The application of cell-derived extracellular matrix (ECM) in tissue engineering has gained increasing interest because it can provide a naturally occurring, complex set of physiologically functional signals for cell growth. The ECM scaffolds produced from decellularized fibroblast cell sheets contain high amounts of ECM substances, such as collagen, elastin, and glycosaminoglycans. They can serve as cell adhesion sites and mechanically strong supports for tissue-engineered constructs. An efficient method that can largely remove cellular materials while maintaining minimal disruption of ECM ultrastructure and content during the decellularization process is critical. In this study, three decellularization methods were investigated: high concentration (0.5 wt%) of sodium dodecyl sulfate (SDS), low concentration (0.05 wt%) of SDS, and freeze-thaw cycling method. They were compared by characterization of ECM preservation, mechanical properties, in vitro immune response, and cell repopulation ability of the resulted ECM scaffolds. The results demonstrated that the high SDS treatment could efficiently remove around 90% of DNA from the cell sheet, but significantly compromised their ECM content and mechanical strength. The elastic and viscous modulus of the ECM decreased around 80% and 62%, respectively, after the high SDS treatment. The freeze-thaw cycling method maintained the ECM structure as well as the mechanical strength, but also preserved a large amount of cellular components in the ECM scaffold. Around 88% of DNA was left in the ECM after the freeze-thaw treatment. In vitro inflammatory tests suggested that the amount of DNA fragments in ECM scaffolds does not cause a significantly different immune response. All three ECM scaffolds showed comparable ability to support in vitro cell repopulation. The ECM scaffolds possess great potential to be selectively used in different tissue engineering applications according to the practical requirement.

Split thickness skin graft (STSG) implantation is one of the standard therapies for full thickness wound repair when full thickness autologous skin grafts (FTG) or skin flap transplants are inapplicable. Combined transplantation of STSG with dermal substitute could enhance its therapeutic effects but the results remain unsatisfactory due to insufficient blood supply at early stages, which causes graft necrosis and fibrosis. Human mesenchymal stem cell (hMSC) sheets are capable of accelerating the wound healing process. We hypothesized that pre-vascularized hMSC sheets would further improve regeneration by providing more versatile angiogenic factors and pre-formed microvessels. In this work, in vitro cultured hMSC cell sheets (HCS) and pre-vascularized hMSC cell sheets (PHCS) were implanted in a rat full thickness skin wound model covered with an autologous STSG. Results demonstrated that the HCS and the PHCS implantations significantly reduced skin contraction and improved cosmetic appearance relative to the STSG control group. The PHCS group experienced the least hemorrhage and necrosis, and lowest inflammatory cell infiltration. It also induced the highest neovascularization in early stages, which established a robust blood micro-circulation to support grafts survival and tissue regeneration. Moreover, the PHCS grafts preserved the largest amount of skin appendages, including hair follicles and sebaceous glands, and developed the smallest epidermal thickness. The superior therapeutic effects seen in PHCS groups were attributed to the elevated presence of growth factors and cytokines in the pre-vascularized cell sheet, which exerted a beneficial paracrine signaling during wound repair. Hence, the strategy of combining STSG with PHCS implantation appears to be a promising approach in regenerative treatment of full thickness skin wounds.

Natural extracellular matrices (ECM) derived from native tissues or cultured cells are extensively employed to fabricate biocompatible scaffolds or living tissue constructs for the application in cellular and tissue engineering. The composition and structure of ECM are not only heterogeneous, but also tissue or cell specific. Recapitulating the unique cell or tissue niche, ECM-based products are promising to quickly integrate with host tissues and accelerate restoration of tissue function. A variety of natural ECM-based scaffolds and tissue constructs have been biomanufactured using different approaches. Native tissue derived ECM is typically grounded into powders that can be further processed into hybrid composites in the form of hydrogels, foams, nanofibers, and 3D-printed complex constructs. Cell-derived ECM follows different biomanufacturing methods. Usually, cells are seeded on a scaffold to deposit ECM resulting in ECM-ornamented materials. The employment of resolvable scaffolds and cell sheet engineering technique enables production of complex 3D constructs exclusively composed of ECM with/without cells. In order to enhance mechanical strength, in vivo stability, and biological performance of ECM-based products, cross-linking reagents or bioactive factors are often used for modification. The major focus of this article is to provide an overview of current biomanufacturing approaches that utilize either native tissue or cell-derived natural ECM in the field of cellular and tissue engineering. Furthermore, the existing challenges for translational application of ECM-based products and the potential resolutions are discussed.

3D tissue based on human mesenchymal stem cell (hMSC) sheets offers many interesting opportunities for regenerating multiple types of connective tissues. Prevascularizing hMSC sheets with endothelial cells (ECs) will improve 3D tissue performance by supporting cell survival and accelerating integration with host tissue. It is hypothesized that hypoxia cultured hMSC sheets can promote microvessel network formation and preserve stemness of hMSCs. This study investigates the vascularization of hMSC sheets under different oxygen tensions. It is found that the HN condition, in which hMSC sheets formed under physiological hypoxia (2% O 2 ) and then cocultured with ECs under normoxia (20% O 2 ), enables longer and more branched microvessel network formation. The observation is corroborated by higher levels of angiogenic factors in coculture medium. Additionally, the hypoxic hMSC sheet is more uniform and less defective, which facilitates fabrication of 3D prevascularized tissue construct by layering the prevascularized hMSC sheets and maturing in rotating wall vessel bioreactor. The hMSCs in the 3D construct still maintain multilineage differentiation ability, which indicates the possible application of the 3D construct for various connective tissues regeneration. These results demonstrate that hypoxia created hMSC sheets benefi t the microvessel growth and it is feasible to construct 3D prevascularized tissue construct using the prevascularized hMSC sheets.

