Arterial grafts exhibiting unprecedented cellular infiltration and remodeling in vivo: The role of cells in the vascular wall

ObjectiveTo engineer and implant vascular grafts in the arterial circulation of a pre-clinical animal model and assess the role of donor medial cells in graft remodeling and function.Approach and resultsVascular grafts were engineered using Small Intestinal Submucosa (SIS)-fibrin hybrid scaffold and implanted interpositionally into the arterial circulation of an ovine model. We sought to demonstrate implantability of SIS-Fibrin based grafts; examine the remodeling; and determine whether the presence of vascular cells in the medial wall was necessary for cellular infiltration from the host and successful remodeling of the implants. We observed no occlusions or anastomotic complications in 18 animals that received these grafts. Notably, the grafts exhibited unprecedented levels of host cell infiltration that was not limited to the anastomotic sites but occurred through the lumen as well as the extramural side, leading to uniform cell distribution. Incoming cells remodeled the extracellular matrix and matured into functional smooth muscle cells as evidenced by expression of myogenic markers and development of vascular reactivity. Interestingly, tracking the donor cells revealed that their presence was beneficial but not necessary for successful grafting. Indeed, the proliferation rate and number of donor cells decreased over time as the vascular wall was dominated by host cells leading to significant remodeling and development of contractile function.ConclusionsThese results demonstrate that SIS-Fibrin grafts can be successfully implanted into the arterial circulation of a clinically relevant animal model, improve our understanding of vascular graft remodeling and raise the possibility of engineering mural cell-free arterial grafts.     

Piezo‐bending actuators for isometric or auxotonic contraction analysis of engineered heart tissue

Engineered heart tissue (EHT) has proven as valuable tool for disease modelling, drug safety screening, and cardiac repair. Especially in combination with the stem cell technology, these in vitro models of the human heart have generated interest not only of basic cardiovascular researchers but also of regulatory authorities responsible for drug safety. A main limitation of 3D‐based assays for evaluating cardiotoxicity is their limited throughput. We integrated piezo‐bending actuators in a 24‐well system for the generation of strip‐like rat and human EHT attached to hollow, elastic silicone posts. Muscle contractions of EHTs induced a measurable electrical current in the piezo‐bending actuators that could be analysed for contraction amplitude, frequency, and contraction and relaxation kinetics. Compared with the standard video‐optical analysis of contractile activity, the new system allows for (a) the analysis of several tissues in parallel, (b) switching between auxotonic and isometric contractions by inserting a stiff metal post in the silicone post opposing the piezo actuator, (c) continuous measurement over days with low data volume (megabyte), (d) automated measurement without the necessity of adjustment of tissue position for video‐optical analysis, (e) reduced complexity and costs, (f) high sensitivity of contraction detection, (g) calculation of absolute contraction force, and (h) suitability for variable tissue geometries. The new set‐up for contraction analysis based on piezo‐bending actuators is a promising new method for the parallel screening of EHT for pharmacological drug effects and other applications of muscle tissue engineering (e.g., skeletal muscle engineering or cardiac repair).     

Tissue‐engineered trachea from a 3D‐printed scaffold enhances whole‐segment tracheal repair in a goat model

Traditional treatment therapies for tracheal stenosis often cause severe post‐operative complications. To solve the current difficulties, novel and more suitable long‐term treatments are needed. A whole‐segment tissue‐engineered trachea (TET) representing the native goat trachea was 3D printed using a poly(caprolactone) (PCL) scaffold engineered with autologous auricular cartilage cells. The TET underwent mechanical analysis followed by in vivo implantations in order to evaluate the clinical feasibility and potential. The 3D‐printed scaffolds were successfully cellularized, as observed by scanning electron microscopy. Mechanical force compression studies revealed that both PCL scaffolds and TETs have a more robust compressive strength than does the native trachea. In vivo implantation of TETs in the experimental group resulted in significantly higher mean post‐operative survival times, 65.00 ± 24.01 days (n = 5), when compared with the control group, which received autologous trachea grafts, 17.60 ± 3.51 days (n = 5). Although tracheal narrowing was confirmed by bronchoscopy and computed tomography examination in the experimental group, tissue necrosis was only observed in the control group. Furthermore, an encouraging epithelial‐like tissue formation was observed in the TETs after transplantation. This large animal study provides potential preclinical evidence around the employment of an orthotopic transplantation of a whole 3D‐printed TET.     

