海藻酸钙水凝胶/聚乳酸复合材料的制备与性能

通过化学发泡-冷冻干燥-粒子滤出复合法制备聚乳酸(PLLA)大孔支架, 然后在大孔内以海藻酸钠(SA)、碳酸钙、葡萄糖酸内酯(GDL)为原料, 通过原位相转变制备海藻酸钙水凝胶/聚乳酸复合材料(CA/PLLA); 分别利用SEM、压缩强度测试和细胞培养对CA/PLLA支架的形貌、力学性能及生物相容性进行了研究。结果表明: PLLA具有直径小于2 mm、孔道相互连通的孔洞, 且在大孔中能够形成均匀的CA。CA/PLLA复合材料的压缩强度(2.74 MPa)远大于单一的海藻酸钙水凝胶的压缩强度(0.10 MPa)。在CA/PLLA复合支架中, 软骨细胞呈簇状圆形生长状态, 与其在天然软骨陷窝里生长状态一致。这种软硬结合、天然与合成高分子杂化的CA/PLLA复合材料的力学强度和生物相容性同时得到提高, 可进一步作为骨和软骨修复材料研究。     

β-磷酸三钙/聚L-乳酸与大鼠骨膜成骨细胞复合骨修复材料的研究

采用溶液浇铸-模压成型-沥滤方法制备了β-TCP/PLLA多孔支架材料, 将支架材料与大鼠骨膜成骨细胞复合获得新型组织工程骨修复材料. 通过抗压强度及压缩模量的表征研究了支架材料的力学性能; 采用SEM观测、MTT法、碱性磷酸酶活性及骨钙素分泌量检测细胞复合材料的体外成骨特性; 通过裸鼠肌袋种植, 以组织学方法评价细胞复合材料的异位成骨能力. 结果表明: β-TCP/PLLA多孔支架材料孔隙率可调, 孔径为100～00μm, 孔道相互贯通; 材料抗压强度和压缩模量随孔隙率的增大而降低, β-TCP复合PLLA后材料的力学性能高于同孔隙率的纯PLLA多孔材料; 复合支架材料适宜骨膜成骨细胞粘附和生长, 无细胞毒性; 骨膜成骨细胞复合β-TCP/PLLA支架材料的体外成骨特性良好, 且具有体内异位成骨能力.     

介孔纳米羟基磷灰石/左旋聚乳酸复合材料的制备及性能

为考察介孔纳米羟基磷灰石(MHA)/左旋聚乳酸(PLLA)复合材料的性能,以十六烷基三甲基溴化铵(CTAB)为模板合成MHA,采用溶液相分离结合粒子沥滤法制备了不同纳米粒子含量的MHA/PLLA多孔支架复合材料,考察了其抗压缩性能和淬断面微观结构。采用溶液浇注法制备了MHA/PLLA复合膜,并对其拉伸性能和拉伸断面微观结构进行了研究。FTIR、XRD、TEM和氮气吸附测试等结果显示:合成的MHA具有典型的晶体结构、介孔结构和较高的比表面积。力学测试结果显示:在发生10%压缩形变时,填料含量为1%、5%和10%的MHA/PLLA多孔支架复合材料的抗压缩强度随填料含量增加而提高,与相应含量的纳米羟基磷灰石(HA)/PLLA多孔支架复合材料相比,分别提高了约37.0%、67.7%和144.7%。在填料含量为5%和10%时,MHA/PLLA复合膜的拉伸强度较HA/PLLA复合膜分别提高约38.7%和46.1%,拉伸模量分别提高约35.4%和14.5%。而且MHA/PLLA复合膜具有更高的断裂伸长率,填料含量为1%、5%和10%时断裂伸长率分别较HA/PLLA复合膜提高约91.3%、79.7%和96.1%。FESEM结果显示:尤其当填料含量较高时,MHA/PLLA多孔支架复合材料或复合膜中填料粒子分布较HA/PLLA中均匀。结果表明:与HA/PLLA复合材料相比,随着MHA含量增加,MHA/PLLA复合材料具有更好的力学性能,MHA在PLLA基体中分布相对更均匀。     

