Mark J. Mondrinos

In vivo pulmonary tissue engineering: contribution of donor-derived endothelial cells to construct vascularization.

Intrapulmonary engraftment of engineered lung tissues could provide a potential therapeutic approach for the treatment of pediatric and adult pulmonary diseases. In working toward this goal, we report here on in vivo generation of vascularized pulmonary tissue constructs utilizing the subcutaneous Matrigel plug model. Mixed populations of murine fetal pulmonary cells (FPCs) containing epithelial, mesenchymal, and endothelial cells (ECs) were isolated from the lungs of embryonic day 17.5 fetuses. FPCs were admixed to Matrigel and injected subcutaneously into the anterior abdominal wall of adult C57/BL6 mice to facilitate in vivo pulmonary tissue construct formation. Vascularization was enhanced by placing fibroblast growth factor 2 (FGF2)-loaded polyvinyl sponges into the hydrogel. After 1 week, routine histology and immunohistochemical staining for donor-derived epithelial cells and ECs as well as analysis of patent vasculature in the constructs following tail vein injection of fluorescein isothiocyanate-conjugated dextran were performed. In the Matrigel-only controls, some level of host infiltrate, but no measurable vascularization, was detected. In the presence of FPCs, the constructs contained ductal epithelial structures and patent vasculature. In the absence of FPCs, exogenous FGF2 induced the formation of numerous patent blood vessels throughout the entire constructs; in combination with FPCs, it resulted in enhanced capillary density and abundant interfacing between developing epithelial and vascular structures. The significant findings of this study are that distal pulmonary epithelial differentiation (as assessed by the expression of prosurfactant protein C) can be maintained in vivo and that donor-derived ECs contribute to the formation of patent vessels that interface tightly with ductal epithelial structures.

Heterogeneous Breast Tumoroids: An In Vitro Assay for Investigating Cellular Heterogeneity and Drug Delivery

Breast tumors are typically heterogeneous and contain diverse subpopulations of tumor cells with differing phenotypic properties. Planar cultures of cancer cell lines are not viable models of investigation of cell-cell and cell-matrix interactions during tumor development. This article presents an in vitro coculture-based 3-dimensional heterogeneous breast tumor model that can be used in drug resistance and drug delivery investigations. Breast cancer cell lines of different phenotypes (MDAMB231, MCF7, and ZR751) were cocultured in a rotating wall vessel bioreactor to form a large number of heterogeneous tumoroids in a single cell culture experiment. Cells in the rotating vessels were labeled with Cell Tracker fluorescent probes to allow for time course fluorescence microscopy to monitor cell aggregation. Histological sections of tumoroids were stained with hematoxylin and eosin, progesterone receptor, E-cadherin (E-cad), and proliferation marker ki67. In vitro tumoroids developed in this study recapture important features of the temporal-spatial organization of solid tumors, including the presence of necrotic areas at the center and higher levels of cell division at the tumor periphery. E-cad-positive MCF7 cells form larger tumoroids than E-cad-negative MDAMB231 cells. In heterogeneous tumors, the irregular surface roughness was mainly due to the presence of MDAMB231 cells, whereas MCF7 cells formed smooth surfaces. Moreover, when heterogeneous tumoroids were placed onto collagen gels, highly invasive MDAMB231 cell-rich surface regions produced extensions into the matrix, whereas poorly invasive MCF7 cells did not. The fact that one can form a large number of 1-mm tumoroids in 1 coculture attests to the potential use of this system at high-throughput investigations of cancer drug development and drug delivery into the tumor.

Electrospun Blends of Natural and Synthetic Polymers as Scaffolds for Tissue Engineering

Engineering functional three-dimensional (3-D) tissue constructs for the replacement and/or repair of damaged native tissues using cells and scaffolds is one of the ultimate goals of tissue engineering. In this study, non-woven fibrous scaffolds were electrospun from the synthetic biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) and natural proteins, gelatin (denatured collagen) and elastin. In the absence of cross-linking agent, the average PGE fiber diameter increased from 347 plusmn 103 nm to 999 plusmn 123 nm upon wetting as measured by scanning electron microscopy. Rat bone marrow stromal cells (rBMSC) were used paradigmatically to study the 3-D cell culture properties of PGE scaffolds. Consistent with the observed properties of the individual fibers, PGE scaffolds initially swelled in aqueous culture medium, however rBMSC seeded PGE scaffolds contracted to < 50% of original size. Time course histological analysis demonstrated uniform seeding of rBMSC into PGE scaffolds and complete cell penetration into the fibrous architecture over 4 weeks of in vitro culture

Enhanced Re-Endothelialization of Decellularized Rat Lungs.

