Decellularization Procedure Research Articles

A New Detergent for the Effective Decellularization of Bovine and Porcine Pericardia

Human and animal pericardia are among the most widely exploited materials suitable to repair damaged tissues in the cardiovascular surgery context. Autologous, xenogeneic (chemically treated) and homologous pericardia are largely utilized, but they do exhibit some crucial drawbacks. Any tissue treated with glutaraldehyde is known to be prone to calcification in vivo, lacks regeneration potential, has limited durability, and can result in cytotoxicity. Moreover, autologous tissues have limited availability. Decellularized biological tissues represent a promising alternative: decellularization removes cellular and nuclear components from native tissues and makes them suitable for repopulation by autologous cells upon implantation into the body. The present work aims to assess the effects of a new detergent, i.e., Tergitol, for decellularizing bovine and porcine pericardia. The decellularization procedure successfully removed cells, while preserving the histoarchitecture of the extracellular matrix. No cytotoxic effect was observed. Therefore, decellularized pericardia showed potential to be used as scaffold for cardiovascular tissue regeneration.

Bioactive Cell-Derived ECM Scaffold Forms a Unique Cellular Microenvironment for Lung Tissue Engineering.

Chronic lung diseases are one of the leading causes of death worldwide. Lung transplantation is currently the only causal therapeutic for lung diseases, which is restricted to end-stage disease and limited by low access to donor lungs. Lung tissue engineering (LTE) is a promising approach to regenerating a replacement for at least a part of the damaged lung tissue. Currently, lung regeneration is limited to a simplified local level (e.g., alveolar–capillary barrier) due to the sophisticated and complex structure and physiology of the lung. Here, we introduce an extracellular matrix (ECM)-integrated scaffold using a cellularization–decellularization–recellularization technique. This ECM-integrated scaffold was developed on our artificial co-polymeric BETA (biphasic elastic thin for air–liquid interface cell culture conditions) scaffold, which were initially populated with human lung fibroblasts (IMR90 cell line), as the main generator of ECM proteins. Due to the interconnected porous structure of the thin (<5 µm) BETA scaffold, the cells can grow on and infiltrate into the scaffold and deposit their own ECM. After a mild decellularization procedure, the ECM proteins remained on the scaffold, which now closely mimicked the cellular microenvironment of pulmonary cells more realistically than the plain artificial scaffolds. We assessed several decellularization methods and found that 20 mM NH4OH and 0.1% Triton X100 with subsequent DNase treatment completely removed the fibroblasts (from the first cellularization) and maintains collagen I and IV as the key ECM proteins on the scaffold. We also showed the repopulation of the primary fibroblast from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o−) cells on the ECM-integrated BETA scaffold. With this technique, we developed a biomimetic scaffold that can mimic both the physico-mechanical properties and the native microenvironment of the lung ECM. The results indicate the potential of the presented bioactive scaffold for LTE application.

Towards the development of osteochondral allografts with reduced immunogenicity

Nowadays, repair and replacement of hyaline articular cartilage still challenges orthopedic surgery. Using a graft of decellularized articular cartilage as a structural scaffold is considered as a promising therapy. So far, successful cell removal has only been possible for small samples with destruction of the macrostructure or loss of biomechanics. Our aim was to develop a mild, enzyme-free chemical decellularization procedure while preserving the biomechanical properties of cartilage.Porcine osteochondral cylinders (diameter: 12 mm; height: 10 mm) were divided into four groups: Native plugs (NA), decellularized plugs treated with PBS, Triton-X-100 and SDS (DC), and plugs additionally treated with freeze-thaw-cycles of −20 °C, −80 °C or shock freezing in nitrogen (N2) before decellularization. In a non-decalcified HE stain the decellularization efficiency (cell removal, cell size, depth of decellularization) was calculated. For biomechanics the elastic and compression modulus, transition and failure strain as well as transition and failure stress were evaluated.The −20 °C, −80 °C, and N2 groups showed a complete decellularization of the superficial and middle zone. In the deep zone cells could not be removed in any experimental group. The biomechanical analysis showed only a reduced elastic modulus in all decellularized samples. No significant differences were found for the other biomechanical parameters.

