Short-peptide-based molecular hydrogels: novel gelation strategies and applications for tissue engineering and drug delivery

Molecular hydrogels hold big potential for tissue engineering and controlled drug delivery. Our lab focuses on short-peptide-based molecular hydrogels formed by biocompatible methods and their applications in tissue engineering (especially, 3D cell culture) and controlled drug delivery. This feature article firstly describes our recent progresses of the development of novel methods to form hydrogels, including the strategy of disulfide bond reduction and assistance with specific protein-peptide interactions. We then introduce the applications of our hydrogels in fields of controlled stem cell differentiation, cell culture, surface modifications of polyester materials by molecular self-assembly, and anti-degradation of recombinant complex proteins. A novel molecular hydrogel system of hydrophobic compounds that are only formed by hydrolysis processes was also included in this article. The hydrogels of hydrophobic compounds, especially those of hydrophobic therapeutic agents, may be developed into a carrier-free delivery system for long term delivery of therapeutic agents. With the efforts in this field, we believe that molecular hydrogels formed by short peptides and hydrophobic therapeutic agents can be practically applied for 3D cell culture and long term drug delivery in near future, respectively.     

Thermoresponsive thin hydrogel-grafted surfaces for biomedical applications

Thin poly(N-isopropylacrylamide) (PIPAAm) hydrogels were introduced onto biomaterials surfaces for accelerating the kinetics of the swelling and shrinking for PIPAAm hydrogels in response to temperature changes. Thin PIPAAm hydrogels on the biomaterials surfaces exhibit rapid and reversible phase transitions and act as switching sequences to regulate the interaction between the surfaces and biological materials by external temperature-induced changes. By utilizing the temperature-dependent changes of thin PIPAAm hydrogels grafted onto surfaces, our laboratory has pursued unique approaches for developing useful biomedical materials as new types of chromatographic matrices and cell culture surfaces.Aqueous thermoresponsive chromatography systems using PIPAAm-grafted stationary phases enable us to separate biomolecules with high biological activity. Additionally, thermoresponsive cell culture surfaces allow for the recovery of confluent cell monolayers, which have been clinically applied to ophthalmological treatments, dilated cardiomyopathy, esophageal ulcerations, periodontal disease, and cartilage injury. Furthermore, next-generation thermoresponsive cell culture surfaces for large-scale cell cultivation and the capture of specific cells have been considered a key technology for expanding small quantities of stem cells and isolating the resulting differentiated cells for therapeutic use.     

Design of polymeric materials for culturing human pluripotent stem cells: Progress toward feeder-free and xeno-free culturing

This review describes recent developments regarding the use of natural and synthetic polymers to support the propagation of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) while maintaining pluripotency in feeder-free and xeno-free cultures. The development of methods for culturing these cells without using mouse embryonic fibroblasts (MEFs) as a feeder layer will enable more reproducible culture conditions and reduce the risk of xenogenic contaminants, thus increasing the potential clinical applications of differentiated hPSCs. Human or recombinant fibronectin, laminin-511, and vitronectin, which are components of the extracellular matrix (ECM), have been used instead of Matrigel for the feeder-free growth of undifferentiated hPSCs. Successful hPSC cultures have been described for the following conditions: on oligopeptide-immobilized surfaces derived from vitronectin, on microcarriers prepared from synthetic polymers, and encapsulated within three-dimensional (3D) hydrogels composed of alginate and other hydrophilic natural polymers. Recently, synthetic biomaterials that allow hPSCs to maintain pluripotency by secreting endogenous ECM components have been designed. The combination of human ECM proteins or cell adhesion molecules (e.g., oligopeptides and poly-d-lysine) and synthetic biomaterials with well-designed surfaces and/or structures (e.g., scaffolds, hydrogels, microcarriers, microcapsules, or microfibers) in the presence of a chemically defined medium containing recombinant growth factors would offer a xeno-free alternative to feeder cells for culturing hPSCs and maintaining their pluripotency.     

Polymeric design of cell culture materials that guide the differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the potential to differentiate into many cell types that originate from the three germ layers, such as dopamine-secreting cells and insulin-secreting cells for the treatment of Alzheimer's disease and diabetes, respectively. However, it is challenging to guide hPSC differentiation into desired cell lineages due to their varying differentiation ability. A reasonable strategy is to mimic the stem cell microenvironment for the differentiation of hPSCs into specific cell lineages using optimal polymeric biomaterials for hPSC culture. This review summarizes various methods for differentiating hPSCs cultured on polymeric biomaterials and discusses the optimal methods and cell culture polymeric biomaterials for hPSC differentiation into specific cell lineages. The recent trend in protocols avoids embryoid body (EB, aggregated cells) formation because EBs contain different types of cells. The combination of appropriate differentiation protocols and cell culture polymeric biomaterials for the differentiation of hPSCs into specific cell lineages will produce a large quantity of highly pure GMP-grade differentiated cells for use in translational medicine.     

