Hydrogels with Dynamically Controllable Mechanics and Biochemistry for 3D Cell Culture Platforms

Many cell-matrix interaction studies have proved that dynamic changes in the extracellular matrix(ECM)are crucial to maintain cellular properties and behaviors.Thus,developing materials that can recapitulate the dynamic attributes of the ECM is highly desired for threedimensional(3 D)cell culture platforms.To this end,we sought to develop a hydrogel system that would enable dynamic and reversible turning of its mechanical and biochemical properties,thus facilitating the control of cell culture to imitate the natural ECM.Herein,a hydrogel with dynamic mechanics and a biochemistry based on an addition-fragmentation chain transfer(AFCT)reaction was constructed.Thiol-modified hyaluronic acid(HA)and allyl sulfide-modifiedε-poly-L-lysine(EPL)were synthesized to form hydrogels,which were non-swellable and biocompatible.The reversible modulus of the hydrogel was first achieved through the AFCT reaction;the modulus can also be regulated stepwise by changing the dose of UVA irradiation.Dynamic patterning of fluorescent markers in the hydrogel was also realized.Therefore,this dynamically controllable hydrogel has great potential as a 3 D cell culture platform for tissue engineering applications.     

Gelatin—Hyaluronic Acid Hydrogels with Tuned Stiffness to Counterbalance Cellular Forces and Promote Cell Differentiation

Cells interact mechanically with their environment, exerting mechanical forces that probe the extracellular matrix (ECM). The mechanical properties of the ECM determine cell behavior and control cell differentiation both in 2D and 3D environments. Gelatin (Gel) is a soft hydrogel into which cells can be embedded. This study shows significant 3D Gel shrinking due to the high traction cellular forces exerted by the cells on the matrix, which prevents cell differentiation. To modulate this process, Gel with hyaluronic acid (HA) has been combined in an injectable crosslinked hydrogel with controlled Gel–HA ratio. HA increases matrix stiffness. The addition of small amounts of HA leads to a significant reduction in hydrogel shrinking after cell encapsulation (C2C12 myoblasts). We show that hydrogel stiffness counterbalanced traction forces of cells and this was decisive in promoting cell differentiation and myotube formation of C2C12 encapsulated in the hybrid hydrogels.

     

Injectable Hyaluronic Acid/Poly(γ-glutamic acid) Hydrogel with Step-by-step Tunable Properties for Soft Tissue Engineering

Injectable hydrogels as an important class of biomaterials have gained much attention in tissue engineering. However, their crosslinking degree is difficult to be controlled after being injected into body. As we all know, the crosslinking degree strongly influences the physicochemical properties of hydrogels. Therefore, developing an injectable hydrogel with tunable crosslinking degree in vivo is important for tissue engineering. Herein, we present a dual crosslinking strategy to prepare injectable hydrogels with step-by-step tunable crosslinking degree using Schiff base reaction and photopolymerization. The developed hyaluronic acid/poly(γ-glutamic acid)(HA/γ-PGA) hydrogels exhibit step-bystep tunable swelling behavior, enzymatic degradation behavior and mechanical properties. Mechanical performance tests show that the storage moduli of HA/γ-PGA hydrogels are all less than 2000 Pa and the compressive moduli are in kilopascal, which have a good match with soft tissue. In addition, NIH 3 T3 cells encapsulated in HA/γ-PGA hydrogel exhibit a high cell viability, indicating a good cytocompatibility of HA/γ-PGA hydrogel.Therefore, the developed HA/γ-PGA hydrogel as an injectable biomaterial has a good potential in soft tissue engineering.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.     

