Temporal and spatially controllable cell encapsulation using a water-soluble phospholipid polymer with phenylboronic acid moiety

Temporal and spatially controllable cell encapsulation based on a water-soluble phospholipid polymer is reported in this study. Phospholipid polymers, i.e., poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV), were synthesized. A series of hydrogels was prepared between the water-soluble PMBV and other water-soluble polymers having multi-valent alcoholic groups, such as poly(vinyl alcohol) (PVA). The PMBV/PVA hydrogels were formed not only in water, but also in a cell culture medium, and dissociated by the excess addition of low molecular weight di-valent hydroxyl compounds, such as d-glucose. The PMBV/PVA hydrogel was applied as a cell-container which has three-dimensional matrices for the reversible encapsulation of living cells without any response in it. Uniform cell seeding can be achieved using the hydrogels due to the homogenous gel formation of PMBV and PVA in the cell culture medium. Fibroblast cells were encapsulated in the PMBV/PVA hydrogel and maintained for 1 week. After dissociation of the PMBV/PVA hydrogel, the cells were seeded on conventional tissue culture polystyrene. The cells adhered and proliferated as usual on the plate. That is, the PMBV/PVA hydrogel will be useful as a cell-container, which can maintain the cells without any significant adverse effect on the entrapped cells.     

Regulation of cell proliferation by multi-layered phospholipid polymer hydrogel coatings through controlled release of paclitaxel

We fabricated multi-layered hydrogels on titanium alloy (Ti) surfaces by applying alternating layers of a water-soluble phospholipid polymer (PMBV) and polyvinyl alcohol (PVA). This was accomplished by a layer-by-layer (LbL) process that is based on the formation of reversible covalent bonds between the boronic acid subunits in the PMBV and the hydroxyl groups in the PVA. When placed in an aqueous medium, PMBV acquires a polymeric aggregate structure with hydrophobic domains that can effectively solubilize hydrophobic molecules such as the anticancer drug paclitaxel (PTX) used in this study. The PTX-containing PMBV layer acted as a reservoir in the multi-layered hydrogels. To obtain diverse release profiles, the PTX was loaded in either the top layer (top-type) or the bottom layer (bottom-type) of the hydrogels; additional layers of PMBV and PVA, without PTX, functioned as a diffusion-barrier. In cell culture experiments, top-type hydrogels demonstrated excessive suppression of human epidermal carcinoma A431 cell proliferation over 5 days due to the initial high concentration of released PTX. However, bottom-type hydrogels were able to maintain a constant cell number profile. The release of PTX from multi-layered hydrogels was governed by both diffusion through the diffusion-barrier and dissociation of the hydrogel through an exchange reaction of phenylboronic acid subunits with the low-molecular weight d-glucose in the cell culture medium. In the cell culture experiments, the cell cycle was arrested in S and G2/M phases, as expected following PTX-mediated growth inhibition; control hydrogels did not demonstrate any appreciable cell cycle arrest. We concluded that cell proliferation could be controlled by the concentration of PTX released from the multi-layered hydrogels prepared through the LbL process. This system when used to solubilize bioactive agents at an appropriate layer within the hydrogel has potential for localized and surface-mediated delivery of bioactive molecules from biomedical devices.     

Degradable hydrogels for spatiotemporal control of mesenchymal stem cells localized at decellularized bone allografts

