Biomechanics of regenerated esophageal tissue following the implantation of a tissue engineered CellspanTM Esophageal Implant.

The esophagus is a tubular organ with a multi-laminated tissue structure that functions to transport nutrition from the oral cavity to the stomach. Several diseases of the esophagus including congenital disorders require complete surgical esophagectomy. Ideally, segmental removal of the diseased/damaged tissue would spare the unaffected tissue and preserve organ function. To this end, a novel tissue engineered implant, the CellspanTM Esophageal Implant (CEI) was used to repair the esophagus following segmental resection of the thoracic esophagus in a porcine model. The current study investigated the mechanical strength and the associated tissue architecture of the CEI-stimulated tissue. The CEI bridged the proximal and distal native esophageal ends to restore the conduit by stimulating a regeneration process that progressed from a fibrovascular scar at 30-days to a fully epithelialized lumen at 90-days, followed by submucosal regeneration and regeneration of a 'laminated' adventitia with smooth muscle development in the 365-day cohort. The mechanical strength of the newly developed tissue as well as the flanking native tissue were assessed using a probe-burst pressure test (ASTM D6797-15). The burst pressures at all three time points were comparable to the native tissue flanking the implant. In addition, the overall pressure required to burst through both the native and regenerated tissues increased with increasing time post-implantation.

Esophageal regeneration following surgical implantation of a tissue engineered esophageal implant in a pediatric model.

Diseases of the esophagus, damage of the esophagus due to injury or congenital defects during fetal esophageal development, i.e., esophageal atresia (EA), typically require surgical intervention to restore esophageal continuity. The development of tissue engineered tubular structures would improve the treatment options for these conditions by providing an alternative that is organ sparing and can be manufactured to fit the exact dimensions of the defect. An autologous tissue engineered Cellspan Esophageal ImplantTM (CEI) was surgically implanted into piglets that underwent surgical resection of the esophagus. Multiple survival time points, post-implantation, were analyzed histologically to understand the tissue architecture and time course of the regeneration process. In addition, we investigated CT imaging as an "in-life" monitoring protocol to assess tissue regeneration. We also utilized a clinically relevant animal management paradigm that was essential for long term survival. Following implantation, CT imaging revealed early tissue deposition and the formation of a contiguous tissue conduit. Endoscopic evaluation at multiple time points revealed complete epithelialization of the lumenal surface by day 90. Histologic evaluation at several necropsy time points, post-implantation, determined the time course of tissue regeneration and demonstrated that the tissue continues to remodel over the course of a 1-year survival time period, resulting in the development of esophageal structural features, including the mucosal epithelium, muscularis mucosae, lamina propria, as well as smooth muscle proliferation/migration initiating the formation of a laminated adventitia. Long term survival (1 year) demonstrated restoration of oral nutrition, normal animal growth and the overall safety of this treatment regimen.

First-in-Human Segmental Esophageal Reconstruction Using a Bioengineered Mesenchymal Stromal Cell-Seeded Implant.

INTRODUCTION: Resection and reconstruction of the esophagus remains fraught with morbidity and mortality. Recently, data from a porcine reconstruction model revealed that segmental esophageal reconstruction using an autologous mesenchymal stromal cell-seeded polyurethane graft (Cellspan esophageal implant [CEI]) can facilitate esophageal regrowth and regeneration. To this end, a patient requiring a full circumferential esophageal segmental reconstruction after a complex multiorgan tumor resection was approved for an investigational treatment under the Food and Drug Administration Expanded Access Use (Investigational New Drug 17402). METHODS: Autologous adipose-derived mesenchymal stromal cells (Ad-MSCs) were isolated from the Emergency Investigational New Drug patient approximately 4 weeks before surgery from an adipose tissue biopsy specimen. The Ad-MSCs were grown and expanded under current Good Manufacturing Practice manufacturing conditions. The cells were then seeded onto a polyurethane fiber mesh scaffold (Cellspan scaffold) and cultured in a custom bioreactor to manufacture the final CEI graft. The cell-seeded scaffold was then shipped to the surgical site for surgical implantation. After removal of a tumor mass and a full circumferential 4 cm segment of the esophagus that was invaded by the tumor, the CEI was implanted by suturing the tubular CEI graft to both ends of the remaining native esophagus using end-to-end anastomosis. RESULTS: In this case report, we found that a clinical-grade, tissue-engineered esophageal graft can be used for segmental esophageal reconstruction in a human patient. This report reveals that the graft supports regeneration of the esophageal conduit. Histologic analysis of the tissue postmortem, 7.5 months after the implantation procedure, revealed complete luminal epithelialization and partial esophageal tissue regeneration. CONCLUSIONS: Autologous Ad-MSC seeded onto a tubular CEI tissue-engineered graft stimulates tissue regeneration following implantation after a full circumferential esophageal resection.