Development of injectable high molecular weight hyaluronic acid hydrogels for cartilage regeneration

The study focuses on developing hyaluronic acid (1200 kilo Dalton) hydrogels for cartilage regeneration. In spite of being highly biocompatible; a large amount of water absorption and easily degrading nature restricts the use of hyaluronic acid in the field of tissue regeneration. This can be rectified by crosslinking hyaluronic acid with a crosslinking agent such as divinyl sulfone; which results in a biocompatible hydrogel with superior rheological properties. Different amounts of divinyl sulfone have been used for crosslinking hyaluronic acid to get three types of hydrogels with differing properties. Swelling studies, rheology analysis, enzymatic degradation and scanning electron microscopic analysis were conducted on all the different types of hydrogels prepared. Viscoelastic properties of the hydrogel were analyzed so that a hydrogel with better elastic property and stability is obtained. Scanning electron microscopy was used to study the morphology of the HA hydrogels. The cytotoxicity testing was conducted to prove the non-toxic nature of the hydrogels and cell culture studies using adipose mesenchymal stem cells showed better adhesion and proliferation properties in all the three hydrogels. Thus hyaluronic acid hydrogel makes a promising material for cartilage regeneration.     

Effect of Composition of Lung Biomimetic Niche on the Mesenchymal Stem Cell Differentiation toward Alveolar Type II Pneumocytes

Pulmonary niche dynamically orchestrates the signals, such as proliferation or differentiation of mesenchymal stem cells (MSCs), which allows inducing tissue repair. Lung niche includes extracellular matrix (ECM), comprising hyaluronic acid (HA) and collagen (COLL), and several types of MSCs. Impaired ECM, in lung pathologies, makes the promising therapies based on MSCs ineffective, as it results in a reduced attachment and homing of MSCs, precluding their differentiation and viability. To overcome this problem, in this study a pulmonary biomimetic niche based on HA and COLL hydrogel is developed, with the specific aim to elucidate the role of COLL and HA/COLL semi-interpenetrating polymer networks (SIPNs) in directing the differentiation of MSCs into Alveolar Type II (ATII) cells. The effect of low (L), medium (M), and high (H) molecular weight (MW) HA is investigated, both like structural component of the SIPNs hydrogel and like trophic factor in cell culture media solution. HA in the culture media significantly improves surfactant protein (SP)-C expression (≈2 ng mL⁻¹), without showing difference in the MW tested, compared to control only (≈1 ng mL⁻¹). Furthermore, LMWHA/COLL hydrogel promotes the SPC expression (approximately two times) compared to COLL, MMWHA/COLL, and HMWHA/COLL hydrogels.     

Self-Forming Norbornene-Tetrazine Hydrogels with Independently Tunable Properties

Although photopolymerization reactions are commonly used to form hydrogels, these strategies rely on light and may not be suitable for delivering therapeutics in a minimally invasive manner. Here, hyaluronic acid (HA) macromers are modified with norbornene (Nor) or tetrazine (Tet) and upon mixing click into covalently crosslinked Nor-Tet hydrogels via a Diels–Alder reaction. By incorporating a high degree of Nor and Tet substitution, Nor-Tet hydrogels with a broad range in elastic moduli (5 to 30 kPa) and fast gelation times (1 to 5 min) are achieved. By pre-coupling methacrylated HANor macromers with thiolated peptides via a Michael addition reaction, Nor-Tet hydrogels are peptide-functionalized without affecting their physical properties. Mesenchymal stem cells (MSCs) on RGD-functionalized Nor-Tet hydrogels adhere and exhibit stiffness-dependent differences in matrix mechanosensing. Fluid properties of Nor-Tet hydrogel solutions allow for injections through narrow syringe needles and can locally deliver viable cells and peptides. Substituting HA with enzymatically degradable gelatin also results in cell-responsive Nor-Tet hydrogels, and MSCs encapsulated in Nor-Tet hydrogels preferentially differentiate into adipocytes or osteoblasts, based on 3D cellular spreading regulated by stable (HA) and degradable (gelatin) macromers.     

Tunable biohybrid hydrogels from coacervation of hyaluronic acid and PEO-based block copolymers

Accurately tuning the macroscopic properties of biopolymer-based hydrogels remains challenging due to the ill-defined molecular architecture of the natural building blocks. Here, we report a biohybrid coacervate hydrogel, combining the biocompatibility and biodegradability of naturally occurring hyaluronic acid (HA) with the tunability of a synthetic polyethylene oxide (PEO) -based ABA-triblock copolymer. Coacervation of the cationic ammonium or guanidinium-functionalized copolymer A-blocks with the anionic HA leads to hydrogel formation. Both mechanical properties and water content of the self-healing hydrogels can be controlled independently by altering the copolymer structure. By controlling the strength of the interaction between the polymer network and small-molecule cargo, both release rate and maximum release are controlled. Finally, we show that coacervation of HA and the triblock copolymer leads to increased biostability upon exposure to hyaluronidase. We envision that noncovalent crosslinking of HA hydrogels through coacervation is an attractive strategy for the facile synthesis of tunable hydrogels for biomedical applications.     

