Zichen Qian

Increasing Mechanical Strength of Gelatin Hydrogels by Divalent Metal Ion Removal

The usage of gelatin hydrogel is limited due to its instability and poor mechanical properties, especially under physiological conditions. Divalent metal ions present in gelatin such as Ca2+ and Fe2+ play important roles in the gelatin molecule interactions. The objective of this study was to determine the impact of divalent ion removal on the stability and mechanical properties of gelatin gels with and without chemical crosslinking. The gelatin solution was purified by Chelex resin to replace divalent metal ions with sodium ions. The gel was then chemically crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Results showed that the removal of divalent metal ions significantly impacted the formation of the gelatin network. The purified gelatin hydrogels had less interactions between gelatin molecules and form larger-pore network which enabled EDC to penetrate and crosslink the gel more efficiently. The crosslinked purified gels showed small swelling ratio, higher crosslinking density and dramatically increased storage and loss moduli. The removal of divalent ions is a simple yet effective method that can significantly improve the stability and strength of gelatin hydrogels. The in vitro cell culture demonstrated that the purified gelatin maintained its ability to support cell attachment and spreading.

A Moldable Nanocomposite Hydrogel Composed of a Mussel‐Inspired Polymer and a Nanosilicate as a Fit‐to‐Shape Tissue Sealant

The engineering of bioadhesives to bind and conform to the complex contour of tissue surfaces remains a challenge. We have developed a novel moldable nanocomposite hydrogel by combining dopamine-modified poly(ethylene glycol) and the nanosilicate Laponite, without the use of cytotoxic oxidants. The hydrogel transitioned from a reversibly cross-linked network formed by dopamine-Laponite interfacial interactions to a covalently cross-linked network through the slow autoxidation and cross-linking of catechol moieties. Initially, the hydrogel could be remolded to different shapes, could recover from large strain deformation, and could be injected through a syringe to adhere to the convex contour of a tissue surface. With time, the hydrogel solidified to adopt the new shape and sealed defects on the tissue. This fit-to-shape sealant has potential in sealing tissues with non-flat geometries, such as a sutured anastomosis.

Hypoxia Created Human Mesenchymal Stem Cell Sheet for Prevascularized 3D Tissue Construction

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% O2 ) and then cocultured with ECs under normoxia (20% O2 ), 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 benefit the microvessel growth and it is feasible to construct 3D prevascularized tissue construct using the prevascularized hMSC sheets.

Aligned Nanofibrous Cell-Derived Extracellular Matrix for Anisotropic Vascular Graft Construction

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 is critical to their functional performance. The objective of this study is 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 direct the initial cell orientation. The subsequent maturation under circumferential stress generated by a rotating wall vessel (RWV) bioreactor further promotes anisotropic structural and mechanical properties in the graft. The circumferential tensile strength is significantly higher than longitudinal strength in bioreactor samples. Expression of smooth muscle cell specific genes, α-smooth muscle actin and calponin, in hMSCs is greatly enhanced in bioreactor samples without any biochemical stimulation. In addition, employment of premade ECM and RWV bioreactor significantly reduces the graft fabrication time to three weeks. Mimicking the ECM composition, cell phenotype, structural and mechanical anisotropy, the vascular graft presented in this study is promising for vascular reconstruction surgery or in vitro tissue model applications.

Prevascularization of natural nanofibrous extracellular matrix for engineering completely biological three-dimensional prevascularized tissues for diverse 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% O2) 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% O2) 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.

Bioactive polydimethylsiloxane surface for optimal human mesenchymal stem cell sheet culture

Human mesenchymal stem cell (hMSC) sheets hold great potential in engineering three-dimensional (3D) completely biological tissues for diverse applications. Conventional cell sheet culturing methods employing thermoresponsive surfaces are cost ineffective, and rely heavily on available facilities. In this study, a cost-effective method of layer-by-layer grafting was utilized for covalently binding a homogenous collagen I layer on a commonly used polydimethylsiloxane (PDMS) substrate surface in order to improve its cell adhesion as well as the uniformity of the resulting hMSC cell sheet. Results showed that a homogenous collagen I layer was obtained via this grafting method, which improved hMSC adhesion and attachment through reliable collagen I binding sites. By utilizing this low-cost method, a uniform hMSC sheet was generated. This technology potentially allows for mass production of hMSC sheets to fulfill the demand of thick hMSC constructs for tissue engineering and biomanufacturing applications.

Bioengineering Scaffolds for Regenerative Engineering

Tissue engineering and regenerative medicine have been vastly developing since the past decade. Cell, scaffold, and signal are the tissue engineering triad. In that, the scaffold is critical to supporting tissue regeneration. The purpose of scaffolding is to mimic the structure and function of extracellular matrix in native tissues, which replaces, or partially replaces, damaged or diseased tissues. This article will provide a broad summary of scaffolding materials and fabrication techniques. Different scaffolding materials including synthetic, natural, and hybrid materials are introduced in detail. Fabrication techniques including decellularization, particulate leaching, gas foaming, phase separation, electrospinning, and three-dimensional printing are evaluated as methods to effectively recreate the architecture of target tissues. Examples of scaffold applications in regenerative therapies are given, while the specific requirements of architecture, material properties, and biochemical properties are discussed.