Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 6: 3D hydrogels with positive and negative surface charges and polyelectrolyte complexes in spinal cord injury repair

Macroporous hydrogels are artificial biomaterials commonly used in tissue engineering, including central nervous system (CNS) repair. Their physical properties may be modified to improve their adhesion properties and promote tissue regeneration. We implanted four types of hydrogels based on 2-hydroxyethyl methacrylate (HEMA) with different surface charges inside a spinal cord hemisection cavity at the Th8 level in rats. The spinal cords were processed 1 and 6 months after implantation and histologically evaluated. Connective tissue deposition was most abundant in the hydrogels with positively-charged functional groups. Axonal regeneration was promoted in hydrogels carrying charged functional groups; hydrogels with positively charged functional groups showed increased axonal ingrowth into the central parts of the implant. Few astrocytes grew into the hydrogels. Our study shows that HEMA-based hydrogels carrying charged functional groups improve axonal ingrowth inside the implants compared to implants without any charge. Further, positively charged functional groups promote connective tissue infiltration and extended axonal regeneration inside a hydrogel bridge.     

Scaffolds for central nervous system tissue engineering

Traumatic injuries to the brain and spinal cord of the central nervous system (CNS) lead to severe and permanent neurological deficits and to date there is no universally accepted treatment. Owing to the profound impact, extensive studies have been carried out aiming at reducing inflammatory responses and overcoming the inhibitory environment in the CNS after injury so as to enhance regeneration. Artificial scaffolds may provide a suitable environment for axonal regeneration and functional recovery, and are of particular importance in cases in which the injury has resulted in a cavitary defect. In this review we discuss development of scaffolds for CNS tissue engineering, focusing on mechanism of CNS injuries, various biomaterials that have been used in studies, and current strategies for designing and fabricating scaffolds.     

中枢神经系统损伤修复生物材料研究进展

中枢神经系统损伤导致神经细胞死亡、组织破坏,造成神经功能永久性缺失,是长期困扰生物医学界的一大难题,目前尚无有效疗法。组织工程技术不仅能通过纳米生物材料为神经细胞和神经纤维生长提供结构支持,还能同时递送各种有利于神经再生修复的活性信号分子,有望在促进中枢神经损伤组织修复的同时,实现神经功能的重建,为中枢神经损伤再生修复带来希望。结合国内外有关中枢神经系统组织工程研究的最新进展,对中枢神经修复生物材料设计的主要策略、以及包括天然生物大分子和合成高分子在内的多种中枢神经修复生物材料的应用进行了详细综述。     

Mechanical properties and in vitro behavior of nanofiber-hydrogel composites for tissue engineering applications

Hydrogel-based biomaterial systems have great potential for tissue reconstruction by serving as temporary scaffolds and cell delivery vehicles for tissue engineering (TE). Hydrogels have poor mechanical properties and their rapid degradation limits the development and application of hydrogels in TE. In this study, nanofiber reinforced composite hydrogels were fabricated by incorporating electrospun poly(ε-caprolactone) (PCL)/gelatin 'blend' or 'coaxial' nanofibers into gelatin hydrogels. The morphological, mechanical, swelling and biodegradation properties of the nanocomposite hydrogels were evaluated and the results indicated that the moduli and compressive strengths of the nanofiber reinforced hydrogels were remarkably higher than those of pure gelatin hydrogels. By increasing the amount of incorporated nanofibers into the hydrogel, the Young's modulus of the composite hydrogels increased from 3.29 ± 1.02 kPa to 20.30 ± 1.79 kPa, while the strain at break decreased from 66.0 ± 1.1% to 52.0 ± 3.0%. Compared to composite hydrogels with coaxial nanofibers, those with blend nanofibers showed higher compressive strength and strain at break, but with lower modulus and energy dissipation properties. Biocompatibility evaluations of the nanofiber reinforced hydrogels were carried out using bone marrow mesenchymal stem cells (BM-MSCs) by cell proliferation assay and immunostaining analysis. The nanocomposite hydrogel with 25 mg ml(-1) PCL/gelatin 'blend' nanofibers (PGB25) was found to enhance cell proliferation, indicating that the 'nanocomposite hydrogels' might provide the necessary mechanical support and could be promising cell delivery systems for tissue regeneration.     

A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations. In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide (SAP) RADA (AcN-RADARADARADARADA-CONH₂) nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.     