Gap closure of different shape wounds: In vitro and in vivo experimental models in the presence of engineered protein adhesive hydrogel

The present study emphasizes the role of engineered protein (gallic acid engineered gelatin [GEG]) on the closure of wound gaps of different shapes assessed under in vitro (fibroblast cell line) and in vivo (rat) experimental models. Circular, triangle, rectangle, and square are the shapes selected for the study. Intending engineered protein (GEG) augments the cell migration in rectangle and triangle shapes and reduces the gap space significantly compared with circular and square shapes. Similar observations were made with in vivo model study, and it was observed that the wound closure starts along the wound edges. In circular and square shapes, the cell movement follow a purse‐string mechanism/the mixed pattern. Thus, the present study suggested that for faster wound healing, the cell migration along the wound edge may be found beneficial, and the external healing agent in the form of engineered protein hydrogel accelerate the healing accordingly.     

Successful development and clinical translation of a novel anterior lamellar artificial cornea

Blindness due to corneal diseases is a common pathology affecting up to 23 million individuals worldwide. The tissue‐engineered anterior human cornea, which is currently being tested in a Phase I/II clinical trial to treat severe corneal trophic ulcers with preliminary good feasibility and safety results. This bioartificial cornea is based on a nanostructured fibrin–agarose biomaterial containing human allogeneic stromal keratocytes and cornea epithelial cells, mimicking the human native anterior cornea in terms of optical, mechanical, and biological behavior. This product is manufactured as a clinical‐grade tissue engineering product, fulfilling European requirements and regulations. The clinical translation process included several phases: an initial in vitro and in vivo preclinical research plan, including preclinical advice from the Spanish Medicines Agency followed by additional preclinical development, the adaptation of the biofabrication protocols to a good manufacturing practice manufacturing process, including all quality controls required, and the design of an advanced therapy clinical trial. The experimental development and successful translation of advanced therapy medicinal products for clinical application has to overcome many obstacles, especially when undertaken by academia or SMEs. We expect that our experience and research strategy may help future researchers to efficiently transfer their preclinical results into the clinical settings.     

From the Cover: Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States

Invasive organisms pose a global threat and are exceptionally difficult to eradicate after they become abundant in their new habitats. We report a successful multitactic strategy for combating the pink bollworm (Pectinophora gossypiella), one of the world’s most invasive pests. A coordinated program in the southwestern United States and northern Mexico included releases of billions of sterile pink bollworm moths from airplanes and planting of cotton engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). An analysis of computer simulations and 21 y of field data from Arizona demonstrate that the transgenic Bt cotton and sterile insect releases interacted synergistically to reduce the pest’s population size. In Arizona, the program started in 2006 and decreased the pest’s estimated statewide population size from over 2 billion in 2005 to zero in 2013. Complementary regional efforts eradicated this pest throughout the cotton-growing areas of the continental United States and northern Mexico a century after it had invaded both countries. The removal of this pest saved farmers in the United States $192 million from 2014 to 2019. It also eliminated the environmental and safety hazards associated with insecticide sprays that had previously targeted the pink bollworm and facilitated an 82% reduction in insecticides used against all cotton pests in Arizona. The economic and social benefits achieved demonstrate the advantages of using agricultural biotechnology in concert with classical pest control tactics.