Engineered Tissue-Stent Biocomposites as Tracheal Replacements.

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.

New Functional Tools for Antithrombogenic Activity Assessment of Live Surface Glycocalyx.

OBJECTIVE: It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators' ability to assess the role of the glycocalyx in vascular health. APPROACH AND RESULTS: We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface's functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm(2) on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography-mass spectrometry analysis. CONCLUSIONS: Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine.

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.

Fate of distal lung epithelium cultured in a decellularized lung extracellular matrix.

Type II cells are the defenders of the alveolus. They produce surfactant to prevent alveolar collapse, they actively transport water to prevent filling of the air sacs that would otherwise prevent gas exchange, and they differentiate to type I epithelial cells. They are an indispensable component of functional lung tissue. To understand the functionality of type II cells in isolation, we sought to track their fate in decellularized matrices and to assess their ability to contribute to barrier function by differentiation to type I alveolar epithelial cells. Rat type II cells were isolated from neonatal rat lungs by labeling with the RTII-70 surface marker and separation using a magnetic column. This produced a population of ∼50% RTII-70-positive cells accompanied by few type I epithelial cells or α-actin-positive mesenchymal cells. This population was seeded into decellularized rat lung matrices and cultured for 1 or 7 days. Culture in Dulbecco's modified Eagle's medium +10% fetal bovine serum (FBS) resulted in reduced expression of epithelial markers and increased expression of mesenchymal markers. By 7 days, no epithelial markers were visible by immunostaining; nearly all cells were α-actin positive. Gene expression for the mesenchymal markers, α-actin, vimentin, and TGF-ßR, was significantly upregulated on day 1 (p=0.0005, 0.0005, and 2.342E-5, respectively). Transcript levels of α-actin and TGF-ßR remained high at 7 days (p=1.364E-10 and 0.0002). Interestingly, human type II cells cultured under the same conditions showed a similar trend in the loss of epithelial markers, but did not display high expression of mesenchymal markers. Rat cells additionally showed the ability to produce and degrade the basement membrane and extracellular matrix components, such as fibronectin, collagen IV, and collagen I. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed significant increases in expression of the fibronectin and matrix metalloprotease-2 (MMP-2) genes after 1 day in culture (p=0.0135 and 0.0128, respectively) and elevated collagen I expression at 7 days (p=0.0016). These data suggest that the original type II-enriched population underwent a transition to increased expression of mesenchymal markers, perhaps as part of a survival or wound-healing program. These results suggest that additional medium components and/or the application of physiologically appropriate stimuli such as ventilation may be required to promote lung-specific epithelial phenotypes.

Click-coated, heparinized, decellularized vascular grafts.

