Detergent-enzymatic Treatment Research Articles

Development of the Acellular Dermal Matrix and Experimental Substantiation of Its Use in the Anterior Abdominal Wall hernia Repair

Objective: To develop a biological implant that is an acellular dermal matrix (ADM), evaluate its use as a support material in tensionfree hernioplasty for ventral hernia, and compare it to that of the commercially available implant PermacolTM. Materials and methods: ADM was derived from the porcine dermis (Landrace breed) decellularized using detergents and enzymes. The quality of devitalization was assessed in vitro. We performed sublay hernioplasty in 4-month-old Landrace pigs using ADM (experimental group) and PermacolTM (control group). The specimens were explanted on day 120 of the experiment for histological and immunohistochemical examination. Results: All cellular elements were removed by the detergent-enzymatic treatment of the dermis; the native architecture of the dermis was slightly disrupted. The specimens of the commercially available chemically cross-linked biomaterial PermacolTM had better mechanical properties than ADM specimens; however, there were no significant differences in terms of cytotoxicity. The state of the tissues after the explantation (number of fibroblasts and endothelial cells) showed no differences in the result of using ADM and PermacolTM. In 120 days, the materials integrated into the tissues without the formation of adhesions or inflammation. Conclusions: Our findings show that ADM does not have cytotoxic properties, has adequate biomechanical parameters to effectively reinforce supporting soft tissues, does not cause an inflammatory response during implantation, and integrates fully into tissues. Our study demonstrates the effectiveness and utility of the developed ADM in surgical treatment of anterior abdominal wall defects.

Development and systematic evaluation of decellularization protocols in different application models for diaphragmatic tissue engineering

BackgroundTissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage. MethodsWe decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy. ResultsAll protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET. ConclusionDET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects.

Results of Experimental Hernioplasty with Acellular Dermal Matrix

We performed a comprehensive assessment of the acellular dermal matrix obtained during the detergent-enzymatic treatment of the porcine dermis. Acellular dermal matrix was used for the experimental treatment of a hernial defect in a pig using the sublay method. Sixty days after the surgery, biopsy specimens were obtained from the area of hernia repair. The acellular dermal matrix can be easily modeled depending on the size and shape of the defect during surgical procedures, can eliminate the defect of the anterior abdominal wall, and is resistant to cutting by the suture material. Histological examination demonstrated replacement of the acellular dermal matrix with newly formed connective tissue.

Development of a porcine acellular bladder matrix for tissue-engineered bladder reconstruction

PurposeEnterocystoplasty is adopted for patients requiring bladder augmentation, but significant long-term complications highlight need for alternatives. We established a protocol for creating a natural-derived bladder extracellular matrix (BEM) for developing tissue-engineered bladder, and investigated its structural and functional characteristics.MethodsPorcine bladders were de-cellularised with a dynamic detergent–enzymatic treatment using peristaltic infusion. Samples and fresh controls were evaluated using histological staining, ultrastructure (electron microscopy), collagen, glycosaminoglycans and DNA quantification and biomechanical testing. Compliance and angiogenic properties (Chicken chorioallantoic membrane [CAM] assay) were evaluated. T test compared stiffness and glycosaminoglycans, collagen and DNA quantity. p value of < 0.05 was regarded as significant.ResultsHistological evaluation demonstrated absence of cells with preservation of tissue matrix architecture (collagen and elastin). DNA was 0.01 μg/mg, significantly reduced compared to fresh tissue 0.13 μg/mg (p < 0.01). BEM had increased tensile strength (0.259 ± 0.022 vs 0.116 ± 0.006, respectively, p < 0.0001) and stiffness (0.00075 ± 0.00016 vs 0.00726 ± 0.00216, p = 0.011). CAM assay showed significantly increased number of convergent allantoic vessels after 6 days compared to day 1 (p < 0.01). Urodynamic studies showed that BEM maintains or increases capacity and compliance.ConclusionDynamic detergent–enzymatic treatment produces a BEM which retains structural characteristics, increases strength and stiffness and is more compliant than native tissue. Furthermore, BEM shows angiogenic potential. These data suggest the use of BEM for development of tissue-engineered bladder for patients requiring bladder augmentation.