An Edible, Decellularized Plant Derived Cell Carrier for Lab Grown Meat

Rapidly expanding skeletal muscle satellite cells with cost-effective methods have been presented as a solution for meeting the growing global demand for meat. A common strategy for scaling cell proliferation employs microcarriers, small beads designed to support anchorage-dependent cells in suspension-style bioreactors. No carrier has yet been marketed for the cultivation of lab-grown meat. The objective of this study was to demonstrate a rapid, food safe, decellularization procedure to yield cell-free extracellular matrix scaffolds and evaluate them as cell carriers for lab grown meat. Broccoli florets were soaked in SDS, Tween-20, and bleach for 48 h. The decellularization process was confirmed via histology, which showed an absence of cell nuclei, and DNA quantification (0.0037 ± 0.00961 μg DNA/mg tissue). Decellularized carriers were sorted by cross sectional area (7.07 ± 1.74 mm2, 3.03 ± 1.15 mm2, and 0.49 ± 0.3 mm2) measured for eccentricity (0.73 ± 0.16). Density measurements of decellularized carriers (1.01 ± 0.01 g/cm) were comparable to traditional microcarriers. Primary bovine satellite cells were inoculated into and cultured within a reactor containing decellularized carriers. Cell adhesion was observed and cell death was limited to 2.55 ± 1.09%. These studies suggested that broccoli florets may serve as adequate edible carrier scaffolds for satellite cells.

Decellularized Three‐Dimensional Biomimetic Scaffolds as In Vitro Model For Tendon And Meniscus Regeneration

Traumatic and degenerative injuries to the meniscus and tendon still pose challenges to treatments available in the fields of orthopedics and sports medicine. In search of more effective therapeutic procedures based on biological strategy, regenerative medicine advances like medical science carrying‐out preclinical and clinical research necessary for establishing therapeutic efficacy and safety. This research aimed to establish the new concept of three‐dimensional decellularized biomimetic scaffolds as in vitro models to investigate the tendon and meniscus regenerative medicine approaches. For this purpose, the menisci of the swine's knee and the tendons of the deep flexor muscles of the bovine's fingers were resected, cleaned, fragmented and frozen (‐20°C) for 48 hours. After this, the fragments were thawed and decellularized by immersion in sodium dodecyl sulfate (0.5%) and Triton‐X100 (1%) solution for 72 hours. To faithfully simulate a degenerative injury, 0.05 mL of collagenase type‐1 (1mg.mL‐1/37°C/1hour) was injected into the central region of the meniscal samples. To simulate the traumatic injury, incisions were made on the surface of the samples with a scalpel. The fragments were washed in running water (3 days), immersed in formaldehyde (4%), washed again, stored in ethanol (70%), frozen (‐30°C), and submitted to the lyophilization process or embedding in paraffin for histological procedures. For microscopic characterization of tissue and injury sites, histological sections were stained with Hematoxylin and Eosin (HE) or Picrosirius and analyzed under a light microscope with a polarization filter. The results, based on qualitative analysis, demonstrate the preservation of macroscopic and microscopic architecture of the scaffold matrix, the efficacy of the decellularization procedure, and the accuracy in creating the traumatic and degenerative sites of injury. These results allow us to conclude the success of this research in establishing the concept of decellularized three‐dimensional scaffolds as a new biomimetic models applicable for in vitro assays evolving regeneration of traumatic and degenerative injuries of tendons and menisci, contributing to the advancement of preclinical research on regenerative medicine approaches.

Novel Decellularization Method for Tissue Slices.

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies.

A Fast and Efficient Decellularization Method for Tissue Slices.

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue's vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2-3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies. This protocol was validated in: Front Bioeng Biotechnol (2022), DOI: 10.3389/fbioe.2022.832178.

