Silvia Baiguera

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study

BACKGROUND Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

The first tissue-engineered airway transplantation: 5-year follow-up results

BACKGROUND In 2008, the first transplantation of a tissue-engineered trachea in a human being was done to replace an end-staged left main bronchus with malacia in a 30-year-old woman. We report 5 year follow-up results. METHODS The patient was followed up approximately every 3 months with multidetector CT scan and bronchoscopic assessment. We obtained mucosal biopsy samples every 6 months for histological, immunohistochemical, and electron microscopy assessment. We also assessed quality of life, respiratory function, cough reflex test, and production and specificity of recipient antibodies against donor human leucocyte antigen. FINDINGS By 12 months after transplantation, a progressive cicatricial stenosis had developed in the native trachea close to the tissue-engineered trachea anastomosis, which needed repeated endoluminal stenting. However, the tissue-engineered trachea itself remained open over its entire length, well vascularised, completely re-cellularised with respiratory epithelium, and had normal ciliary function and mucus clearance. Lung function and cough reflex were normal. No stem-cell-related teratoma formed and no anti-donor antibodies developed. Aside from intermittent bronchoscopic interventions, the patient had a normal social and working life. INTERPRETATION These clinical results provide evidence that a tissue-engineering strategy including decellularisation of a human trachea, autologous epithelial and stem-cell culture and differentiation, and cell-scaffold seeding with a bioreactor is safe and promising. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, ALF Medicine.

Tissue engineered human tracheas for in vivo implantation

Two years ago we performed the first clinical successful transplantation of a fully tissue engineered trachea. Despite the clinically positive outcome, the graft production took almost 3 months, a not feasible period of time for patients with the need of an urgent transplantation. We have then improved decellularization process and herein, for the first time, we completely describe and characterize the obtainment of human tracheal bioactive supports. Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed and quantitative PCR confirmed it. SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea, and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties. Moreover immunohistological staining showed the preservation of angiogenic factors and angiogenic assays demonstrated that acellular human tracheal scaffolds exert an in vitro chemo-active action and induce strong in vivo angiogenic response (CAM analysis). We are now able to obtained, in a short and clinically useful time (approximately 3 weeks), a bioengineered trachea that is structurally and mechanically similar to native trachea, which exert chemotactive and pro-angiogenic properties and which could be successfully used for clinical tissue engineered airway clinical replacements.

Endothelialization approaches for viable engineered tissues

One of the main limitation in obtaining thick, 3-dimensional viable engineered constructs is the inability to provide a sufficient and functional blood vessel system essential for the in vitro survival and the in vivo integration of the construct. Different strategies have been proposed to simulate the ingrowth of new blood vessels into engineered tissue, such as the use of growth factors, fabrication scaffold technologies, in vivo prevascularization and cell-based strategies, and it has been demonstrated that endothelial cells play a central role in the neovascularization process and in the control of blood vessel function. In particular, different “environmental” settings (origin, presence of supporting cells, biomaterial surface, presence of hemodynamic forces) strongly influence endothelial cell function, angiogenic potential and the in vivo formation of durable vessels. This review provides an overview of the different techniques developed so far for the vascularization of tissue-engineered constructs (with their advantages and pitfalls), focusing the attention on the recent development in the cell-based vascularization strategy and the in vivo applications.

Tissue‐Engineered Airway: A Regenerative Solution

The use of synthetic degradable or permanent polymers and biomaterials has not yet helped to achieve successful clinical whole‐airway replacement. A novel, clinically successful approach involves tissue engineering (TE) replacement using three‐dimensional biologic scaffolds composed of allogeneic extracellular scaffolds derived from nonautologous sources and recellularized with autologous stem cells or differentiated cells. In this paper, we discuss this novel approach and review information that can lead to a better understanding of stem cell recruitment and/or mobilization and site‐specific tissue protection, which can be pharmacologically boosted in humans.

Development of bioengineered human larynx.

To date, only two human laryngeal allotransplants have been reported and, although they were successful, both patients required life-long immunosuppression. A bioengineered human larynx could represent a possible alternative to allotransplantation. Human larynxes were decellularized enzymatically to obtain acellular matrices. Histological and molecular analysis demonstrated that all cellular components and nuclear material were removed. SEM showed that decellularized matrices retained the hierarchical structures of the native larynx, and mechanical tests demonstrated that the decellularization did not significantly impaired the biomechanically properties of the obtained matrices. Immunohistochemical staining found residual angiogenic factors after decellularization, and CAM analysis demonstrated that acellular laryngeal scaffolds induce a strong in vivo angiogenic response. Using a decellularization method, we are now able to obtain, in a short and clinically useful time, natural bioengineered laryngeal scaffolds which could be use for partial or total implantation in humans.

Ghrelin inhibits FGF-2-mediated angiogenesis in vitro and in vivo.