Extracellular Matrix From Decellularized Wharton's Jelly Improves the Behavior of Cells From Degenerated Intervertebral Disc.

Regenerative therapies for intervertebral disc (IVD) injuries are currently a major challenge that is addressed in different ways by scientists working in this field. Extracellular matrix (ECM) deriving from decellularized non-autologous tissues has been established as a biomaterial with remarkable regenerative capacity and its potential as a therapeutic agent is rising. In the present study, we investigated the potential of decellularized Wharton’s jelly matrix (DWJM) from human umbilical cord to act as an ECM-based scaffold for IVD cell culturing. An efficient detergent-enzymatic treatment (DET) was used to produce DWJM maintaining its native microarchitecture. Afterward, immunofluorescence, biochemical assays and electron microscopy analysis showed that DWJM was able to produce sizeable 3D cell aggregates, when combined with human mesenchymal stromal cells isolated from WJ (MSCs) and IVD cells. These latter cells are characterized by the loss of their chondrocyte-like phenotype since they have been isolated from degenerated IVD and in vitro expanded to further de-differentiate. While the effect exerted by DWJM on MSCs was essentially the induction of proliferation, conversely, on IVD cells the DWJM promoted cell differentiation toward a discogenic phenotype. Notably, for the first time, the ability of DWJM to improve the degenerated phenotype of human IVD cells was demonstrated, showing that the mere presence of the matrix maintained the viability of the cells, and positively affected the expression of critical regulators of IVD homeostasis, such as SOX2, SOX9, and TRPS1 transcription factors at specific culture time. Our data are in line with the hypothesis that the strengthening of cell properties in terms of viability and expression of specific proteins at precise times represents an important condition in the perspective of guiding the recovery of cellular functionality and triggering regenerative potential. Currently, there are no definitive surgical or pharmacological treatments for IVD degeneration (IDD) able to restore the disc structure and function. Therefore, the potential of DWJM to revert degenerated IVD cells could be exploited in the next future an ECM-based intradiscal injectable therapeutic.

Recellularized Colorectal Cancer Patient-derived Scaffolds as in vitro Pre-clinical 3D Model for Drug Screening.

Colorectal cancer (CRC) shows highly ineffective therapeutic management. An urgent unmet need is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III CRC patients without any predictive factor of efficacy. In the field of drug discovery, a critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We proposed a patient-derived 3D preclinical model for drug evaluation that could mimic in vitro the patient’s disease. Surgically resected CRC tissue and adjacent healthy colon mucosa were decellularized by a detergent-enzymatic treatment. Scaffolds were recellularized with HT29 and HCT116 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences, scanning electron microscopy, and DNA amount quantification. A chemosensitivity test was performed using an increasing concentration of 5-fluorouracil (5FU). In vivo studies were carried out using zebrafish (Danio rerio) animal model. Permeability test and drug absorption were also determined. The decellularization protocol allowed the preservation of the original structure and ultrastructure. Five days after recellularization with HT29 and HCT116 cell lines, the 3D CRC model exhibited reduced sensitivity to 5FU treatments compared with conventional 2D cultures. Calculated the half maximal inhibitory concentration (IC50) for HT29 treated with 5FU resulted in 11.5 µM in 3D and 1.3 µM in 2D, and for HCT116, 9.87 µM in 3D and 1.7 µM in 2D. In xenograft experiments, HT29 extravasation was detected after 4 days post-injection, and we obtained a 5FU IC50 fully comparable to that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that the drug diffused through the repopulated 3D CRC scaffolds and co-localized with the cell nuclei. The bioengineered CRC 3D model could be a reliable preclinical patient-specific platform to bridge the gap between in vitro and in vivo drug testing assays and provide effective cancer treatment.

Optimizing the decellularization process of human maxillofacial muscles for facial reconstruction using a detergent-only approach.

