MMP-2 sensitive, VEGF-bearing bioactive hydrogels for promotion of vascular healing

We sought to develop bioactive hydrogels to facilitate arterial healing, e.g., after balloon angioplasty. Toward this end, we developed a new class of proteolytically sensitive, biologically active polyethylene glycol (PEG)-peptide hydrogels that can be formed in situ to temporarily protect the arterial injury from blood contact. Furthermore, we incorporated endothelial cell-specific biological signals with the goal of enhancing arterial reendothelialization. Here we demonstrate efficient endothelial cell anchorage and activation on PEG hydrogel matrices modified by conjugation with both the cell adhesive peptide motif RGD and an engineered variant of vascular endothelial growth factor (VEGF). By crosslinking peptide sequences for cleavage by MMP-2 into the polymer backbone, the hydrogels became sensitive to proteolytic degradation by cell-derived matrix metalloproteinases (MMPs). Analysis of molecular hallmarks associated with endothelial cell activation by VEGF-RGD hydrogel matrices revealed a 70% increase in production of the latent MMP-2 zymogen compared with PEG-peptide hydrogels lacking VEGF. By additional provision of transforming growth factor beta1 (TGF-beta1) within the PEG-peptide hydrogel, conversion of the latent MMP zymogen into its active form was demonstrated. As a result of MMP-2 activation, strongly enhanced hydrogel degradation by activated endothelial cells was observed. Our data illustrate the critical importance of growth factor activities for remodeling of synthetic biomaterials into native tissue, as it is desired in many applications of regenerative medicine. Functionalized PEG-peptide hydrogels could help restore the native vessel wall and improve the performance of angioplasty procedures.     

Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing

Organ or tissue printing, a novel approach in tissue engineering, creates layered, cell-laden hydrogel scaffolds with a defined three-dimensional (3D) structure and organized cell placement. In applying the concept of tissue printing for the development of vascularized bone grafts, the primary focus lies on combining endothelial progenitors and bone marrow stromal cells (BMSCs). Here we characterize the applicability of 3D fiber deposition with a plotting device, Bioplotter, for the fabrication of spatially organized, cell-laden hydrogel constructs. The viability of printed BMSCs was studied in time, in several hydrogels, and extruded from different needle diameters. Our findings indicate that cells survive the extrusion and that their subsequent viability was not different from that of unprinted cells. The applied extrusion conditions did not affect cell survival, and BMSCs could subsequently differentiate along the osteoblast lineage. Furthermore, we were able to combine two distinct cell populations within a single scaffold by exchanging the printing syringe during deposition, indicating that this 3D fiber deposition system is suited for the development of bone grafts containing multiple cell types.     

Hydrogel macroporosity and the prolongation of transgene expression and the enhancement of angiogenesis

The utility of hydrogels for regenerative medicine can be improved through localized gene delivery to enhance their bioactivity. However, current systems typically lead to low-level transgene expression located in host tissue surrounding the implant. Herein, we investigated the inclusion of macropores into hydrogels to facilitate cell ingrowth and enhance gene delivery within the macropores in?vivo. Macropores were created within PEG hydrogels by gelation around gelatin microspheres, with gelatin subsequently dissolved by incubation at 37?°C. The macropores were interconnected, as evidenced by homogeneous cell seeding in?vitro and complete cell infiltration in?vivo. Lentivirus loaded within hydrogels following gelation retained its activity relative to the unencapsulated control virus. In?vivo, macroporous PEG demonstrated sustained, elevated levels of transgene expression for 6 weeks, while hydrogels without macropores had transient expression. Transduced cells were located throughout the macroporous structure, while non-macroporous PEG hydrogels had transduction only in the adjacent host tissue. Delivery of lentivirus encoding for VEGF increased vascularization relative to the control, with vessels throughout the macropores of the hydrogel. The inclusion of macropores within the hydrogel to enhance cell infiltration enhances transduction and influences tissue development, which has implications for multiple regenerative medicine applications.     