骨软骨组织工程仿生梯度支架研究进展

骨软骨缺损是导致关节发病和残疾的重要原因,骨软骨组织工程是修复骨软骨缺损的方法之一。骨软骨组织工程方法涉及仿生梯度支架的制造,该支架需模仿天然骨软骨组织的生理特性（例如从软骨表面到软骨下骨之间的梯度过渡）。在许多研究中骨软骨仿生梯度支架表现为离散梯度或连续梯度,用于模仿骨软骨组织的特性,例如生物化学组成、结构和力学性能。连续型骨软骨梯度支架的优点是其每层之间没有明显的界面,因此更相似地模拟天然骨软骨组织。到目前为止,骨软骨仿生梯度支架在骨软骨缺损修复研究中已经取得了良好的实验结果,但是骨软骨仿生梯度支架与天然骨软骨组织之间仍然存在差异,其临床应用还需要进一步研究。本文首先从骨软骨缺损的背景、微尺度结构与力学性能、骨软骨仿生梯度支架制造相关的材料与方法等方面概述了离散和连续梯度支架的研究进展。其次,由于3D打印骨软骨仿生梯度支架的方法能够精确控制支架孔的几何形状和力学性能,因此进一步介绍了计算仿真模型在骨软骨组织工程中的应用,例如采用仿真模型优化支架结构和力学性能以预测组织再生。最后,提出了骨软骨缺损修复相关的挑战以及骨软骨组织再生未来研究的展望。例如,连续型骨软骨仿生梯度支架需要更相似地模拟天然骨软骨组织单元的结构,即力学性能和生化性能的过渡更加自然地平滑。同时,虽然大多数骨软骨仿生梯度支架在体内外实验中均取得了良好的效果,但临床研究和应用仍然需要进行进一步深入研究。     

激光熔融静电纺丝法制备PLLA及PLLA/nHA纤维支架及性能研究

利用激光熔融静电纺丝方法制备了PLLA和PLLA/nHA纤维支架,采用FTIR和DSC测试对支架材料的结构和热学性能进行表征,通过熔融电纺对PLLA和PLLA/nHA纤维支架进行体外降解实验,研究了失重率与降解时间的关系。同时对激光熔融和一般溶液电纺得到的PLLA和PLLA/nHA纤维支架进行细胞相容性实验,对两种方法所得支架的安全性进行评价。结果表明:nHA对PLLA的结构和晶型产生影响,并减缓PLLA的降解速度,激光熔融电纺支架更具安全性,其更适合组织工程应用。     

分级多孔壳聚糖/聚乳酸复合支架的制备及骨修复性能

为了仿生莲藕内部的贯穿大孔结构,以生物相容性好的壳聚糖(CS)作为基质材料,利用冰粒致孔、石蜡模具和冰模具成型3种成型方法制备了分级多孔CS支架材料,然后与力学强度较高的聚乳酸(PLLA)复合,制备网络互穿CS/PLLA复合支架。通过SEM、压缩强度测试和兔股骨髁骨缺损模型对CS/PLLA复合材料的形貌、力学强度和骨修复性能进行了表征。结果表明:利用冰模具制备的CS/PLLA复合支架能可控、批量制备,具有微米-毫米分级多孔结构,大孔孔径约为2mm,内部均匀分布着孔径约为60μm的贯穿微孔,并在微孔内形成密集的PLLA絮状网络结构。干态复合材料的压缩强度和模量分别比纯CS支架的提高了6倍和15倍。体内植入实验结果表明,CS/PLLA复合材料能够促进骨缺损的愈合,并随着新骨的形成,复合材料逐渐被降解吸收。     