Effect of a synthetic link N peptide nanofiber scaffold on the matrix deposition of aggrecan and type II collagen in rabbit notochordal cells

Self-assembling peptide nanofiber scaffolds have been studied extensively as biological materials for 3-dimensional cell culture and repairing tissue defects in animals. However, few studies have applied peptide nanofiber scaffolds in the tissue engineering of intervertebral discs (IVDs). In this study, a novel functionalized peptide scaffold was specifically designed for IVD tissue engineering, and notochordal cells (NCs) as an alternative cell source for IVD degeneration were selected to investigate the bioactive scaffold material. The novel RADA16-Link N self-assembling peptide scaffold material was designed by direct coupling to a bioactive motif link N. The link N nanofiber scaffold (LN-NS) material was obtained by mixing pure RADA16-I and RADA16-Link N (1:1) designer peptide solutions. Although live/dead cell assays showed that LN-NS and RADA16-I scaffold materials were both biocompatible with NCs, the LN-NS material significantly promoted NC adhesion compared with that of the pure RADA16-I SAP scaffold material. The depositions of aggrecan and type II collagen, which are significant markers for IVD cells, were remarkably increased. Furthermore, the results indicated that the link N motif, the matrix analog of the nucleus pulposus, significantly promoted the accumulation of other extracellular matrices in vitro. We conclude that the novel LN-NS material is a promising biological scaffold material, and may have a broad range of applications in IVD tissue engineering.     

Systematic studies of a self-assembling peptide nanofiber scaffold with other scaffolds

A designer self-assembling peptide nanofiber scaffold has been systematically studied with 10 commonly used scaffolds in a several week study using neural stem cells (NSC), a potential therapeutic source for cellular transplantations in nervous system injuries. These cells not only provide a good in vitro model for the development and regeneration of the nervous system, but may also be helpful in testing for cytotoxicity, cellular adhesion, and differentiation properties of biological and synthetic scaffolds used in medical practices. We tested the self-assembling peptide nanofiber scaffold with the most commonly used scaffolds for tissue engineering and regenerative medicine including PLLA, PLGA, PCLA, collagen I, collagen IV, and Matrigel. Additionally, each scaffold was coated with laminin in order to evaluate the utility of this surface treatment. Each scaffold was evaluated by measuring cell viability, differentiation and maturation of the differentiated stem cell progeny (i.e. progenitor cells, astrocytes, oligodendrocytes, and neurons) over 4 weeks. The optimal scaffold should show high numbers of living and differentiated cells. In addition, it was demonstrated that the laminin surface treatment is capable of improving the overall scaffold performance. The designer self-assembling peptide RADA16 nanofiber scaffold represents a new class of biologically inspired material. The well-defined molecular structure with considerable potential for further functionalization and slow drug delivery makes the designer peptide scaffolds a very attractive class of biological material for a number of applications. The peptide nanofiber scaffold is comparable with the clinically approved synthetic scaffolds. The peptide scaffolds are not only pure, but also have the potential to be further designed at the molecular level, thus they promise to be useful for cell adhesion and differentiation studies as well as for future biomedical and clinical studies.     

Efficient Delivery of Nerve Growth Factors to the Central Nervous System for Neural Regeneration

The central nervous system (CNS) plays a central role in the control of sensory and motor functions, and the disruption of its barriers can result in severe and debilitating neurological disorders. Neurotrophins are promising therapeutic agents for neural regeneration in the damaged CNS. However, their penetration across the blood–brain barrier remains a formidable challenge, representing a bottleneck for brain and spinal cord therapy. Herein, a nanocapsule‐based delivery system is reported that enables intravenously injected nerve growth factor (NGF) to enter the CNS in healthy mice and nonhuman primates. Under pathological conditions, the delivery of NGF enables neural regeneration, tissue remodeling, and functional recovery in mice with spinal cord injury. This technology can be utilized to deliver other neurotrophins and growth factors to the CNS, opening a new avenue for tissue engineering and the treatment of CNS disorders and neurodegenerative diseases.     

Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering

In this paper, hyaluronic acid hydrogels with open porous structure have been developed for scaffold of brain tissue engineering. A short peptide sequence of arginine–glycine–aspartic acid (RGD) was immobilized on the backbone of the hydrogels. Both unmodified hydrogels and those modified with RGD were implanted into the defects of cortex in rats and evaluated for their ability to improve tissue reconstruction. After 6 and 12 weeks, sections of brains were processed for DAB and Glees staining. They were also labeled with GFAP and ED1 antibodies, and observed under the SEM for ultrastructral examination. After implanting into the lesion of cortex, the porous hydrogels functioned as a scaffold to support cells infiltration and angiogenesis, simultaneously inhibitting the formation of glial scar. In addition, HA hydrogels modified with RGD were able to promote neurites extension. Our experiments showed that the hyaluronic acid-RGD hydrogel provided a structural, three-dimensional continuity across the defect and favoured reorganization of local wound-repair cells, angiogenesis and axonal growth into the hydrogel scaffold, while there was little evidence of axons regeneration in unmodified hydrogel.     

