Biological and MRI characterization of biomimetic ECM scaffolds for cartilage tissue regeneration

Osteoarthritis is the most common joint disorder affecting millions of people. Most scaffolds developed for cartilage regeneration fail due to vascularization and matrix mineralization. In this study we present a chondrogenic extracellular matrix (ECM) incorporated collagen/chitosan scaffold (chondrogenic ECM scaffold) for potential use in cartilage regenerative therapy. Biochemical characterization showed that these scaffolds possess key pro-chondrogenic ECM components and growth factors. MRI characterization showed that the scaffolds possess mechanical properties and diffusion characteristics important for cartilage tissue regeneration. In vivo implantation of the chondrogenic ECM scaffolds with bone marrow derived mesenchymal stem cells (MSCs) triggered chondrogenic differentiation of the MSCs without the need for external stimulus. Finally, results from in vivo MRI experiments indicate that the chondrogenic ECM scaffolds are stable and possess MR properties on par with native cartilage. Based on our results, we envision that such ECM incorporated scaffolds have great potential in cartilage regenerative therapy. Additionally, our validation of MR parameters with histology and biochemical analysis indicates the ability of MRI techniques to track the progress of our ECM scaffolds non-invasively in vivo; highlighting the translatory potential of this technology.     

胶原水凝胶在软骨与骨组织工程中的研究进展

胶原水凝胶因其具有优良的生物相容性、生物力学性能，在软骨与骨组织工程、生物填充材料、创伤修复、药物缓释和细胞培养等医学领域获得广泛的关注和应用。本文重点介绍了胶原水凝胶在软骨与骨组织工程方面的研究进展，详细阐述了胶原水凝胶的性能、交联方法和类型，并对胶原水凝胶在软骨与骨组织工程中的研究现状进行了讨论，对其应用前景进行了展望。     

Self-crosslinked oxidized alginate/gelatin hydrogel as injectable,adhesive biomimetic scaffolds for cartilage regeneration

Biopolymeric hydrogels that mimic the properties of extracellular matrix have great potential in promoting cellular migration and proliferation for tissue regeneration. The authors reported earlier that rapidly gelling, biodegradable, injectable hydrogels can be prepared by self-crosslinking of periodate oxidized alginate and gelatin in the presence of borax, without using any toxic crosslinking agents. The present paper investigates the suitability of this hydrogel as a minimally invasive injectable, cell-attractive and adhesive scaffold for cartilage tissue engineering for the treatment of osteoarthritis. Time and frequency sweep rheology analysis confirmed gel formation within 20 s. The hydrogel integrated well with the cartilage tissue, with a burst pressure of 70 ± 3 mmHg, indicating its adhesive nature. Hydrogel induced negligible inflammatory and oxidative stress responses, a prerequisite for the management and treatment of osteoarthritis. Scanning electron microscopy images of primary murine chondrocytes encapsulated within the matrix revealed attachment of cells onto the hydrogel matrix. Chondrocytes demonstrated viability, proliferation and migration within the matrix, while maintaining their phenotype, as seen by expression of collagen type II and aggrecan, and functionality, as seen by enhanced glycosoaminoglycan (GAG) deposition with time. DNA content and GAG deposition of chondrocytes within the matrix can be tuned by incorporation of bioactive signaling molecules such as dexamethasone, chondroitin sulphate, platelet derived growth factor (PDGF-BB) and combination of these three agents. The results suggest that self-crosslinked oxidized alginate/gelatin hydrogel may be a promising injectable, cell-attracting adhesive matrix for neo-cartilage formation in the management and treatment of osteoarthritis.     

Semi-interpenetrating networks of hyaluronic acid in degradable PEG hydrogels for cartilage tissue engineering

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9 × 10⁴ and 2 × 10⁶ Da) and concentration (0.5 and 5 mg g⁻¹) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4 weeks. A steady release of HA was observed over the course of the study with 90% released by 4 weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8 days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4 weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.     

Injectable alginate hydrogels for cell delivery in tissue engineering

Alginate hydrogels are extremely versatile and adaptable biomaterials, with great potential for use in biomedical applications. Their extracellular matrix-like features have been key factors for their choice as vehicles for cell delivery strategies aimed at tissue regeneration. A variety of strategies to decorate them with biofunctional moieties and to modulate their biophysical properties have been developed recently, which further allow their tailoring to the desired application. Additionally, their potential use as injectable materials offers several advantages over preformed scaffold-based approaches, namely: easy incorporation of therapeutic agents, such as cells, under mild conditions; minimally invasive local delivery; and high contourability, which is essential for filling in irregular defects. Alginate hydrogels have already been explored as cell delivery systems to enhance regeneration in different tissues and organs. Here, the in vitro and in vivo potential of injectable alginate hydrogels to deliver cells in a targeted fashion is reviewed. In each example, the selected crosslinking approach, the cell type, the target tissue and the main findings of the study are highlighted.     