Trauma, congenital diseases, and cancer resection cause muscle deformities of the human facial muscle. Muscle defects are either treated with local or distal flaps if direct closure is not possible. However, such surgical interventions are limited by donor morbidity and limited tissue availability. Decellularized scaffolds provide alternative strategies for replacing and restoring missing facial muscle by creating scaffolds that mimic the native tissue. This study aimed to develop a protocol to decellularize human zygomaticus major muscle (ZMM) and masseter muscle (MM). Three protocols were assessed including a detergent-only treatment (DOT), detergent-enzymatic treatment (DET) protocol, and a third nondetergent nonenzymatic treatment protocol. Scaffolds were then characterized via histological, immunofluorescent, and quantitative techniques to assess which protocol provided optimal decellularization and maintenance of the extracellular matrix (ECM). The results demonstrated three cycles of DOT protocol consisting of 2% sodium dodecyl sulfate for 4 hr was optimal for decellularization for both ZMM and MM. After three cycles, DNA content was significantly reduced compared with native ZMM and MM (p < .05) with preservation of collagen and glycosaminoglycan content and ECM on histological analysis. DET and nondetergent nonenzymatic treatment protocols were unsuccessful in decellularizing the ZMM and MM with residual DNA content after four cycles and caused ECM disruption on histological analysis. All protocols did not impair the mechanical properties and supported human fibroblast growth. In conclusion, the DOT protocol is effective in producing human decellularized muscle scaffolds that maintain the ECM. Further investigation of detergent only decellurization techniques should be explored as a first step to create effective scaffolds for muscle tissue engineering.

Whole rat stomach decellularisation using a detergent-enzymatic protocol

BackgroundConditions leading to reduced gastric volume are difficult to manage and are associated to poor quality-of-life. Stomach augmentation using a tissue-engineered stomach is a potential solution to restore adequate physiology and food reservoir. Aim of this study was to evaluate the decellularisation of whole rat stomach using a detergent-enzymatic protocol.MethodsStomachs harvested from rats were decellularised through luminal and vascular cannulation using 24-h detergent-enzymatic treatment and completely characterized by appropriate staining, DNA and Extracellular matrix -component quantifications.ResultsThe detergent-enzymatic protocol allows a complete decellularisation of the gastric tissue, with a complete removal of the DNA with two cycles as confirmed by both quantifications and histological analysis. Extracellular matrix components, collagen, fibronectin, laminin and elastin, were optimally preserved by the treatment, while glycosaminoglycans were reduced.ConclusionGastric tissue can be efficiently decellularised. Scaffolds retained original structure and important components that could enhance integration with other tissues for in vivo transplant. The use of naturally derived material could be potentially considered for the treatment of both congenital and acquired conditions.

Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application.

Oesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for short- and long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergent-enzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes.

Heterotopic Implantation of Decellularized Pulmonary Artery Homografts In A Rodent Model: Technique Description and Preliminary Report

ABSTRACTPurpose: Despite a substantial amount of literature on tissue-guided regeneration, decellularization process, repopulation time points and stem cell turnover, more in-depth study on the argument is required. Currently, there are plenty of reports involving large animals, as well as clinical studies facing cardiac repair with decellularized homografts, but no exhaustive rodent models are described. The purpose of this study was to develop such a model in rats; preliminary results are also herein reported. Material and Methods: Fresh or decellularized pulmonary homografts from wild type rats were implanted in the abdominal aorta of green fluorescent protein positive rats. Three experimental groups were build up: sham, fresh homograft recipients and decellularized homograft recipients. The homograft decellularization process was performed with three cycles of detergent-enzymatic treatment protocol. Surgical technique of pulmonary homograft implantation and postoperative ultrasonographic evaluation were also reported; gross, histology and immunohistochemistry analysis on unimplanted and postoperative homografts were also carried out. Results: The median total recipient operating time was 148 minutes, with a surgical success rate of 82%. The decellularization protocol resulted effective and showed a complete decellularization with intact extracellular matrix. At 15 days from surgery, the implanted decellularized pulmonary homografts exhibited cell repopulation in the outer media wall and partial endothelial lining in absence of rejection. Conclusions: Our technique is a feasible and reproducible model that can be fundamental for building a valid study for further exploitation on the field. Even in a short-term follow up, the decellularized pulmonary homografts showed autologous repopulation in absence of rejection.

