ABSTRACT: Decellularization techniques have been widely used as an alternative strategy for organ reconstruction. This study investigated the mechanical, pro-angiogenic and in vivo biocompatibility properties of decellularized airway matrices cross-linked with genipin. New Zealand rabbit tracheae were decellularized and cross-linked with genipin, a naturally derived agent. The results demonstrated that, a significant (p < 0.05) increase in the secant modulus was computed for the cross-linked tracheae, compared to the decellularized samples. Angiogenic assays demonstrated that decellularized tracheal scaffolds and cross-linked tracheae treated with 1% genipin induce strong in vivo angiogenic responses (CAM analysis). Seven, 15 and 30 days after implantation, decreased (p < 0.01) inflammatory reactions were observed in the xenograft models for the genipin cross-linked tracheae matrices compared with control tracheae, and no increase in the IgM or IgG content was observed in rats. In conclusion, treatment with genipin improves the mechanical properties of decellularized airway matrices without altering the pro-angiogenic properties or eliciting an in vivo inflammatory response.
Project description:Decellularized xenogeneic whole-liver matrices are plausible biomedical materials for the bioengineering of liver transplantation. A common method to reduce the inflammatory potential of xenogeneic matrices is crosslinking. Nevertheless, a comprehensive analysis of the immunogenic features of cross-linked decellularized tissue is still lacking. We aimed to reduce the immunogenicity of decellularized porcine whole-liver matrix through crosslinking with glutaraldehyde or genipin, a new natural agent, and investigated the mechanism of the immune-mediated responses. The histologic assessment of the host's immune reaction activated in response to these scaffolds, as well as the M1/M2 phenotypic polarization profile of macrophages, was studied in vivo. The genipin-fixed scaffold elicited a predominantly M2 phenotype response, while the glutaraldehyde-fixed scaffold resulted in disrupted host tissue remodeling and a mixed macrophage polarization profile. The specific subsets of immune cells involved in the responses to the scaffolds were identified in vitro. Crosslinking alleviated the host response by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. Therefore, we conclude that the natural genipin crosslinking could lower the immunogenic potential of xenogeneic decellularized whole-liver scaffolds.
Project description:Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials. Stem Cells Translational Medicine 2017;6:1458-1464.
Project description:The extracellular matrix (ECM) creates the microenvironment of the tissue; an altered ECM in the asthmatic airway may be central in airway inflammation and remodelling. Tumstatin is a collagen IV-derived matrikine reduced in the asthmatic airway wall that reverses airway inflammation and remodelling in small and large animal models of asthma. This study hypothesized that the mechanisms underlying the broad asthma-resolving effects of tumstatin were due to autocrine remodelling of the ECM. Neutrophils and endothelial cells were seeded on decellularized ECM of non-asthmatic (NA) or asthmatic (A) airway smooth muscle (ASM) cells previously exposed to tumstatin in the presence or absence of a broad matrix metalloproteinase inhibitor, Marimastat. Gene expression in NA and A ASM induced by tumstatin was assessed using RT-PCR arrays. The presence of tumstatin during ECM deposition affected neutrophil and endothelial cell properties on both NA and A ASM-derived matrices and this was only partly due to MMP activity. Gene expression patterns in response to tumstatin in NA and A ASM cells were different. Tumstatin may foster an anti-inflammatory and anti-angiogenic microenvironment by modifying ASM-derived ECM. Further work is required to examine whether restoring tumstatin levels in the asthmatic airway represents a potential novel therapeutic approach.
