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。     

Injectable glycopolypeptide hydrogels as biomimetic scaffolds for cartilage tissue engineering

Glycopolypeptides are an emerging class of bioinspired polymers that mimic naturally occurring glycopeptides or glycoproteins, and therefore are expected to exhibit great potential for biomedical applications. In this study, a glycopolypeptide was synthesized by conjugation of poly(γ-propargyl-l-glutamate) (PPLG) with azido-modified mannose and 3-(4-hydroxyphenyl) propanamide (HPPA), via click chemistry. Injectable hydrogels based on the glycopolypeptide were developed through enzymatic crosslinking reaction in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The physicochemical properties of the hydrogels, such as gelation time, storage modulus, swelling and degradation time, could be controlled by varying the concentrations of HRP and H₂O₂. The glycopolypetide copolymer as well as the extracts of the glycopolypetide hydrogels displayed good cytocompatibility in vitro. After subcutaneous injection into rats, the glycopolypeptide hydrogels were rapidly formed in situ, and exhibited acceptable biocompatibility accompanying the degradation of the hydrogels in vivo. The rabbit chondrocytes inside the glycopolypeptide hydrogels showed spherical morphology with high viability during the incubation period of 3 weeks in vitro, and exhibited a higher proliferation rate than within the hydrogel counterparts of PPLG grafted with 2-(2-(2-methoxyethoxy)ethoxy)ethane (MEO₃) and HPPA. Biochemical analysis demonstrated that the production of glycosaminoglycans (GAG) and type II collagen were significantly enhanced after incubation for 2 and 3 weeks in vitro. Moreover, the chondrocyte-containing glycopolypeptide hydrogels in subcutaneous model of nude mice maintained chondrocyte phenotype and produced the cartilaginous specific matrix. These results indicated that the biomimetic glycopolypeptide-based hydrogels hold potential as three-dimensional scaffolds for cartilage tissue engineering.     

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.     

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.     

自体骨软骨移植修复兔膝关节软骨的早期生物力学评价

Biomechanical properties of the repaired tissue of rabbit knee articular cartilage by autologous osteochondral transplantation     

蚕丝组织工程化肌腱的生物力学研究

目的探讨用蚕丝与同种异体肌腱细胞联合培养植入体内,构建组织工程化肌腱的生物力学指标。方法实验分2组,一组是植入附着了肌腱细胞的蚕丝材料组,另一组是单纯植入蚕丝材料组。分别在术后的第2,4,6,8周进行随机取材,在每次取材时每组分别取20只,对材料进行生物力学测定。所得数据均采用SPSS13．0统计软件进行处理和分析。结果在第2,4,6,8周进行取材,生物力学的测定结果显示在同时间点内,细胞组的结果明显优于非细胞组（P≮0．05）,细胞组自身在不同时间点的比较中,发现除第8周以外（P〉O．05）,时间越长,力学的结果越优秀（P〈O．05）;而在非细胞组则只有第8周的结果与前3次测定结果的差异有统计学意义（P〈0．05）。结论本实验的结果说明蚕丝材料对肌腱细胞的黏附性好,生物力学性能优越,附着肌腱细胞后可以构成组织工程化肌腱。经更深入的实验和研究,蚕丝材料可能会在肌腱缺损的治疗方面具有良好的应用前景。     

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.     

Composite hydrogel scaffolds incorporating decellularized adipose tissue for soft tissue engineering with adipose-derived stem cells

An injectable tissue-engineered adipose substitute that could be used to deliver adipose-derived stem cells (ASCs), filling irregular defects and stimulating natural soft tissue regeneration, would have significant value in plastic and reconstructive surgery. With this focus, the primary aim of the current study was to characterize the response of human ASCs encapsulated within three-dimensional bioscaffolds incorporating decellularized adipose tissue (DAT) as a bioactive matrix within photo-cross-linkable methacrylated glycol chitosan (MGC) or methacrylated chondroitin sulphate (MCS) delivery vehicles. Stable MGC- and MCS-based composite scaffolds were fabricated containing up to 5 wt% cryomilled DAT through initiation with long-wavelength ultraviolet light. The encapsulation strategy allows for tuning of the 3-D microenvironment and provides an effective method of cell delivery with high seeding efficiency and uniformity, which could be adapted as a minimally-invasive in situ approach. Through in vitro cell culture studies, human ASCs were assessed over 14 days in terms of viability, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, adipogenic gene expression and intracellular lipid accumulation. In all of the composites, the DAT functioned as a cell-supportive matrix that enhanced ASC viability, retention and adipogenesis within the gels. The choice of hydrogel also influenced the cell response, with significantly higher viability and adipogenic differentiation observed in the MCS composites containing 5 wt% DAT. In vivo analysis in a subcutaneous Wistar rat model at 1, 4 and 12 weeks showed superior implant integration and adipogenesis in the MCS-based composites, with allogenic ASCs promoting cell infiltration, angiogenesis and ultimately, fat formation.     

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.     

Micro-CT-based screening of biomechanical and structural properties of bone tissue engineering scaffolds

The development of successful scaffolds for bone tissue engineering requires a concurrent engineering approach that combines different research fields. In order to limit in vivo experiments and reduce trial and error research, a scaffold screening technique has been developed. In this protocol seven structural and three biomechanical properties of potential scaffold materials are quantified and compared to the desired values. The property assessment is done on computer models of the scaffolds, and these models are based on micro-CT images. As a proof of principle, three porous scaffolds were evaluated with this protocol: stainless steel, hydroxyapatite, and titanium. These examples demonstrate that the modelling technique is able to quantify important scaffold properties. Thus, a powerful technique for automated screening of bone tissue engineering scaffolds has been developed that in a later stage may be used to tailor the scaffold properties to specific requirements.     