The transplantation of cells, such as mesenchymal stem cells (MSCs), has numerous applications in the field of regenerative medicine. For cell transplantation strategies to be successful therapeutically, cellular localization and persistence must be controlled to maximize cell-mediated contributions to healing. Herein, we demonstrate that hydrolytic degradation of poly(ethylene glycol) (PEG) hydrogels can be used to spatiotemporally control encapsulated MSC localization to decellularized bone allografts, both in vitro and in vivo. By altering the number of hydrolytically degradable lactide repeat units within PEG-d,l-lactide-methacrylate macromers, a series of hydrogels was synthesized that degraded over ∼1, 2 and 3 weeks. MSCs were encapsulated within these hydrogels formed around decellularized bone allografts, and non-invasive, longitudinal fluorescence imaging was used to track cell persistence both in vitro and in vivo. Spatiotemporal localization of MSCs to the exterior of bone allograft surfaces was similar to in vitro hydrogel degradation kinetics despite hydrogel mesh sizes being ∼2–3 orders of magnitude smaller than MSC size throughout the degradation process. Thus, localized, cell-mediated degradation and MSC migration from the hydrogels are suspected, particularly as ∼10% of the total transplanted MSC population was shown to persist in close proximity (within ∼650 μm) to grafts 7 weeks after complete hydrogel degradation. This work demonstrates the therapeutic utility of PEG-based hydrogels for controlling spatiotemporal cell transplantation for a myriad of regenerative medicine strategies.     

Human mesenchymal stem cell culture on heparin-based hydrogels and the modulation of interactions by gel elasticity and heparin amount

Human adipose-derived stem cells (hADSCs) are a promising cell source for tissue engineering and regenerative medicine with no ethnical issue and easy access of large quantities. Conventional surfaces for hADSC culture, such as tissue culture plates (TCPs), do not provide optimal environmental cues, leading to limited expansion, loss of pluripotency and undesirable differentiation of stem cells. The present study demonstrated that heparin-based hydrogels without additional modification provided an excellent surface for adhesion and proliferation of hADSCs, which were further tunable by both the amount of heparin (in a positive way) and the elasticity of hydrogel (in a negative way). The optimized heparin-based hydrogel could selectively modulate the adhesion of hADSCs and human bone marrow stem cells (but not all kinds of cells), and resulted in a significant increase in cell proliferation compared to TCP. Furthermore, in terms of the maintenance of pluripotency and specific differentiation, heparin-based hydrogel was much superior to TCP. The selective binding and proliferation of human mesenchymal stem cells on heparin-based hydrogel over other hydrogels were largely mediated by integrin β1 and selectin, and these superior characteristics were observed regardless of the presence of serum proteins in the culture medium. Consequently, heparin-based hydrogel could be a powerful platform for cultivation of mesenchymal stem cells in various applications.     

The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels

Synthetic hydrogel scaffolds that can be used as culture systems that mimic the natural stem cell niche are of increased importance for stem cell biology and regenerative medicine. These artificial niches can be utilized to control the stem cell fate and will have potential applications for expanding/differentiating stem cells in vitro, delivering stem cells in vivo, as well as making tissue constructs. In this study, we synthesized hyaluronic acid (HA) hydrogels that could be degraded through a combination of cell-released enzymes and used them to culture mouse mesenchymal stem cells (mMSC). To form the hydrogels, HA was modified to contain acrylate groups and crosslinked through Michael addition chemistry using non-degradable, plasmin degradable or matrix metalloproteinase (MMP) degradable crosslinkers. Using this hydrogel we found that mMSC proliferation occurred in the absence of cell spreading, that mMSCs could only spread when both RGD and MMP degradation sites were present in the hydrogel and that mMSCs in hydrogels with high density of RGD (1000 μm) spread and migrated faster and more extensively than in hydrogels with low density of RGD (100 μm).     

Three-dimensional hypoxic culture of human mesenchymal stem cells encapsulated in a photocurable,biodegradable polymer hydrogel: A potential injectable cellular product for nucleus pulposus regeneration