Project description:Decellularized tissues have proven to be versatile matrices for the engineering of tissues and organs. These matrices usually consist of collagens, matrix-specific proteins, and a set of largely undefined growth factors and signaling molecules. Although several decellularized tissues have found their way to clinical applications, their use in the engineering of cartilage tissue has only been explored to a limited extent. We set out to generate hydrogels from several tissue-derived matrices, as hydrogels are the current preferred cell carriers for cartilage repair. Equine cartilage, meniscus, and tendon tissue was harvested, decellularized, enzymatically digested, and functionalized with methacrylamide groups. After photo-cross-linking, these tissue digests were mechanically characterized. Next, gelatin methacrylamide (GelMA) hydrogel was functionalized with these methacrylated tissue digests. Equine chondrocytes and mesenchymal stromal cells (MSCs) (both from three donors) were encapsulated and cultured in vitro up to 6 weeks. Gene expression (COL1A1, COL2A1, ACAN, MMP-3, MMP-13, and MMP-14), cartilage-specific matrix formation, and hydrogel stiffness were analyzed after culture. The cartilage, meniscus, and tendon digests were successfully photo-cross-linked into hydrogels. The addition of the tissue-derived matrices to GelMA affected chondrogenic differentiation of MSCs, although no consequent improvement was demonstrated. For chondrocytes, the tissue-derived matrix gels performed worse compared to GelMA alone. This work demonstrates for the first time that native tissues can be processed into crosslinkable hydrogels for the engineering of tissues. Moreover, the differentiation of encapsulated cells can be influenced in these stable, decellularized matrix hydrogels.
Project description:In 2010, a tissue-engineered trachea was transplanted into a 10-year-old child using a decellularized deceased donor trachea repopulated with the recipient's respiratory epithelium and mesenchymal stromal cells. We report the child's clinical progress, tracheal epithelialization and costs over the 4 years. A chronology of events was derived from clinical notes and costs determined using reference costs per procedure. Serial tracheoscopy images, lung function tests and anti-HLA blood samples were compared. Epithelial morphology and T cell, Ki67 and cleaved caspase 3 activity were examined. Computational fluid dynamic simulations determined flow, velocity and airway pressure drops. After the first year following transplantation, the number of interventions fell and the child is currently clinically well and continues in education. Endoscopy demonstrated a complete mucosal lining at 15 months, despite retention of a stent. Histocytology indicates a differentiated respiratory layer and no abnormal immune activity. Computational fluid dynamic analysis demonstrated increased velocity and pressure drops around a distal tracheal narrowing. Cross-sectional area analysis showed restriction of growth within an area of in-stent stenosis. This report demonstrates the long-term viability of a decellularized tissue-engineered trachea within a child. Further research is needed to develop bioengineered pediatric tracheal replacements with lower morbidity, better biomechanics and lower costs.
Project description:Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.
Project description:Here we report the creation of a novel tracheal construct in the form of an engineered, acellular tissue-stent biocomposite trachea (TSBT). Allogeneic or xenogeneic smooth muscle cells are cultured on polyglycolic acid polymer-metal stent scaffold leading to the formation of a tissue comprising cells, their deposited collagenous matrix, and the stent material. Thorough decellularization then produces a final acellular tubular construct. Engineered TSBTs were tested as end-to-end tracheal replacements in 11 rats and 3 nonhuman primates. Over a period of 8 weeks, no instances of airway perforation, infection, stent migration, or erosion were observed. Histological analyses reveal that the patent implants remodel adaptively with native host cells, including formation of connective tissue in the tracheal wall and formation of a confluent, columnar epithelium in the graft lumen, although some instances of airway stenosis were observed. Overall, TSBTs resisted collapse and compression that often limit the function of other decellularized tracheal replacements, and additionally do not require any cells from the intended recipient. Such engineered TSBTs represent a model for future efforts in tracheal regeneration.
Project description:Genipin, a natural cross-linking reagent extracted from the fruits of Gardenia jasminoides, can be effectively employed in tissue engineering applications due to its low cytotoxicity and high biocompatibility. The cross-linking of collagen hydrogels with genipin was followed with one-photon fluorescence spectroscopy, second harmonic generation, fluorescence and transmission electron microscopy. The incubation with genipin induced strong auto-fluorescence within the collagen hydrogels. The fluorescence emission maximum of the fluorescent adducts formed by genipin exhibit a strong dependence on the excitation wavelength. The emission maximum is at 630 nm when we excite the cross-linked samples with 590 nm light and shifts to 462 nm when we use 400 nm light instead. The fluorescence imaging studies show that genipin induces formation of long aggregated fluorescent strands throughout the depth of samples. The second harmonic generation (SHG) imaging studies suggest that genipin partially disaggregates 10 ?m "fiberlike" collagen structures because of the formation of these fluorescent cross-links. Transmission electron microscopy (TEM) studies reveal that genipin largely eliminates collagen's characteristic native fibrillar striations. Our study is the first one to nondestructively follow and identify the structure within collagen hydrogels in situ and to sample structures formed on both micro- and nanoscales. Our findings suggest that genipin cross-linking of collagen follows a complex mechanism and this compound modifies the structure within the collagen hydrogels in both micro- and nanoscale.