Decellularized lung tissue has been recognized as a potential platform to engineer whole lung organs suitable for transplantation or for modeling a variety of lung diseases. However, many technical hurdles remain before this potential may be fully realized. Inability to efficiently re-endothelialize the pulmonary vasculature with a functional endothelium appears to be the primary cause of failure of recellularized lung scaffolds in early transplant studies. Here, we present an optimized approach for enhanced re-endothelialization of decellularized rodent lung scaffolds with rat lung microvascular endothelial cells (ECs). This was achieved by adjusting the posture of the lung to a supine position during cell seeding through the pulmonary artery. The supine position allowed for significantly more homogeneous seeding and better cell retention in the apex regions of all lobes than the traditional upright position, especially in the right upper and left lobes. Additionally, the supine position allowed for greater cell retention within large diameter vessels (proximal 100-5000 μm) than the upright position, with little to no difference in the small diameter distal vessels. EC adhesion in the proximal regions of the pulmonary vasculature in the decellularized lung was dependent on the binding of EC integrins, specifically α1β1, α2β1, and α5β1 integrins to, respectively, collagen type-I, type-IV, and fibronectin in the residual extracellular matrix. Following in vitro maturation of the seeded constructs under perfusion culture, the seeded ECs spread along the vascular wall, leading to a partial reestablishment of endothelial barrier function as inferred from a custom-designed leakage assay. Our results suggest that attention to cellular distribution within the whole organ is of paramount importance for restoring proper vascular function.

Designing Intelligent Polymeric Scaffolds for Tissue Engineering: Blending and Co-Electrospinning Synthetic and Natural Polymers

There is a growing interest in blending natural and synthetic polymers as biomaterials for generating complex fibrous scaffolds for tissue engineering purposes. In this talk we will report on co-electrospinning binary and tertiary blends of gelatin (denatured collagen) with either a conductive polymer, polyaniline (PANi), or with a mixture of polylactic acid / polyglycolic acid, and elastin (PGE) Finally, we will demonstrate the usefulness of elastin-based fibrous scaffolds for pulmonary tissue engineering

Manufacturing System Development for Fabrication of Bone Scaffold

Musculoskeletal conditions are a major health concern in United States because of a large aging population and increased occurrence of sport-related injuries. The need for bone substitutes is especially important. Traditional treatments of bone-defect have many of limitations. Bone tissue engineering may offer a less painful alternative to traditional bone grafts with lower risk of infection. This research integrates biomimetic modeling, solid freeform fabrication (SFF), systems and control, and tissue engineering in one intelligent system for structured, highly porous biomaterials, which will be applied to bone scaffolds. Currently a new SFF-based fabrication system has been developed, which uses a pressurized extrusion to print highly biocompatible and water soluble sucrose bone scaffold porogens. To date, this system can build simple bone structures. In parallel we are utilizing a commercial rapid prototyping (RP) machine to fabricate thermoplastic porogens of various designs to determine the feasibility of injecting a highly viscous scaffold material into porogens. Materials which have been successfully used to make scaffolds by injection include calcium phosphate cement (CPC), molten poly-caprolactone (PCL), 90/10 and 80/20 (v/v %) composite of PCL and calcium phosphate (CaPO4 ,). Results presented for the injection method include characterization of attainable feature resolution of the RP machine, as well as preliminary cell-biomaterial interaction data demonstrating biocompatibility of CPC scaffolds. The preliminary results using a commercial rapid prototyping machine have demonstrated that the indirect porogen technique can improve 2–4 folds the resolution of SFF system in fabricating bone scaffolds. The resultant scaffolds demonstrate that the defined porous structures will be suitable for tissue engineering applications.© 2005 ASME