Nicholas C. Pashos

A Review of Cellularization Strategies for Tissue Engineering of Whole Organs

With the advent of whole organ decellularization, extracellular matrix scaffolds suitable for organ engineering were generated from numerous tissues, including the heart, lung, liver, kidney, and pancreas, for use as alternatives to traditional organ transplantation. Biomedical researchers now face the challenge of adequately and efficiently recellularizing these organ scaffolds. Herein, an overview of whole organ decellularization and a thorough review of the current literature for whole organ recellularization are presented. The cell types, delivery methods, and bioreactors employed for recellularization are discussed along with commercial and clinical considerations, such as immunogenicity, biocompatibility, and Food and Drug Administartion regulation.

Nonhuman primate lung decellularization and recellularization using a specialized large-organ bioreactor.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organs natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use. The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Characterization of an Acellular Scaffold for a Tissue Engineering Approach to the Nipple-Areolar Complex Reconstruction

A significant number of patients undergo mastectomies and breast reconstructions every year using many surgical-based techniques to reconstruct the nipple-areolar complex (NAC). Described herein is a tissue engineering approach that may permit a human NAC onlay graft during breast reconstruction procedures. By applying decellularization, which is the removal of cellular components from tissue, to an intact whole donor NAC, the extracellular matrix (ECM) structure of the NAC is preserved. This creates a biologically derived scaffold for cells to repopulate and regenerate the NAC. A detergent-based decellularization method was used to derive whole NAC scaffolds from nonhuman primate rhesus macaque NAC tissue. Using both histological and quantitative analyses for the native and decellularized tissues, the derived ECM graft was assessed. The bioactivity of the scaffold was evaluated following cell culture with bone marrow-derived mesenchymal stem cells (BMSCs). The data presented here demonstrate that scaffolds are devoid of cells and retain ECM integrity and a high degree of bioactivity. The content of collagen and glycosaminoglycans were not significantly altered by the decellularization process, whereas the elastin content was significantly decreased. The proliferation and apoptosis of seeded BMSCs were found to be approximately 65 and <1.5%, respectively. This study characterizes the successful decellularization of NAC tissue as compared to native NACs based on structural protein composition, lubricating protein retention, the maintenance of adhesion molecules, and bioactivity when reseeded with cells. These histological and quantitative analyses provide the foundation for a novel approach to NAC reconstruction.

Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells

Effective re‐endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re‐endothelialized scaffolds that haemorrhage and become thrombogenic upon implantation. Herein, gravity‐driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells were seeded into the pulmonary artery by either gravity‐driven, variable flow perfusion seeding or pump‐driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re‐lining of the vascular walls while perfusion pump‐driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells and rat pulmonary vein endothelial cells attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells deposited mostly in microvessels. Combination seeding of these cells led to positive vascular endothelial cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re‐endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary–alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. Copyright

640. A Tissue Engineered Nipple and Areola Complex

There are more than 2.8 million breast cancer survivors in the United States, many of who have undergone reconstructive surgery. Approximately 36% of patients with early stage diagnoses and 60% of patients with late stage diagnoses undergo mastectomies. Moreover, immediate breast reconstruction following mastectomies has become more common, significantly increasing at an average rate of 5% per year, from 20.8% in 1998 to 37.8% in 2008. This increasing trend is not surprising as breast reconstruction likely provides psychological benefits for women who undergo mastectomies. There is evidence to suggest that nipple and areola complex (NAC) reconstruction affects psychological wellbeing by enhancing body image and selfesteem, or decreasing the feeling of distress felt by female patients with mastectomies. Due to this, there exists a need for a reproducible and more naturally aesthetic architecture for NAC reconstruction. Current strategies for NAC reconstruction are limited to surgical techniques that create a NAC-like structure from existing local tissue, secondary site grafting, 3D tattooing, or using commercially available acellular dermal matrix sheets, such as Alloderm. Generating a tissue engineered, biocompatible NAC implant, made of decellularized whole NAC, for use in place of surgically created NAC structures is a promising approach to NAC reconstruction following mastectomies.To date, no tissue engineering and cellular therapy strategies have been developed focused on NAC reconstruction. The application of decellularization to the whole, semi-glandular NAC can create a non-immunogenic NAC that retains the microarchitecture and gross structures of a native NAC. This tissue engineering approach to whole NAC structure regeneration allows for the effective removal of cellular material, the retention of the extracellular matrix components and structure, as well as cell adhesion molecules. Once decellularized, NAC scaffolds would be seeded with autologous cells to create a graft that is patient-specific. Preliminary studies have shown, using tissues from a Rhesus Macaque Non- Human Primate animal model, that biologically derived scaffolds were able to be reproducibly isolated with effective removal of nuclear material—less than ≈50ng of 200bp DNA per mg of sample remaining. Through histological analysis of the NAC scaffolds it was shown that the presence of extracellular matrix and adhesion proteins were maintained after the decellularization process. Additionally, bioactivity of the scaffolds were assessed using rhesus bone marrow-derived stem cells for one week, under dynamic cell culture conditions. Herein, a tissue engineered, regenerative medicine approach to reconstruct the nipple and areola complex using a biologically derived scaffold and autologous cell sources is described.