Nucleus pulposus (NP) tissue damage can induce detrimental mechanical stresses and strains on the intervertebral disc, leading to disc degeneration. This study demonstrates the potential of a novel, photo-curable, injectable, synthetic polymer hydrogel (pHEMA-co-APMA grafted with polyamidoamine (PAA)) to encapsulate and differentiate human mesenchymal stem cells (hMSC) towards a NP phenotype under hypoxic conditions which could be used to restore NP tissue function and mechanical properties. Encapsulated hMSC cultured in media (hMSC and chondrogenic) displayed good cell viability up to day 14. The genotoxicity effects of ultraviolet (UV) on hMSC activity confirmed the acceptability of 2.5 min of UV light exposure to cells. Cytotoxicity investigations revealed that hMSC cultured in media containing p(HEMA-co-APMA) grafted with PAA degradation product (10% and 20% v/v concentration) for 14 days significantly decreased the initial hMSC adhesion ability and proliferation rate from 24 hrs to day 14. Successful differentiation of encapsulated hMSC within hydrogels towards chondrogenesis was observed with elevated expression levels of aggrecan and collagen II when cultured in chondrogenic media under hypoxic conditions, in comparison with culture in hMSC media for 14 days. Characterization of the mechanical properties revealed a significant decrease in stiffness and modulus values of cellular hydrogels in comparison with acellular hydrogels at both day 7 and day 14. These results demonstrate the potential use of an in vivo photo-curable injectable, synthetic hydrogel with encapsulated hMSC for application in the repair and regeneration of NP tissue.     

Chondrogenesis of Synovium-Derived Mesenchymal Stem Cells in Photopolymerizing Hydrogel Scaffolds

Recently, tissues adjacent to the wound sites are regarded as a promising therapeutic cell source for curing and repairing purpose. Specifically, therapeutic stem cells have been identified in synovial tissue, a tissue adjacent to articular cartilage. The purpose of this study was to explore therapeutic chondrogenesis with rabbit synovium-derived mesenchymal stem cells (SMSCs) encapsulated in photopolymerized hydrogels. A non-degradable poly(ethylene glycol) diacrylate (PEGDA)-based hydrogel and biodegradable phosphoester–poly(ethylene glycol) (PhosPEG)-based hydrogel were both applied as 3-D scaffolds mediating SMSC chondrogenesis in vitro. The viability of SMSCs in both hydrogels was assessed by fluorescent Live/Dead assay and WST-1 assay. Levels of genes and proteins specific to SMSC chondrogenesis were evaluated by real-time RT-PCR, biochemical analysis and immunohistochemical analysis, respectively. The results demonstrated that SMSCs continue to have a high viability when encapsulated in the hydrogel. By treatment with transforming growth factor (TGF)-β1 or TGF-β3, positive SMSC chondrogenesis was successfully achieved in both gels, with the best outcome in the PEGDA system. It can be concluded that both PEGDA and PhosPEG hydrogels are appropriate cell-delivery vehicles for SMSC chondrogenesis. Especially as a biodegradable material, PhosPEG hydrogel displayed great potentials in future applications for articular cartilage regeneration coupling with SMSCs.     

Sustained localized presentation of RNA interfering molecules from in situ forming hydrogels to guide stem cell osteogenic differentiation

To date, RNA interfering molecules have been used to differentiate stem cells on two-dimensional (2D) substrates that do not mimic three-dimensional (3D) microenvironments in the body. Here, in situ forming poly(ethylene glycol) (PEG) hydrogels were engineered for controlled, localized and sustained delivery of RNA interfering molecules to differentiate stem cells encapsulated within the 3D polymer network. RNA interfering molecules were released from the hydrogels in a sustained and controlled manner over the course of 3–6 weeks, and exhibited high bioactivity. Importantly, it was demonstrated that the delivery of siRNA and/or miRNA from the hydrogel constructs enhanced the osteogenic differentiation of encapsulated stem cells. Prolonged delivery of siRNA and/or miRNA from this polymeric scaffold permitted extended regulation of cell behavior, unlike traditional siRNA experiments performed in vitro. This approach presents a powerful new methodology for controlling cell fate, and is promising for multiple applications in tissue engineering and regenerative medicine.     