A novel method enabling the engineering of a dense and appropriately oriented heparin-containing layer on decellularized aortas has been developed. Amino groups of decellularized aortas were first modified to azido groups using 3-azidobenzoic acid. Azide-clickable dendrons were attached onto the azido groups through "alkyne-azide" click chemistry, affording a tenfold amplification of adhesions sites. Dendron end groups were finally decorated with end-on modified heparin chains. Heparin chains were oriented like heparan sulfate groups on native endothelial cells surface. X-ray photoelectron spectroscopy, nuclear magnetic resonance imaging, mass spectrometry and Fourier transform infrared FTIR spectroscopy were used to characterize the synthesis steps, building the final heparin layered coatings. The continuity of the heparin coating was verified using fluorescent microscopy and histological analysis. The efficacy of heparin linkage was demonstrated with factor Xa anti-thrombogenic assay and platelet adhesion studies. The results suggest that oriented heparin immobilization to decellularized aortas may improve the in vivo blood compatibility of decellularized aortas and vessels.

Tissue-engineered vascular grafts created from human induced pluripotent stem cells.

The utility of human induced pluripotent stem cells (hiPSCs) to create tissue-engineered vascular grafts was evaluated in this study. hiPSC lines were first induced into a mesenchymal lineage via a neural crest intermediate using a serum-free, chemically defined differentiation scheme. Derived cells exhibited commonly known mesenchymal markers (CD90, CD105, and CD73 and negative marker CD45) and were shown to differentiate into several mesenchymal lineages (osteogenic, chondrogenic, and adipogenic). Functional vascular grafts were then engineered by culturing hiPSC-derived mesenchymal progenitor cells in a pulsatile bioreactor system over 8 weeks to induce smooth muscle cell differentiation and collagenous matrix generation. Histological analyses confirmed layers of calponin-positive smooth muscle cells in a collagen-rich matrix. Mechanical tests revealed that grafts had an average burst pressure of 700 mmHg, which is approximately half that of native veins. Additionally, studies revealed that karyotypically normal mesenchymal stem cell clones led to generation of grafts with predicted features of engineered vascular grafts, whereas derived clones having chromosomal abnormalities generated calcified vessel constructs, possibly because of cell apoptosis during culture. Overall, these results provide significant insight into the utility of hiPS cells for vascular graft generation. They pave the way for creating personalized, patient-specific vascular grafts for surgical applications, as well as for creating experimental models of vascular development and disease.

Strategies for whole lung tissue engineering.

Recent work has demonstrated the feasibility of using decellularized lung extracellular matrix scaffolds to support the engineering of functional lung tissue in vitro. Rendered acellular through the use of detergents and other reagents, the scaffolds are mounted in organ-specific bioreactors where cells in the scaffold are provided with nutrients and appropriate mechanical stimuli such as ventilation and perfusion. Though initial studies are encouraging, a great deal remains to be done to advance the field and transition from rodent lungs to whole human tissue engineered lungs. To do so, a variety of hurdles must be overcome. In particular, a reliable source of human-sized scaffolds, as well as a method of terminal sterilization of scaffolds, must be identified. Continued research in lung cell and developmental biology will hopefully help identify the number and types of cells that will be required to regenerate functional lung tissue. Finally, bioreactor designs must be improved in order to provide more precise ventilation stimuli and vascular perfusion in order to avoid injury to or death of the cells cultivated within the scaffold. Ultimately, the success of efforts to engineer a functional lung in vitro will critically depend on the ability to create a fully endothelialized vascular network that provides sufficient barrier function and alveolar-capillary surface area to exchange gas at rates compatible with healthy lung function.

Small-diameter vascular graft engineered using human embryonic stem cell-derived mesenchymal cells.

Despite the progress made thus far in the generation of small-diameter vascular grafts, cell sourcing still remains a problem. Human embryonic stem cells (hESCs) present an exciting new cell source for the regeneration applications due to their high proliferative and differentiation capabilities. In this study, the feasibility of creating small-diameter vascular constructs using smooth muscle cells (SMCs) differentiated from hESC-derived mesenchymal cells was evaluated. In vitro experiments confirmed the ability of these cells to differentiate into smooth muscle actin- and calponin-expressing SMCs in the presence of known inducers, such as transforming growth factor beta. Human vessel walls were constructed by culturing these cells in a bioreactor system under pulsatile conditions for 8 weeks. Histological analysis showed that vessel grafts had similarities to their native counterparts in terms of cellularity and SMC marker expression. However, markers of cartilage and bone tissue were also detected, thus raising questions about stable lineage commitment during differentiation and calling for more stringent analysis of differentiating cell populations.

