC and DISC.ResultsWe found the expression of the four main ECM components (collagen type I and IV, fibronectin, and laminin) gradually increased with the progression of SCI compared to NSC, peaking at 14 days of injury then slightly decreasing at 21 days, and the distribution of the four ECM proteins in the ISC also changed dynamically. H&E staining, residual DNA content determination, and IHC showed decellularization removed cellular components and preserved an intact ECM. The results of co-cultured DRG with NSCs, ISCs, DNSCs, and DISCs showed DNSCs and DISCs had a stronger ability in supporting neurite outgrowth than NSC and ISC. We found through proteomics that decellularization could remove proteins associated with inflammatory responses, scarring, and other pathological factors, while completely retaining the ECM proteins.ConclusionsTaken together, our findings demonstrate decellularization can optimize the imbalanced microenvironment after SCI by removing components that inhibit spinal cord regeneration, providing a theoretical basis for clinical application of acellular tissue transplantation to repair SCI.

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.

Biochemical Traits, 1H NMR Profile and Residual DNA Content of 'Asprinio', White Wine from Campania Region (Southern Italy).

‘Asprinio’ is a white dry wine characteristic for its acidity and aromatic flavour, known as emerging DOP wine in Southern Italy. Nevertheless, little information is available on the metabolomic profile of this wine. Thus, in this paper we evaluated the colourimetric parameters, 1H NMR profiles and free amino acids content of ‘Asprinio’ wines, bottled by two different wineries (hereafter ‘Asprinio_A’ and ‘Asprinio_B’) collected in 2019 and 2020, using ‘Greco di Tufo’ for comparison. The colourimetric parameters are similar for both ‘Asprinio’ wines and differ from ‘Greco di Tufo’ wines. On the other hand, both 1H NMR and free amino acid content profiles show different chemometric profiles among the three wines analysed, although the profiles are similar for both vintages. Moreover, the multivariate analyses carried out highlight differences between ‘Asprinio_A’ and ‘Asprinio_B’, which exbibit also different residual yeast and plant DNA. Overall, considering that the two-manufacturing wineries use 100% ‘Asprinio’ grape, the difference retrieved between the two ‘Asprinio’ wines could be explained by the different grapevine training systems: ‘vite maritata’ (training system inherited from Etruscans) for ‘Asprinio_A’ and ‘guyot’ for ‘Asprinio_B’.

Serum-free purified Vero rabies vaccine is safe and immunogenic in children: Results of a randomized phase II pre-exposure prophylaxis regimen study

BackgroundA serum-free, highly purified Vero rabies vaccine (PVRV-NG) has been developed with no animal or human components and low residual DNA content. A phaseII randomized clinical study aimed to demonstrate the non-inferiority of the immune response and assess the safety profile of PVRV-NG versus a licensed human diploid cell culture rabies vaccine (HDCV) in a pre-exposure regimen in healthy children and adolescents in the Philippines. MethodologyChildren aged 2–11 years and adolescents aged 12–17 years were randomized (2:1) to receive three injections of either PVRV-NG or HDCV (on day [D] 0, D7 and D28). Rabies virus-neutralizing antibodies (RVNA) were measured at D0, D42 and 6 months after the first injection (month [M] 6). Safety was assessed during the vaccination period and up to 28 days after the last vaccination. Serious adverse events were followed until 6 months after last vaccination. Principal findings342 healthy participants (171 children and 171 adolescents) were randomized and followed for 6 months after the last dose. All participants in both groups had an RVNA titer ≥ 0.5 IU/ml at D42, demonstrating non-inferiority in seroconversion rate for PVRV-NG versus HDCV. Over 90% of participants had RVNA titer ≥ 0.5 IU/ml at M6. PVRV-NG was well tolerated after each vaccination and up to 6 months following the last dose. There were no major safety concerns during the study, and the type and severity of solicited adverse events was similar for both treatment groups. ConclusionsThis study demonstrated the non-inferior immune profile of PVRV-NG compared with HDCV in a pre-exposure setting within a pediatric population. PVRV-NG was well tolerated with no safety concerns. This study is registered at ClinicalTrials.gov (NCT01930357) and EU Clinical Trials Register (2015–003203-30).