组织工程材料仿生化构建的研究进展

组织工程是通过细胞和生物材料的组合重建组织的,涉及细胞分化和细胞增殖。无论何种材料作为组织工程支架,只有提供仿生的组织环境才能保持细胞正常的生长状态。通过探索细胞与基质之间的相互关系,模拟体内细胞的生存条件,为工程化组织的构建的发展起到关键的作用。就有关进展做一综述。     

关节软骨组织工程的研究进展

介绍了关节软骨组织工程中种子细胞、支架材料的研究现状以及生长因子在关节软骨组织工程中的应用进展，阐明了随着对细胞行为、支架材料特性、细胞与材料的组合构建研究的深入，组织工程在关节软骨修复方面应用前景十分广阔。     

A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin,hyaluronic acid and chondroitin sulfate

The development of hydrogels tailored for cartilage tissue engineering has been a research and clinical goal for over a decade. Directing cells towards a chondrogenic phenotype and promoting new matrix formation are significant challenges that must be overcome for the successful application of hydrogels in cartilage tissue therapies. Gelatin–methacrylamide (Gel-MA) hydrogels have shown promise for the repair of some tissues, but have not been extensively investigated for cartilage tissue engineering. We encapsulated human chondrocytes in Gel-MA-based hydrogels, and show that with the incorporation of small quantities of photocrosslinkable hyaluronic acid methacrylate (HA-MA), and to a lesser extent chondroitin sulfate methacrylate (CS-MA), chondrogenesis and mechanical properties can be enhanced. The addition of HA-MA to Gel-MA constructs resulted in more rounded cell morphologies, enhanced chondrogenesis as assessed by gene expression and immunofluorescence, and increased quantity and distribution of the newly synthesized extracellular matrix (ECM) throughout the construct. Consequently, while the compressive moduli of control Gel-MA constructs increased by 26 kPa after 8 weeks culture, constructs with HA-MA and CS-MA increased by 114 kPa. The enhanced chondrogenic differentiation, distribution of ECM, and improved mechanical properties make these materials potential candidates for cartilage tissue engineering applications.     

细胞外基质来源支架在软骨组织工程中的应用

BACKGROUND: At present, a variety of extracellular matrix-derived scaffolds have been successfully applied for cartilage tissue engineering in experiment and clinical practice. OBJECTIVE: To summarize the application and research status of extracellular matrix-derived scaffolds in cartilage tissue engineering. METHODS: A computer-based online search in PubMed, CNKI, CqVip and WanFang databases was performed using the keywords of “tissue engineering, cartilage, extracellular matrix, scaffolds” in English and Chinese, respectively. A total of 1 140 literatures were retrieved, and finally 65 eligible literatures were included. RESULTS AND CONCLUSION: In terms of the components, extracellular matrix-derived scaffolds are divided into monomeric natural polymers, mixed natural polymers, natural polymers compositing with synthetic polymers as well as acellular extracellular matrix-derived materials. Extracellular matrix-derived scaffolds hold good biocompatibility and degradability, and can promote proliferation and differentiation of choncrodytes; therefore, they as good bionic scaffolds have been applied for cartilage tissue engineering in clinical practice, However, poor mechanical properties and difficulty to molding should never be ignored. Further research should focus on improving the preparation technology by combining synthetic materials with extracellular matrix-derived scaffolds for cartilage tissue engineering.      

计算机辅助组织工程在组织支架仿生建模和设计中的应用

计算机辅助组织工程(CATE)可以帮助进行复杂组织支架的建模,设计和制造,使很多用于改善替代材料力学及生物学性能的新方法得以实施。CATE通过获取组织的生物学、生物力学及生物化学信息,进行界面的设计、模拟和组织的制作。本文将讲述CATE在骨组织工程支架仿生设计中的应用:介绍运用CATE进行仿生建模,解剖结构重建,组织支架设计,定量CT分析,有限元分析和支架的自由挤压沉积制作。     

牙髓牙本质组织工程生物支架材料的研究进展

背景：组织工程方法中选择合适的支架是关键性的步骤。 目的：回顾分析牙髓牙本质组织工程中支架材料的应用研究。 方法：由第一作者检索1993至2012年 PubMed数据及万方数据库有关牙髓牙本质组织工程中支架材料应用研究等方面的文献。 结果与结论：在牙髓牙本质组织工程中有包括天然生物、人工合成材料和复合材料在内的大量生物材料可供选择,每一种材料都有各自的生物学特点。其中胶原、聚酯、羟基磷灰石等是研究较多的支架材料。自组装多肽水凝胶是由氨基酸制成的新型支架材料,满足理想牙髓牙本质组织工程支架材料的大部分要求,是一种前景广阔的牙髓牙本质组织工程支架材料。     