利用水凝胶设计组织再生微环境

水凝胶如何为细胞、组织的生长提供三维微环境已成为组织工程和再生医学领域的研究热点,生物活性分子修饰获得的智能水凝胶是能够促进组织再生的重要生物材料。根据水凝胶的来源,可将其分为天然和合成水凝胶两种类型。水凝胶的设计策略主要包括凝胶降解敏感位点的设计、生物黏附性的获得、生长因子和细胞因子对凝胶的修饰以及再生组织的血管重建。此外,本文以智能水凝胶在软骨组织工程、神经组织工程等方面的应用为例,阐述了水凝胶在组织工程和再生医学领域的突出研究进展。     

智能水凝胶在骨类硬组织再生和修复中的应用

BACKGROUND: Intelligent hydrogel as a new material is widely used in biological medicine, tissue engineering, memory element switch, biological enzyme immobilization and other related fields, and exhibits good biological characteristics. Intelligent hydrogels provide a new approach for regeneration and repair of bone and other hard tissues.  OBJECTIVE: To summarize the latest developments of intelligent hydrogel in the biological medicine and tissue engineering in order to find out new methods for regeneration and repair of bone and other hard tissues. METHODS: A computer-based research of CNKI, PubMed and EBSCO-MEDLINE databases was performed to retrieve relevant literatures about the application of intelligent hydrogel in regeneration and repair of bone and other hard tissues published from 2000 to 2015. The keywords were “hydrogel, bone tissue engineering, bone defect, regeneration, repair” in Chinese and English, respectively. RESULTS AND CONCLUSION: Intelligent hydrogels are classified into pH-sensitive, temperature-sensitive, light-sensitive, multiple-sensitive and other sensitive hydrogels. In order to improve the mineralization ability of the hydrogel and construct the three-dimensional polymer scaffold of hydrogel, the main structure of the hydrogel materials can be mixed with various signal factors, thus achieving the multi-utility and multi-function of the material system, which will become the development trend of tissue engineering construction.      

The role of biomaterials in stem cell differentiation: applications in the musculoskeletal system

The capabilities of stem cells continue to be revealed, leading to excitement regarding potential new therapies. Regenerative medicine is an area in which stem cells hold great promise for overcoming the challenge of limited cell sources for tissue repair. Biomaterials play an important role in directing tissue growth and may provide another tool to manipulate and control stem cell behavior. Biomaterials are made from natural or synthetic polymers and can be processed into three-dimensional scaffolds designed to promote cell proliferation and/or differentiation that ultimately produces new tissue. Stem cells will have a significant impact on the fields of regenerative medicine and tissue engineering as a powerful cell source that will work, in conjunction with biomaterials, to treat tissue and organ loss. Herein, we survey our latest research on applying embryonic stem (ES) cells to hydrogel biomaterials for engineering musculoskeletal tissues, emphasizing the unique biomaterial requirements of ES cells for differentiation and tissue development.     

Scaffold porosity and oxygenation of printed hydrogel constructs affect functionality of embedded osteogenic progenitors

Insufficient supply of oxygen and nutrients throughout the graft is considered one of the principal limitations in development of large, tissue-engineered bone grafts. Organ or tissue printing by means of three-dimensional (3D) fiber deposition is a novel modality in regenerative medicine that combines pore formation and defined cell placement, and is used here for development of cell-laden hydrogel structures with reproducible internal architecture to sustain oxygen supply and to support adequate tissue development. In this study we tested the effect of porosity on multipotent stromal cells (MSCs) embedded in hydrogel constructs printed with a 3D fiber deposition (3DF) machine. For this, porous and solid alginate hydrogel scaffolds, with MSCs homogeneously dispersed throughout the construct, were printed and analyzed in vitro for the presence of hypoxia markers, metabolism, survival, and osteogenic differentiation. We demonstrated that porosity promotes oxygenation of MSCs in printed hydrogel scaffolds and supported the viability and osteogenic differentiation of embedded cells. Porous and solid printed constructs were subsequently implanted subcutaneously in immunodeficient mice to analyze tissue formation in relation to hypoxia responses of embedded cells. Implantation of printed grafts resulted in ingrowth of vascularized tissue and significantly enhanced oxygenation of embedded MSCs. In conclusion, the introduction of pores significantly enhances the conductive properties of printed hydrogel constructs and contributes to the functionality of embedded osteogenic progenitors.     

