Macrophage phenotype in response to ECM bioscaffolds.

Macrophage presence and phenotype are critical determinants of the healing response following injury. Downregulation of the pro-inflammatory macrophage phenotype has been associated with the therapeutic use of bioscaffolds composed of extracellular matrix (ECM), but phenotypic characterization of macrophages has typically been limited to small number of non-specific cell surface markers or expressed proteins. The present study determined the response of both primary murine bone marrow derived macrophages (BMDM) and a transformed human mononuclear cell line (THP-1 cells) to degradation products of two different, commonly used ECM bioscaffolds; urinary bladder matrix (UBM-ECM) and small intestinal submucosa (SIS-ECM). Quantified cell responses included gene expression, protein expression, commonly used cell surface markers, and functional assays. Results showed that the phenotype elicited by ECM exposure (MECM) is distinct from both the classically activated IFNγ+LPS phenotype and the alternatively activated IL-4 phenotype. Furthermore, the BMDM and THP-1 macrophages responded differently to identical stimuli, and UBM-ECM and SIS-ECM bioscaffolds induced similar, yet distinct phenotypic profiles. The results of this study not only characterized an MECM phenotype that has anti-inflammatory traits but also showed the risks and challenges of making conclusions about the role of macrophage mediated events without consideration of the source of macrophages and the limitations of individual cell markers.

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 © 2016 John Wiley & Sons, Ltd.

The Influence of Extracellular RNA on Cell Behavior in Health, Disease and Regeneration.

PURPOSE OF REVIEW: An overview of the role of extracellular RNAs (exRNA) in the regulation of homeostasis, disease progression, and regeneration is provided herein. Several exRNAs have been identified as potential biomarkers for disease and disease progression. In addition, the potential of exRNAs as a therapeutic modality is discussed. RECENT FINDINGS: Fibrotic diseases of the lung, liver, and heart, among other organs share a number of identical exRNAs which play key roles in disease pathogenesis. Though regeneration is limited to only a few tissues in humans, small RNAs (e.g. microRNA) have been shown to be involved in the regenerative process of tissues such as liver and bone. The regulation of healing versus disease appears to be balanced by small RNAs. Because small RNAs are critical to health, they are being investigated as drug targets in multiple ongoing clinical trials. Preclinical studies suggest that promoting or blocking specific small RNAs can provide a novel therapeutic approach. SUMMARY: exRNA can be utilized for both detection and treatment of disease. Natural and synthetic RNA carriers are being investigated as delivery methods for small RNA molecules. Current and future investigations are likely to lead to expanded applications for exRNAs.

Extracellular Matrix Bioscaffolds as Immunomodulatory Biomaterials.

Suppression of the recipient immune response is a common component of tissue and organ transplantation strategies and has also been used as a method of mitigating the inflammatory and scar tissue response to many biomaterials. It is now recognized, however, that long-term functional tissue replacement not only benefits from an intact host immune response but also depends upon such a response. The present article reviews the limitations associated with the traditionally held view of avoiding the immune response, the ability of acellular biologic scaffold materials to modulate the host immune response and promote a functional tissue replacement outcome, and current strategies within the fields of tissue engineering and biomaterials to develop immune-responsive and immunoregulatory biomaterials.

Matrix-Bound Nanovesicles Recapitulate Extracellular Matrix Effects on Macrophage Phenotype.

The early macrophage response to biomaterials has been shown to be a critical and predictive determinant of downstream outcomes. When properly prepared, bioscaffolds composed of mammalian extracellular matrix (ECM) have been shown to promote a transition in macrophage behavior from a proinflammatory to a regulatory/anti-inflammatory phenotype, which in turn has been associated with constructive and functional tissue repair. The mechanism by which ECM bioscaffolds promote this phenotypic transition, however, is poorly understood. The present study shows that matrix-bound nanovesicles (MBV), a component of ECM bioscaffolds, are capable of recapitulating the macrophage activation effects of the ECM bioscaffold from which they are derived. MBV isolated from two different source tissues, porcine urinary bladder and small intestinal submucosa, were found to be enriched in miRNA125b-5p, 143-3p, and 145-5p. Inhibition of these miRNAs within macrophages was associated with a gene and protein expression profile more consistent with a proinflammatory rather than an anti-inflammatory/regulatory phenotype. MBV and their associated miRNA cargo appear to play a significant role in mediating the effects of ECM bioscaffolds on macrophage phenotype.

Regenerative Medicine: lessons from Mother Nature.

Regenerative medicine strategies for the restoration of functional tissue have evolved from the concept of ex vivo creation of engineered tissue toward the broader concept of in vivo induction of functional tissue reconstruction. Multidisciplinary approaches are being investigated to achieve this goal using evolutionarily conserved principles of stem cell biology, developmental biology and immunology, current methods of engineering and medicine. This evolution from ex vivo tissue engineering to the manipulation of fundamental in vivo tenets of development and regeneration has the potential to capitalize upon the incredibly complex and only partially understood ability of cells to adapt, proliferate, self-organize and differentiate into functional tissue.

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.

Hypertensive rat lungs retain hallmarks of vascular disease upon decellularization but support the growth of mesenchymal stem cells.

There are an insufficient number of donor organs available to meet the demand for lung transplantation. This issue could be addressed by regenerating functional tissue from diseased or damaged lungs that would otherwise be deemed unsuitable for transplant. Detergent-mediated whole-lung decellularization produces a three-dimensional natural scaffold that can be repopulated with various cell types. In this study, we investigated the decellularization and initial recellularization of diseased lungs using a rat model of monocrotaline-induced pulmonary hypertension (MCT-PHT). Decellularization of control and MCT-PHT Sprague-Dawley rat lungs was accomplished by treating the lungs with a combination of Triton X-100, sodium deoxycholate, NaCl, and DNase. The resulting acellular matrices were characterized by DNA quantification, Western blotting, immunohistochemistry, and proteomic analyses revealing that decellularization was able to remove cells while leaving the extracellular matrix (ECM) components and lung ultrastructure intact. Decellularization significantly reduced DNA content (∼30-fold in MCT-PHT lungs and ∼50-fold in the control lungs) and enriched ECM components (>60-fold in both the control and MCT-PHT lungs) while depleting cellular proteins. MicroCT visualization of MCT-PHT rat lungs indicated that the vasculature was narrowed as a result of MCT treatment, and this characteristic was unchanged by decellularization. Mean arterial vessel diameter of representative decellularized MCT-PHT and control scaffolds was estimated to be 0.152±0.134 mm and 0.247±0.160 mm, respectively. Decellularized MCT-PHT lung scaffolds supported attachment and survival of rat adipose-derived stem cells (rASCs), seeded into the airspace or the vasculature, for at least 2 weeks. The cells seeded in MCT-PHT lung scaffolds proliferated and underwent apoptosis similar to control scaffolds; however, the initial percentage of apoptotic cells was slightly higher in MCT-PHT lungs (2.79±2.03% vs. 1.05±1.02% of airway-seeded rASCs, and 4.47±1.21% vs. 2.66±0.10% of vascular seeded rASCs). The ECM of cell-seeded scaffolds showed no signs of degradation by the cells after 14 days in culture. These data suggest that diseased hypertensive lungs can be efficiently decellularized similar to control lungs and have the potential to be recellularized with mesenchymal stem cells with the ultimate goal of generating healthy, functional pulmonary tissue.

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 organ's 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.