Due to the indispensable role of periosteum in bone defect healing and regeneration, a promising method to enhance osteogenesis of bone grafts by using an engineered biomimetic periosteum would be highly beneficial. The stromal microenvironment of periosteum is composed of various highly organized extracellular matrix (ECM) fibers, so an aligned natural ECM sheet, derived from the human dermal fibroblast cell sheet, may be advantageous when applied for artificial periosteum fabrication. Human mesenchymal stem cells (hMSCs) have been used to replace the osteoprogenitor cell population in native periosteum due to hMSCs' great osteogenic potential and fast in vitro expansion capacity. The objective of this work is to investigate if the natural ECM sheet and the substrate alignment can promote in vitro osteogenesis of hMSCs. The conventional cell culture substrates collagen I-coated polydimethylsiloxane (PDMS) and tissue culture plastic (TCP) were used as controls. It was found that the ECM sheet significantly increased alkaline phosphatase activity and calcium deposition. The enhanced osteogenic potential was further confirmed by increased bone-specific gene expression. The ECM sheet can bind significantly higher amounts of growth factors including ANG-1, TGF-β1, bFGF, and VEGF, as well as calcium phosphate nanoparticles, which contributed to high osteogenesis of the hMSCs on ECM sheet. However, the alignment of the substrates did not show significant influence on osteogenic activity and growth factor binding. These results demonstrated the great potential of hMSC-seeded ECM sheet as a biomimetic periosteum to improve critical sized bone regeneration.

There is a large demand for tissue engineered vascular grafts for the application of vascular reconstruction surgery or in vitro drug screening tissue model. The extracellular matrix (ECM) composition, along with the structural and mechanical anisotropy of native blood vessels are critical to their functional performance. The objective of this study was to develop a biomimetic vascular graft recapitulating the anisotropic features of native blood vessels by employing nanofibrous aligned fibroblast-derived ECM and human mesenchymal stem cells (hMSCs). The nanotopographic cues of aligned ECM directed the initial cell orientation The subsequent maturation under circumferential stress generated by a rotating wall vessel (RWV) bioreactor further promoted anisotropic structural and mechanical properties in the graft. The circumferential tensile strength was significantly higher than longitudinal strength in bioreactor samples. Expression of smooth muscle cell specific genes, α-smooth muscle actin and calponin, in hMSCs was greatly enhanced in bioreactor samples without any biochemical stimulation. In addition, employment of pre-made ECM and RWV bioreactor significantly reduced the graft fabrication time to 3 weeks. Mimicking the ECM composition, cell phenotype, structural and mechanical anisotropy, the vascular graft presented in our study is promising for vascular reconstruction surgery or in vitro tissue model applications.

Self-sustainability after implantation is one of the critical obstacles facing large engineered tissues. A preformed functional vascular network provides an effective solution for solving the mass transportation problem. With the support of mural cells, endothelial cells (ECs) can form microvessels within engineered tissues. As an important mural cell, human mesenchymal stem cells (hMSCs) not only stabilize the engineered microvessel network, but also preserve their multi-potency when grown under optimal culture conditions. A prevascularized hMSC/extracellular matrix (ECM) sheet fabricated by the combination of hMSCs, ECs and a naturally derived nanofibrous ECM scaffold offers great opportunity for engineering mechanically strong and completely biological three-dimensional prevascularized tissues. The objective of this study was to create a prevascularized hMSC/ECM sheet by co-culturing ECs and hMSCs on a nanofibrous ECM scaffold. Physiologically low oxygen (2% O ) was introduced during the 7 day hMSC culture to preserve the stemness of hMSCs and thereby their capability to secrete angiogenic factors. The ECs were then included to form microvessels under normal oxygen (20% O ) for up to 7 days. The results showed that a branched and mature vascular network was formed in the co-culture condition. Angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiopoietin-1 (Ang-1) were significantly increased by low-oxygen culture of hMSCs, which further stabilized and supported the maturation of microvessels. A differentiation assay of the prevascularized ECM scaffold demonstrated a retained hMSC multi-potency in the hypoxia cultured samples. The prevascularized hMSC/ECM sheet holds great promise for engineering three-dimensional prevascularized tissues for diverse applications.

Engineered skeletal muscle grafts have made great progress during the past decades, benefiting from a growing understanding of mechanobiology and stem cell differentiation. Current techniques are widely varied, ranging from in vitro methods following the classical tissue engineering paradigm to in situ approaches such as host cell recruitment. In different ways, all of these try to supply mechanical toughness while providing the necessary signals for differentiation and maturation of the engineered skeletal muscle.