Human-engineered auricular reconstruction (hEAR) by 3D-printed molding with human-derived auricular and costal chondrocytes and adipose-derived mesenchymal stem cells.

Microtia is a small, malformed external ear, which occurs at an incidence of 1-10 per 10 000 births. Autologous reconstruction using costal cartilage is the most widely accepted surgical microtia repair technique. Yet, the method involves donor-site pain and discomfort and relies on the artistic skill of the surgeon to create an aesthetic ear. This study employed novel tissue engineering techniques to overcome these limitations by developing a clinical-grade, 3D-printed biodegradable auricle scaffold that formed stable, custom-made neocartilage implants. The unique scaffold design combined strategically reinforced areas to maintain the complex topography of the outer ear and micropores to allow cell adhesion for the effective production of stable cartilage. The auricle construct was computed tomography (CT) scan-based composed of a 3D-printed clinical-grade polycaprolactone scaffold loaded with patient-derived chondrocytes produced from either auricular cartilage or costal cartilage biopsies combined with adipose-derived mesenchymal stem cells. Cartilage formation was measured within the constructin vitro, and cartilage maturation and stabilization were observed 12 weeks after its subcutaneous implantation into a murine model. The proposed technology is simple and effective and is expected to improve aesthetic outcomes and reduce patient discomfort.

GLUT4-overexpressing engineered muscle constructs as a therapeutic platform to normalize glycemia in diabetic mice.

Skeletal muscle insulin resistance is a main defect in type 2 diabetes (T2D), which is associated with impaired function and content of glucose transporter type 4 (GLUT4). GLUT4 overexpression in skeletal muscle tissue can improve glucose homeostasis. Therefore, we created an engineered muscle construct (EMC) composed of GLUT4-overexpressing (OEG4) cells. The ability of the engineered implants to reduce fasting glucose levels was tested in diet-induced obesity mice. Decrease and stabilization of basal glucose levels were apparent up to 4 months after implantation. Analysis of the retrieved constructs showed elevated expression of myokines and proteins related to metabolic processes. In addition, we validated the efficiency of OEG4-EMCs in insulin-resistant mice. Following high glucose load administration, mice showed improved glucose tolerance. Our data indicate that OEG4-EMC implant is an efficient mode for restoring insulin sensitivity and improving glucose homeostasis in diabetic mice. Such procedure is a potential innovative modality for T2D therapy.

Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks.

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-ß signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

The effect of endothelial cells on hESC-derived pancreatic progenitors in a 3D environment.

Generating transplantable ß-like-cells from human embryonic stem cells (hESC) could serve as an ideal cell-based therapy for treatment of type 1 diabetes, which is characterized by the destruction of insulin-secreting pancreatic ß-cells. There are several protocols for differentiating hESCs into pancreatic or endocrine precursors. However, so far, production of mature, functional ß-like-cells has been achieved mainly by transplanting hESC derived pancreatic progenitors (PPs) and allowing several months for maturation to occur in vivo. One approach, believed to have potential in promoting differentiation into ß-like-cells prior to transplantation, is culturing PPs alongside blood vessels. Endothelium and blood vessels have been shown to direct pancreatic development during embryogenesis and also induce endocrine differentiation in vitro. Here we designed a three-dimensional (3D) construct utilizing highly porous polymeric scaffolds that mimic natural conditions and provide cells with mechanical support, and used it in the differentiation protocol. Clusters of hESC derived pancreatic precursor cells were embedded within the scaffolds along with human endothelial cells (ECs) and fibroblasts forming vessel-like networks. Culturing these clusters with ECs for one week significantly increased the population of PPs, characterized by co-expression of the pancreatic markers Pdx1 and Nkx6.1 and also highly induced Ngn3 expression which indicates commitment to endocrine fate. The presence of fibroblasts, however, reduced this cell population. Three months upon implantation of constructs containing clusters and ECs or clusters alone, implanted mice retained normal blood glucose levels after treatment with STZ, while un-implanted mice became diabetic. These findings may lay the foundation for creating an optimal tissue-construct that will support PPs' maturation in vitro and enhance graft function upon implantation.

Infantile aphakic glaucoma: a proposed etiologic role of IL-4 and VEGF.

PURPOSE: To identify the factors secreted by lens epithelial cells (LECs) responsible for the altered trabecular meshwork (TM) cells and to compare their effect on monocultured TM cells with that of TM cells co-cultured with LECs. METHODS: Such factors were isolated using cytokine antibody array membranes, and their effect on TM cells was assessed by analyzing changes in morphology and gene expression. In addition, inhibition of the isolated factors was performed in the co-culture model by adding specific antibodies to the cell culture media. RESULTS: Transforming growth factor beta-2, interleukin-4 (IL-4), and vascular endothelial growth factor (VEGF) are presented as candidate cytokines responsible for the observed changes in LEC-TM co-cultures. Culturing TM cells in the presence of VEGF and IL-4 triggered alterations closely reflecting those observed in the LEC-TM co-culture model, where their inhibition significantly hindered the alteration of the TM cells. CONCLUSION: These findings suggest a possible explanation for the development of infantile aphakic glaucoma, based on residual LECs secreting IL-4 and VEGF after removal of congenital cataract, which then alter trabecular meshwork cell morphology and gene expression.

Interactions between trabecular meshwork cells and lens epithelial cells: a possible mechanism in infantile aphakic glaucoma.

PURPOSE: Infantile aphakic glaucoma may develop as a postoperative complication of early childhood cataract surgery. It has been associated with risk factors including surgery in early life and retained lens material; however, its cause and mechanism are poorly understood. This study focused on the potential role of retained lens material (specifically, exposed lens epithelial cells [LECs]) in undesired changes of the trabecular meshwork (TM) structure and function. METHODS: Interactions between LECs and TM cells were studied by analyzing structural changes and differential gene and protein expression in TM cells cocultured with LECs. RESULTS: Subjecting normal TM cells to the presence of LECs resulted in changes in their structural features (such as increase in volume and size, and decrease in cell-cell interactions), as well as in their protein expression (mainly cytoskeletal) and gene expression (such as genes related to organ and cell morphogenesis, inflammatory response, response to stimulus, ion homeostasis, and several signaling pathways). CONCLUSIONS: Many of the changes observed in TM cells after exposure to LECs resemble alternations seen in primary open-angle glaucoma. This strengthens the suspected role of LECs in the development of aphakic glaucoma.