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).     

Light activated cell migration in synthetic extracellular matrices

Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels.     

喷墨打印技术同步打印细胞和生物支架材料及在组织工程中的应用

背景：喷墨打印技术是将墨滴喷射到接受体形成图像或文字的非接触性打印技术。 目的：总结并讨论喷墨打印技术的研究进展及其在组织工程中的运用。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：1995/2010)和PubMed数据库(1995/2010),检索词分别为“喷墨打印技术,细胞打印,生物材料,细胞因子”和“inkjet printing,cell printing,biomaterial,cytokine,organ printing”。共检索到352篇文章,按纳入和排除标准对文献进行筛选,共纳入35篇文章。从喷墨打印技术研究进展及其在组织工程中的运用2个方面进行总结,对喷墨打印技术在细胞打印、生物支架材料打印、细胞活性因子打印及细胞-生物支架材料同步打印的应用等方面进行介绍。 结果和结论：喷墨打印技术作为一种新型组织工程技术,在组织与器官打印研究中有广泛的运用前景。该技术可以打印多种细胞,打印后的细胞能够存活和保持原有生物学活性,而且喷墨打印技术通过打印水凝胶支架材料,构建具有良好的三维结构的3D水凝胶支架。此外,运用喷墨打印技术可以打印细胞活性因子,并保持其生物学活性。通过同步打印细胞和生物支架材料,喷墨打印技术有望构建3D细胞-生物支架材料复合的仿生组织和器官。     

Organ printing: Tissue spheroids as building blocks

Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion — a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with “built-in” perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.     

Synthesis and characterization of tunable poly(ethylene glycol): gelatin methacrylate composite hydrogels

Poly(ethylene glycol) (PEG) hydrogels are popular for cell culture and tissue-engineering applications because they are nontoxic and exhibit favorable hydration and nutrient transport properties. However, cells cannot adhere to, remodel, proliferate within, or degrade PEG hydrogels. Methacrylated gelatin (GelMA), derived from denatured collagen, yields an enzymatically degradable, photocrosslinkable hydrogel that cells can degrade, adhere to and spread within. To combine the desirable features of each of these materials we synthesized PEG-GelMA composite hydrogels, hypothesizing that copolymerization would enable adjustable cell binding, mechanical, and degradation properties. The addition of GelMA to PEG resulted in a composite hydrogel that exhibited tunable mechanical and biological profiles. Adding GelMA (5%-15% w/v) to PEG (5% and 10% w/v) proportionally increased fibroblast surface binding and spreading as compared to PEG hydrogels (p<0.05). Encapsulated fibroblasts were also able to form 3D cellular networks 7 days after photoencapsulation only within composite hydrogels as compared to PEG alone. Additionally, PEG-GelMA hydrogels displayed tunable enzymatic degradation and stiffness profiles. PEG-GelMA composite hydrogels show great promise as tunable, cell-responsive hydrogels for 3D cell culture and regenerative medicine applications.     

Evaluation of skeletal tissue repair,Part 1: Assessment of novel growth-factor-releasing hydrogels in an ex vivo chick femur defect model

Current clinical treatments for skeletal conditions resulting in large-scale bone loss include autograft or allograft, both of which have limited effectiveness. In seeking to address bone regeneration, several tissue engineering strategies have come to the fore, including the development of growth factor releasing technologies and appropriate animal models to evaluate repair. Ex vivo models represent a promising alternative to simple in vitro systems or complex, ethically challenging in vivo models. We have developed an ex vivo culture system of whole embryonic chick femora, adapted in this study as a critical size defect model to investigate the effects of novel bone extracellular matrix (bECM) hydrogel scaffolds containing spatio-temporal growth factor-releasing microparticles and skeletal stem cells on bone regeneration, to develop a viable alternative treatment for skeletal degeneration. Alginate/bECM hydrogels combined with poly (d,l-lactic-co-glycolic acid) (P_DLLGA)/triblock copolymer (10–30% P_DLLGA–PEG–P_DLLGA) microparticles releasing VEGF, TGF-β3 or BMP-2 were placed, with human adult Stro-1+ bone marrow stromal cells, into 2 mm central segmental defects in embryonic chick femurs. Alginate/bECM hydrogels loaded with HSA/VEGF or HSA/TGF-β3 demonstrated a cartilage-like phenotype, with minimal collagen I deposition, comparable to HSA-only control hydrogels. The addition of BMP-2 releasing microparticles resulted in enhanced structured bone matrix formation, evidenced by increased Sirius red-stained matrix and collagen expression within hydrogels. This study demonstrates delivery of bioactive growth factors from a novel alginate/bECM hydrogel to augment skeletal tissue formation and the use of an organotypic chick femur defect culture system as a high-throughput test model for scaffold/cell/growth factor therapies for regenerative medicine.     