Hyaluronic acid-human blood hydrogels for stem cell transplantation

Tissue engineering-based approaches have the potential to improve stem cell engraftment by increasing cell delivery to the myocardium. Our objective was to develop and characterize a naturally-derived, autologous, biodegradable hydrogel in order to improve acute stem cell retention in the myocardium. HA-blood hydrogels (HA-BL) were synthesized by mixing in a 1:1(v/v) ratio, lysed whole blood and hyaluronic acid (HA), whose carboxyl groups were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA-NHS). We performed physical characterization and measured survival/proliferation of cardiosphere-derived cells (CDCs) encapsulated in the hydrogels. Hydrogels were injected intra-myocardially or applied epicardially in rats. NHS-activated carboxyl groups in HA react with primary amines present in blood and myocardium to form amide bonds, resulting in a 3D hydrogel bound to tissue. HA-blood hydrogels had a gelation time of 58?±?12?s, swelling ratio of 10?±?0.5, compressive and elastic modulus of 14?±?3 and 1.75?±?0.6?kPa respectively. These hydrogels were not degraded at 4wks by hydrolysis alone. CDC encapsulation promoted their survival and proliferation. Intra-myocardial injection of CDCs encapsulated in these hydrogels greatly increased acute myocardial retention (p?=?0.001). Epicardial application of HA-blood hydrogels improved left ventricular ejection fraction following myocardial infarction (p?=?0.01). HA-blood hydrogels are highly adhesive, biodegradable, promote CDC survival and increase cardiac function following epicardial application after myocardial infarction.     

低聚乙二醇富马酸酯水凝胶中骨髓来源间充质干细胞的生物学行为

文章快速阅读：  文题释义：水凝胶：是以水为分散介质的凝胶,具有网状交联结构的水溶性高分子中引入一部分疏水基团和亲水残基,亲水残基与水分子结合,将水分子连接在网状内部,而疏水残基遇水膨胀的交联聚合物。是一种高分子网络体系,性质柔软,能保持一定的形状,能吸收大量的水。凡是水溶性或亲水性的高分子,通过一定的化学交联或物理交联,都可以形成水凝胶。这些高分子按其来源可分为天然和合成两大类。天然的亲水性高分子包括多糖类(淀粉、纤维素、海藻酸、透明质酸,壳聚糖等)和多肽类(胶原、聚L-赖氨酸、聚L-谷胺酸等)。合成的亲水高分子包括醇、 丙烯酸及其衍生物类(聚丙烯酸,聚甲基丙烯酸,聚丙烯酰胺,聚N-聚代丙烯酰胺等)。低聚乙二醇富马酸酯水凝胶：是由聚乙二醇和延胡索酸酯与聚乙二醇二丙烯酸酯化学交联而成,具有良好的组织相容性和生物可降解性。实验证明低聚乙二醇富马酸酯水凝胶随着相对分子质量的升高其溶胀度降增加,溶胀比增加引起水凝胶降解速度加快及力学强度减弱。故研制与筛选具有合适溶胀比的低聚乙二醇富马酸酯水凝胶材料是进行骨组织工程支架材料研究的前提。   背景：低聚乙二醇富马酸酯水凝胶是一种具有良好生物相容性及可注射性和可降解性的生物材料。不同相对分子质量水凝胶之间的特性有所差异,将骨髓间充质干细胞包裹其中并诱导细胞成骨分化,相对分子质量相当的水凝胶更有利于细胞增殖和分化,所以采用该材料为骨组织工程支架提供了新的选择。目的：探讨不同相对分子质量的低聚乙二醇富马酸酯水凝胶材料体外包裹大鼠骨髓间充质干细胞的增殖和分化的影响。方法：低聚乙二醇富马酸酯通过氧化还原基团引发系统产生交联,制备出相对分子质量为1000,3000,10000,35 000的低聚乙二醇富马酸酯水凝胶,对水凝胶的溶胀和降解性能进行检测。将骨髓间充质干细胞包裹到4种相对分子质量的水凝胶中,在成骨培养液中诱导1-3周,通过组织学染色(苏木精-伊红染色和茜素红染色)和免疫荧光染色检测水凝胶材料对骨髓间充质干细胞形态的影响以及成骨分化的效果。 结果与结论：①随着水凝胶相对分子质量的增加,成胶时间变短,凝胶的溶胀度明显增加,且随着时间的推移,水凝胶的降解速率与相对分子质量成正比;②细胞复合水凝胶支架材料的组织学与免疫荧光染色结果表明,细胞经过诱导后,在具有适当溶胀与降解特性的相对分子质量为3 000与10 000的水凝胶中所形成的矿化结节数量显著多于在其它两种相对分子质量中的,说明有利于细胞的增殖与分化;③结果表明,低聚乙二醇富马酸酯水凝胶具有良好的生物相容性,且相对分子质量为3 000与     10 000的水凝胶对间充质干细胞的成骨分化有一定的良性调节作用。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程 ORCID:0000-0002-0616-3754(魏丽君)     