N-组氨酸壳聚糖/左旋聚乳酸支架的制备及表征

在冷冻诱导相分离制备N-组氨酸壳聚糖支架(NHCS)的基础上进行二次相分离,利用不同取代度的NHCS和不同N-组氨酸壳聚糖/聚乳酸质量比制备一系列N-组氨酸壳聚糖/聚乳酸(NHCS/PLLA)支架。通过红外光谱、广角X射线衍射、热分析和扫描电镜等来表征NHCS/PLLA支架。结果表明,同一种N-组氨酸壳聚糖支架(50kD-NHCS-3),N-组氨酸壳聚糖/聚乳酸质量比减小,支架孔隙率减小,密度增大。支架材料的孔尺寸约在12~25μm,孔隙率均大于92%,抗压强度和弹性模量分别在0.33~0.78 MPa和1.75~5.28 MPa之间,有望适用于软骨组织工程支架。     

氧化石墨烯增强的骨仿生静电纺左旋聚乳酸纳米纤维支架的制备及表征

利用静电纺丝技术制备了左旋聚乳酸/氧化石墨烯(PLLA/GO)复合纳米纤维毡。通过扫描电子显微镜(SEM)、透射电子显微镜(TEM)、孔隙率测试、傅里叶红外光谱分析(FTIR)以及拉伸测试分别对PLLA/GO纳米纤维的形貌结构、孔隙率及力学性能进行了研究。将小鼠骨髓间充质干细胞(MSCs)种植在TSF/PLLA纳米纤维上,通过荧光显微镜分析和碱性磷酸酶(ALP)测试、SEM观察细胞在材料表面的生长以及矿物沉积情况评价复合纳米纤维的生物学性能。结果表明,与纯的PLLA静电纺纳米纤维支架相比,PLLA/GO复合纳米纤维支架的纤维直径显著减小,孔隙率增大,力学性能明显得到改善,拉伸强度和杨氏模量均高于纯PLLA纳米纤维支架将近3倍,而且能够更好地促进MSCs的粘附、增殖和分化。     

溶剂自扩散法制备聚L-乳酸/β-磷酸三钙复合多孔支架材料

采用溶剂自扩散原理从聚L-乳酸(PLLA)/β-磷酸三钙(β-TCP)氯仿液中沉积得到PLLA/β-TCP复合颗粒,研究了不同扩散介质对该过程的影响.研究表明制备复合颗粒以丙酮/无水乙醇混合液为扩散介质效果最佳,以其为扩散介质沉积速率快、沉积充分,且得到的复合颗粒可以经模压成型、粒子沥滤工艺制备PLLA/β-TCP多孔复合支架.对多孔支架进行了SEM、孔隙率、力学性能及有机溶剂残留量测试,结果表明制备的多孔支架孔结构三维贯通,孔隙率60.3%,抗压强度4.40MPa,氯仿、丙酮、无水乙醇残留量分别为3.630×10-5、2.07 × 10-6、2.517×10-5,满足组织工程支架材料要求.     

聚吡咯/碳纤维纸电热复合材料的制备及性能

在碳纤维纸（CPP）上采用气相聚合法使吡咯单体发生聚合生成聚吡咯（PPY）,制备聚吡咯/碳纤维（PPY/CF）纸导电发热复合材料。通过FTIR、SEM、XRD对PPY/CF纸复合材料进行测试分析,并探究了CPP中CF的最佳质量分数及FeCl₃的最优溶度,研究了PPY/CF纸复合材料的力学性能和电热性能。结果表明,成功制备了PPY/CF纸复合材料,且PPY主要附着在芳纶浆粕上;采用CF质量分数为10 wt%的CPP且FeCl₃浓度为1.2 mol/L时,制备的PPY/CF纸复合材料具有最低的电阻率,为0.139 Ω·cm;PPY/CF纸复合材料与CPP相比不仅力学性能大幅提高,而且在低电压下,PPY/CF纸复合材料具有导电性能稳定、发热效果显著、热稳定性好等优点。      

Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.     