Efficient differentiation of stem cells encapsulated in a cytocompatible phospholipid polymer hydrogel with tunable physical properties

A large number of lineage-committed progenitor cells are required for advanced regenerative medicine based on cell engineering. Due to their ability to differentiate into multiple cells lines, multipotent stem cells have emerged as a vital source for generating transplantable cells for use in regenerative medicine. Increment in differentiation efficiency of the mesenchymal stem cell was obtained by using hydrogel to adjust the proliferation cycle of encapsulated cells to signal sensitive phase. Three dimensional (3-D) polymer networks composed of poly(2-methacyloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-vinylphenylboronic acid (VPBA)) (PMBV) and poly(vinyl alcohol) (PVA) were prepared as a hydrogel. The proliferation of cells encapsulated in the PMBV/PVA hydrogel was highly sensitive to the storage modulus (G′) of the hydrogel. That is, when the G′ value of the hydrogel was higher than 1.0 kPa, the cell proliferation was ceased and the proliferation cycle of cells was converged to G1 phase, whereas when the G′ value was below 1.0 kPa, cell proliferation proceeded. By changing the G′ value of hydrogels under encapsulation the cells, proliferation cycle of encapsulated mesenchymal stem cells was regulated to G1 phase and thus signal sensitivity were increased. 3-D polymer networks as hydrogels with tunable physical properties can be effectively used to control proliferation and lineage-restricted differentiation of stem cells.     

Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering

Injectable, biodegradable scaffolds are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural polysaccharides are ideal scaffolds as they resemble the extracellular matrices of tissues comprised of various glycosaminoglycans (GAGs). Here, we report a new class of biocompatible and biodegradable composite hydrogels derived from water-soluble chitosan and oxidized hyaluronic acid upon mixing, without the addition of a chemical crosslinking agent. The gelation is attributed to the Schiff base reaction between amino and aldehyde groups of polysaccharide derivatives. In the current work, N-succinyl-chitosan (S-CS) and aldehyde hyaluronic acid (A-HA) were synthesized for preparation of the composite hydrogels. The polysaccharide derivatives and composite hydrogels were characterized by FTIR spectroscopy. The effect of the ratio of S-CS and A-HA on the gelation time, microstructure, surface morphology, equilibrium swelling, compressive modulus, and in vitro degradation of composite hydrogels was examined. The potential of the composite hydrogel as an injectable scaffold was demonstrated by the encapsulation of bovine articular chondrocytes within the composite hydrogel matrix in vitro. The results demonstrated that the composite hydrogel supported cell survival and the cells retained chondrocytic morphology. These characteristics provide a potential opportunity to use the injectable, composite hydrogels in tissue engineering applications.     

Neural tissue formation within porous hydrogels implanted in brain and spinal cord lesions: ultrastructural, immunohistochemical, and diffusion studies

