Improvement of Decellularization Efficiency of Porcine Aorta Using Dimethyl Sulfoxide as a Penetration Enhancer.

Decellularization of tissues and organs enables researchers to obtain extracellular matrix (ECM) with the natural conformation and chemical composition of specific tissues. However, drawbacks exist such as the structural alteration of ECM or loss of some important components in ECM due to overexposure to chemicals during the decellularization process. In this study, porcine aorta was decellularized by sodium dodecyl sulfate (SDS). Dimethyl sulfoxide (DMSO) was used as a penetration enhancer in the decellularization process to enhance the penetration of SDS, consequently reducing the exposure time of SDS to treated tissues. It is revealed that by addition of DMSO to the decellularization process 64.4% more DNA was removed when compared with just SDS exposure within a 3 h reaction. Cross-validation by DAPI staining showed that, in the presence of DMSO, the penetration of SDS was improved and almost all cells were removed from the aorta within the 3 h exposure time. Collagen staining revealed that just SDS treatment showed less polarized collagen fibers, while the DMSO addition groups revealed denser and organized collagen fibers. Moreover 77% glycosaminoglycan content was preserved by addition of DMSO in resultant tissues. Scanning electron microscopy analysis of decellularized aortic matrix showed that ECM components remained in the adventitia layer with the addition of DMSO treatment, while the layer was removed with just SDS treatment. Biocompatibility assays proved that after washing the decellularized samples with media supplemented with 3% antibiotic and antimycotic solution for 2 days there was no cytotoxic effect related to the SDS + DMSO decellularization protocol. This study demonstrates that the new decellularization protocol not only improves the removal efficiency of cellular components but also protects the crucial ECM components.

Biofabrication of Poly(glycerol sebacate) Scaffolds Functionalized with a Decellularized Bone Extracellular Matrix for Bone Tissue Engineering.

The microarchitecture of bone tissue engineering (BTE) scaffolds has been shown to have a direct effect on the osteogenesis of mesenchymal stem cells (MSCs) and bone tissue regeneration. Poly(glycerol sebacate) (PGS) is a promising polymer that can be tailored to have specific mechanical properties, as well as be used to create microenvironments that are relevant in the context of BTE applications. In this study, we utilized PGS elastomer for the fabrication of a biocompatible and bioactive scaffold for BTE, with tissue-specific cues and a suitable microstructure for the osteogenic lineage commitment of MSCs. In order to achieve this, the PGS was functionalized with a decellularized bone (deB) extracellular matrix (ECM) (14% and 28% by weight) to enhance its osteoinductive potential. Two different pore sizes were fabricated (small: 100-150 µm and large: 250-355 µm) to determine a preferred pore size for in vitro osteogenesis. The decellularized bone ECM functionalization of the PGS not only improved initial cell attachment and osteogenesis but also enhanced the mechanical strength of the scaffold by up to 165 kPa. Furthermore, the constructs were also successfully tailored with an enhanced degradation rate/pH change and wettability. The highest bone-inserted small-pore scaffold had a 12% endpoint weight loss, and the pH was measured at around 7.14. The in vitro osteogenic differentiation of the MSCs in the PGS-deB blends revealed a better lineage commitment of the small-pore-sized and 28% (w/w) bone-inserted scaffolds, as evidenced by calcium quantification, ALP expression, and alizarin red staining. This study demonstrates a suitable pore size and amount of decellularized bone ECM for osteoinduction via precisely tailored PGS elastomer BTE scaffolds.

Bilayered extracellular matrix derived scaffolds with anisotropic pore architecture guide tissue organization during osteochondral defect repair.