Phospholipid Polymer Hydrogel Matrices with Dually Immobilized Cytokines for Accelerating Secretion of the Extracellular Matrix by Encapsulated Cells

Construction of 3D tissues by various types of cells with specific characteristics is an important and fundamental technology in tissue reconstruction medicine and animal‐free diagnosis system. To do so, an excellent extracellular matrix (ECM) is needed for encapsulation of cells and maintaining cell activity. Spontaneously forming hydrogel matrix is used by complexation between two water‐soluble polymers, 2‐methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups and poly(vinyl alcohol). Two cytokines for cell proliferation are immobilized in the hydrogel matrix to control the activities of the encapsulated cells. The cytokine‐immobilized hydrogel matrix can encapsulate both L929 fibroblasts and normal human dermal fibroblasts under mild condition. The physical properties of the hydrogel matrix can follow the proliferation process of the encapsulated cells. The encapsulated cells secrete ECM in the polymer hydrogel networks upon 3D culturing for 7 days. Consequently, the tissue‐mimicking ECM hybrid hydrogels are fabricated successfully.     

Preparation and properties of redox responsive modified hyaluronic acid hydrogels for drug release

A series of oxidized hyaluronic acid (oxi‐HA)/3,3′‐dithiobis (propionohydrazide) (DTP) redox responsive hydrogels by Schiff base reaction under physiological conditions were designed and prepared. The influence of the concentration of oxi‐HA and DTP on rheological properties, equilibrium swelling ratio, and degradation rate were investigated. All oxi‐HA/DTP hydrogels exhibited good rheological properties, high equilibrium swelling ratio, low degradation rate, and sustainable drug release properties, and the comprehensive performance of oxi‐HA5/DTP6 hydrogel was better than that of others. The redox responsiveness was evaluated by means of degradation and in vitro bovine serum albumin release behavior investigation with the stimulus of different concentration of dithiothreitol as reducing agent. The intelligent hydrogels could be potentially applied in the fields of drug delivery system, tissue engineering, or cell scaffold materials. Copyright © 2017 John Wiley & Sons, Ltd.     

Comprehensive Examination of Mechanical and Diffusional Effects on Cell Behavior Using a Decoupled 3D Hydrogel System

Hydrogels possess several physical and chemical properties suitable for engineering cellular environments for biomedical applications. Despite recent advances in hydrogel systems for cell culture, it is still a significant challenge to independently control the mechanical and diffusional properties of hydrogels, both of which are well known to influence various cell behaviors when using hydrogels as 3D cell culture systems. Controlling the crosslinking density of a hydrogel system to tune the mechanical properties inevitably affects their diffusional properties, as the crosslinking density and diffusion are often inversely correlated. In this study, a polymeric crosslinker is demonstrated that allows for the adjustment of the degree of substitution of reactive functional groups. By using this polymeric crosslinker, the rigidity of the resulting hydrogel is controlled in a wide range without changing the polymer concentration. Furthermore, their diffusional properties, as characterized by their swelling ratios, pore diameters, and drug release rates, are not significantly affected by the changes in the degree of substitution. 3D cell studies using this hydrogel system successfully demonstrate the varying effects of mechanical properties on different cell types, whereas those in a conventional hydrogel system are more significantly influenced by changes in diffusional properties.     

TEMPO/TCC as a Chemo Selective Alternative for the Oxidation of Hyaluronic Acid

Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer’s backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker.     

聚(L-谷氨酸)水凝胶介导羟基磷灰石的生物矿化

以1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)为羧基活化剂, 己二酸二酰肼(ADH)为交联剂, 制备了生物活性聚(L-谷氨酸)(PLGA)水凝胶. 通过X射线衍射和扫描电子显微镜等表征了在不同浓度模拟体液(SBF)中羟基磷灰石(HA)的形成和生长. PLGA水凝胶的表面和内部均可观察到HA的形成和生长. 同时探讨了PLGA水凝胶矿化前后的力学性能. 将矿化前后PLGA水凝胶用于脂肪干细胞(ASCs)的培养, 研究其细胞相容性.     