Three‐dimensional hydrogel encapsulated embryonic stem and carcinoma cells as culture platforms for cytotoxicity studies

The utility of alginate hydrogels for three‐dimensional (3‐D) culture of mouse embryonic stem cells (mESCs) and future development of 3‐D stem cell culture‐based in vitro screens of toxicity is described. Using alginate hydrogels of various stiffness, we first evaluated the impact of substrate modulus on mESC viability, proliferation, as well as expression of pluripotency and germ‐layer markers and observed that low concentration alginate hydrogels (0.5% and 1% alginate) were most suitable for long‐term culture of mESCs. These results were not unique to mESCs; long‐term viability and proliferation of mouse embryonic carcinoma cells (mECCs) was also best supported by similar conditions. Finally, we determined cytotoxic responses of alginate encapsulated cells to commercially available chemicals and interestingly observed similar responses for mESCs and mECCs, thereby suggesting that mECCs can predict stem cell responses to chemicals. These studies will facilitate future design of optimal stem cell‐based platforms of organ‐specific and developmental toxicity. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3180–3184, 2015     

Synthesis of polymer materials for use as cell culture substrates

Up to today, several techniques have been used to maintain cells in culture for studying many aspects of cell biology and physiology. More often, cell culture is dependent on proper anchorage of cells to the growth surface. Thus, poly-l-lysine, fibronectin or laminin are the most commonly used substrates. In this study, electrosynthesized biocompatible polymer films are proposed as an alternative to these standard substrates. The electrosynthesized polymers tested were polyethylenimine, polypropylenimine and polypyrrole. Then, the adhesion, proliferation and morphology of rat neuronal cell lines were investigated on these polymer substrates in an attempt to develop new and efficient polymer materials for cell culture. During their growth on the polymers, the evolution of the cell morphology was monitored using both confocal microscopy and immunohistochemistry, leading to the conclusion of a normal development. An estimation of the adhesion and proliferation rates of rat neuronal cell cultures indicated that polyethylenimine and polypropylenimine were the best substrates for culturing olfactory neuronal cells. A method to favour the differentiation of the neuronal cells was also developed since the final aim of this work is to develop a biosensor for odour detection using differentiated neuronal cells as transducers. Consequently, a biosensor was microfabricated using silicon technology. This microsystem allowed us to culture the cells on a silicon wafer and to position the cells on certain parts of the silicon wafer.     

In vitro nanoparticle dosimetry for adherent growing cell monolayers covering bottom and lateral walls

Background

Even though a continuously high number of in vitro studies on nanoparticles are being published, the issue of correct dose matter is often not sufficiently taken into account. Due to their size, the diffusion of nanoparticles is slower, as compared to soluble chemicals, and they sediment slowly. Therefore, the administered dose of particles in in vitro experiments is not necessarily the same (effective) dose that comes into contact with the cellular system. This can lead to misinterpretations of experimental toxic effects and disturbs the meaningfulness of in vitro studies. In silico calculations of the effective nanoparticle dose can help circumventing this problem.

Results

This study addresses more complex in vitro models like the human intestinal cell line Caco-2 or the human liver cell line HepaRG, which need to be differentiated over a few weeks to reach their full complexity. During the differentiation time the cells grow up the wall of the cell culture dishes and therefore a three-dimensional-based in silico model of the nanoparticle dose was developed to calculate the administered dose received by different cell populations at the bottom and the walls of the culture dish. Moreover, the model can perform calculations based on the hydrodynamic diameter which is measured by light scattering methods, or based on the diffusion coefficient measured by nanoparticle tracking analysis (NTA). This 3DSDD (3D-sedimentation-diffusion-dosimetry) model was experimentally verified against existing dosimetry models and was applied to differentiated Caco-2 cells incubated with silver nanoparticles.

Conclusions

The 3DSDD accounts for the 3D distribution of cells in in vitro cell culture dishes and is therefore suitable for differentiated cells. To encourage the use of dosimetry calculating software, our model can be downloaded from the supporting information.