While some clinical advances in cartilage repair have occurred, osteochondral (OC) defect repair remains a significant challenge, with current scaffold-based approaches failing to recapitulate the complex, hierarchical structure of native articular cartilage (AC). To address this need, we fabricated bilayered extracellular matrix (ECM)-derived scaffolds with aligned pore architectures. By modifying the freeze-drying kinetics and controlling the direction of heat transfer during freezing, it was possible to produce anisotropic scaffolds with larger pores which supported homogenous cellular infiltration and improved sulfated glycosaminoglycan deposition. Neo-tissue organization in vitro could also be controlled by altering scaffold pore architecture, with collagen fibres aligning parallel to the long-axis of the pores within scaffolds containing aligned pore networks. Furthermore, we used in vitro and in vivo assays to demonstrate that AC and bone ECM derived scaffolds could preferentially direct the differentiation of mesenchymal stromal cells (MSCs) towards either a chondrogenic or osteogenic lineage respectively, enabling the development of bilayered ECM scaffolds capable of spatially supporting unique tissue phenotypes. Finally, we implanted these scaffolds into a large animal model of OC defect repair. After 6 months in vivo, scaffold implantation was found to improve cartilage matrix deposition, with collagen fibres preferentially aligning parallel to the long axis of the scaffold pores, resulting in a repair tissue that structurally and compositionally was more hyaline-like in nature. These results demonstrate how scaffold architecture and composition can be spatially modulated to direct the regeneration of complex interfaces such as the osteochondral unit, enabling their use as cell-free, off-the-shelf implants for joint regeneration. STATEMENT OF SIGNIFICANCE: The architecture of the extracellular matrix, while integral to tissue function, is often neglected in the design and evaluation of regenerative biomaterials. In this study we developed a bilayered scaffold for osteochondral defect repair consisting of tissue-specific extracellular matrix (ECM)-derived biomaterials to spatially direct stem/progenitor cell differentiation, with a tailored pore microarchitecture to promote the development of a repair tissue that recapitulates the hierarchical structure of native AC. The use of this bilayered scaffold resulted in improved tissue repair outcomes in a large animal model, specifically the ability to guide neo-tissue organization and therefore recapitulate key aspects of the zonal structure of native articular cartilage. These bilayer scaffolds have the potential to become a new therapeutic option for osteochondral defect repair.

Use of supercritical CO2 in soft tissue decellularization.

Supercritical carbon dioxide (scCO2) is being used as an alternative approach to the traditional methods for the decellularization of tissues. This chapter describes the use of scCO2 for the decellularization of optic nerve, myocardium, and cornea tissues. The main goal of this method is to burst the cells with high-pressure, remove them from the tissues and to maintain the extracellular matrix structure of the native tissues. For this purpose, several scCO2-assisted decellularization protocols were developed and optimized according to the requirements of these tissues. Efficiencies of the utilized decellularization protocols were determined via histological and morphological analysis. The decrease in the DNA content and the preserved glycosaminoglycan (GAG) amounts were also used as assessment parameters.

Use of cyclic strain bioreactor for the upregulation of key tenocyte gene expression on Poly(glycerol-sebacate) (PGS) sheets.

The inadequate donor source and the difficulty of using natural grafts in tendon repair and regeneration has led researchers to develop biodegradable and biocompatible synthetic based tissue equivalents. Poly(glycerol sebacate) (PGS) is a surface-erodible bioelastomer and has been increasingly investigated in a variety of biomedical applications. In this study, PGS elastomeric sheets were prepared by using a facile microwave method and used as elastomeric platform for the first time under mechanical stimulation to induct the tenocyte gene expression. It is revealed that elastomeric PGS sheets promote progenitor tendon cell structure by increasing proliferation and gene expression with regard to tendon extracellular matrix components. Human tenocytes were seeded onto poly(glycerol-sebacate) sheets and were cultured two days prior to transfer to dynamic culture in a bioreactor system. Cell culture studies were carried out for 12 days under 0%, 3% and 6% strain at 0.33 Hz. The PGS-cell constructs were examined by using Scanning Electron Microscopy (SEM), cell viability via live/dead staining using confocal microscopy, and GAG/DNA analysis. In addition, gene expression was examined using real-time polymerase chain reaction (RT-PCR). Tenocytes cultured upon PGS scaffolds under 6% cyclic strain exhibited tendon-like gene expression profile compared to 3% and 0% strain groups. The results of this study show that PGS is a suitable material in promoting tendon tissue formation under dynamic conditions.