Project description:Importance:Airway transplantation could be an option for patients with proximal lung tumor or with end-stage tracheobronchial disease. New methods for airway transplantation remain highly controversial. Objective:To establish the feasibility of airway bioengineering using a technique based on the implantation of stented aortic matrices. Design, Setting, and Participants:Uncontrolled single-center cohort study including 20 patients with end-stage tracheal lesions or with proximal lung tumors requiring a pneumonectomy. The study was conducted in Paris, France, from October 2009 through February 2017; final follow-up for all patients occurred on November 2, 2017. Exposures:Radical resection of the lesions was performed using standard surgical techniques. After resection, airway reconstruction was performed using a human cryopreserved (-80°C) aortic allograft, which was not matched by the ABO and leukocyte antigen systems. To prevent airway collapse, a custom-made stent was inserted into the allograft. In patients with proximal lung tumors, the lung-sparing intervention of bronchial transplantation was used. Main Outcomes and Measures:The primary outcome was 90-day mortality. The secondary outcome was 90-day morbidity. Results:Twenty patients were included in the study (mean age, 54.9 years; age range, 24-79 years; 13 men [65%]). Thirteen patients underwent tracheal (n = 5), bronchial (n = 7), or carinal (n = 1) transplantation. Airway transplantation was not performed in 7 patients for the following reasons: medical contraindication (n = 1), unavoidable pneumonectomy (n = 1), exploratory thoracotomy only (n = 2), and a lobectomy or bilobectomy was possible (n = 3). Among the 20 patients initially included, the overall 90-day mortality rate was 5% (1 patient underwent a carinal transplantation and died). No mortality at 90 days was observed among patients who underwent tracheal or bronchial reconstruction. Among the 13 patients who underwent airway transplantation, major 90-day morbidity events occurred in 4 (30.8%) and included laryngeal edema, acute lung edema, acute respiratory distress syndrome, and atrial fibrillation. There was no adverse event directly related to the surgical technique. Stent removal was performed at a postoperative mean of 18.2 months. At a median follow-up of 3 years 11 months, 10 of the 13 patients (76.9%) were alive. Of these 10 patients, 8 (80%) breathed normally through newly formed airways after stent removal. Regeneration of epithelium and de novo generation of cartilage were observed within aortic matrices from recipient cells. Conclusions and Relevance:In this uncontrolled study, airway bioengineering using stented aortic matrices demonstrated feasibility for complex tracheal and bronchial reconstruction. Further research is needed to assess efficacy and safety. Trial Registration:clinicaltrials.gov Identifier: NCT01331863.
Project description:Abdominal pumping in caterpillars has only been documented during molting. Using synchrotron X-ray imaging in conjunction with high-speed flow-through respirometry, we show that Manduca sexta caterpillars cyclically contract their bodies in response to hypoxia, resulting in significant compressions of the tracheal system. Compression of tracheae induced by abdominal pumping drives external gas exchange, as evidenced by the high correlation between CO2 emission peaks and body movements. During abdominal pumping, both the compression frequency and fractional change in diameter of tracheae increased with body mass. However, abdominal pumping and tracheal compression were only observed in larger, older caterpillars (>0.2 g body mass), suggesting that this hypoxic response increases during ontogeny. The diameters of major tracheae in the thorax increased isometrically with body mass. However, tracheae in the head did not scale with mass, suggesting that there is a large safety margin for oxygen delivery in the head in the youngest animals. Together, these results highlight the need for more studies of tracheal system scaling and suggest that patterns of tracheal investment vary regionally in the body.