Controlled drug release from multilayered phospholipid polymer hydrogel on titanium alloy surface

Here we describe the functionalization of a multilayered hydrogel layer on a Ti alloy with an antineoplastic agent, paclitaxel (PTX). The multilayered hydrogel was synthesized via layer-by-layer self-assembly (LbL) using selective intermolecular reactions between two water-soluble polymers, phospholipid polymer (PMBV) containing a phenylboronic acid unit and poly(vinyl alcohol) (PVA). Reversible covalent bonding between phenylboronic acid and the polyol provided the driving force for self-assembly. Poorly water-soluble PTX dissolves in PMBV aqueous solutions because PMBV is amphiphilic. Therefore, our multilayered hydrogel could be loaded with PTX at different locations to control the release profile and act as a drug reservoir. The amount of PTX incorporated in the hydrogel samples increased with the number of layers but was not directly proportional to the number of layers. However, as the step for making layers was repeated, the concentration of PTX in the PMBV layers increased. The different solubilities of PTX in PMBV and PVA aqueous solutions allow for the production of multilayered hydrogels loaded with PTX at different locations. In vitro experiments demonstrated that the location of PTX in the multilayered hydrogel influences the start and profile of PTX release. We expect that this rapid and facile LbL synthesis of multilayered hydrogels and technique for in situ loading with PTX, where the location of loading controls the release pattern, will find applications in biomedicine and pharmaceutics as a promising new technique.     

Creating stiffness gradient polyvinyl alcohol hydrogel using a simple gradual freezing–thawing method to investigate stem cell differentiation behaviors

Polyvinyl alcohol (PVA) cylindrical hydrogel with a stiffness gradient was prepared using a simple liquid nitrogen (LN₂)-contacting gradual freezing and thawing method in order to investigate the effects of substrate stiffness on stem cell differentiation into specific cell types. The prepared cylindrical PVA hydrogel showed a gradually increasing stiffness along the longitudinal direction from the top at approximately 1 kPa to the bottom (LN₂ contacted side) at approximately 24 kPa. From the in vitro culture of bone marrow stem cells, it was observed that each soft (∼1 kPa) and stiff (∼24 kPa) hydrogel section promotes effective neurogenesis and osteogenesis of the cells, respectively, with the tendency to gradually decrease toward the opposing characteristic's side. The stiffness gradient cylindrical PVA hydrogel fabricated using this simple gradual freezing and thawing method can be a useful tool for basic studies, including the determination of optimum stiffness ranges for a variety of stem cell differentiations, as well as the investigation of cell migration in terms of substrate stiffness.     