Fabrication and cell affinity of biomimetic structured PLGA/articular cartilage ECM composite scaffold

An ideal scaffold for cartilage tissue engineering should be biomimetic in not only mechanical property and biochemical composition, but also the morphological structure. In this research, we fabricated a composite scaffold with oriented structure to mimic cartilage physiological morphology, where natural nanofibrous articular cartilage extracellular matrix (ACECM) was used to mimic the biochemical composition, and synthetic PLGA was used to enhance the mechanical strength of ACECM. The composite scaffold has well oriented structure and more than 89% of porosity as well as about 107 μm of average pore diameter. The composite scaffold was compared with ACECM and PLGA scaffolds. Cell proliferation test showed that the number of MSCs in ACECM and composite scaffolds was noticeably bigger than that in PLGA scaffold, which was coincident with results of SEM observation and cell viability staining. The water absorption of ACECM and composite scaffolds were 22.1 and 10.2 times respectively, which was much higher than that of PLGA scaffolds (3.8 times). The compressive modulus of composite scaffold in hydrous status was 1.03 MPa, which was near 10 times higher than that of hydrous ACECM scaffold. The aforementioned results suggested that the composite scaffold has the potential for application in cartilage tissue engineering.     

A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

Abstract

The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.     

Nanosized fibers' effect on adult human articular chondrocytes behavior

Tissue engineering with chondrogenic cell based therapies is an expanding field with the intention of treating cartilage defects. It has been suggested that scaffolds used in cartilage tissue engineering influence cellular behavior and thus the long-term clinical outcome. The objective of this study was to assess whether chondrocyte attachment, proliferation and post-expansion re-differentiation could be influenced by the size of the fibers presented to the cells in a scaffold. Polylactic acid (PLA) scaffolds with different fiber morphologies were produced, i.e. microfiber (MS) scaffolds as well as nanofiber-coated microfiber scaffold (NMS). Adult human articular chondrocytes were cultured in the scaffolds in vitro up to 28 days, and the resulting constructs were assessed histologically, immunohistochemically, and biochemically. Attachment of cells and serum proteins to the scaffolds was affected by the architecture. The results point toward nano-patterning onto the microfibers influencing proliferation of the chondrocytes, and the overall 3D environment having a greater influence on the re-differentiation. In the efforts of finding the optimal scaffold for cartilage tissue engineering, studies as the current contribute to the knowledge of how to affect and control chondrocytes behavior.     

Structurally and Functionally Optimized Silk‐Fibroin–Gelatin Scaffold Using 3D Printing to Repair Cartilage Injury In Vitro and In Vivo

Articular cartilage repair remains a great challenge for clinicians and researchers. Recently, there emerges a promising way to achieve one‐step cartilage repair in situ by combining endogenic bone marrow stem cells (BMSCs) with suitable biomaterials using a tissue engineering technique. To meet the increasing demand for cartilage tissue engineering, a structurally and functionally optimized scaffold is designed, by integrating silk fibroin with gelatin in combination with BMSC‐specific‐affinity peptide using 3D printing (3DP) technology. The combination ratio of silk fibroin and gelatin greatly balances the mechanical properties and degradation rate to match the newly formed cartilage. This dually optimized scaffold has shown superior performance for cartilage repair in a knee joint because it not only retains adequate BMSCs, due to efficient recruiting ability, and acts as a physical barrier for blood clots, but also provides a mechanical protection before neocartilage formation and a suitable 3D microenvironment for BMSC proliferation, differentiation, and extracellular matrix production. It appears to be a promising biomaterial for knee cartilage repair and is worthy of further investigation in large animal studies and preclinical applications. Beyond knee cartilage, this dually optimized scaffold may also serve as an ideal biomaterial for the regeneration of other joint cartilages.     

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。     

Gelatin/chitosan/hyaluronan ternary complex scaffold containing basic fibroblast growth factor for cartilage tissue engineering