A biocompatible heterogeneous hydrogel of poly [N-(2-hydroxypropyl) methacrylamide] (PHPMA), was evaluated for its ability to promote tissue repair and enhance axonal regrowth across lesion cavities in the brain and spinal cord in adult and juvenile (P17 P21) rats. Incorporation of PHPMA hydrogels into surrounding host tissue was examined at the ultrastructural level and using immunohistochemical techniques. In addition, and in parallel to these studies, diffusion parameters (volume fraction and tortuosity of the gel network) of the PHPMA hydrogels were evaluated pre- to postimplantation using an in vivo real-time iontophoretic method. The polymer hydrogels were able to bridge tissue defects created in the brain or spinal cord, and supported cellular ingrowth, angiogenesis, and axonogenesis within the structure of the polymer network. As a result, a reparative tissue grew within the porous structure of the gel, composed of glial cells, blood vessels, axons and dendrites, and extracellular biological matrices, such as laminin and/or collagen. Consistent with matrix deposition and tissue formation within the porous structure of the PHPMA hydrogels, there were measurable changes in the diffusion characteristics of the polymers. Extracellular space volume decreased and tortuosity increased within implanted hydrogels, attaining values similar to that seen in developing neural tissue. PHPMA polymer hydrogel matrices thus show neuroinductive and neuroconductive properties. They have the potential to repair tissue defects in the central nervous system by replacing lost tissue and by promoting the formation of a histotypic tissue matrix that facilitates and supports regenerative axonal growth. () ()     

PEG hydrogels formed by thiol-ene photo-click chemistry and their effect on the formation and recovery of insulin-secreting cell spheroids

Hydrogels provide three-dimensional frameworks with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. While recent research efforts have created diverse macromer chemistry to form hydrogels, the mechanisms of hydrogel polymerization for in situ cell encapsulation remain limited. Hydrogels prepared from chain-growth photopolymerization of poly(ethylene glycol) diacrylate (PEGDA) are commonly used to encapsulate cells. However, free radical associated cell damage poses significant limitation for this gel platform. More recently, PEG hydrogels formed by thiol-ene photo-click chemistry have been developed for cell encapsulation. While both chain-growth and step-growth photopolymerizations offer spatial-temporal control over polymerization kinetics, step-growth thiol-ene hydrogels offer more diverse and preferential properties. Here, we report the superior properties of step-growth thiol-ene click hydrogels, including cytocompatibility of the reactions, improved hydrogel physical properties, and the ability for 3D culture of pancreatic β-cells. Cells encapsulated in thiol-ene hydrogels formed spherical clusters naturally and were retrieved via rapid chymotrypsin-mediated gel erosion. The recovered cell spheroids released insulin in response to glucose treatment, demonstrating the cytocompatibility of thiol-ene hydrogels and the enzymatic mechanism of cell spheroids recovery. Thiol-ene click reactions provide an attractive means to fabricate PEG hydrogels with superior gel properties for in situ cell encapsulation, as well as to generate and recover 3D cellular structures for regenerative medicine applications.     

In vivo osteogenic differentiation of human turbinate mesenchymal stem cells in an injectable in situ-forming hydrogel

Human turbinate mesenchymal stromal cells (hTMSCs) are an alternate source of adult stem cells for regenerative medicine. In this work, we demonstrated that hTMSCs are easily harvested from turbinate tissue using a minimal surgical procedure. hTMSCs showed positive expression of mesenchymal stem cell markers and proliferated at a high rate. The specific surface proteins of harvested hTMSCs were relatively tolerant of ex vivo manipulation in culture. hTMSCs exhibited osteogenic differentiation in vitro in the presence of osteogenic factors. To examine osteogenic differentiation of hTMSCs in vivo in an injectable hydrogel, cells were incorporated into a methoxy polyethylene glycol–polycaprolactone block copolymer (MPEG–PCL (MP)) solution simply by mixing. hTMSC-loaded MP solutions exhibited a temperature-dependent solution-to-gel phase transition. The hTMSC attached and grew well on in vitro- and in vivo-formed MP hydrogels. hTMSC-loaded MP solutions formed a hydrogel almost immediately upon injection into animals and the cells remained viable, even after 12 weeks. Injected hTMSCs in in situ-formed MP hydrogels differentiated into osteogenic cells, mainly in the presence of osteogenic factors. Differentiated osteoblasts were identified by Alizarin Red S, von Kossa, and alkaline phosphatase (ALP) staining, and osteonectin, osteopontin, and osteocalcin mRNA expression. To the best of our knowledge, this is the first study to show hTMSCs undergoing osteogenic differentiation in in vivo-formed MP hydrogels. In conclusion, hTMSCs could serve as adult stem cell sources and, when embedded in an in situ-formed hydrogel, may provide numerous benefits as a noninvasive alternative for bone tissue engineering applications.