Endocrine disruptors and the tumor microenvironment: A new paradigm in breast cancer biology

Breast cancer is one of the most frequently diagnosed malignancies in women and is characterized by predominantly estrogen dependent growth. Endocrine disruptors (EDCs) have estrogenic properties which have been shown to increase breast cancer risk. While the direct effects of EDCs on breast cancer cell biology and tumor progression have been well studied, the roles for EDCs on tumor microenvironment composition, signaling and structure are incompletely defined. Estrogen targeting of tumor stromal cells can drive paracrine signaling to breast cancer cells regulating tumorigenesis and progression. Additionally, estrogen and estrogen receptor signaling has been shown to alter breast architecture and extracellular matrix component synthesis. Unsurprisingly, EDCs have been shown to induce structural changes in the mammary gland as well as increased collagen fibers in the tissue stroma. Previous work demonstrates that human mesenchymal stem cells (hMSC) are essential components of the tumor microenvironment and are direct targets of both estrogens and EDCs. Furthermore, estrogen-stem cell cross talk has been implicated in breast cancer progression and results in increased tumor cell proliferation, angiogenesis and invasion. This review aims to dissect the possible relationship and mechanisms between EDCs, the tumor microenvironment, and breast cancer progression.

Therapeutic Potential of Adipose Stem Cells

Adipose stem cells (ASCs) have gained attention in the fields of stem cells regenerative medicine due to their multifaceted therapeutic capabilities. Promising preclinical evidence of ASCs has supported the substantial interest in the use of these cells as therapy for human disease. ASCs are an adult stem cell resident in adipose tissue with the potential to differentiation along mesenchymal lineages. They also are known to be recruited to sites of inflammation where they exhibit strong immunomodulatory capabilities to promote wound healing and regeneration. ASCs can be isolated from adipose tissue at a relatively high yield compared to their mesenchymal cell counterparts: bone marrow-derived mesenchymal stem cells (BM-MSCs). Like BM-MSCs, ASCs are easily culture expanded and have a reduced immunogenicity or are perhaps immune privileged, making them attractive options for cellular therapy. Additionally, the heterogeneous cellular product obtained after digestion of adipose tissue, called the stromal vascular fraction (SVF), contains ASCs and several populations of stromal and immune cells. Both the SVF and culture expanded ASCs have the potential to be therapeutic in various diseases. This review will focus on the preclinical and clinical evidence of SVF and ASCs, which make them potential candidates for therapy in regenerative medicine and inflammatory disease processes.

Comparative proteomic analyses of human adipose extracellular matrices decellularized using alternative procedures: COMPARATIVE PROTEOMIC ANALYSES OF HUMAN ADIPOSE ECM

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting, and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for deoxyribose nucleic acid depletion, extracellular matrix composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy. Mass spectrometry based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP+ transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds.

Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope. In the present study, lungs from wild type or α-Gal knockout pigs were harvested, decellularized, and implanted subcutaneously in a non-human primate model to evaluate the host immune response. The decellularized porcine implants were compared to a sham surgery control, as well as native porcine and decellularized macaque lung implants. The results demonstrated differential profiles of circulating and infiltrating immune cell subsets and histological outcomes depending on the implanted tissue source. Upon implantation, the decellularized α-Gal knockout lung constructs performed similarly to the decellularized wild type lung constructs. However, upon re-implantation into a chronic exposure model, the decellularized wild type lung constructs resulted in a greater proportion of infiltrating CD45+ cells, including CD3+ and CD8+ cytotoxic T-cells, likely mediated by an increase in production of Gal-specific antibodies. The results suggest that removal of the Gal epitope can potentially reduce adverse inflammatory reactions associated with chronic exposure to engineered organs containing xenogeneic components.

Abstract 5096: Development of a decellularized tumor model for the evaluation of breast carcinomas

Breast cancer recurrence is a clinical manifestation of tumor progression in patients which has been shown to significantly increase mortality rates. While up to one-fifth of breast cancer patients will experience recurrence, the mechanisms underlying this process remain poorly understood. Recently, gene expression profiling of human tumor metastasis and recurrence demonstrated that the extracellular matrix (ECM)-receptor interactions pathway was enhanced. In breast carcinomas, the ECM is known to vary with cancer progression. The ECM acts as a chemical reservoir, structural component, and mediator of tumorigenesis to tumors. To date, the ECM of breast carcinomas has been evaluated within the context of a seeded cellular population, i.e. intact tumor. Here we demonstrate for the first time a mechanism to evaluate the ECM devoid of breast cancer cells. We have employed a model of tissue decellularization to breast cancer cell line derived tumors and demonstrate that tumor ECM architecture remains intact when devoid of cells. Validation of loss of cell content is evident through DNA quantification, nuclear DAPI stain, and HE 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5096.