Extracellular Matrix-Derived Hydrogels to Augment Dermal Wound Healing: A Systematic Review

Chronic, non-healing, dermal wounds form a worldwide medical problem with limited and inadequate treatment options and high societal burden and costs. With the advent of regenerative therapies exploiting extracellular matrix (ECM) components, its efficacy to augment wound healing is to be explored. This systematic review was performed to assess and compare the current therapeutic efficacy of ECM hydrogels on dermal wound healing. The electronic databases of Embase, Medline Ovid, and Cochrane Central were searched for in vivo and clinical studies on the therapeutic effect of ECM-composed hydrogels on dermal wound healing (April 13, 2021). Two reviewers selected studies independently. Studies were assessed based on ECM content, ECM hydrogel composition, additives, and wound healing outcomes, such as wound size, angiogenesis, and complications. Of the 2102 publications, 9 rodent-based studies were included while clinical studies were not published at the time of the search. Procedures to decellularize tissue or cultured cells and subsequently generate hydrogels were highly variable and in demand of standardization. ECM hydrogels with or without additives reduced wound size and also seem to enhance angiogenesis. Serious complications were not reported. To date, preclinical studies preclude to draw firm conclusions on the efficacy and working mechanism of ECM-derived hydrogels on dermal wound healing. The use of ECM hydrogels can be considered safe. Standardization of decellularization protocols and implementation of quality and cytotoxicity controls will enable obtaining a generic and comparable ECM product. Impact statement Extracellular matrix (ECM)-based hydrogels are biocompatible and harbor growth factors that can instruct tissue healing. Their application is a novelty in (pre)clinical wound healing treatment. This systematic review provides an overview of the current evidence for ECM hydrogels in enhancing wound healing and an extensive overview of the decellularization procedures used. Lastly, challenges and future directions to standardize decellularization procedures and implement quality controls are proposed.

Decellularized bovine aorta as a promising 3D elastin scaffold for vascular tissue engineering applications.

Aim: To evaluate the suitability of using aorta elastin scaffold, in combination with human adipose-derived mesenchymal stem cells (hAd-MSCs), as an approach for cardiovascular tissue engineering. Materials & Methods: Human adipose-derived MSCs were seeded on elastin samples of decellularized bovine aorta. The samples were cultured in vitro to investigate the inductive effects of this scaffold on the cells. The results were evaluated using histological, and immunohistochemical methods, as well as MTT assay, DNA content, reverse transcription-PCR and scanning electron microscopy. Results: Histological staining and DNA content confirmed the efficacy of decellularization procedure (82% DNA removal). MTT assay showed the construct's ability to support cell viability and proliferation. Cell differentiation was confirmed by reverse transcription-PCR and positive immunohistochemistry for alfa smooth muscle actin and von Willebrand. Conclusion: The prepared aortic elastin samples act as a potential scaffold, in combination with MSCs, for applications in cardiovascular tissue engineering. Further experiments in animal models are required to confirm this.

Decellularization of ram cardiac tissue via supercritical CO2

The decellularization method efficacy varies depending on the tissue type. There are commonly used physical, chemical and enzymatic methods. Most of the methods affect the extracellular matrix properties that will in turn alter the cell fate when the biological scaffold is recellularised. We aimed to optimize the decellularization of ram cardiac via scCO2 extraction to preserve the moisture and the components of the ECM during the decellularization procedure. In literature decellularization studies focus on heart vessels. In this study, we aimed to decellularize and recellularize ram cardiac tissue for the first time. Besides we obtained a decellularized tissue without cells on it with a mechanical strength similar to native ECM. Decellularized ram cardiac tissue is expected to be animal-based biomaterial for candidate biomedical applications in vitro and in vivo further studies.

Haemocompatibility testing allows selective adaption of GA-free SULEEI-preparation strategy for bovine pericardium

