In Situ Forming Hydrogel Based on Chondroitin Sulfate–Hydroxyapatite for Bone Tissue Engineering

In this research, tissue engineered hydrogels consist of chondroitin sulfate and hydroxyapatite capable of light curing and cell encapsulation were prepared. Chemical and physical characteristics were analyzed by FTIR spectroscopy, compressive strength and water uptake measurements. Observations with fluorescence microscopy as well as MTT viability assay revealed no toxicity for encapsulated cells. Moreover, alkaline phosphatase activity, calcium deposition, and gene expression analysis were determined to prove the osteogenic potential of the prepared hydrogel. In conclusion, this in situ forming hydrogel can be utilized as filler for bone deficiencies as well as carrier for cell encapsulation and osteogenic differentiation.     

以鸡胸软骨为原料提取硫酸软骨素的研究

以鸡胸软骨为原料,在60℃下用木瓜蛋白酶水解除去蛋白质,再用乙醇沉淀获得鸡胸软骨多糖。色谱分析和聚丙烯酰胺凝胶电泳鉴别显示,鸡胸软骨多糖主要是硫酸软骨素,表明鸡胸软骨是可行的硫酸软骨素新来源。     

Alginate Based Scaffolds for Cartilage Tissue Engineering: A Review

Abstract

Many people, especially old and middle-aged, suffer from pain and disabilities caused by cartilage degradation. There are many surgical methods for cartilage treatment, however, none of them have shown acceptable results in long-term. Tissue engineering would be an acceptable approach for cartilage treatment. This includes cells, a carrier such as a matrix scaffold and signaling molecule. An ideal scaffold for cartilage tissue engineering should meet some requirements includes biocompatibility, biodegradability, and sufficient mechanical characteristic. While there are many suitable scaffolds made by natural and synthesis polymers, alginate- a natural polymer- has received good attention. Alginate offers many advantages for cartilage treatment; it has great potential in having tunable mechanical properties and easy manufacturing process. In the present paper, focusing on alginate as main scaffold constructive component, different studies on alginate based scaffolds in the form of physically, chemically and biologically crosslinked hydrogel, sponge, fiber, micro/nano particles and 3?D printed for articular cartilage tissue engineering are discussed and reviewed.     

HPLC—DAD法测定食品样品中的硫酸软骨素

应用反相液相色谱法测试硫酸软骨素,方法简便快速,易于操作,适合于食品样品的测试,易于在普通的食品实验室推广。该方法在HPLC上的线性范围广,相关系数R〉0．9999,重复性好,有较满意的检出低限。该方法的样品回收率为81．6％-99．3％。     

聚氨酯材料在生物支架组织工程上的应用

概述了医用聚氨酯材料的结构特点及其特性,介绍了国外主要医用聚氨酯产品生产现状,综述了聚氨酯材料在生物支架组织工程如人工心脏及心脏辅助装置、心导管、颌面修复材料和人工软骨等方面的应用,指出医用聚氨酯材料存在的问题以及未来的发展方向。     

Tissue Engineering of Cartilage Using a Random Positioning Machine

Articular cartilage is a skeletal tissue of avascular nature and limited self-repair capacity. Cartilage-degenerative diseases, such as osteoarthritis (OA), are difficult to treat and often necessitate joint replacement surgery. Cartilage is a tough but flexible material and relatively easy to damage. It is, therefore, of high interest to develop methods allowing chondrocytes to recolonize, to rebuild the cartilage and to restore joint functionality. Here we studied the in vitro production of cartilage-like tissue using human articular chondrocytes exposed to the Random Positioning Machine (RPM), a device to simulate certain aspects of microgravity on Earth. To screen early adoption reactions of chondrocytes exposed to the RPM, we performed quantitative real-time PCR analyses after 24 h on chondrocytes cultured in DMEM/F-12. A significant up-regulation in the gene expression of IL6, RUNX2, RUNX3, SPP1, SOX6, SOX9, and MMP13 was detected, while the levels of IL8, ACAN, PRG4, ITGB1, TGFB1, COL1A1, COL2A1, COL10A1, SOD3, SOX5, MMP1, and MMP2 mRNAs remained unchanged. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis demonstrated among others the importance of these differentially regulated genes for cartilage formation. Chondrocytes grown in DMEM/F-12 medium produced three-dimensional (3D) spheroids after five days without the addition of scaffolds. On day 28, the produced tissue constructs reached up to 2 mm in diameter. Using specific chondrocyte growth medium, similar results were achieved within 14 days. Spheroids from both types of culture media showed the typical cartilage morphology with aggrecan positivity. Intermediate filaments form clusters under RPM conditions as detected by vimentin staining after 7 d and 14 d. Larger meshes appear in the network in 28-day samples. Furthermore, they were able to form a confluent chondrocyte monolayer after being transferred back into cell culture flasks in 1 g conditions showing their suitability for transplantation into joints. Our results demonstrate that the cultivation medium has a direct influence on the velocity of tissue formation and tissue composition. The spheroids show properties that make them interesting candidates for cellular cartilage regeneration approaches in trauma and OA therapy.     