Biomechanical study of the edge outgrowth phenomenon of encapsulated chondrocytic isogenous groups in the surface layer of hydrogel scaffolds for cartilage tissue engineering

In cartilage tissue engineering, hydrogel is widely used as the scaffold for hosting and culturing chondrocyte suspension during neo-tissue formation. In order to develop cultured chondrocytes into a functional cartilage equivalent, the hydrogel must provide an ideal microenvironment for the rapidly proliferating chondrocytes. At the same time, the essential functions of chondrocytes, such as the secretion of type II collagen and glycosaminoglycans, must be maintained. In these studies, we quantitatively characterize the mechanobiology underlying a newly discovered “edge flourish” phenomenon of cultured chondrocytes within a three-dimensional agarose hydrogel, which may ultimately nurture scaffold-free cartilaginous tissue regeneration. First, real-time microscopy was used to track the spatiotemporal distributions of chondrocytes at different focal planes. The chondrocytes were observed to exhibit abundant neo-tissue outgrowth and significant cartilaginous phenotype at the edge of the hydrogel compared to those inside the hydrogel bulk. Secondly, the hydrogel surface stresses induced by the encapsulated chondrocytes were characterized quantitatively in real time using the finite-element method. Finally, the real-time three-dimensional matrix deformations of agarose hydrogel under the influence of chondrocytes were measured using a multiple-particle tracking assay. Our results indicate that the mechanism of the “edge flourish” phenomenon is induced by the oriented outgrowth of chondrocytic isogenous groups located at the edge of hydrogel. These isogenous groups exhibit directed outgrowth towards the surface of the hydrogel and eventually generate substantial surface tension on the interface of hydrogel and medium. Ultimately, the encapsulated chondrocytes closest to the hydrogel/medium interface will spontaneously sprout out of the hydrogel and form a layer of rich proliferative and chondrocytic extracellular matrix secreting chondrocytes at the surface of the hydrogel.     

Dermis isolated adult stem cells for cartilage tissue engineering

Adult stem cells from the dermal layer of skin are an attractive alternative to primary cells for meniscus engineering, as they may be easily obtained and used autologously. Recently, chondroinducible dermis cells from caprine skin have shown promising characteristics for cartilage tissue engineering. In this study, their multilineage differentiation capacity is determined, and methods of expanding and tissue engineering these cells are investigated. It was found that these cells could differentiate along adipogenic, osteogenic, and chondrogenic lineages, allowing them to be termed dermis isolated adult stem cells (DIAS cells). Focusing on cartilage tissue engineering, it was found that passaging these cells in chondrogenic medium and forming them into self-assembled tissue engineered constructs caused upregulation of collagen type II and COMP gene expression. Further investigation showed that applying transforming growth factor β1 (TGF-β1) or bone morphogenetic protein 2 (BMP-2) to DIAS constructs caused increased sulfated glycosaminoglycan content. Additionally, TGF-β1 treatment caused significant increases in compressive properties and construct contraction. In contrast, BMP-2 treatment resulted in the largest constructs, but did not increase compressive properties. These results show that DIAS cells can be easily manipulated for cartilage tissue engineering strategies, and may also be a useful cell source for other mesenchymal tissues.     

Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue engineering

State-of-the-art bioactive hydrogels can easily and efficiently be formed by enzyme-catalyzed mild-crosslinking reactions in situ. Yet this cell-friendly and substrate-specific method remains under explored. Hydrogels prepared by using enzyme systems like tyrosinases, transferases and lysyl oxidases show interesting characteristics as dynamic scaffolds and as systems for controlled release. Increased attention is currently paid to hydrogels obtained via crosslinking of precursors by transferases or peroxidases as catalysts. Enzyme-mediated crosslinking has proven its efficiency and attention has now shifted to the development of enzymatically crosslinked hydrogels with higher degrees of complexity, mimicking extracellular matrices. Moreover, bottom-up approaches combining biocatalysts and self-assembly are being explored for the development of complex nano-scale architectures. In this review, the use of enzymatic crosslinking for the preparation of hydrogels as an innovative alternative to other crosslinking methods, such as the commonly used UV-mediated photo-crosslinking or physical crosslinking, will be discussed. Photo-initiator-based crosslinking may induce cytotoxicity in the formed gels, whereas physical crosslinking may lead to gels which do not have sufficient mechanical strength and stability. These limitations can be overcome using enzymes to form covalently crosslinked hydrogels. Herewith, we report the mechanisms involved and current applications, focusing on emerging strategies for tissue engineering and regenerative medicine.     