Cellulose-based hydrogels with excellent microstructural replication ability and cytocompatibility for microfluidic devices

In the face of challenges in the development of excellent biocompatible materials for microfluidic device fabrication, we demonstrated that cross-linked cellulose (RCC) hydrogel can be used as the bulk material for microchips. The cellulose hydrogel was prepared from cellulose solution dissolved in an 8 wt% LiOH/15 wt% urea aqueous system with cooling by crosslinking with epichlorohydrin. Collagen as a key extracellular matrix component for promoting cell cultivation was cross-linked in the cellulose hydrogel to obtain cellulose–collagen (RCC/C) hybrid hydrogels. The experimental results revealed that cellulose-based hydrogel microchips with well-defined 2D or 3D microstructures possessed excellent structural replication ability, good mechanical properties, and cytocompatibility for cell culture as well as excellent dimensional stability at elevated temperature. The hydrogel, as a transparent microchip material, had no effect on the fluorescence behaviors of FITC-dextran and rhodamine-dextran, leading to the good conjunction with fluorescent detection and imaging. Moreover, collagen could be immobilized in the RCC/C hydrogel scaffold for promoting cell growth and generating stable chemical concentration gradients, leading to superior cytocompatibility. This work provides new hydrogel materials for the microfluidic technology field and mimicks a 3D cell culture microenvironment for cell-based tissue engineering and drug screening.     

Synthetic hydrogels as scaffolds for manipulating endothelium cell behaviors

Synthetic hydrogels can be used as scaffolds that not only favor endothelial cells（ECs） proliferation but also manipulate the behaviors and functions of the ECs.In this review paper,the effect of chemical structure,Young’s modulus （E） and zeta potential（ξ） of synthetic hydrogel scaffolds on static cell behaviors,including cell morphology,proliferation, cytoskeleton structure and focal adhesion,and on dynamic cell behaviors,including migration velocity and morphology oscillation,as well as on EC function such as anti-platelet adhesion,are reported.It was found that negatively charged hydrogels,poly（2-acrylamido-2-methylpropanesulfonic sodium）（PNaAMPS） and poly（sodium p-styrene sulphonate） （PNaSS）,can directly promote cell proliferation,with no need of surface modification by any cell-adhesive proteins or peptides at the environment of serum-containing medium.In addition,the Young’s modulus（E） and zeta potential（ξ） of hydrogel scaffolds are quantitatively tuned by copolymer hydrogels,poly（NaAMPS-co-DMAAm） and poly（NaSS-co-DMAAm）, in which the two kinds of negatively charged monomers NaAMPS and NaSS are copolymerized with neutral monomer,N,N-dimethylacrylamide（DMAAm）.It was found that the critical zeta potential of hydrogels manipulating EC morphology,proliferation,and motility isξ_critical= -20.83 mV andξ_critical= -14.0 mV for poly（NaAMPS-co-DMAAm） and poly（NaSS-co-DMAAm）,respectively.The above mentioned EC behaviors well correlate with the adsorption of fibronectin, a kind of cell-adhesive protein,on the hydrogel surfaces.Furthermore,adhered platelets on the EC monolayers cultured on the hydrogel scaffolds obviously decreases with an increase of the Young’s modulus（E） of the hydrogels,especially when E>60 kPa.Glycocalyx assay and gene expression of ECs demonstrate that the anti-platelet adhesion well correlates with the EC-specific glycocalyx.The above investigation suggests that understanding the relationship between physic-chemical properties of synthetic hydrogels and cell responses is essential to design optimal soft and wet scaffolds for tissue engineering.     