Mouse Skeletal Muscle Decellularization.

Natural acellular matrices obtained from decellularization procedures are biocompatible and non-immunogenic materials considered promising tools for regenerative medicine purposes. Before in vivo implantation, these matrices must be efficiently decellularized, removing all the cellular components to avoid any immunogenic reaction. At the same time, it is important to maintain the original three-dimensional structure of the specific tissue. Here we describe a method: (1) to decellularize mouse quadriceps using a detergent-enzymatic treatment (DET) and (2) to assess decellularization efficiency and scaffold properties.

Dry acellular oesophageal matrix prepared by supercritical carbon dioxide

Recently, the use of acellular matrices in tissue engineering has become extremely significant as tissue substitute for organ/tissue reconstruction. In this clinical scenario, banking decellularised organs ready for transplantation would be mandatory for patients with emergency needs.In this work a new process based on supercritical carbon dioxide (SC-CO2) drying technique was investigated for obtaining a dry/preserved decellularized oesophagus. Experiments were performed coupling a detergent enzymatic treatment with two different protocols: (i) SC-CO2 drying; (ii) dehydration in ethanol and a subsequent SC-CO2 drying. The efficiency of the treatments was investigated by monitoring the loss of weight of the treated samples and the maintenance of the extracellular matrix (ECM) architecture, composition and mechanical properties after rehydration.A successful dry acellular matrix was reached in a shorter time using the combined ethanol and SC-CO2 treatment. Histological analysis reported the maintenance of the tissue matrix architecture and the collagen content for all the treated samples, while the preservation of ultrastructural features were confirmed by scanning electron microscopy (SEM). Tensile tests did not show significant differences in terms of fracture strength before and after the supercritical process. Furthermore, the scaffolds demonstrated good biocompatible properties in terms of cell culture viability in vitro. Overall, the results highlighted the potential of this novel technology to obtain a dried acellular matrix for oesophageal regeneration, preserving the extracellular matrix structure of the native tissue.

OP‐4 ADVANCES IN TISSUE ENGINEERING TO REGENERATE THE INTESTINAL EPITHELIUM

Introduction:Irreversible intestinal failure (IF) due to anatomical or functional loss is a devastating condition is associated with significant morbidity and mortality. Patients referred for intestinal transplantation suffer low survival rates due to the high graft‐rejection rate and sepsis as a consequence long‐term immunosuppression. These clinical challenges reveal an urgent need for research into new treatments for IF. Here, we present recent advances in our work on intestinal tissue engineering.Methods:Ethical approval is in place to collect intestinal tissue from patients at Great Ormond Street Hospital. Intestinal crypts were isolated and organoids were cultured as previously described. Decellularization of whole intestinal sections was performed using 2 cycles of the Detergent Enzymatic Treatment (DET) as previously reported. Organoids were cultured on the epithelial surface of the acellular matrix in vitro for 10 days. Cell survival was analysed by histology, immunofluorescence, immunohistochemistry and electron microscopy techniques.Results:Intestinal crypts from the small intestine and colon were successfully isolated from endoscopic biopsies and cultured in order to generate 3 dimensional organoids. The presence of goblet cells and enterocyte cells were confirmed on immunohistochemistry. Whole sections of intestine of varying lengths were successfully decellularized and assessed using H&amp;E staining and scanning electron microscopy. Organoids were seeded onto the epithelial surface of the acellular intestinal matrices and allowed to engraft for a period of 10 days in vitro. The formation of a neo‐epithelium with evidence of a microvillus brush border was evident after 10 days in culture. Furthermore, the cells had successfully migrated into the crypt domains of the matrix.Conclusions:We demonstrate the potential of tissue engineering for intestinal failure by combining organoid cultures with decellularization expertise. Our results confirm human intestinal organoids to be the ideal source of progenitor cells to regenerate the intestinal epithelium, with potential clinical applications for transplantation.