Highly Organized Porous Gelatin-Based Scaffold by Microfluidic 3D-Foaming Technology and Dynamic Culture for Cartilage Tissue Engineering

A gelatin-based hydrogel scaffold with highly uniform pore size and biocompatibility was fabricated for cartilage tissue engineering using microfluidic 3D-foaming technology. Mainly, bubbles with different diameters, such as 100 μm and 160 μm, were produced by introducing an optimized nitrogen gas and gelatin solution at an optimized flow rate, and N₂/gelatin bubbles were formed. Furthermore, a cross-linking agent (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, EDC) was employed for the cross-linking reaction of the gelatin-based hydrogel scaffold with uniform bubbles, and then the interface between the close cells were broken by degassing. The pore uniformity of the gelatin-based hydrogel scaffolds was confirmed by use of a bright field microscope, conjugate focus microscope and scanning electron microscope. The in vitro degradation rate, mechanical properties, and swelling rate of gelatin-based hydrogel scaffolds with highly uniform pore size were studied. Rabbit knee cartilage was cultured, and its extracellular matrix content was analyzed. Histological analysis and immunofluorescence staining were employed to confirm the activity of the rabbit knee chondrocytes. The chondrocytes were seeded into the resulting 3D porous gelatin-based hydrogel scaffolds. The growth conditions of the chondrocyte culture on the resulting 3D porous gelatin-based hydrogel scaffolds were evaluated by MTT analysis, live/dead cell activity analysis, and extracellular matrix content analysis. Additionally, a dynamic culture of cartilage tissue was performed, and the expression of cartilage-specific proteins within the culture time was studied by immunofluorescence staining analysis. The gelatin-based hydrogel scaffold encouraged chondrocyte proliferation, promoting the expression of collagen type II, aggrecan, and sox9 while retaining the structural stability and durability of the cartilage after dynamic compression and promoting cartilage repair.     

组织工程支架材料研究进展

组织工程支架材料在组织工程研究中起中心作用，不仅为特定的细胞提供结构支撑作用，而且还起到模板作用，引导组织再生和控制组织结构。寻找一种既有良好生物相容性和生物降解性又具有特定形状和连通三维多孔结构的支架材料是组织工程的重要方面。本文概述了几种常用的组织工程支架材料，并对组织工程支架材料目前存在的问题作了分析、对其发展趋势进行了展望。     

Advances in Growth Factor Delivery for Bone Tissue Engineering

Shortcomings related to the treatment of bone diseases and consequent tissue regeneration such as transplants have been addressed to some extent by tissue engineering and regenerative medicine. Tissue engineering has promoted structures that can simulate the extracellular matrix and are capable of guiding natural bone repair using signaling molecules to promote osteoinduction and angiogenesis essential in the formation of new bone tissues. Although recent studies on developing novel growth factor delivery systems for bone repair have attracted great attention, taking into account the complexity of the extracellular matrix, scaffolding and growth factors should not be explored independently. Consequently, systems that combine both concepts have great potential to promote the effectiveness of bone regeneration methods. In this review, recent developments in bone regeneration that simultaneously consider scaffolding and growth factors are covered in detail. The main emphasis in this overview is on delivery strategies that employ polymer-based scaffolds for spatiotemporal-controlled delivery of both single and multiple growth factors in bone-regeneration approaches. From clinical applications to creating alternative structural materials, bone tissue engineering has been advancing constantly, and it is relevant to regularly update related topics.     

Polymeric Microspheres/Cells/Extracellular Matrix Constructs Produced by Auto-Assembly for Bone Modular Tissue Engineering