Gelatin, chitosan and hyaluronan with a weight ratio of 82.6%, 16.5% and 0.1% were chosen as a scaffold material to mimic the composition of natural cartilage matrix for cartilage tissue engineering. Water soluble carbodiimide was added into the biomacromolecule solution with a concentration of 5% to crosslink the complex. Following a freeze-drying procedure, a porous scaffold (control) was then prepared. To enhance chondrogenesis, heparin was covalently immobilized onto the scaffold by carbodiimide chemistry, through which basic fibroblast growth factor (bFGF) was further incorporated by a bioaffinity force. Incubation in phosphate buffered saline (PBS, pH 7.4) at 37 °C caused the weight loss of all kinds of the scaffolds, which could be brought by both the degradation and dissolution of the biomacromolecules. Compared with the control, however, the heparinized scaffold showed stronger ability to resist the weight loss, implying that a higher crosslinking degree was achieved by incorporation of the heparin. Rabbit auricular chondrocytes were seeded onto the ternary complex scaffold containing bFGF to assess cell response. Chondrocytes could adhere and proliferate in all kinds of the scaffold, regardless of the existence of bFGF. No significant difference on glycosaminoglycan (GAG) secretion was recorded between these scaffolds after cultured for 7 and 21 days too, although the absolute value from the Scaffold-heparin-bFGF was somewhat higher. However, chondrocytes seeded in the Scaffold-heparin-bFGF indeed showed significant higher viability than that on the control scaffold. These results reveal that the ternary complex scaffolds, in particular the one containing bFGF, are a potential candidate for cartilage tissue engineering.     

A polylactide/fibrin gel composite scaffold for cartilage tissue engineering: fabrication and an in vitro evaluation

A composite scaffold for cartilage tissue engineering was fabricated by filling a porous poly (l-lactide) (PLLA) scaffold with fibrin gel. The porous PLLA scaffold prepared by a method of thermally induced phase separation has an average pore diameter of 200 μm and a porosity of 93%. Incorporation of fibrin gel into the scaffold was achieved by dropping a fibrinogen and thrombin mixture solution onto the scaffold. For a couple of minutes the fibrin gel was in situ formed within the scaffold. The filling efficiency was decreased along with the increase of the fibrinogen concentration. After fibrin gel filling, the compressive modulus and the yield stress increased from 5.94 MPa and 0.37 MPa (control PLLA scaffold in a hydrated state) to 7.21 MPa and 0.53 MPa, respectively. While the fibrin gel lost its weight in phosphate buffered saline up to ~50% within 3 days, 85% and 70% of the fibrin gel weight in the composite scaffold was remained within 3 and 35 days, respectively. A consistent significant higher level of rabbit auricular chondrocyte viability, cell number and glycosaminoglycan was measured in the composite scaffold than that in the control PLLA scaffold. Rabbit auricular chondrocytes with round morphology were also observed in the composite scaffold by confocal microscopy and scanning electron microscopy. Altogether with the features of better strength and cytocompatibility, this type of composite scaffold may have better performance as a matrix for cartilage tissue engineering.     

Preparation and characterization of collagen/PLA,chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.     

A silk fibroin/chitosan scaffold in combination with bone marrow-derived mesenchymal stem cells to repair cartilage defects in the rabbit knee

Bone marrow-derived mesenchymal stem cells (BMSCs) were seeded in a three-dimensional scaffold of silk fibroin (SF) and chitosan (CS) to repair cartilage defects in the rabbit knee. Totally 54 rabbits were randomly assigned to BMSCs + SF/CS scaffold, SF/CS scaffold and control groups. A cylindrical defect was created at the patellofemoral facet of the right knee of each rabbit and repaired by scaffold respectively. Samples were prepared at 4, 8 and 12 weeks post-surgery for gross observation, hematoxylin–eosin and toluidine blue staining, type II collagen immunohistochemistry, Wakitani histology. The results showed that differentiated BMSCs proliferated well in the scaffold. In the BMSCs + SF/CS scaffold group, the bone defect was nearly repaired, the scaffold was absorbed and immunohistochemistry was positive. In the SF/CS scaffold alone group, fiber-like tissues were observed, the scaffold was nearly degraded and immunohistochemistry was weakly positive. In the control group, the defect was not well repaired and positive immunoreactions were not detected. Modified Wakitani scores were superior in the BMSCs + SF/CS scaffold group compared with those in other groups at 4, 8 and 12 weeks (P < 0.05). A SF/CS scaffold can serve as carrier for stem cells to repair cartilage defects and may be used for cartilage tissue engineering.