可降解水凝胶作为关节软骨修复材料的研究进展

可降解水凝胶因其良好的生物相容性和生物降解性被广泛用于关节软骨的修复和再生。本文以可降解水凝胶在软骨组织工程中的三类应用策略为主线,概述了用于原位成型可注射水凝胶的蛋白多糖类材料及纳米复合类材料;系统总结了传统工艺制造组织工程支架的优缺点及多种工艺结合的制备方法;重点归纳了近年来3D打印组织工程支架从纯软骨到骨/软骨一体化、从单层到多层的研究进展;最后分析了可降解水凝胶作为关节软骨支架材料在微观定向结构和生物活性功能化方面的局限性,并作出展望：未来开展多材料、多尺度、多诱导的高仿生梯度支架是关节软骨组织工程的一个重要研究方向。     

Multifunctional Nanoengineered Hydrogels Consisting of Black Phosphorus Nanosheets Upregulate Bone Formation

Tissue‐engineered hydrogels have received extensive attention as their mechanical properties, chemical compositions, and biological signals can be dynamically modified for mimicking extracellular matrices (ECM). Herein, the synthesis of novel double network (DN) hydrogels with tunable mechanical properties using combinatorial screening methods is reported. Furthermore, nanoengineered (NE) hydrogels are constructed by addition of ultrathin 2D black phosphorus (BP) nanosheets to the DN hydrogels with multiple functions for mimicking the ECM microenvironment to induce tissue regeneration. Notably, it is found that the BP nanosheets exhibit intrinsic properties for induced CaP crystal particle formation and therefore improve the mineralization ability of NE hydrogels. Finally, in vitro and in vivo data demonstrate that the BP nanosheets, mineralized CaP crystal nanoparticles, and excellent mechanical properties provide a favorable ECM microenvironment to mediate greater osteogenic cell differentiation and bone regeneration. Consequently, the combination of bioactive chemical materials and excellent mechanical stimuli of NE hydrogels inspire novel engineering strategies for bone‐tissue regeneration.     

Electrospun PCL/PLA/HA based nanofibers as scaffold for osteoblast-like cells

Polycaprolactone (PCL), poly (lactic acid) (PLA) and hydroxyapatite (HA) are frequently used as materials for tissue engineering. In this study, PCL/PLA/HA nanofiber mats with different weight ratio were prepared using electrospinning. Their structure and morphology were studied by FTIR and FESEM. FTIR results demonstrated that the HA particles were successfully incorporated into the PCL/PLA nanofibers. The FESEM images showed that the surface of fibers became coarser with the introduction of HA nanoparticles into PCL/PLA system. Furthermore, the addition of HA led to the decreasing of fiber diameter. The average diameters of PCL/PLA/HA nanofiber were in the range of 300-600 nm, while that of PCL/PLA was 776 +/- 15.4 nm. The effect of nanofiber composition on the osteoblast-like MC3T3-E1 cell adhesion and proliferation were investigated as the preliminary biological evaluation of the scaffold. The MC3T3-E1 cell could be attached actively on all the scaffolds. The MTT assay revealed that PCL/PLA/HA scaffold shows significantly higher cell proliferation than PCL/PLA scaffolds. After 15 days of culture, mineral particles on the surface of the cells was appeared on PCL/PLA/HA nanofibers while normal cell spreading morphology on PCL/PLA nanofibers. These results manifested that electrospun PCL/PLA/HA scaffolds could enhance bone regeneration, showing their marvelous prospect as scaffolds for bone tissue engineering.     

Neurogenic differentiation of human umbilical cord mesenchymal stem cells on aligned electrospun polypyrrole/polylactide composite nanofibers with electrical stimulation

Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Recent medical cell therapy using polymeric biomaterialloaded stem cells with the capability of differentiation to specific neural population has directed focuses toward the recovery of CNS. Fibers that can provide topographical, biochemical and electrical cues would be attractive for directing the differentiation of stem cells into electro-responsive cells such as neuronal cells. Here we report on the fabrication of an electrospun polypyrrole/polylactide composite nanofiber film that direct or determine the fate of mesenchymal stem cells (MSCs), via combination of aligned surface topography, and electrical stimulation (ES). The surface morphology, mechanical properties and electric properties of the film were characterized. Comparing with that on random surface film, expression of neurofilament-lowest and nestin of human umbilical cord mesenchymal stemcells (huMSCs) cultured on film with aligned surface topography and ES were obviously enhanced. These results suggest that aligned topography combining with ES facilitates the neurogenic differentiation of huMSCs and the aligned conductive film can act as a potential nerve scaffold.     