Formulation of Dermal Tissue Matrix Bioink by a Facile Decellularization Method and Process Optimization for 3D Bioprinting toward Translation Research.

Decellularized extracellular matrices (ECMs) are being extensively used for tissue engineering purposes and detergents are predominantly used for this. A facile detergent-free decellularization method is developed for dermal matrix and compared it with the most used detergent-based decellularization methods. An optimized, single-step, cost-effective Hypotonic/Hypertonic (H/H) Sodium Chloride (NaCl) solutions-based method is employed to decellularize goat skin that resulted in much higher yield than other methods. The ECM composition, mechanical property, and cytocompatibility are evaluated and compared with other decellularization methods. Furthermore, this H/H-treated decellularized dermal ECM (ddECM) exhibits a residual DNA content of <50ngmg-1 of dry tissue. Moreover, 85.64±3.01% of glycosaminoglycans and 65.53±2.9% collagen are retained compared to the native tissue, which is higher than the ddECMs prepared by other methods. The cellular response is superior in ddECM (H/H) than other ddECMs prepared by detergent-based methods. Additionally, a bioink is formulated with the ddECM (H/H), showing good shear thinning and shear recovery properties. Process optimization in terms of print speed, flow rate, and viscosity is done to obtain a bioprinting window for ddECM bioink. The printed constructs with optimized parameters have adequate mechanical and cell adhesive properties and excellent isotropic cellular alignment.

Optimal delipidation solvent to secure extracellular matrix from human perirenal adipose tissue.

The objective of this study was to select the optimal delipidation solvent for preparation of human perirenal adipose tissue-derived extracellular matrix (ECM). Human perirenal adipose tissue can be obtained in large amounts during surgery, and it can be an alternative source of human ECM. Delipidation is an essential procedure for the ECM preparation, because lipid strongly inhibits regeneration of target tissue. Isopropanol has been widely used as a delipidation solvent for adipose tissue. However, because adipose tissue is mostly composed of nonpolar lipid, a nonpolar solvent might be more effective for delipidation. We evaluated the delipidation efficiency of acetone, chloroform, methanol, ether, ethanol, isopropanol, water, chloroform/methanol, ethanol/heptane, ether/methanol, hexane/ethanol, and butanol/methanol solvents for ECM extraction from human perirenal adipose tissue. Among them, acetone-treated adipose tissue showed the greatest delipidation efficiency (93.05%), significantly lower residual DNA content, and the greatest residual collagen concentration (42.49 ± 0.05 μg/g). In addition, acetone-treated tissue also had well-preserved ultrastructure with high porosity and significantly low in vitro cytotoxicity. These results suggested that acetone may be an optimal delipidation solvent for extraction of ECM from human perirenal adipose tissue.

Acellular Porcine Cornea Produced by Supercritical Carbon Dioxide Extraction: A Potential Substitute for Human Corneal Regeneration.

The aim of this study was to develop a non-cytotoxic, biocompatible innovative acellular porcine cornea (APC) for corneal wound healing and corneal blindness treatment. APC was produced by using supercritical carbon dioxide (SCCO2) to decellularize the porcine cornea. Decellularization of the porcine cornea was examined by hematoxylin and eosin staining and 4,6-diamidino-2-phenylindole, dihydrochloride staining. The residual DNA content of APC was analyzed in comparison with the native porcine cornea. Virus inactivation up to at least 6 log10 was confirmed for the stepwise process of APC for 4 different model viruses. In addition, a series of in vitro and in vivo tests in accordance with ISO-10993 biocompatibility assay and animal performance tests were performed. APC produced by the SCCO2 process revealed complete decellularization, without any residual non-collagenous proteins. The scanning electron microscopy structural features of the decellularized cornea were similar to those of human. APC was found to be nontoxic and exhibited excellent biocompatibility in both in vitro and in vivo studies. The animal performance test proved that APC exerted excellent adaptability on the cornea and no sign of irritation and good compatibility in lamellar corneal transplantation. APC manufactured by SCCO2 technology revealed complete cells and non-collagenous protein removal compared with the Triton-sodium dodecyl sulfate decellularization process. APC showed excellent biocompatibility in rabbit lamellar corneal transplantation with a follow-up to 1 year. APC can be a potential substitute for human-donated cornea for corneal transplantation in the near future.