OC-032 Regenerative medicine for intestinal failure: demonstrating the potential for intestinal organoids to repopulate the epithelium of the intestine

<h3>Introduction</h3> Irreversible intestinal failure (IF) is associated with significant morbidity and mortality. Parenteral nutrition (PN) services have improved over the last decade, yet the life expectancy of patients on PN remains far below enterally fed patients.¹Patients suffering complications of PN are referred for intestinal transplantation however survival amongst those receiving a transplant is limited due to high graft-rejection rate, sepsis and risks of long-term immunosuppression. All these challenges reveal a persisting urgent need for new treatments for IF. Here, we present advances in our work on intestinal stem cell biology and tissue engineering that provide a realistic alternative to current available treatments for IF. <h3>Method</h3> Ethical approval was obtained from the NRES Committee London–Bloomsbury to collect intestinal tissue from patients at Great Ormond Street Hospital. Intestinal organoids were cultured as previously described.²Decellularization of whole intestinal sections was performed with 2 cycles of Detergent Enzymatic Treatment (DET).³Organoids were cultured on epithelial surfaces of acellular matrices <i><i>in vitro</i></i>for 10 days before fixation. Cell survival was analysed by histology, immunofluorescence and electron microscopy techniques. <h3>Results</h3> Three dimensional epithelial organoids were successfully cultured from endoscopic biopsies (Figure 1). Organoids were expanded to generate sufficient cells to repopulate the matrix at the required density. Whole sections of intestine were successfully decellularized and assessed using H&amp;E staining and scanning electron microscopy. Organoids were seeded onto the epithelial surface of the matrices and allowed to engraft for a period of 10 days. The formation of a neo-epithelium with evidence of a microvillus brush border was evident after 10 days in culture. Furthermore, cells had successfully migrated into the crypt domains of the matrix. <h3>Conclusion</h3> We demonstrate the potential of tissue engineering for intestinal failure by combining organoid cultures with decellularization expertise. Our results confirm intestinal organoids to be the ideal source of progenitor cells to regenerate the intestinal epithelium, with potential clinical applications for transplantation. <h3>Disclosure of interest</h3> None Declared. <h3>References</h3> Pironi L, Joly F, Forbes A, <i>et al</i>. Long-term follow up of patients on home parenteral nutrition in Europe: implications for intestinal transplantation. Gut 2011;60:17–25 Sato T, Stange DE, Ferrante M, <i>et al</i>. Long term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma and Barrett’s epithelium. Gastroenterology 2011;141(5):1762–72 Totonelli G, Maghsoudlou P, Garriboli M, <i>et al</i>. A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration. Biomaterials 2012;33(12):3401–10

PS-292 Tissue Engineering In Oesophageal Atresia: Generation Of Decellularized Animal Scaffolds And Comparison Of Different Storage Methods

Introduction Oesophageal atresia has an incidence of 1:3000–1:5000 births and is associated with significant morbidity after conventional surgery (30–40%). Tissue engineering could represent a safer alternative. Animal oesophagi can be decellularised to create scaffolds that maintain the 3D structure and the extracellular matrix (ECM) information. Acellular scaffolds are biocompatible and thought not do provoke chronic rejection reactions. They can be used as a matrix for reseeding prior to implantation, but a storage protocol is required to allow off-the-shelf production and increase their availability for transplant. Aim Optimise oesophageal decellularization in large animals and assess different storage techniques. Materials and methods Rabbit oesophagi (n = 30) were decellularized with different cycles of detergent-enzymatic treatment (DET). Scaffolds were evaluated by histology, immunofluorescence, scanning electron microscopy (SEM), synchrotron imaging, biomechanical testing and ECM component quantification. Scaffolds were stored in: i) 4°C in PBS (4C), ii) -20°C after freeze-drying (FD), iii) -80°C after snap-freezing (SF), iv) medium with 10%DMSO cooled at -1°C/min then stored in liquid nitrogen (MD). Samples were analysed at 2 and 4 weeks, 3 and 6 months with ECM quantification, SEM and biomechanical testing. Results 3 DET cycles preserve the microarchitecture while removing cellular material. 6 months storage at 4C ruined scaffolds architecture. Oesophageal architecture was preserved in SF and MD groups, although elastin fibres were hyper-contracted in SF group. Oesophageal layers in FD group were completely collapsed. Conclusions Optimal decellularization is achieved after 3 DET cycles. MD is the optimal storing method.