Controlling Stem Cell Fate with Material Design

Advances in our understanding of stem cell interactions with their environment are leading to the development of new materials‐based approaches to control stem cell behavior toward cellular culture and tissue regeneration applications. Materials can provide cues based on chemistry, mechanics, structure, and molecule delivery that control stem cell fate decisions and matrix formation. These approaches are helping to advance clinical translation of a range of stem cell types through better expansion techniques and scaffolding for use in tissue engineering approaches for the regeneration of many tissues. With this in mind, this progress report covers basic concepts and recent advances in the use of materials for manipulating stem cells.     

The role of the surface on microglia function: implications for central nervous system tissue engineering

In tissue engineering, it is well accepted that a scaffold surface has a decisive impact on cell behaviour. Here we focused on microglia—the resident immune cells of the central nervous system (CNS)—and on their response to poly(trimethylene carbonate-co-ε-caprolactone) (P(TMC-CL)) fibrous and flat surfaces obtained by electrospinning and solvent cast, respectively. This study aims to provide cues for the design of instructive surfaces that can contribute to the challenging process of CNS regeneration. Cell morphology was evidently affected by the substrate, mirroring the surface main features. Cells cultured on flat substrates presented a round shape, while cells with elongated processes were observed on the electrospun fibres. A higher concentration of the pro-inflammatory cytokine tumour necrosis factor-α was detected in culture media from microglia on fibres. Still, astrogliosis is not exacerbated when astrocytes are cultured in the presence of microglia-conditioned media obtained from cultures in contact with either substrate. Furthermore, a significant percentage of microglia was found to participate in the process of myelin phagocytosis, with the formation of multinucleated giant cells being observed only on films. Altogether, the results presented suggest that microglia in contact with the tested substrates may contribute to the regeneration process, putting forward P(TMC-CL) substrates as supporting matrices for nerve regeneration.     

25th Anniversary Article: Engineering Hydrogels for Biofabrication

With advances in tissue engineering, the possibility of regenerating injured tissue or failing organs has become a realistic prospect for the first time in medical history. Tissue engineering – the combination of bioactive materials with cells to generate engineered constructs that functionally replace lost and/or damaged tissue – is a major strategy to achieve this goal. One facet of tissue engineering is biofabrication, where three‐dimensional tissue‐like structures composed of biomaterials and cells in a single manufacturing procedure are generated. Cell‐laden hydrogels are commonly used in biofabrication and are termed “bioinks”. Hydrogels are particularly attractive for biofabrication as they recapitulate several features of the natural extracellular matrix and allow cell encapsulation in a highly hydrated mechanically supportive three‐dimensional environment. Additionally, they allow for efficient and homogeneous cell seeding, can provide biologically‐relevant chemical and physical signals, and can be formed in various shapes and biomechanical characteristics. However, despite the progress made in modifying hydrogels for enhanced bioactivation, cell survival and tissue formation, little attention has so far been paid to optimize hydrogels for the physico‐chemical demands of the biofabrication process. The resulting lack of hydrogel bioinks have been identified as one major hurdle for a more rapid progress of the field. In this review we summarize and focus on the deposition process, the parameters and demands of hydrogels in biofabrication, with special attention to robotic dispensing as an approach that generates constructs of clinically relevant dimensions. We aim to highlight this current lack of effectual hydrogels within biofabrication and initiate new ideas and developments in the design and tailoring of hydrogels. The successful development of a “printable” hydrogel that supports cell adhesion, migration, and differentiation will significantly advance this exciting and promising approach for tissue engineering.     