QS4: Optimizing The Decellularization Of The Rodent Epigastric Free Flap: A Comparison Of Automated SDS-based Protocols

Purpose:Raising flaps to cover complex wounds with exposed critical structures are lengthy operations that result in donor site morbidity. Tissue engineering research is developing with great promise to build replacement tissues without morbidity. Decellularization removes whole cells and cell debris, and is the initial step to create a scaffold with an intact vascular network. Benchmark measurement of the overall cellular level is quantification of the DNA content, where 50 ng/mg is classically considered as a threshold. Although perfusion decellularization and recellularization approaches have shown exceptional promise in whole organ engineering, there is minimal crossover into the microsurgical field. Sodium dodecyl sulfate (SDS)-based protocols are known to have deleterious effects on the ultrastructure and capillary network of the scaffolds, but remain the predominant choice for decellularization protocols. This study aims to optimize the SDS exposure protocol for automated decellularization by comparing different SDS perfusion times to gain better understanding of the balance between decellularization and scaffold preservation.Methods:A 3D-printed closed-system bioreactor capable of continuously perfusing fluid throughout the vasculature was used for decellularization of free flaps. 2x2 cm fasciocutaneous free flaps from the epigastric region of the rat were harvested, and the vascular pedicle was isolated as a single artery and vein. Three flaps were evaluated in each group. The artery (inflow) and vein (outflow) were cannulated to monitor preservation of the vasculature. 1% SDS solution was perfused in different durations (3, 5 and 10 days) based on several protocols found in the literature. Automated SDS perfusion was followed by 1 day of 1% Triton X-100 and 1 day of 1xPhosphate-buffered saline (PBS), all of which were at the perfusion pressure of 120 mmHg.Results:For vasculature analysis, continual perfusion into the artery and out of the vein within the bioreactor was assessed throughout the decellularization process. H&E, Masson’s trichrome, and Verhoeff-Van Gieson staining were performed to assess architecture and locale of residual nuclei. Residual DNA was quantified by the fluorescent marker PicoGreen. 5 days of 1% SDS solution had the least residual DNA content (1.309±0.807 ng/mg) followed by 10 days (12.684±14.184 ng/mg) and 3 days (82.387±71.595 ng/mg), p<0.001. The DNA content ratio of skin over subcutaneous tissues was consistent across all protocols with skin having twice as much residual DNA after each protocol. The vascular network was visualized for qualitative assessment with the perfusion of hardening cast to create contrast against soft tissue to visualize with microCT imaging.Conclusion:A decellularization protocol of 5 days of 1% SDS solution followed by 1 day of 1% Triton X-100 and 1 day of 1xPBS was the most successful to keep the residual DNA content at a minimum, while preserving the structural integrity of the tissues. The bioreactor is capable of running automated and repeatable protocols using several solutions and continuously perfusing fluid throughout the vasculature of a free flap. This compact and integrated system can decrease hands-on time and be used in the future for further recellularization of scaffolds to bioengineer soft tissue replacement without donor site morbidity.