     

基于互穿网络技术构筑的氧化海藻酸钠/纤维素纳米晶/聚丙烯酰胺-明胶复合水凝胶与性能

为了提高海藻酸盐水凝胶的生物应用性,采用互穿网络技术、纤维素纳米晶(CNCs)补强和明胶表面覆积相结合的方法构建了氧化海藻酸盐/纤维素纳米晶/聚丙烯酰胺-明胶 (OSA/CNCs/PAM-GT) 复合水凝胶。通过FT-IR、TGA、 XRD、溶胀性和降解性实验以及细胞相容性测试考察了CNCs含量对OSA/CNCs/PAM-GT复合水凝胶结构和性能的影响。实验结果表明,CNCs能够与基体中的聚合物产生相互作用力。并且随着CNCs含量的增加,OSA/CNCs/PAM-GT 复合水凝胶的孔隙率下降,力学性能提高。而且它们的溶胀性和生物降解性虽然受CNCs含量增加而呈现下降的趋势,但是幅度较小,说明CNCs能够在一定程度上调控复合水凝胶的物化性能。同时,OSA/CNCs/PAM-GT 复合水凝胶展现出较好的细胞粘附、增殖和分化性能。当CNCs的含量在0.5%时,细胞增殖的效果最佳,而CNCs的含量为1.5%时,细胞分化效果最显著。因此,将CNCs掺杂到OSA/PAM互穿网络基体中能够有效地调控其生物性能,使其适用于生物医学领域。     

Maintenance of hemiround colonies and undifferentiated state of mouse induced pluripotent stem cells on carbon nanotube-coated dishes

Induced pluripotent stem (iPS) cells have attracted worldwide interest. However, there have been only a few studies investigating effective culture substrates for feeder-free culturing for the maintenance of iPS cells. In this study, we cultured mouse iPS cells under feeder-free conditions on carbon nanotube (CNT)-coated dishes and then evaluated the colony morphology and differentiation state of the cells on the dishes. After 5 d of cultivation in a medium containing 15% fetal bovine serum (FBS) and leukemia inhibitory factor (LIF), the colonies on thick films of multi-walled CNTs (MWCNTs) were observed to be hemiround; further, the cells expressed early undifferentiation markers. On the other hand, the colonies on a cell culture polystyrene dish and a collagen-coated polystyrene dish showed indistinct outline and spread well, and most spreading cells only weakly expressed early undifferentiation markers. These results indicate that the thick films of MWCNTs could maintain hemiround colonies and undifferentiated state of mouse iPS cells cultured under feeder-free conditions.     

大鼠骨髓间充质干细胞的体外分离培养与鉴定

目的建立大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外分离培养及鉴定的方法 ,为MSCs的系列研究奠定基础。方法采用全骨髓直接贴壁筛选法分离培养MSCs并传代,倒置相差显微镜下观察细胞形态,以MTT法检测细胞增殖水平并绘制生长曲线。取第3代MSCs,流式细胞术检测细胞周期和细胞表型,应用成骨细胞诱导液和脂肪样细胞诱导液诱导MSCs定向分化,鉴定其分化能力。结果全骨髓细胞培养5d,镜下可见贴壁细胞增殖明显,细胞形态较均一,大部分呈梭形,7d左右可传代,经2～3次传代后细胞呈单一梭形的成纤维样细胞,即MSCs;细胞生长曲线呈S形;经流式细胞仪检测,MSCs细胞76.01%处于G0/G1期,7.13%处于G2/M期,16.86%处于S期;MSCs表面不表达CD34;在特定诱导液作用下,MSCs可分别向成骨样细胞及脂肪样细胞分化。结论已成功建立了分离培养及鉴定MSCs的方法 ,可用来评价体外培养的MSCs。     

Design and application of stimulus-responsive peptide systems

The ability of peptides and proteins to change conformations in response to external stimuli such as temperature, pH and the presence of specific small molecules is ubiquitous in nature. Exploiting this phenomenon, numerous natural and designed peptides have been used to engineer stimulus-responsive systems with potential applications in important research areas such as biomaterials, nanodevices, biosensors, bioseparations, tissue engineering and drug delivery. This review describes prominent examples of both natural and designed synthetic stimulus-responsive peptide systems. While the future looks bright for stimulus-responsive systems based on natural and rationally engineered peptides, it is expected that the range of stimulants used to manipulate such systems will be significantly broadened through the use of combinatorial protein engineering approaches such as directed evolution. These new proteins and peptides will continue to be employed in exciting and high-impact research areas including bionanotechnology and synthetic biology.     