Marine macromolecules cross-linked hydrogel scaffolds as physiochemically and biologically favorable entities for tissue engineering applications

Marine biopolymer composite materials provide a technological platform for launching biomedical applications. Biomaterials demand good biocompatibility without the possibility of inflammation or foreign body reactions. In this study, we prepared two biocomposite hydrogels namely; HAC (hydroxyapatite, alginate & chitosan) and HACF (hydroxyapatite, alginate, chitosan & fucoidan) followed by calcium chloride cross linking. The prepared scaffolds were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Porosity measurement, swelling, biodegradation, hemolysis, RBC aggregation, plasma protein adsorption and cytotoxicity studies were also done. The hydrogel scaffold HACF possessed a well-defined porous architecture, sufficient water holding capacity, better hemocompatibility and biodegradability. The biocompatibility was confirmed through in vitro cytotoxicity studies such as MTT assay, Neutral red uptake, DAPI staining, Trypan blue dye exclusion test and direct contact assay in L929 mouse fibroblast cells. In addition, immunomodulatory and anti-inflammatory properties of both of these scaffolds were revealed by the mRNA expressions of major inflammatory marker genes in cytotoxic condition such as TNF-α, IL-6 and NF-κB. The physiochemical characterization and biological responses of HACF hydrogel signifies its suitability for various tissue engineering applications.     

生物陶瓷与骨组织工程

为修复创伤及病理因素导致的骨缺损 ,骨组织工程是一项迅速发展、不断革新的课题。本文重点综述了国外就生物陶瓷的材料学及其作为骨组织工程细胞支架方面的研究。在分子水平对生物陶瓷理化性质、生物功能以及与细胞间相互作用的认识 ,势必加深对生物陶瓷本身及骨组织工程技术的理解。     

Proliferation of chondrocytes on porous poly(DL-lactide)/chitosan scaffolds

Porous poly(DL-lactide)(PDLLA)/chitosan scaffolds with well-controlled pore structures and desirable mechanical characteristics were fabricated via a combination of solvent extraction, phase separation and freeze-drying. These scaffolds were further evaluated for the proliferation of isolated rabbit chondrocytes in vitro for various incubation periods up to 4 weeks in order to finally use them for the cartilage tissue engineering. MTT assay data revealed that the number of cells grown on PDLLA/chitosan scaffolds measurably increased with the weight ratio of the chitosan component and was significantly higher than those collected from pure PDLLA scaffolds for the entire incubation period. Scanning electron microscopy examinations, histological observations and proteoglycan measurements indicated that the resulting PDLLA/chitosan scaffolds exhibited increasing ability to promote the attachment and proliferation of chondrocytes, and also helped seeded chondrocytes spread through the scaffolds and distribute homogeneously inside compared to pure PDLLA scaffolds. Immunohistochemical staining verified that these PDLLA/chitosan scaffolds could preserve the phenotype of chondrocyte and effectively support the production of type II collagen.     

骨髓间充质干细胞用于软骨组织工程的研究进展

细胞移植技术治疗软骨损伤已成为一项新兴的组织工程学研究热点.骨髓间充质干细胞由于其具有扩增快、便于分离提纯、可以体外诱导分化成为软骨细胞的特性,有可能成为组织工程化软骨的新型种子细胞.随着骨髓间充质干细胞应用于软骨组织工程研究的深入,结合近年的研究文献和成果,就骨髓间充质干细胞的诱导微环境和诱导方式的研究进展进行综述,探讨骨髓间充质干细胞作为种子细胞在构建组织工程软骨中的优越性.     

Development of arginine-glycine-aspartate-immobilized 3D printed poly(propylene fumarate) scaffolds for cartilage tissue engineering

Abstract

Poly(propylene fumarate) (PPF) has known to be a good candidate material for cartilage tissue regeneration because of its excellent mechanical properties during its degradation processes. Here, we describe the potential application of PPF-based materials as 3D printing bioinks to create macroporous cell scaffolds using micro-stereolithography. To improve cell-matrix interaction of seeded human chondrocytes within the PPF-based 3D scaffolds, we immobilized arginine-glycine-aspartate (RGD) peptide onto the PPF scaffolds. We also evaluated various cellular behaviors of the seeded chondrocytes using MTS assay, microscopic and histological analyses. The results indicated that PPF-based biocompatible scaffolds with immobilized RGD peptide could effectively support initial adhesion and proliferation of human chondrocytes. Such a 3D bio-printable scaffold can offer an opportunity to promote cartilage tissue regeneration.     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.