Smooth muscle and other cell sources for human blood vessel engineering.

Despite substantial progress in the field of vascular tissue engineering over the past decades, transition to human models has been rather challenging. The limited replicative life spans of human adult vascular cells, and their slow rate of collagenous matrix production in vitro, have posed important hurdles in the development of mechanically robust and biologically functional engineered grafts. With the more recent advances in the field of stem cells, investigators now have access to a plethora of new cell source alternatives for vascular engineering. In this paper, we review various alternative cell sources made available more recently for blood vessel engineering and also present some recent data on the derivation of smooth muscle cells from human induced pluripotent stem cells.

Interplay of polyethyleneimine molecular weight and oligonucleotide backbone chemistry in the dynamics of antisense activity.

The widespread utilization of gene silencing techniques, such as antisense, is impeded by the poor cellular delivery of oligonucleotides (ONs). Rational design of carriers for enhanced ON delivery demands a better understanding of the role of the vector on the extent and time course of antisense effects. The aim of this study is to understand the effects of polymer molecular weight (MW) and ON backbone chemistry on antisense activity. Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries. We measured their physico-chemical properties and evaluated their ability to deliver ONs to cells, leading to an antisense response. Our key finding is that the antisense activity is not determined solely by PEI MW or by ON chemistry, but rather by the interplay of both factors. While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry. Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly. We also present a mathematical model of the antisense process to highlight the importance of ON delivery to antisense down-regulation.

Oligonucleotide structure influences the interactions between cationic polymers and oligonucleotides.

We examined the effect of oligodeoxynucleotide (ODN) structure on the interactions between cationic polymers and ODNs. Unstructured and hairpin structured ODNs were used to form complexes with the model cationic polymer, poly-L-lysine (pLL), and the characteristics of these polymer-ODN interactions were subsequently examined. We found that hairpin structured ODNs formed complexes with pLL at slightly lower pLL:ODN charge ratios as compared to unstructured ODNs and that, at high charge ratios, greater fractions of the hairpin ODNs were complexed, as measured by dye exclusion. The dissociation of pLL-ODN interactions was tested further by challenge with heparin, which induced complex disruption. Both the kinetics and heparin dose response of ODN release were determined. The absolute amount and the kinetic rate of ODN release from the complexes of pLL and unstructured ODN were greater, as compared to hairpin ODNs. Our results therefore highlight the role of ODN structure on the association-dissociation behavior of polymer-ODN complexes. These findings have implications for the selection of ODN sequences and design of polymeric carriers used for cellular delivery of ODNs.

Engineering synthetic vectors for improved DNA delivery: insights from intracellular pathways.

Significant progress has been made in the area of nonviral gene delivery to date. Yet, synthetic vectors remain less efficient by orders of magnitude than their viral counterparts. Research continues toward unraveling and overcoming various barriers to the efficient delivery of DNA, whether in plasmid form encoding a gene or as an oligonucleotide for the selective inhibition of target gene expression. Novel components for overcoming these hurdles are continually being incorporated into the design of synthetic vectors, leading to increasingly more virus-like particles. Despite these advances, general principles defining the design of synthetic vectors are yet to be developed fully. A more quantitative analysis of the cellular uptake and intracellular processing of these vectors is required for the rational manipulation of vector design. Mathematical frameworks with a more conceptual basis will help obtain an integrated perspective on these complex systems. In this review, we critically examine the progress made toward the improved design of synthetic vectors by the strategic exploitation of intracellular mechanisms and explore newer possibilities to overcome obstacles in the practical realization of this field.