New bioactive motifs and their use in functionalized self-assembling peptides for NSC differentiation and neural tissue engineering

Developing functionalized biomaterials for enhancing transplanted cell engraftment in vivo and stimulating the regeneration of injured tissues requires a multi-disciplinary approach customized for the tissue to be regenerated. In particular, nervous tissue engineering may take a great advantage from the discovery of novel functional motifs fostering transplanted stem cell engraftment and nervous fiber regeneration. Using phage display technology we have discovered new peptide sequences that bind to murine neural stem cell (NSC)-derived neural precursor cells (NPCs), and promote their viability and differentiation in vitro when linked to LDLK12 self-assembling peptide (SAPeptide). We characterized the newly functionalized LDLK12 SAPeptides via atomic force microscopy, circular dichroism and rheology, obtaining nanostructured hydrogels that support human and murine NSC proliferation and differentiation in vitro. One functionalized SAPeptide (Ac-FAQ), showing the highest stem cell viability and neural differentiation in vitro, was finally tested in acute contusive spinal cord injury in rats, where it fostered nervous tissue regrowth and improved locomotor recovery. Interestingly, animals treated with the non-functionalized LDLK12 had an axon sprouting/regeneration intermediate between Ac-FAQ-treated animals and controls. These results suggest that hydrogels functionalized with phage-derived peptides may constitute promising biomimetic scaffolds for in vitro NSC differentiation, as well as regenerative therapy of the injured nervous system. Moreover, this multi-disciplinary approach can be used to customize SAPeptides for other specific tissue engineering applications.     

A review on thermoresponsive cell culture systems based on poly(N-isopropylacrylamide) and derivatives

The thermoresponsive poly(N-isopropylacrylamide) and matching thermal-liftoff method are widely employed for cell culture. There is already growing evidence that this special cell culture system not only provides an appropriate growth environment for anchorage-dependent cells at physiologic conditions, but also supports mild and efficient cell harvesting by cooling treatment. This review elaborates the internal and external factors affecting two-dimensional surface thermosensitivity and cell behavior, and then briefly discusses three-dimensional cell culture systems involved in thermosensitive microcarriers, scaffolds, hydrogels, and hollow fiber membranes, and finally presents their potential applications including large-scale cell expansion as well as tissue repair and reconstruction.     

人脂肪干细胞复合胶原/壳聚糖支架在生物反应器中扩增的研究

研究表明脂肪组织是多潜能干细胞的又一新来源,脂肪组织来源的干细胞可被用于细胞治疗及组织工程。然而,传统的培养方法很难满足临床需求。为获得大量的脂肪干细胞以满足临床需求,现将脂肪干细胞接种到胶原/壳聚糖支架上,比较细胞在静态环境和在转瓶中动态扩增的情况。通过CCK-8检测细胞增殖情况;并分析葡萄糖和乳酸的代谢情况;14d后扫描电镜观察细胞在支架内的生长情况;流式细胞仪检测干细胞相关表面标记表达;RT-PCR检测干细胞相关转录因子的表达;并检测扩增后的干细胞的多向分化潜能。结果表明:在动态微环境中扩增14d后,与静态条件下相比,支架内的细胞具有更强的增殖活性和更好的多向分化潜能。所扩增的细胞能够保持原有干细胞的特性。结论:所设计的支架-转瓶培养系统是一个简便有效的扩增脂肪干细胞的方法。     

体外诱导微环境对骨髓间充质干细胞成神经分化的影响及分化后的自发逆转现象

目的体外定向诱导大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)成神经分化,并探讨诱导微环境对其分化的影响及分化后的自发逆转现象。方法体外分离培养大鼠MSCs,流式细胞仪检测细胞表面标志。采用改良神经元诱导液[Modified neuronal induction media(MNM)]定向诱导MSCs,免疫荧光检测神经细胞表面标志。观察胎牛血清(FBS)浓度、细胞密度、MNM剂量、新鲜与使用过的MNM等不同诱导微环境对MSCs成神经分化的影响。结果 MSCs经MNM诱导后,6h即可见尼氏体,表达神经元特异性表面标志神经元特异性烯醇化酶(NSE)、巢蛋白(Nestin)和微管相关蛋白-2(MAP-2)。随着诱导微环境的改变,MSCs成神经分化率及神经元表面标志表达亦发生改变,且分化后的神经元样细胞可自发逆转。结论 MSCs能够在MNM微环境中定向成神经分化,但诱导微环境的改变可以从量和质两个层面影响MSCs定向分化。     

Hydrogels with Reversible Crosslinks for Improved Localised Stem Cell Retention: A Review