Recent evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is highly expressed in the cardiovascular system, and in this study we addressed the possibility that ghrelin may affect angiogenesis in vitro and in vivo. Reverse transcription-polymerase chain reaction showed that human umbilical vein endothelial cells (HUVECs) express ghrelin and GHS-R mRNAs. Ghrelin inhibited FGF-2-induced proliferation of HUVECs cultured in vitro, the maximal effective concentration being 10(-8) M, and this effect was annulled by the GHS-R antagonist D-Lys3-growth hormone releasing peptide-6. FGF-2 stimulated HUVEC cultured on Matrigel to form capillary-like structures, and ghrelin (10(-8) M) suppressed this effect. In the chick embryo chorioallantoic membrane in vivo assay, FGF-2 induced a strong angiogenic response, which was counteracted by ghrelin (500 ng). Taken together, these findings suggest that ghrelin acts as an angiostatic molecule and indicate that its activity is comparable to that of a well-known angiostatic agent, i.e., vinblastine. The antiangiogenic activity of ghrelin deserves further investigations, alone or together with other antiangiogenic agents, for the treatment of pathological conditions characterized by enhanced angiogenesis.

Effects of Hyperbaric Oxygen on Proliferative and Apoptotic Activities and Reactive Oxygen Species Generation in Mouse Fibroblast 3T3/J2 Cell Line

Background Hyperbaric oxygen (HBO) therapy is widely used to treat problem wounds associated with pathologic conditions compromising blood supply and tissue oxygenation because increased tissue oxygen levels enhance collagen synthesis, cell proliferation, and angiogenesis. However, little is known about the dose of hyperoxia needed to achieve optimal therapeutic effects. Moreover, HBO, by enhancing the production of reactive oxygen species (ROS), may also exert cytotoxic effects. In vitro models are simplified systems that may aid the development of treatment protocols with HBO. Hence, we have investigated the effects of HBO on the growth and ROS production of the 3T3/J2 fibroblast cell line in relation to the pressure and the duration of exposure. Methods 3T3/J2 fibroblasts were plated (5 × 103 cells/cm2) on six-well microtiter plates in phosphate buffered saline (PBS), put in a compression chamber, and exposed to 100% oxygen at a pressure of 1.0 or 2.5 atmosphere absolute (ATA) for 15, 30, 60, or 120 minutes. Then the cells were incubated in Dulbeccos modified minimum essential medium (DMEM) for 24, 48, or 72 hours, and at the end of the post-HBO incubation period, their number was determined. In other experiments, cells were detached just after HBO exposure, seeded on 60 mm Petri dishes, and cultured for 10 days in DMEM, and the colony forming units were counted. The effects of HBO exposure (2.5 ATA) on the apoptotic rate of cultured cells were investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and enzyme-linked immunosorbent assays. To measure ROS production, 60 minutes before HBO exposure, 2′,7′-dichlorofluorescin (DCF) diacetate (200 nmol/mL) was added to PBS, and after HBO exposure (2.5 ATA), cells were lysated, and fluorescence-emission intensity was measured and converted to μmol DCF/μg protein. Results At 1.0 ATA, all HBO exposures increased the proliferation rate of cultured fibroblasts and their clonal growth efficiency. At 2.5 ATA, 15-minute exposure to HBO was ineffective, whereas 30- and 60-minute exposures raised the proliferation rate and clonal growth efficiency. Conversely, a 120-minute exposure significantly decreased these parameters compared with control cultures. The exposure of cells to HBO at 2.5 ATA for 120 minutes raised the apoptotic rate of cultured fibroblasts, whereas shorter exposure times were ineffective. All exposure periods to HBO at 2.5 ATA enhanced ROS production from cultured fibroblasts. Conclusions Collectively, our findings allow us to conclude that (1) all of the exposure periods to HBO at 1.0 ATA or 30- and 60-minute periods at 2.5 ATA enhance cell growth, (2) 120-minute exposure to HBO at 2.5 ATA exerts a marked proapoptotic effect, and (3) no evident relationships occur between the effects of HBO on cell growth and ROS production.

The concept of in vivo airway tissue engineering.

We investigated whether decellularized pig tracheas could regenerate in vivo, without being recellularized before transplantation, using the own body as bioreactor. Decellularized pig tracheal scaffolds were intraoperative conditioned with mononuclear cells and growth and differentiation factors. During the postoperative period, the in situ regeneration was boosted by administering bioactive molecules to promote peripheral mobilization and differentiation of stem/progenitor cells and ultimately the regenerative process. Results revealed, after 2 weeks, a nearly normal trachea, with respiratory epithelium and a double-banded cartilage but without any mechanical differences compared to the native tissue. The growth factor administration resulted in a mobilization of progenitor and stem cells into the peripheral circulation and in an up-regulation of anti-apoptotic genes. Isolated stem/progenitor cells could be differentiated in vitro into several cell types, proving their multipotency. We provide evidence that the own body can be used as bioreactor to promote in vivo tissue engineering replacement. Moreover, we demonstrated the beneficial effect of additional pharmaceutical intervention for an improved engraftment of the transplant.

Long-term changes to in vitro preserved bioengineered human trachea and their implications for decellularized tissues

Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro. Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture - in particular of collagenous and elastic fiber structure -and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo, having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.