Evaluation of collagen foam, poly(l-lactic acid) nanofiber mesh, and decellularized matrices for corneal regeneration.

Corneal tissue engineering efforts to obtain corneal tissue matrices through various types of materials for the replacement of damaged tissues. In this study, three different corneal constructs were prepared and evaluated in terms of morphological, optical, and biological characteristics. Type-I collagen was used to obtain collagen foam scaffolds through dehydrothermal crosslinking, while poly(l-lactic acid) (PLLA) was used to produce both random and aligned oriented electrospun corneal constructs. Bovine corneas were decellularized as third matrix. Software analyses showed that average pore size of collagen scaffolds was 88.207 ± 29.7 µm, while the average fiber diameter of aligned and random PLLA scaffolds were 0.69 ± 0.03 and 0.65 ± 0.03 µm, respectively. Degradation profiles revealed that collagen foam exhibits high degradation (20% mass loss) while electrospun PLLA scaffolds hold low degradation (9% mass loss) rates at day-28. Transmittance values of the obtained scaffolds were calculated as 92, 80, and 70% for collagen, PLLA, and decellularized cornea constructs, respectively. The evaluation of stromal keratocyte behavior on the constructs revealed that the cells exhibited their own morphology mostly on the aligned PLLA constructs, while they were mostly active on random PLLA electrospun corneal scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2157-2168, 2018.

Hybrid Aorta Constructs via In Situ Crosslinking of Poly(glycerol-sebacate) Elastomer Within a Decellularized Matrix.

Decellularization of tissues and organs has high potential to obtain unique conformation and composition as native tissue structure but may result in weakened tissue mechanical strength. In this study, poly(glycerol-sebacate) (PGS) elastomers were combined with decellularized aorta fragments to investigate the changes in mechanical properties. PGS prepolymer was synthesized via microwave irradiation and then in situ crosslinked within the decellularized aorta extracellular matrix (ECM). Tensile strength (σ) values were found comparable as 0.44 ± 0.10 MPa and 0.57 ± 0.18 MPa for native and hybrid aorta samples, respectively, while elongation at break (ɛ) values were 261% ± 17%, 7.5% ± 0.57%, and 22.18% ± 2.48% for wet control (native), decellularized dried aortae, and hybrid matrices, showing elastic contribution. Young's modulus data indicate that there was a threefold decrease in stiffness compared to decellularized samples once PGS is introduced into the ECM structure. Scanning electron microscopy (SEM) analysis of hybrid grafts revealed that the construct preserves porosity in medial layer of the vessel. Biocompatibility analyses showed no cytotoxic effects on human abdominal aorta smooth muscle cells. Cell studies showed 98% activity in hybrid graft extracts. As a control, collagen coating of the hybrid grafts was performed in the recellularization stage. SEM analysis of recellularized hybrid grafts revealed that cells were attached to the surface of the hybrid graft and proliferated during the 14 days of culture in both groups. This study shows that introducing an elastomer into the native ECM structure following decellularization process can be a useful approach for the preparation of mechanically enhanced composites for soft tissues.

Supercritical Carbon Dioxide-Assisted Decellularization of Aorta and Cornea.

Tissue engineering approaches utilize both natural and synthetic materials in the repair and regeneration processes. A naturally sourced material for this purpose is required to be free from any antigenic matter such as cells or cellular components. Decellularization of tissues may be achieved through chemical or physical removal agents. Supercritical carbon dioxide (sc-CO2) has been used on the purpose of removing bioburden from tissues and offers an alternative to the traditionally used treatment methods. In addition to many advantages it offers with regard to the successful decellularization of tissues, it is known to have a sterilization effect. This study provides an insight into sc-CO2-assisted decellularization trials of corneal and aortic tissues. Results showed that high pressure of the fluid bursts the cells during the treatment and rapid depressurization was found to be effective in the removal of the cells from the tissues. sc-CO2 decellularization offers significantly reduced treatment times, complete decellularization, and preserved extracellular matrix structure.