Derivation of epithelial-like cells from eyelid fat-derived stem cells in thermosensitive hydrogel

Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation. In the study, the thermosensitive chitosan/gelatin/β-glycerol phosphate (C/G/GP) disodium salt hydrogels were designed and investigated by different analyses. The eye fat-derived stem cells were used to evaluate the biocompatibility of hydrogels based on their phenotypic profile, viability, proliferation, and attachment ability. The results show that the sol/gel transition temperature of the C/G/GP hydrogel was in the range of 31.1–33.8?°C at neutral pH value, the gelation time was shortened, and the gel strength also improved at body temperature when compared with the C/GP hydrogel. In vitro cell culture experiments with eyelid fat-derived stem cells in hydrogel showed beneficial effects on the cell phenotypic morphology, proliferation, and differentiation. Microscopic figures showed that the eyelid fat stem cell were firmly anchored to the substrates and were able to retain a normal stem cell phenotype. Immunocytochemistry (ICC) and real-time–PCR results revealed change in the expression profile of eyelid fat stem cells grown with hydrogels when compared to those grown on control in epithelial induction condition. This study indicates that using chitosan/gelatin/β-glycerol phosphate hydrogel for cell culture is feasible and may apply in minimal invasive surgery in the future.     

Poly(acrylamide) Spheroids with Tunable Elasticity for Scalable Cell Culture Applications

Many biological tissues resemble hydrogels and display Young's moduli below 50 kPa, corresponding to most cell types for the natural environmental conditions to grow. Contrastingly, conventional cell culture usually involves rigid substrates resulting in stiff priming effects, which are of increasing concern when it comes to scalable culturing of adhesive cells for regenerative purposes. As a solution to this problem, the employment of synthetic poly(acryl amide) (PAAm)-based hydrogel beads with tissue-matched mechanical properties are proposed as soft matrix for culture modes suitable for tidal bioreactor culture. Herein, technology is described to generate spherical, mm-scaled PAAm hydrogel beads with adjustable, soft elastic properties that are produced by a continuous microfluidic approach. A simple and robust method is demonstrated to functionalize the spheroids with protein ligands, and the suitability of the matrix for cell cultivation is successfully demonstrated with three different cell types (murine mesenchymal stem cells, renal carcinoma cells, and human induced pluripotent stem cells) in model experiments and in a tidal bioreactor system. This versatile approach will pave the way toward novel cell culture systems based on bioreactors that allow scalable, soft carrier-based expansion of cells on matrices with tissue-matched elasticity.     

Transfer stamping of human mesenchymal stem cell patches using thermally expandable hydrogels with tunable cell-adhesive properties

Development of stem cell delivery system with ability of control over mutilineage differentiation and improved engraft efficiency is imperative in regenerative medicine. We herein report transfer stamping of human mesenchymal stem cells (hMSCs) patches using thermally expandable hydrogels with tunable cell-adhesive properties. The hydrogels were prepared from functionalized four arm copolymer of Tetronic^®, and the cell adhesion on the hydrogel was modulated by incorporation of fibronectin (FN) or cell-adhesive peptide (RGD). The resulting hydrogels showed spontaneous expansion in size within 10 min in response to the temperature reduction from 37 to 4°C. The adhesion and proliferation of hMSCs on FN-hydrogels were positively tunable in proportion to the amount of FN within hydrogels with complete monolayer of hMSCs (hMSC patch) being successfully achieved. The hMSC patch on the hydrogel was faced to the target substrate, which was then easily detached and re-attached to the target when the temperature was reduced from 37°C up to 4°C. We found that the transfer stamping of cell patch was facilitated at lower temperature of 4°C relative to 25°C, with the use of thinner hydrogels (0.5 mm in thickness relatively to 1.0 or 1.5 mm) and longer transfer time (>15 min). Notably, the hMSC patch was simply transferred from the hydrogel to the subcutaneous mouse skin tissue within 15 min with cold saline solution being dropped to the hydrogel. The hMSC patch following osteogenic or adipogenic commitment was also achieved with long-term culture of hMSCs on the hydrogel, which was successfully detached to the target surface. These results suggest that the hydrogels with thermally expandable and tunable cell-adhesive properties may serve as a universal substrate to harvest hMSC patch in a reliable and effective manner, which could potentially be utilized in many cell-sheet based therapeutic applications.     