Comparative analysis of protocols for decellularization of corneal lenticular tissue

Shortage of donor corneas is a burning issue in ophthalmology. That is why there is a search for new alternative ways for treating corneal diseases. Decellularization technologies make it possible to create corneal tissue-engineered constructs that can adrress the issue of donor corneal shortage. Objective: to conduct a comparative analysis of effective methods for treating the corneal lenticula and to create an optimized and standardized decellularization protocol. Materials and methods. Corneal stromal lenticules obtained after ReLEx SMILE surgery were chosen for the study. Lenticule parameters: thickness 77–120 microns, diameter 6.5 mm. We used 3 protocols for the treatment of lenticules: 1) treatment with 1.5 M sodium chloride with nucleases (NaCl); 2) 0.1% SDS (SDS); 3) treatment with Trypsin-EDTA solution, followed by double washing in a hypotonic Tris buffer solution with nucleases (Trypsin-EDTA). Optical properties of lenticles were determined spectrophotometrically, where the samples before decellularization served as a control. Fluorescence imaging of nuclear material in the original cryosections was performed using Hoechst dye. The state of collagen fiber ultrastructure was assessed by scanning electron microscopy. The quantitative DNA content in fresh lenticules and in lenticules after treatment was analyzed. Results. All three decellularization protocols effectively removed nuclear and cellular material; the residual DNA content was &lt; 50 ng/mg. However, the Trypsin-EDTA protocol led to significant damage to the extracellular matrix structure, which negatively affected the transparency of corneal tissue-engineered constructs. Transparency of samples for the NaCl protocol was close to native lenticules. Conclusion. To create a corneal tissue-engineered construct, NaCl decellularization protocols appear to be optimized and can be used to treat various corneal diseases.

Impact of Porcine Pancreas Decellularization Conditions on the Quality of Obtained dECM.

Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.

Biomechanical Properties of the Porcine Trachea before and after Decellularization for Airway Transplantation

<h3>Purpose</h3> Tracheal replacement based on a tissue engineered tracheal scaffold is an alternative for extensive benign or malignant tracheal disease. The mechanics of the scaffold depends on the decellularization protocol used. This study focuses on the mechanical properties of tracheal scaffolds obtained from the decellularization protocol used in our laboratory. <h3>Methods</h3> The protocol was approved by the Animal Ethics Committee (CEUA-FMUSP). Fresh tracheas (24) were obtained from adult Landrace pigs (100kg). Twelve were decellularized and 12 were tested <i>in natura</i>. Decellularization protocol included 10 cycles of detergent (DS 2%) followed by 3 washings with PBS for 10 min and agitation for 48h in an incubator (36°C). Decellularization of the scaffold was assessed by measuring residual DNA contents. The scaffolds were then submitted to freeze-drying, immersion in DNAse and Proteinase K for 12h each. The DNA extraction was done with the phenol-chloroform technique and quantified by absorbance in a spectrophotometer. Tracheal mechanical properties were assessed using a tensile test system (INSTROM Model 3365, Norwood, MA) in two axes according to the norm of the American Society for Testing and Material (ASTM D638-14). Data is represented as mean± STD. Comparison between data used Student t test and Mann-Whitney. Significance was assumed for p<0.05. <h3>Results</h3> There was no difference in the Young's (elastic) module in both axes between decellularized scaffolds and tracheas <i>in natura</i>. The maximum load increased in the longitudinal axis and decreased in the transverse axis of the decellularized scaffolds reflecting the changes in the extracellular matrix in the cartilage (transverse) and connective tissue (longitudinal) (table). <h3>Conclusion</h3> The mechanical properties of the scaffolds obtained after the decellularization protocol suggest that despite the changes in the extracellular matrix the decellularized scaffolds will be able to retain its physiological properties.

Comparing various protocols of human and bovine ovarian tissue decellularization to prepare extracellular matrix-alginate scaffold for better follicle development in vitro