Modular tissue engineering (MTE) is a novel “bottom-up” approach to create engineered biological tissues from microscale repeating units. Our aim was to obtain microtissue constructs, based on polymer microspheres (MSs) populated with cells, which can be further assembled into larger tissue blocks and used in bone MTE. Poly(L-lactide-co-glycolide) MS of 165 ± 47 µm in diameter were produced by oil-in-water emulsification and treated with 0.1 M NaOH. To improve cell adhesion, MSs were coated with poly-L-lysine (PLL) or human recombinant collagen type I (COL). The presence of oxygenated functionalities and PLL/COL coating on MS was confirmed by X-ray photoelectron spectroscopy (XPS). To assess the influence of medium composition on adhesion, proliferation, and osteogenic differentiation, preosteoblast MC3T3-E1 cells were cultured on MS in minimal essential medium (MEM) and osteogenic differentiation medium (OSG). Moreover, to assess the potential osteoblast–osteoclast cross-talk phenomenon and the influence of signaling molecules released by osteoclasts on osteoblast cell culture, a medium obtained from osteoclast culture (OSC) was also used. To impel the cells to adhere and grow on the MS, anti-adhesive cell culture plates were utilized. The results show that MS coated with PLL and COL significantly favor the adhesion and growth of MC3T3-E1 cells on days 1 and 7, respectively, in all experimental conditions tested. On day 7, three-dimensional MS/cell/extracellular matrix constructs were created owing to auto-assembly. The cells grown in such constructs exhibited high activity of early osteogenic differentiation marker, namely, alkaline phosphatase. Superior cell growth on PLL- and COL-coated MS on day 14 was observed in the OSG medium. Interestingly, deposition of extracellular matrix and its mineralization was particularly enhanced on COL-coated MS in OSG medium on day 14. In our study, we developed a method of spontaneous formation of organoid-like MS-based cell/ECM constructs with a few millimeters in size. Such constructs may be regarded as building blocks in bone MTE.     

热致相分离技术制备组织工程支架

用生物可降解材料制备细胞生长支架是组织工程的关键技术之一,而热致相分离技术是制备生物可降解三维多孔支架的重要方法.综述了凝胶浇铸、热致凝胶化、乳化/冷冻干燥、固液相分离和液液相分离等几种相分离技术的原理及应用,并预测了相分离技术的应用前景.     

展进新法方定测素骨软酸硫化酸硫多中钠素肝染污

介绍了污染肝素钠中多硫酸化硫酸软骨素的结构、性质及来源,并对其测定方法进行了综述.     

基于快速成型技术的组织工程支架制备进展

介绍了组织工程支架的重要性和基本要求,综述了组织工程支架制备中的新技术：三雏打印技术、熔融沉积模型以及选择性激光烧结等三种快速成型技术的基本原理及应用情况,指出了各种技术的特点并对其应用前景进行了展望。     

Piezoelectric Electrospun Fibrous Scaffolds for Bone,Articular Cartilage and Osteochondral Tissue Engineering

Osteochondral tissue (OCT) related diseases, particularly osteoarthritis, number among the most prevalent in the adult population worldwide. However, no satisfactory clinical treatments have been developed to date to resolve this unmet medical issue. Osteochondral tissue engineering (OCTE) strategies involving the fabrication of OCT-mimicking scaffold structures capable of replacing damaged tissue and promoting its regeneration are currently under development. While the piezoelectric properties of the OCT have been extensively reported in different studies, they keep being neglected in the design of novel OCT scaffolds, which focus primarily on the tissue’s structural and mechanical properties. Given the promising potential of piezoelectric electrospun scaffolds capable of both recapitulating the piezoelectric nature of the tissue’s fibrous ECM and of providing a platform for electrical and mechanical stimulation to promote the regeneration of damaged OCT, the present review aims to examine the current state of the art of these electroactive smart scaffolds in OCTE strategies. A summary of the piezoelectric properties of the different regions of the OCT and an overview of the main piezoelectric biomaterials applied in OCTE applications are presented. Some recent examples of piezoelectric electrospun scaffolds developed for potentially replacing damaged OCT as well as for the bone or articular cartilage segments of this interfacial tissue are summarized. Finally, the current challenges and future perspectives concerning the use of piezoelectric electrospun scaffolds in OCT regeneration are discussed.     

层状结构磷酸钙骨水泥组织工程支架材料的制备与表征

采用可溶粒子造孔法结合冷等静压成型技术,模拟扁骨的结构,制备了一种新型层状结构的多孔磷酸钙骨水泥组织工程支架材料,并用XRD和SEM等手段对其组成和结构进行了表征,用万能材料试验机测定了支架的抗压强度.结果表明,材料由致密层和多孔层构成,具有与扁骨类似的结构.其中致密层起到了增强作用,可以显著提高支架的强度.支架多孔层的孔隙率(77.26±1.99)%,孔隙直径在100～400 μm,决定于可溶盐晶粒的大小;致密层的孔隙率(20.78±0.56)%,主要是磷酸钙骨水泥固化过程中产生的微孔.     