Screening of Hydrogels for Human Pluripotent Stem Cell–Derived Neural Cells: Hyaluronan‐Polyvinyl Alcohol‐Collagen‐Based Interpenetrating Polymer Network Provides an Improved Hydrogel Scaffold

There is a clear need for novel in vitro models, especially for neuronal applications. Development of in vitro models is a multiparameter task consisting of cell‐, biomaterial‐, and environment‐related parameters. Here, three different human origin neuronal cell sources are studied and cultured in various hydrogel 3D scaffolds. For the efficient evaluation of complex results, an indexing method for data is developed and used in principal component analysis (PCA). It is found that no single hydrogel is superior to other hydrogels, and collagen I (Col1) and hyaluronan–poly(vinyl alcohol) (HA1‐PVA) gels are combined into an interpenetrating network (IPN) hydrogel. The IPN gel combines cell supportiveness of the collagen gel and stability of the HA1‐PVA gel. Moreover, cell adhesion is studied in particular and it is found that adhesion of neurons differs from that observed for fibroblasts. In conclusion, the HA1‐PVA‐col1 hydrogel is a suitable scaffold for neuronal cells and supports adhesion formation in 3D.     

温敏性半乳糖基化壳聚糖水凝胶的制备及其对HL-7702细胞增殖行为影响的研究

以壳聚糖(CS)为原料,在1-乙基-3-(3-二甲胺丙基)碳-二亚胺盐酸盐(EDC.HCL)和N-羟基琥珀酰亚胺(NHS)的活化作用下,合成了半乳糖基化壳聚糖(GC)单体,并与N-异丙基丙烯酰胺(NIPAAm)反应,制备了温敏性半乳糖基化壳聚糖N-异丙基丙烯酰胺共聚水凝胶(Gal-CS-g-PNIPAAm).通过红外光谱(FTIR)、光电子能谱(XPS)和扫描电子显微镜(SEM)等测试方法对其成分和结构进行了表征,并对其溶胀率和表面亲疏水性进行了研究.在Gal-CS-g-PNIPAAm凝胶表面培养人正常肝细胞系(HL-7702),研究其生长、脱附及转载(再增殖)行为.结果表明Gal-CS-g-PNIPAAm水凝胶具有良好的温度响应性和生物相容性,与PNIPAAm水凝胶相比,Gal-CS-g-PNIPAAm凝胶表面更有利于HL-7702细胞增殖.将温度降低至临界温度(LCST,32.5℃)以下,细胞可以从凝胶表面自发脱附,与酶消化脱附相比,细胞损伤更少.Gal-CS-g-PNIPAAm凝胶表面脱附的细胞比PNIPAAm凝胶表面脱附的细胞活性更高,表明PNIPAAm水凝胶引入GC单体后,凝胶的生物相容性得到改善,且脱附后细胞的增殖活力明显增加.     

聚L-谷氨酸可注射水凝胶的制备及性能

通过活化改性聚L-谷氨酸（PLGA）制备酰肼化PLGA（PLGA-ADH）和3-氨基-1,2-丙二醇改性的PLGA（PLGA-OH）,PLGA-OH经高碘酸钠氧化制得醛基化PLGA（PLGA-CHO）,以PLGA-ADH和PLGA-CHO为前驱体,通过席夫碱交联反应构建了PLGA可注射水凝胶.研究了酰肼化和醛基化改性前后PLGA的结构变化,考察了固含量对水凝胶成胶时间、溶胀行为、机械性能、体外降解性能、药物释放行为及微观形貌等的影响,并进行了初步的细胞培养实验及裸鼠皮下注射成胶实验.结果表明,该PLGA可注射水凝胶在组织工程领域具有良好的应用前景.     

Preparation and characterization of PVA/SA/HA composite hydrogels for wound dressing

A composite hydrogel based on, by introducing, polyvinyl alcohol, sodium alginate, and hyaluronic acid was fabricated using CaCl₂ as a cross-linker. The physical properties including morphology, water vapor transmission rate, and hydrophilicity were investigated. All PVA/SA/HA composite hydrogels with different compositions had highly homogeneous and interconnected pores, and the morphologies of the PVA/SA/HA hydrogels ranged from fibrous structure to irregular structure with increasing content of SA. The introduction of sodium alginate enhanced the hydrophilicity and water vapor transmission capacity of the hydrogel; however, the hydrophilicity of the composite hydrogels decreased with the increasing cross-linker content.