Invasive life forms pose a major global threat and are especially difficult to eradicate after they become widespread and abundant in their new habitats (1–4). The pink bollworm (Pectinophora gossypiella), one of the world’s most invasive insects, is a voracious lepidopteran pest of cotton that was first detected in the United States in 1917 (5–8). For most of the past century, it was particularly destructive in the southwestern United States, including Arizona, where its larvae fed almost exclusively on cotton, consuming the seeds inside bolls and disrupting lint production (6, 8). In 1969, its peak seasonal density at an Arizona study site was 1.8 million larvae per hectare (ha), which translates to over 200 billion larvae in the 126,000 ha of cotton planted statewide that year (9, 10). In 1990, this pest cost Arizona cotton growers $48 million, including $32 million damage to cotton despite $16 million spent for insecticides sprayed to control it (11). In several field trials, mass releases of sterile pink bollworm moths to mate with wild moths reduced progeny production somewhat, yet did not suppress established populations because the sterile moths did not sufficiently outnumber the wild moths (6, 12–14).Pink bollworm control was revolutionized in 1996 by the introduction of cotton genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Bt proteins kill some major insect pests yet are not toxic to most nontarget organisms, including people and many beneficial insects (15–17). Transgenic Bt cotton helped to reduce the total annual cost of pink bollworm damage and insecticide treatments to $32 million in the United States (18). Although Bt cotton kills essentially 100% of susceptible pink bollworm larvae (19–21), this pest rapidly evolved resistance to Bt proteins in laboratory selection experiments in Arizona and in Bt cotton fields in India (20–24). To delay the evolution of resistance to Bt cotton, farmers in Arizona planted “refuges” of non-Bt cotton that yielded abundant susceptible moths to mate with the rare resistant moths emerging from Bt cotton (Fig. 1A). The refuge strategy, which has been mandated in the United States and many other countries, but was not adopted widely by farmers in India, helped preserve pink bollworm susceptibility to Bt cotton in Arizona from 1996 to 2005 (24).Open in a separate windowFig. 1.Management strategies. (A) The refuge strategy is the primary approach adopted worldwide to delay the evolution of pest resistance to Bt crops and was used in Arizona from 1996 to 2005. Refuges of non-Bt cotton planted near Bt cotton produce abundant susceptible moths (blue) to mate with the rare resistant moths (red) emerging from Bt cotton. If the inheritance of resistance to Bt cotton is recessive, as in pink bollworm, the heterozygous offspring from matings between resistant and susceptible moths die when they feed on Bt cotton bolls as larvae (24). (B) Bt cotton and sterile moth releases were used together in Arizona from 2006 to 2014 as part of a multitactic program to eradicate the pink bollworm. Susceptible sterile moths (brown) were released from airplanes to mate with the rare resistant moths emerging from Bt cotton. The few progeny produced by such matings (48) are expected to be heterozygous for resistance and to die when they feed on Bt cotton bolls as larvae.As part of a coordinated, multitactic effort to eradicate the pink bollworm from the southwestern United States and northern Mexico, a new strategy largely replacing refuges with mass releases of sterile pink bollworm moths was initiated in Arizona during 2006 (Fig. 1B; 24–27). To enable this novel strategy, the US Environmental Protection Agency granted a special exemption from the refuge requirement, which allowed Arizona cotton growers to plant up to 100% of their cotton with Bt cotton (28). We previously reported data from 1998 to 2009 showing that this innovative strategy sustained susceptibility of pink bollworm to Bt cotton while reducing the pest’s population density (25). Here, to test the idea of eradicating pink bollworm with the combination of Bt cotton and sterile releases, we conducted computer simulations and analyzed field data collected in Arizona from 1998 to 2018.     