生物反应器在组织工程中的应用

组织工程是迅速发展的交叉学科，细胞及组织的体外培养是其中的关键环节，而生物反应器的应用则是廉价，大量地获得具有临床实用价值的细胞和组织的有效手段，在此为背景，本文评述了生物反应器在组织工程中用应用的最新进展。     

Three-Dimensional Culture of Cartilage Tissue on Nanogel-Cross-Linked Porous Freeze-Dried Gel Scaffold for Regenerative Cartilage Therapy: A Vibrational Spectroscopy Evaluation

This study presents a set of vibrational characterizations on a nanogel-cross-linked porous freeze-dried gel (NanoCliP-FD gel) scaffold for tissue engineering and regenerative therapy. This scaffold is designed for the in vitro culture of high-quality cartilage tissue to be then transplanted in vivo to enable recovery from congenital malformations in the maxillofacial area or crippling jaw disease. The three-dimensional scaffold for in-plate culture is designed with interface chemistry capable of stimulating cartilage formation and maintaining its structure through counteracting the dedifferentiation of mesenchymal stem cells (MSCs) during the formation of cartilage tissue. The developed interface chemistry enabled high efficiency in both growth rate and tissue quality, thus satisfying the requirements of large volumes, high matrix quality, and superior mechanical properties needed in cartilage transplants. We characterized the cartilage tissue in vitro grown on a NanoCliP-FD gel scaffold by human periodontal ligament-derived stem cells (a type of MSC) with cartilage grown by the same cells and under the same conditions on a conventional (porous) atelocollagen scaffold. The cartilage tissues produced by the MSCs on different scaffolds were comparatively evaluated by immunohistochemical and spectroscopic analyses. Cartilage differentiation occurred at a higher rate when MSCs were cultured on the NanoCliP-FD gel scaffold compared to the atelocollagen scaffold, and produced a tissue richer in cartilage matrix. In situ spectroscopic analyses revealed the cell/scaffold interactive mechanisms by which the NanoCliP-FD gel scaffold stimulated such increased efficiency in cartilage matrix formation. In addition to demonstrating the high potential of human periodontal ligament-derived stem cell cultures on NanoCliP-FD gel scaffolds in regenerative cartilage therapy, the present study also highlights the novelty of Raman spectroscopy as a non-destructive method for the concurrent evaluation of matrix quality and cell metabolic response. In situ Raman analyses on living cells unveiled for the first time the underlying physiological mechanisms behind such improved chondrocyte performance.     

Suture Fiber Reinforcement of a 3D Printed Gelatin Scaffold for Its Potential Application in Soft Tissue Engineering

Gelatin has excellent biological properties, but its poor physical properties are a major obstacle to its use as a biomaterial ink. These disadvantages not only worsen the printability of gelatin biomaterial ink, but also reduce the dimensional stability of its 3D scaffolds and limit its application in the tissue engineering field. Herein, biodegradable suture fibers were added into a gelatin biomaterial ink to improve the printability, mechanical strength, and dimensional stability of the 3D printed scaffolds. The suture fiber reinforced gelatin 3D scaffolds were fabricated using the thermo-responsive properties of gelatin under optimized 3D printing conditions (−10 °C cryogenic plate, 40–80 kPa pneumatic pressure, and 9 mm/s printing speed), and were crosslinked using EDC/NHS to maintain their 3D structures. Scanning electron microscopy images revealed that the morphologies of the 3D printed scaffolds maintained their 3D structure after crosslinking. The addition of 0.5% (w/v) of suture fibers increased the printing accuracy of the 3D printed scaffolds to 97%. The suture fibers also increased the mechanical strength of the 3D printed scaffolds by up to 6-fold, and the degradation rate could be controlled by the suture fiber content. In in vitro cell studies, DNA assay results showed that human dermal fibroblasts’ proliferation rate of a 3D printed scaffold containing 0.5% suture fiber was 10% higher than that of a 3D printed scaffold without suture fibers after 14 days of culture. Interestingly, the supplement of suture fibers into gelatin biomaterial ink was able to minimize the cell-mediated contraction of the cell cultured 3D scaffolds over the cell culture period. These results show that advanced biomaterial inks can be developed by supplementing biodegradable fibers to improve the poor physical properties of natural polymer-based biomaterial inks.     

The Regeneration of Large-Sized and Vascularized Adipose Tissue Using a Tailored Elastic Scaffold and dECM Hydrogels

A dome-shaped elastic poly(l-lactide-co-caprolactone) (PLCL) scaffold with a channel and pore structure was fabricated by a combinative method of 3D printing technology and the gel pressing method (13 mm in diameter and 6.5 mm in thickness) for patient-specific regeneration. The PLCL scaffold was combined with adipose decellularized extracellular matrix (adECM) and heart decellularized extracellular matrix (hdECM) hydrogels and human adipose-derived stem cells (hADSCs) to promote adipogenesis and angiogenesis. These scaffolds had mechanical properties similar to those of native adipose tissue for improved tissue regeneration. The results of the in vitro real-time PCR showed that the dECM hydrogel mixture induces adipogenesis. In addition, the in vivo study at 12 weeks demonstrated that the tissue-engineered PLCL scaffolds containing the hydrogel mixture (hdECM/adECM (80:20)) and hADSCs promoted angiogenesis and adipose tissue formation, and suppressed apoptosis. Therefore, we expect that our constructs will be clinically applicable as material for the regeneration of patient-specific large-sized adipose tissue.