Abstract Background/Introduction Bovine pericardium is the main natural source for patches or valve substitutes in cardiac surgery, but still, long-term durability is limited. An alternative, glutaraldehyde (GA)-free preparation combining decellularization, riboflavin/UVA crosslinking, and low-energy electron irradiation (SULEEI) procedure was established to avoid this. Protocol optimization is ongoing to further improve the final material. Haemocompatibility in vitro testing with fresh, human whole blood has advantages for material analysis, as it provides essential information on the activation of the complement system and immune cells therefore substantiating and possibly reducing the amount of animal testing. Purpose The haemocompatibility testing system was applied to reveal advantageous protocol adaptation of GA-free SULEEI preparation of pericardia. Methods Decellularized bovine pericardia were UVA-crosslinked in riboflavin/dextran solution. Protocol SULEEI B adaption includes the addition of trypsin in decellularization buffer and a 10x reduced riboflavin/dextran concentration during prolonged UVA irradiation with reduced intensity. Electron irradiation with 36 kGy was retained. Pericardia were incubated in blood chambers with heparinized human blood (2h, 37°C, GA-fixed/native controls). Inflammation (granulocyte loss and activation [CD11b]), complement activation (C5a) and haemostasis parameters (F1+2, PF4, granulocyte/platelet conjugates, platelet loss) were analyzed via flow cytometry or ELISA. Surface cell adhesion was investigated immunohistologically and via REM-analyses. Nuclear fragments were quantified in HE-stained sections. Results Haemostasis parameters F1+2 and PF4 were significantly higher in SULEEI A-pericardium (133±25 nmol/l and 1507±677 U/ml) vs. GA-fixation (2.9±2.9 nmol/l and 320±160 U/ml). Also, granulocyte/platelet conjugates (76.3±18.8%) and platelet loss (40.4±29.7%) were significantly higher after SULEEI A preparation in comparison to GA-fixation (39.3±9.9% and 20.5±6.4%). SULEEI B treated pericardia did not differ significantly. C5a complement activation was significantly lower in SULEEI A samples but comparable in SULEEI B to GA-fixed material. This is in contrast to a higher rate of nuclear fragments in SULEEI A tissue HE stained sections. Granulocyte activation of SULEEI pericardium (A: 89.8±29.7%; B: 106.2±18.8%) was significantly higher compared to native (46.1±22.6%) and for SULEEI B higher than in GA-fixed samples (67.2±9%). Granulocyte loss was comparable to GA-fixed pericardium after SULEEI B protocol. REM-analyses and histological evaluation visualized a dense blood cell and fibrin covering particular of SULEEI A-pericardium. Conclusions Haemocompatibility testing reveals advantageous properties of SULEEI B-pericardium. The impact of treatment substances such as dextran on high inflammatory response and dense surface covering in SULEEI A protocol is focus in ongoing experiments. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): EU/EFRE

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine.

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

Fabrication and characterization of an acellular annulus fibrosus scaffold with aligned porous construct for tissue engineering.

Scaffolds mimicking the native annulus fibrosus (AF) extracellular matrix (ECM) structure are crucial to guide the seeding cells to regenerate aligned tissue, while fabricating such a scaffold by synthetic material is challengeable. Native acellular scaffolds derived from AF tissue certainly possess the advantages of natural structure and composition. Based on previous studies, we modified decellularization procedure and especially compared two drying methods, including gradient dehydration and freeze-drying. The decellularization process can effectively remove the host cells and antigens such as α-Gal, while maintaining the original ECM including GAG and collagen I. Compared with gradient dehydration, freeze-drying not only rendered the decellularized scaffold in dry state for storage but also gave the scaffold more aligned porous structure and hydrophilicity. And, the acellular porous scaffold manifested better capacity of supporting cell ingrowth when seeded human bone marrow mesenchymal stem cells (hBMSCs) or implanted in vivo. Furthermore, this optimized freeze-dried scaffold showed similar mechanical elastic modulus as native AF and demonstrated rare inflammatory granuloma and immune rejection as observed in HE staining and immunohistochemistry staining (IHC) of CD8 and MAC387 epitopes when implanted subcutaneously in vivo. To sum up, through our decellularization and freeze-drying procedure, an aligned porous three-dimensional scaffold derived from the natural AF ECM was successfully fabricated with good retention of ECM components and benign biocompatibility. It will be a promising scaffold for AF tissue engineering.