软骨组织工程生物反应器的研究进展

体外软骨构建是软骨组织工程产业化发展及临床应用的重要手段。然而,采用现有的体外构建技术无法构建功能接近正常的软骨。生物反应器能够在一定程度上模拟体内环境,有望弥补现有体外构建技术的弊端。研究发现,流体剪切力、静态液压力和直接压缩力是体内软骨发育和成熟的重要力学因素,常用软骨生物反应器均据此设计而产生。由于不同类型生物反应器各具特点,研究和开发新型复合式生物反应器将成为未来的发展方向。对目前软骨组织工程生物反应器的研究现状做一综述。     

智能水凝胶在组织工程中的应用

背景：与传统水凝胶相比,智能水凝胶能够对外界刺激诸如温度、pH值、光、磁场等作出不同的应答表现,产生二级结构甚至化学结构的变化,自发组装形成有序的超分子结构,最终形成具有三维结构的凝胶。 目的：综述智能水凝胶的研究现状及其在组织工程的应用。 方法：应用计算机检索中国知网及PubMed 数据库从建库至2014年有关智能水凝胶在组织工程中应用的文献,检索关键词为“水凝胶,组织工程学,hydrogel,Tissue engineering”。 结果与结论：智能水凝胶中包括温度敏感性、pH敏感性、光敏感性、磁敏感性及温度/pH双重敏感性水凝胶,其对于外界环境变化能自动感知并能作出响应性的反应,在药物递送系统、药物释放,修复和改善缺损组织等领域表现出一系列传统材料所没有的突出性能,尤其是在组织工程方面表现出相当的优越性：低免疫原性,减少了炎症和排斥作用;具备生物可降解性;能真正在三维尺度上模拟细胞所处微环境,从而利于细胞黏附、生长、迁移及分化等。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

细胞外基质作为组织工程支架的最新进展

目前存在很多由于先天缺陷、创伤或肿瘤切除等导致人体完整性缺陷的现象,故近年来组织工程成为研究热点。支架材料是组织工程的要素之一。人脂肪组织细胞外基质粉末呈三维空间结构,能促进干细胞增殖、分化、新生血管形成及生长因子释放等,已成为组织工程支架的新材料。     

Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration

Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p < 0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs.     

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.     

提高骨组织工程支架材料生物相容性的关键影响因素

骨组织工程领域除了对支架材料本身的构成和性能加以研究之外，其研究范围还包括：对支架材料的孔径、孔隙率及三维相通性的研究；种子细胞的筛选、生物活性因子的参与以及生物复合材料的构建等相关因素的研究，这些因素对支架材料的生物相容性以及体内的骨传导性和骨诱导性都至关重要。从这些方面人手，有可能使骨组织工程支架材料的发展取得长足的进步。     

组织工程中生物材料表面修饰的研究

为了提高生物材料的生物相容性和细胞亲和性 ,需对生物材料进行表面修饰。本文对生物材料表面修饰的方法进行了综述 ,并提出今后尚待解决的问题和发展趋势     

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.     

掺锶复合PVA/生物活性玻璃水凝胶的制备及其体外软骨修复性能研究

目的研究以聚乙烯醇(PVA)、生物活性玻璃(BG)及氯化锶为主要原料,制备的PVA水凝胶、PVA/生物活性玻璃水凝胶、掺锶复合PVA/生物活性玻璃水凝胶的可降解性能、离子释放性能和促软骨修复性能。方法PVA溶液与BG溶胶凝胶溶液在加热搅拌下生成PB水凝胶,PVA溶液与Sr-BG溶胶凝胶溶液加热搅拌生成PBSr水凝胶,将PB和PB-Sr水凝胶浸泡于磷酸盐缓冲液(PBS)中,研究其体外降解性能、离子释放性能和结构变化。在水凝胶上培养软骨细胞,经细胞增殖能力实验和细胞荧光染色观察细胞增殖情况。结果 PB和PB-Sr水凝胶在PBS溶液中逐渐降解,28 d后PB水凝胶降解率为25%,PB-Sr水凝胶降解率为16%,水凝胶表面均有羟基磷灰石形成。细胞实验结果显示培养7 d后PB-Sr水凝胶的OD值为0.76±0.04,PB水凝胶的OD值为0.52±0.02,均显著高于对照组,PVA水凝胶的OD值0.45±0.04,差异具有统计学意义(0.05)。结论该掺锶复合PVA/生物活性玻璃水凝胶具有良好的降解性能和离子释放性能,能有效促进软骨细胞增殖。     

3D打印骨组织工程支架的制备技术

近年来,随着3D打印技术的飞速发展,人们开始通过3D打印技术去不断完善适合不同需求的定制骨组织工程支架。由于组织工程制造的支架是需要植入生物体内的,这就对支架有着极为严苛的要求。3D打印技术作为一种新兴制备骨组织工程支架的技术,其最大的优点是可以依照需求来定制个性化形状、结构,良好的宏微观结构、润湿性、机械强度和细胞反应的新型骨组织工程支架。本文回顾了2014―2019年间对骨组织工程支架的研究,对3D打印骨组织工程支架进行了总结,并且介绍了在多功能骨组织工程支架设计与制作中的理念与研究。