BackgroundNowadays, the number of cancer survivors is significantly increasing as a result of efficient chemo/radio therapeutic treatments. Female cancer survivors may suffer from decreased fertility. In this regard, different fertility preservation techniques were developed. Artificial ovary is one of these methods suggested by several scientific groups. Decellularized ovarian cortex has been introduced as a scaffold in the field of human fertility preservation. This study was carried out to compare decellularization of the ovarian scaffold by various protocols and evaluate the follicle survival in extracellular matrix (ECM)-alginate scaffold.ResultsThe micrographs of H&E and DAPI staining confirmed successful decellularization of the ovarian cortex in all experimental groups, but residual DNA content in SDS-Triton group was significantly higher than other groups (P < 0.05). SEM images demonstrated that complex fiber network and porosity structure were maintained in all groups. Furthermore, elastin and collagen fibers were observed in all groups after decellularization process. MTT test revealed higher cytobiocompatibility of the SDS-Triton-Ammonium and SDS-Triton decellularized scaffolds compared with SDS groups. Compared to the transferred follicles into the sodium alginate (81%), 85.9% of the transferred follicles into the decellularized scaffold were viable after 7 days of cultivation (P = 0.04).ConclusionAlthough all the decellularization procedures was effective in removal of cells from ovarian cortex, SDS-Triton-Ammonium group showed less residual DNA content with higher cytobiocompatibility for follicles when compared with other groups. In addition, the scaffold made from ovarian tissues decellularized using SDS-Triton-Ammonium and sodium alginate is suggested as a potential 3D substrate for in vitro culture of follicles for fertility preservation.

A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro

Augmentative and reconstructive rhinoplasty surgical procedures use autologous tissue grafts or synthetic grafts to repair the nasal defect and aesthetic reconstruction. Donor site trauma and morbidity are common in autologous grafts. The desperate need for the production of grafted 3D cartilage tissues as rhinoplasty grafts without the adverse effect is the need of the hour. In the present study, we developed a bioactive 3D histotypic construct engineered with the various ratio of adipose-derived stem cells (ADSC) and chondrocytes together with decellularized porcine nasal cartilage graft (dPNCG). We decellularized porcine nasal cartilage using supercritical carbon dioxide (SCCO2) extraction technology. dPNCG was characterized by H&E, DAPI, alcian blue staining, scanning electron microscopy and residual DNA content, which demonstrated complete decellularization. 3D histotypic constructs were engineered using dPNCG, rat ADSC and chondrocytes with different percentage of cells and cultured for 21 days. dPNCG together with 100% chondrocytes produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. H&E and alcian blue staining showed an intact mass, with cartilage granules bound to one another by extracellular matrix and proteoglycan, to form a 3D structure. Besides, the expression of chondrogenic markers, type II collagen, aggrecan and SOX-9 were elevated indicating chondrocytes cultured on dPNCG substrate facilitates the synthesis of type II collagen along with extracellular matrix to produce 3D histotypic cartilage. To conclude, dPNCG is an excellent substrate scaffold that might offer a suitable environment for chondrocytes to produce 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty.

Ex-vivo recellularisation and stem cell differentiation of a decellularised rat dental pulp matrix

Implementing the principles of tissue engineering within the clinical management of non-vital immature permanent teeth is of clinical interest. However, the ideal scaffold remains elusive. The aim of this work was to assess the feasibility of decellularising rat dental pulp tissue and evaluate the ability of such scaffold to support stem cell repopulation. Rat dental pulps were retrieved and divided into control and decellularised groups. The decellularisation protocol incorporated a low detergent concentration and hypotonic buffers. After decellularisation, the scaffolds were characterised histologically, immunohistochemistry and the residual DNA content quantified. Surface topography was also viewed under scanning electron microscopy. Biocompatibility was evaluated using cytotoxicity assays utilising L-929 cell line. Decellularised scaffolds were recellularised with human dental pulp stem cells up to 14 days in vitro. Cellular viability was assessed using LIVE/DEAD stain kit and the recellularised scaffolds were further assessed histologically and immunolabelled using makers for odontoblastic differentiation, cytoskeleton components and growth factors. Analysis of the decellularised scaffolds revealed an acellular matrix with histological preservation of structural components. Decellularised scaffolds were biocompatible and able to support stem cell survival following recellularisation. Immunolabelling of the recellularised scaffolds demonstrated positive cellular expression against the tested markers in culture. This study has demonstrated the feasibility of developing a biocompatible decellularised dental pulp scaffold, which is able to support dental pulp stem cell repopulation. Clinically, decellularised pulp tissue could possibly be a suitable scaffold for use within regenerative (reparative) endodontic techniques.