Successful stem cell applications could have a significant impact on the medical field, where many lives are at stake. However, the translation of stem cells to the clinic could be improved by overcoming challenges in stem cell transplantation and in vivo retention at the site of tissue damage. This review aims to showcase the most recent insights into developing hydrogels that can deliver, retain, and accommodate stem cells for tissue repair. Hydrogels can be used for tissue engineering, as their flexibility and water content makes them excellent substitutes for the native extracellular matrix. Moreover, the mechanical properties of hydrogels are highly tuneable, and recognition moieties to control cell behaviour and fate can quickly be introduced. This review covers the parameters necessary for the physicochemical design of adaptable hydrogels, the variety of (bio)materials that can be used in such hydrogels, their application in stem cell delivery and some recently developed chemistries for reversible crosslinking. Implementing physical and dynamic covalent chemistry has resulted in adaptable hydrogels that can mimic the dynamic nature of the extracellular matrix.     

Receptor-Targeted,Magneto-Mechanical Stimulation of Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Mechanical cues are employed to promote stem cell differentiation and functional tissue formation in tissue engineering and regenerative medicine. We have developed a Magnetic Force Bioreactor (MFB) that delivers highly targeted local forces to cells at a pico-newton level, utilizing magnetic micro- and nano-particles to target cell surface receptors. In this study, we investigated the effects of magnetically targeting and actuating specific two mechanical-sensitive cell membrane receptors—platelet-derived growth factor receptor α (PDGFRα) and integrin α_νβ₃. It was found that a higher mineral-to-matrix ratio was obtained after three weeks of magneto-mechanical stimulation coupled with osteogenic medium culture by initially targeting PDGFRα compared with targeting integrin α_νβ₃ and non-treated controls. Moreover, different initiation sites caused a differentiated response profile when using a 2-day-lagged magneto-mechanical stimulation over culture periods of 7 and 12 days). However, both resulted in statistically higher osteogenic marker genes expression compared with immediate magneto-mechanical stimulation. These results provide insights into important parameters for designing appropriate protocols for ex vivo induced bone formation via magneto-mechanical actuation.     

Application of minimally invasive injectable conductive hydrogels as stimulating scaffolds for myocardial tissue engineering

So far, several methods for myocardial tissue engineering have been developed to regenerate myocardium and even create contractile heart muscles. Among these approaches, hydrogel based methods have attracted much attention due to their ability to mimic the architecture of native extracellular matrix. Injectable hydrogels are a specific class of hydrogels which can be formed in situ by physical and/or chemical crosslinking. Generally, using these hydrogels is more advantageous because they are minimally (less) invasive in comparison with open surgery. Moreover, with respect to the fact that ‘myocardium is a conductive tissue’, utilization of conductive polymers for myocardial tissue engineering has demonstrated promising results. Both the injectable hydrogels and conductive polymers have some merits and demerits, but studies show that using a combination of them has prominently enhanced regeneration of the myocardium. In this review, the focus is on injectable hydrogels, conductive polymers and injectable conductive hydrogels for myocardial tissue engineering. © 2018 Society of Chemical Industry     

SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.     

Temperature‐sensitive poly(N‐tert‐butylacrylamide‐co‐acrylamide) hydrogels bonded on cotton fabrics by coating technique

Different from the conventional method of developing stimuli‐sensitive textiles by graft copolymerization of environmental responsive polymers onto the fabric, the coating technique was applied to bond temperature‐sensitive hydrogels with cotton fabric through chemical covalent in our work. A temperature‐sensitive linear copolymer of N‐tert‐butylacrylamide (NTBA) and acrylamide (AAm) was prepared in methanol. Then, the cotton fabrics were coated using an aqueous solution of this copolymer containing 1,2,3,4‐butanetertracarboxylic acid as a crosslinker and sodium hypophosphite (SHP) as a catalyst, followed by drying and curing. The surface of the cotton fabrics was bonded on more or less coatings of poly (NTBA‐co‐AAm) hydrogels, as verified by Fourier transform infrared spectroscopy and scanning electron microscopy images. The poly(NTBA‐co‐AAm) hydrogels‐coated fabrics exhibited temperature sensitive, and the temperature interval of the deswelling transition was higher than lower critical solution temperature of linear copolymer solution. The coated fabrics presented good water‐impermeable ability because of the swelling of hydrogels bonded, especially when the add‐on was as high as 14.14%. Environmental scanning electron microscopy images revealed that coating hydrogels swelled and covered on the surface as a barrier to prevent water from penetrating once the coated fabric came into contact with water. The findings demonstrate that the temperature‐sensitive hydrogels can be covalently bonded on the cotton fabrics by coating technique and the coated fabrics have potential on immersion fabrics. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009