Assessment of a tissue-engineered gastric wall patch in a rat model

Stenosis or deformity of the remaining stomach can occur after gastrectomy and result in stomach malfunction. The objective of this study is to demonstrate the feasibility of transplanting a tissue-engineered gastric wall patch in a rat model to alleviate the complications after resection of a large area of the gastric wall. Tissue-engineered gastric wall patches were created from gastric epithelial organoid units and biodegradable polymer scaffolds. In the first treatment group, gastric wall defects were created in recipient rats and covered with fresh tissue-engineered gastric wall patches (simultaneous transplantation). In the second treatment group, the tissue-engineered gastric wall patches were frozen for 12weeks, and then transplanted in recipient rats (metachronous transplantation). Tissue-engineered gastric wall patches were successfully used as a substitute of the resected native gastric wall in both simultaneous and metachronous transplantation groups. The defrosted wall patches showed almost the same cell viability as the fresh ones. Twenty-four weeks after transplantation, the defect in the gastric wall was well-covered with tissue-engineered gastric wall patch, and the repaired stomach showed no deformity macroscopically in both groups. Histology showed continuous mucosa and smooth muscle layers at the tissue-engineered stomach wall margin. The feasibility of transplanting a tissue-engineered patch to repair a defect in the native gastric wall has been successfully shown in a rat model, thereby taking one step closer toward the transplantation of an entire tissue-engineered stomach in the future.     

CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer

Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.     

Carbonation Behavior of Engineered Cementitious Composites under Coupled Sustained Flexural Load and Accelerated Carbonation

Engineered cementitious composites (ECCs) belong to a broad class of fibre-reinforced concrete. They incorporate synthetic polyvinyl alcohol (PVA) fibres, cement, fly ash and fine aggregates, and are designed to have a tensile strain capacity typically beyond 3%. This paper presents an investigation on the carbonation behaviour of engineered cementitious composites (ECCs) under coupled sustained flexural load and accelerated carbonation. The carbonation depth under a sustained stress level of 0, 0.075, 0.15, 0.3 and 0.6 relative to flexural strength was measured after 7, 14 and 28 days of accelerated carbonation. Thermogravimetric analysis, mercury intrusion porosimetry and microhardness measurements were carried out to show the coupled influence of sustained flexural load and accelerated carbonation on the changes of the mineral phases, porosity, pore size distribution and microhardness along the carbonation profile. A modified carbonation depth model that can be used to consider the coupled effect of flexural tensile stress and carbonation time was proposed. The results show that an exponential relationship can be observed between stress influence coefficient and flexural tensile stress level in the carbonation depth model of ECC, which is different when using plain concrete. Areas with a higher carbonation degree have greater microhardness, even under a large sustained load level, as the carbonation process refines the pore structure and the fibre bridges the crack effectively.     

Behavior of Engineered Cementitious Composites (ECCs) Subjected to Coupled Sustained Flexural Load and Salt Frost

The performance of engineered cementitious composites (ECCs) under coupled salt freezing and loaded conditions is important for its application on the transportation infrastructure. However, in most of the studies, the specimens were generally loaded prior to the freezing. The influence of sustained load was merely considered. To this end, four sustained deflection levels, i.e., 0%, 10%, 30% and 50% of the deflection at the ultimate flexural strength, and three salt concentrations (1%, 3% and 5%) were applied. Prior to the salt frost resistance test, the fluid absorption of ECC specimens under various conditions were measured. The changes in relative dynamic elastic modulus (RDEM) during the freeze–thaw cycles were captured. The depth and the content profile of free chloride were measured after the coupled sustained load and freezing and thawing cycles. It is shown that 3% NaCl solution leads to the largest deterioration in all cases. There is no visible flaking or damage occurring on the surface. The relationships between locally sustained flexural stress and RDEM loss and also locally sustained flexural stress and free chloride penetration depth were proposed and showed satisfactory results. It is concluded that when ECC is subjected to the FTCs under 1% de-ice salt solution, no depassivation of the steel is expected even under a large deflection level. In terms of 3% and 5% salt solution, the thickness of cover should be no less than 20 mm when a deflection level of 0.5 is applied.