Qi Xing

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model.

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3‐D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3‐D scaffolds have not been fully investigated. In this study, experimental results from a 3‐D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3‐D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 μmol/106 cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher YL/G in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering

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.

Porous biocompatible three-dimensional scaffolds of cellulose microfiber/gelatin composites for cell culture.

Physiological tissues, including brain and other organs, have three-dimensional (3-D) aspects that need to be supported to model them in vitro. Here we report the use of cellulose microfibers combined with cross-linked gelatin to make biocompatible porous microscaffolds for the sustained growth of brain cell and human mesenchymal stem cells (hMSCs) in 3-D structure. Live imaging using confocal microscopy indicated that 3-D microscaffolds composed of gelatin or cellulose fiber/gelatin both supported brain cell adhesion and growth for 16days in vitro. Cellulose microfiber/gelatin composites containing up to 75% cellulose fibers can withstand a higher mechanical load than gelatin alone, and composites also provided linear pathways along which brain cells could grow compared to more clumped cell growth in gelatin alone. Therefore, the bulk cellulose microfiber provides a novel skeleton in this new scaffold material. Cellulose fiber/gelatin scaffold supported hMSCs growth and extracellular matrix formation. hMSCs osteogenic and adipogenic assays indicated that hMSCs cultured in cellulose fiber/gelatin composite preserved the multilineage differentiation potential. As natural, biocompatible components, the combination of gelatin and cellulose microfibers, fabricated into 3-D matrices, may therefore provide optimal porosity and tensile strength for long-term maintenance and observation of cells.

Conductive paper from lignocellulose wood microfibers coated with a nanocomposite of carbon nanotubes and conductive polymers

Composite nanocoating of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) and aqueous dispersion of carbon nanotubes (CNT-PSS) on lignocellulose wood microfibers has been developed to make conductive microfibers and paper sheets. To construct the multilayers on wood microfibers, cationic poly(ethyleneimine) (PEI) has been used in alternate deposition with anionic conductive PEDOT-PSS and solubilized CNT-PSS. Using a Keithley microprobe measurement system, current-voltage measurements have been carried out on single composite microfibers after deposition of each layer to optimize the electrical properties of the coated microfibers. The conductivity of the resultant wood microfibers was in the range of 10(-2)-2 S cm(-1) depending on the architecture of the coated layer. Further, the conductivity of the coated wood microfibers increased up to 20 S cm(-1) by sandwiching multilayers of conductive co-polymer PEDOT-PSS with CNT-PSS through a polycation (PEI) interlayer. Moreover, paper hand sheets were manufactured from these coated wood microfibers with conductivity ranging from 1 to 20 S cm(-1). A paper composite structure consisting of conductive/dielectric/conductive layers that acts as a capacitor has also been fabricated and is reported.

Fabrication and characterization of a nitric oxide-releasing nanofibrous gelatin matrix.

Nitric oxide (NO) plays an important role in cardiovascular homeostasis, immune responses, and wound repair. The pro-angiogenic and antimicrobial properties of NO has stimulated the development of NO-releasing materials for wound dressings. Gelatin, an abundant natural biodegradable polymer derived from collagen, is able to promote wound repair. S-Nitroso-N-acetylpenicillamine (SNAP) can release NO under physiological conditions and when exposed to light. The objective of this project was to fabricate a NO-releasing gelatin-based nanofibrous matrix with precise light-controllable ability. Results showed that under controlled phase separation fabrication conditions, the gelatin formed a highly porous matrix with the nanofiber diameter ranging from 50 to 500 nm. Importantly, the removal of the trace amount of divalent metal ions within gelatin generated a more stable nanofibrous structure. N-acetyl-D-penicillamine (NAP) was functionalized onto the matrix and nitrosated with t-butyl nitrite, yielding a SNAP-gelatin matrix. Analysis of the photoinitiated NO-release showed that the SNAP-gelatin matrices released NO in a highly controllable manner. Application of increasing light intensities yielded increased NO flux from the matrices. In addition, the dried matrices stored in dark at 4 °C maintained stable NO storage capacity, and the purified (ion-removed) gelatin preserved higher NO-releasing capacity than nonpurified gelatin. The antibacterial effect from the SNAP-gelatin matrices was demonstrated by exposing Staphylococcus aureus ( S. aureus ) to a light-triggered NO flux. This controllable NO-releasing scaffold provides a potential antibacterial therapeutic approach to combat drug resistant bacteria.

Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Repair

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.

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.

Nitric oxide regulates cell behavior on an interactive cell‐derived extracellular matrix scaffold

During tissue injury and wound healing process, there are dynamic reciprocal interactions among cells, extracellular matrix (ECM), and mediating molecules which are crucial for functional tissue repair. Nitric oxide (NO) is one of the key mediating molecules that can positively regulate various biological activities involved in wound healing. Various ECM components serve as binding sites for cells and mediating molecules, and the interactions further stimulate cellular activities. Human mesenchymal stem cells (hMSCs) can migrate to the wound site and contribute to tissue regeneration through differentiation and paracrine signaling. The objective of this work was to investigate the regulatory effect of NO on hMSCs in an interactive ECM-rich microenvironment. In order to mimic the in vivo stromal environment in wound site, a cell-derived ECM scaffold that was able to release NO within the range of in vivo wound fluid NO level was fabricated. Results showed that the micro-molar level of NO released from the ECM scaffold had an inhibitory effect on cellular activities of hMSCs. The NO impaired cell growth, altered cell morphology, disrupted the F-actin organization, also decreased the expression of focal adhesion related molecules integrin α5 and paxillin. These results may contribute to the elucidation of how NO acts on hMSCs in wound healing process.

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.

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.