Drop-on-demand printing of cells and materials for designer tissue constructs

Adapting bottom-up approaches to tissue engineering is a real challenge. Since the first application of fused deposition modeling for tissue engineering scaffolds, considerable effort has been focused on printing synthetic biodegradable scaffolds. Concurrently a variety of rapid prototyping techniques have been developed to define macroscopically the shapes of deposited biomaterials, including photolithography, syringe-based gel deposition, and solid freeform fabrication. These designed scaffolds have shown promise in regenerating tissues at least equivalent to other scaffolding methods.An exciting advance in scaffold aided tissue regeneration is presented here, that of cell and organ printing, which allows direct printing of cells and proteins within 3D hydrogel structures. Cell printing opens the possibility to programmed deposition of scaffold structure and cell type, thus controlling the type of tissue that can be regenerated within the scaffold. Several examples of printed tissues will be presented including contractile cardiac hybrids. The hybrid materials have properties that can be tailored in 3D to achieve desired porosities, mechanical and chemical properties. The materials include alginate hydrogels with controlled microshell structures that can be built by spraying cross-linkers onto ungelled alginic acid.Endothelial cells were seen to attach to the inside of these microshells. The cells remained viable in constructs as thick as 1 cm due to the programmed porosity. Finite element modeling was used to predict the mechanical properties and to generate CAD models with properties matching cardiac tissue. These results suggest that the printing method could be used for hierarchical design of functional cardiac patches, balanced with porosity for mass transport and structural support.     

Microstructured,Functional PVA Hydrogels through Bioconjugation with Oligopeptides under Physiological Conditions

In this work, bioconjugation techniques are developed to achieve peptide functionalization of poly(vinyl alcohol), PVA, as both a polymer in solution and within microstructured physical hydrogels, in both cases under physiological conditions. PVA is unique in that it is one of very few polymers with excellent biocompatibility and safety and has FDA approval for clinical uses in humans. However, decades of development have documented only scant opportunities in bioconjugation with PVA. As such, materials derived thereof fail to answer the call for functional biomaterials for advanced cell culture and tissue engineering applications. To address these limitations, PVA is synthesized with terminal thiol groups and conjugated with thiolated peptides using PVA in solution. Further, microstructured, surface‐adhered PVA physical hydrogels are assembled, the available conjugation sites within the hydrogels are quantified, and quantitative kinetic data are collected on peptide conjugation to the hydrogels. The success of bioconjugation in the gel phase is quantified through the use of a cell‐adhesive peptide and visualization of cell adhesion on PVA hydrogels as cell culture substrates. Taken together, the presented data establish a novel paradigm in bioconjugation and functionalization of PVA physical hydrogels. Coupled with an excellent safety profile of PVA, these results deliver a superior biomaterial for diverse biomedical applications.     

Amino Acids and Peptide‐Based Supramolecular Hydrogels for Three‐Dimensional Cell Culture

Supramolecular hydrogels assembled from amino acids and peptide‐derived hydrogelators have shown great potential as biomimetic three‐dimensional (3D) extracellular matrices because of their merits over conventional polymeric hydrogels, such as non‐covalent or physical interactions, controllable self‐assembly, and biocompatibility. These merits enable hydrogels to be made not only by using external stimuli, but also under physiological conditions by rationally designing gelator structures, as well as in situ encapsulation of cells into hydrogels for 3D culture. This review will assess current progress in the preparation of amino acids and peptide‐based hydrogels under various kinds of external stimuli, and in situ encapsulation of cells into the hydrogels, with a focus on understanding the associations between their structures, properties, and functions during cell culture, and the remaining challenges in this field. The amino acids and peptide‐based hydrogelators with rationally designed structures have promising applications in the fields of regenerative medicine, tissue engineering, and pre‐clinical evaluation.     

Multi-functionalized nanofibers with reactive oxygen species scavenging capability and fibrocartilage inductivity for tendon-bone integration

The presence of excessive reactive oxygen species(ROS)after injuries to the enthesis could lead to cellular oxidative damage,high inflammatory response,chronic inflammation,and limited fibrochondral induc-tivity,making tissue repair and functional recovery difficult.Here,a multifunctional silk fibroin nanofiber modified with polydopamine and kartogenin was designed and fabricated to not only effectively reduce inflammation by scavenging ROS in the early stage of the enthesis healing but also enhance fibrocarti-lage formation with fibrochondrogenic induction in the later stages.The in vitro results confirmed the antioxidant capability and the fibrochondral inductivity of the functionalized nanofibers.In vivo studies showed that the multifunctional nanofiber can significantly improve the integration of tendon-bone and accelerate the regeneration of interface tissue,resulting in an excellent biomechanical property.Thus,the incorporation of antioxidant and bio-active molecules into extracellular matrix-like biomaterials in interface tissue engineering provides an integrative approach that facilitates damaged tissue regeneration and functional recovery,thereby improving the clinical outcome of the engineered tissue.