Self-assembling peptide amphiphile nanofibers as a scaffold for dental stem cells

Dental caries remains one of the most prevalent infectious diseases in the world. So far, available treatment methods rely on the replacement of decayed soft and mineralized tissue with inert biomaterials alone. As an approach to develop novel regenerative strategies and engineer dental tissues, two dental stem cell lines were combined with peptide-amphiphile (PA) hydrogel scaffolds. PAs self-assemble into three-dimensional networks of nanofibers, and living cells can be encapsulated. Cell-matrix interactions were tailored by incorporation of the cell adhesion sequence RGD and an enzyme-cleavable site. SHED (stem cells from human exfoliated deciduous teeth) and DPSC (dental pulp stem cells) were cultured in PA hydrogels for 4 weeks using different osteogenic supplements. Both cell lines proliferate and differentiate within the hydrogels. Histologic analysis shows degradation of the gels and extracellular matrix production. However, distinct differences between the two cell lines can be observed. SHED show a spindle-shaped morphology, high proliferation rates, and collagen production, resulting in soft tissue formation. In contrast, DPSC reduce proliferation, but exhibit an osteoblast-like phenotype, express osteoblast marker genes, and deposit mineral. Since the hydrogels are easy to handle and can be introduced into small defects, this novel system might be suitable for engineering both soft and mineralized matrices for dental tissue regeneration.     

Structural and permeability characterization of biosynthetic PVA hydrogels designed for cell-based therapy

Incorporation of extracellular matrix (ECM) components to synthetic hydrogels has been shown to be the key for successful cell encapsulation devices, by providing a biofunctional microenvironment for the encapsulated cells. However, the influence of adding ECM components into synthetic hydrogels on the permeability as well as the physical and mechanical properties of the hydrogel has had little attention. Therefore, the aim of this study was to investigate the effect of incorporated ECM analogues on the permeability performance of permselective synthetic poly(vinyl alcohol) (PVA) hydrogels in addition to examining the physico-mechanical characteristics. PVA was functionalized with a systematically increased number of methacrylate functional groups per chain (FG/c) to tailor the permselectivity of UV photopolymerized hydrogel network. Heparin and gelatin were successfully incorporated into PVA network at low percentage (1%), and co-hydrogels were characterized for network properties and permeability to bovine serum albumin (BSA) and immunoglobulin G (IgG) proteins. Incorporation of these ECM analogues did not interfere with the base PVA network characteristics, as the controlled hydrogel mesh sizes, swelling and compressive modulii remained unchanged. While the permeation profiles of both BSA and IgG were not affected by the addition of heparin and gelatin as compared with pure PVA, increasing the FG/c from 7 to 20 significantly limited the diffusion of the larger IgG. Consequently, biosynthetic hydrogels composed of PVA with high FG/c and low percent ECM analogues show promise in their ability to be permselective for various biomedical applications.     

Fabrication of a live cell-containing multilayered polymer hydrogel membrane with micrometer-scale thickness to evaluate pharmaceutical activity

We propose a spinning-assisted layer-by-layer method for simple fabrication of a multilayered polymer hydrogel membrane that contains living cells. Hydrogel formation occurred based on the spontaneous cross-linking reaction between two polymers in aqueous solution. A water-soluble 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups (PMBV) and poly(vinyl alcohol) (PVA) were used as polymers for hydrogel membrane formation. Changing the number of hydrogel membrane layers, polymer concentration, spinning rate, and processing time for diffusion-dependent gelation of PMBV and PVA facilitated the regulation of the multilayered polymer hydrogel membrane thickness and morphology. We concluded that a multilayered polymer hydrogel membrane prepared using 5.0 wt% PMBV and 5.0 wt% PVA at a spinning rate of 2000 rpm was suitable for precise spatial control of cells in single layers. This multilayered polymer hydrogel membrane was used to prepare a single cell-laden layer to minimize barriers to the diffusion of bioactive compounds while preserving the three-dimensional (3-D) context. The pharmaceutical effects of one of the anticancer agents, paclitaxel, on a human cervical cancer line, HeLa cells, were evaluated in vitro, and the usability of this culture model was demonstrated.     