Assessment of electromechanically stimulated bone marrow stem cells seeded acellular cardiac patch in a rat myocardial infarct model

In this study, we evaluated cardiomyogenic differentiation of electromechanically stimulated rat bone marrow-derived stem cells (rt-BMSCs) on an acellular bovine pericardium (aBP) and we looked at the functioning of this engineered patch in a rat myocardial infarct (MI) model. aBP was prepared using a detergent-based decellularization procedure followed by rt-BMSCs seeding, and electrical, mechanical, or electromechanical stimulations (3 millisecond pulses of 5 V cm−1 at 1 Hz, 5% stretching) to enhance cardiomyogenic differentiation. Furthermore, the electromechanically stimulated patch was applied to the MI region over 3 weeks. After this period, the retrieved patch and infarct region were evaluated for the presence of calcification, inflammatory reaction (CD68), patch to host tissue cell migration, and structural sarcomere protein expressions. In conjunction with any sign of calcification, a higher number of BrdU-labelled cells, and a low level of CD68 positive cells were observed in the infarct region under electromechanically stimulated conditions compared with static conditions. More importantly, MHC, SAC, Troponin T, and N-cad positive cells were observed in both infarct region, and retrieved engineered patch after 3 weeks. In a clear alignment with other results, our developed acellular patch promoted the expression of cardiomyogenic differentiation factors under electromechanical stimulation. Our engineered patch showed a successful integration with the host tissue followed by the cell migration to the infarct region.

Preparation and characterization of decellularized rooster comb as a scaffold for tissue engineering applications

Decellularization is a method that has been widely used in tissue engineering especially in the last 20 years. In this study decellularized rooster comb was prepared and characterized for using it as a tissue scaffold. Treatment of tissues with sodium dodecyl sulfate (SDS) and Triton X-100 as two decellularization procedures in different time points were compared according to different parameters such as cytocompatibility, cell removal, preservation of extracellular matrix (ECM), and mechanical properties to find the optimum technique. Even though there was no difference regarding to efficiency on cell removal, SDS demonstrated better results on protection of tissue morphology in comparison with Triton X-100. Therefore, in general the samples treated with SDS showed higher levels of mechanical properties and cytocompatibility in comparison with Triton X-100 applied tissues. In the cuisines of many countries, rooster comb is discarded as a waste material however, in this study it was demonstrated that decellularized rooster comb could be utilized as a cheap, easily obtainable, and biocompatible scaffold. In conclusion, it was revealed that decellularized rooster comb is a promising biomaterial for using as scaffold and it is expected to be utilized for the further studies in particular on skin tissue engineering.

Image-Based Method to Quantify Decellularization of Tissue Sections.

Tissue decellularization is typically assessed through absorbance-based DNA quantification after tissue digestion. This method has several disadvantages, namely its destructive nature and inadequacy in experimental situations where tissue is scarce. Here, we present an image processing algorithm for quantitative analysis of DNA content in (de)cellularized tissues as a faster, simpler and more comprehensive alternative. Our method uses local entropy measurements of a phase contrast image to create a mask, which is then applied to corresponding nuclei labelled (UV) images to extract average fluorescence intensities as an estimate of DNA content. The method can be used on native or decellularized tissue to quantify DNA content, thus allowing quantitative assessment of decellularization procedures. We confirm that our new method yields results in line with those obtained using the standard DNA quantification method and that it is successful for both lung and heart tissues. We are also able to accurately obtain a timeline of decreasing DNA content with increased incubation time with a decellularizing agent. Finally, the identified masks can also be applied to additional fluorescence images of immunostained proteins such as collagen or elastin, thus allowing further image-based tissue characterization.

Microporous scaffolds via a designed decellularization procedure combined with papain-containing reagent treatments after supercritical fluid of carbon dioxide

Tissue-engineering was important which combine medical applications and engineering materials. A extraction process was employed to remove the tissue and cellular molecules from porcine costal cartilages in this study via pretreatment of supercritical fluid of carbon dioxide. Varying porosities of the decellularized tissues were then created using papain-containing reagent treatments. The author studied characterizations of the designed decellularized scaffolds derived from porcine costal cartilages via supercritical fluid and papain-containing reagent treatments.

Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing

Decellularized scaffolds have been found to be excellent platforms for tissue engineering applications. The attempts are still being made to optimize a decellularization protocol with successful removal of the cells with minimal damages to extracellular matrix components. We examined twelve decellularization procedures using different concentrations of Sodium dodecyl sulfate and Triton X-100 (alone or in combination), and incubation time points of 15 or 30 min. Then, the potential of the decellularized scaffold as a three-dimensional substrate for colony formation capacity of mouse spermatogonial stem cells was determined. The morphological, degradation, biocompatibility, and swelling properties of the samples were fully characterized. The 0.5%/30 SDS/Triton showed optimal decellularization with minimal negative effects on ECM (P ≤ 0.05). The swelling ratios increased with the increase of SDS and Triton concentration and incubation time. Only 0.5%/15 and 30 SDS showed a significant decrease in the SSCs viability compared with other groups (P < 0.05). The SSCs colony formation was clearly observed under SEM and H&E stained slides. The cells infiltrated into the subcutaneously implanted scaffold at days 7 and 30 post-implantation with no sign of graft rejection. Our data suggest the %0.5/30 SDS/Triton as an excellent platform for tissue engineering and reproductive biology applications.

Micro-mechanical properties of corneal scaffolds from two different bio-models obtained by an efficient chemical decellularization

The present study elucidates the impact of detergent-based chemical decellularization on the micro-mechanical properties of porcine and rabbit corneas for the purpose of extracellular matrix (ECM) derived scaffolds. Aiming to optimize the decellularization process, different concentrations of Sodium Dodecyl Sulfate (SDS), Triton X-100 and CHAPS detergents were assessed on their ability to decellularize corneas from both bio-models at incubation periods of 12 and 24h. We evaluated the effect of decellularization on corneal ECM Young's Modulus and various area's roughness parameters (topography features) at a microscale by using Atomic Force Microscopy (AFM). Only SDS presented adequate decellularization properties at the selected concentrations (0.2, 0.5 and 1%) and incubation periods. All topography features displayed by native corneas were preserved after SDS treatments, while no statistically significant differences were identified for the average value of Young's Modulus between the control samples and those treated with 0.2% SDS (rabbit corneas) and 0.5% SDS (porcine corneas) after 12h. In this sense, cornea decellularization procedures can be improved by simultaneously reducing SDS concentration and incubation period. AFM is a useful tool to perform biomechanical analysis of the effect of decellularization on scaffold micro-mechanics. Evaluation of the scaffold mechanical behavior at a microscale could help in understanding cell-scaffold interactions in terms of mechanotransduction, complementing macroscale techniques (e.g. tensile tests) relevant for tissue engineering quality control and decision-making.

Mapping of bovine pericardium to enable a standardized acquirement of material for medical implants

ObjectivesBovine pericardium - native, fixed as well as decellularized - is one of the most common implant materials in modern cardiovascular surgery. Although used in everyday procedures, there are no recommendations in regard to which part of the pericardium to prefer. It was the aim of this study, to identify areas of the pericardium with consistent properties and high durability. MethodsFresh bovine pericardia were collected from a local slaughterhouse. The native pericardia were analyzed at 140 spots in regard to thickness and fiber orientation. Based on these results, five promising areas were selected for further evaluation. The pericardia were decellularized with detergents (0.5% sodiumdesoxycholate/0.5% sodiumdodecylsulfate) and subsequently incubated in DNAse. The two investigation groups native und DC consisted of 20 samples each. The efficiency of the decellularization was evaluated by DNA quantification, as well as DAPI and H&E staining. Biomechanical properties were determined using uniaxial tensile tests. To evaluate the microstructure, scanning electron microscopy, Picrosirius Red- and Movat's Pentachrome staining were utilized. To assess the long-term durability, patches were tested in a high-cycle system for a duration equaling the stress of three months in-vivo. Commercially available, fixed pericardium patches served as control group. ResultsOnly a limited part of the pericardium showed a homogenous and usable thickness. The decellularization removed all cell nuclei, proven by negative DAPI and H&E staining, and also significantly reduced the DNA amount by 84%. The mechanical testing revealed that two investigated areas had an inconsistent tensile strength. Microscopical observations showed that the integrity of the extracellular matrix did not suffer by the decellularization procedure. During the long-term testing, most of the pericardia slowly lost tautness, though none of them got measurably damaged. Especially one area showed no decline of tensile strength after durability testing at all. Decellularized patches and fixed patches achieved comparable results in mechanical testing and microscopical evaluation after the durability testing. ConclusionWe could clearly document significant, location-based differences within single pericardia. Only one area showed consistent properties and a high durability. We highly recommend taking this into account for future implant material selections.