PC.02 Tissue engineering approach with acellular matrix supports affected diaphragm of an atrophic mouse model with activation of resident cells

Myopathies are frequently caused by mutations in genes encoding for extracellular matrix (ECM) proteins, which gets progressively substituted by fibrotic tissue. Spinal muscular atrophy is a disorder caused by mutations...

10.5 Tissue engineering approach with acellular matrix supports affected diaphragm of an atrophic mouse model with activation of resident cells

Myopathies are frequently caused by mutations in genes encoding for extracellular matrix (ECM) proteins, which gets progressively substituted by fibrotic tissue. Spinal muscular atrophy is a disorder caused by mutations...

PFM.57 Decellularization using the detergent enzymatic treatment creates a scaffold that preserves oesophageal microarchitecture

IntroductionTissue engineering of the oesophagus (TEO) has been proposed as a therapeutic alternative to oesophageal substitution. Acellular matrices are ideal for TEO because they contain all the extracellular matrix (ECM)...

Blood vessel-derived acellular matrix for vascular graft application.

To overcome the issues connected to the use of autologous vascular grafts and artificial materials for reconstruction of small diameter (<6 mm) blood vessels, this study aimed to develop acellular matrix- (AM-) based vascular grafts. Rat iliac arteries were decellularized by a detergent-enzymatic treatment, whereas endothelial cells (ECs) were obtained through enzymatic digestion of rat skin followed by immunomagnetic separation of CD31-positive cells. Sixteen female Lewis rats (8 weeks old) received only AM or previously in vitro reendothelialized AM as abdominal aorta interposition grafts (about 1 cm). The detergent-enzymatic treatment completely removed the cellular part of vessels and both MHC class I and class II antigens. One month after surgery, the luminal surface of implanted AMs was partially covered by ECs and several platelets adhered in the areas lacking cell coverage. Intimal hyperplasia, already detected after 1 month, increased at 3 months. On the contrary, all grafts composed by AM and ECs were completely covered at 1 month and their structure was similar to that of native vessels at 3 months. Taken together, our findings show that prostheses composed of AM preseeded with ECs could be a promising approach for the replacement of blood vessels.

Preservation of micro-architecture and angiogenic potential in a pulmonary acellular matrix obtained using intermittent intra-tracheal flow of detergent enzymatic treatment

Tissue engineering of autologous lung tissue aims to become a therapeutic alternative to transplantation. Efforts published so far in creating scaffolds have used harsh decellularization techniques that damage the extracellular matrix (ECM), deplete its components and take up to 5 weeks to perform. The aim of this study was to create a lung natural acellular scaffold using a method that will reduce the time of production and better preserve scaffold architecture and ECM components. Decellularization of rat lungs via the intratracheal route removed most of the nuclear material when compared to the other entry points. An intermittent inflation approach that mimics lung respiration yielded an acellular scaffold in a shorter time with an improved preservation of pulmonary micro-architecture. Electron microscopy demonstrated the maintenance of an intact alveolar network, with no evidence of collapse or tearing. Pulsatile dye injection via the vasculature indicated an intact capillary network in the scaffold. Morphometry analysis demonstrated a significant increase in alveolar fractional volume, with alveolar size analysis confirming that alveolar dimensions were maintained. Biomechanical testing of the scaffolds indicated an increase in resistance and elastance when compared to fresh lungs. Staining and quantification for ECM components showed a presence of collagen, elastin, GAG and laminin. The intratracheal intermittent decellularization methodology could be translated to sheep lungs, demonstrating a preservation of ECM components, alveolar and vascular architecture. Decellularization treatment and methodology preserves lung architecture and ECM whilst reducing the production time to 3 h. Cell seeding and in vivo experiments are necessary to proceed towards clinical translation.