Elastin based cell-laden injectable hydrogels with tunable gelation,mechanical and biodegradation properties

Injectable hydrogels made from extracellular matrix proteins such as elastin show great promise for various biomedical applications. Use of cytotoxic reagents, fixed gelling behavior, and lack of mechanical strength in these hydrogels are the main associated drawbacks. The aim of this study was to develop highly cytocompatible and injectable elastin-based hydrogels with alterable gelation characteristics, favorable mechanical properties and structural stability for load bearing applications. A thermoresponsive copolymer, poly(N-isopropylacrylamide-co-polylactide-2-hydroxyethyl methacrylate-co-oligo(ethylene glycol)monomethyl ether methacrylate, was functionalized with succinimide ester groups by incorporating N-acryloxysuccinimide monomer. These ester groups were exploited to covalently bond this polymer, denoted as PNPHO, to different proteins with primary amine groups such as α-elastin in aqueous media. The incorporation of elastin through covalent bond formation with PNPHO promotes the structural stability, mechanical properties and live cell proliferation within the structure of hydrogels. Our results demonstrated that elastin-co-PNPHO solutions were injectable through fine gauge needles and converted to hydrogels in situ at 37 °C in the absence of any crosslinking reagent. By altering PNPHO content, the gelling time of these hydrogels can be finely tuned within the range of 2–15 min to ensure compatibility with surgical requirements. In addition, these hydrogels exhibited compression moduli in the range of 40–145 kPa, which are substantially higher than those of previously developed elastin-based hydrogels. These hydrogels were highly stable in the physiological environment with the evidence of 10 wt% mass loss in 30 days of incubation in a simulated environment. This class of hydrogels is in vivo bioabsorbable due to the gradual increase of the lower critical solution temperature of the copolymer to above 37 °C due to the cleavage of polylactide from the PNPHO copolymer. Moreover, our results demonstrated that more than 80% of cells encapsulated in these hydrogels remained viable, and the number of encapsulated cells increased for at least 5 days. These unique properties mark elastin-co-PNHPO hydrogels as favorable candidates for a broad range of tissue engineering applications.     

In situ forming hydrogels with long-lasting miR-21 enhances the therapeutic potential of MSC by sustaining stimulation of target gene

Regulating stem cells by microRNA (miRNA) is promising in regenerative medicine. However, non-viral transfection is usually transient while stably lentiviral transfection is accompanied with oncogenic risk. In the study, we explored the feasibility to retain the microRNAs within biopolymer hydrogels for their long-lasting working and sustaining stimulation of target gene. miRNA-21 (MiR-21), a reported microRNA enhancing the therapeutic potential of mesenchymal stem cells (MSCs) was used. We demonstrated that miR-21 could be efficiently retained within collagen hydrogel after forming complex with cationic polymer polyethylenimine (PEI). Due to the electronic interaction with positively charged PEI, the release of miR-21 was largely prevented during 2 week incubation (<20%), while free miR-21 encapsulated in hydrogels was largely released (>50%). When MSCs were cultivated in the PEI/miR-21-incorporated hydrogels, the sustained activation of targeted gene HIF-1α was observed, resulting in the sustaining up-regulation of several downstream therapeutic cytokines. Then, the hydrogels encapsulating miR-21/PEI were coated onto tissue plate for MSC cultivation, which further confirmed the long-lasting retention and efficacy of miR-21 on the plate surface. In addition, under H₂O₂-simulated stress condition, we also demonstrated that the anti-apoptotic capacity of MSCs was significantly improved when growing on miR-21-retained hydrogels. Our study provided a safe and promising method for long-lasting stem cell regulation with miRNAs.