The maturity of tissue-engineered cartilage in vitro affects the repairability for osteochondral defect

Cartilage tissue engineering using cells and biocompatible scaffolds has emerged as a promising approach to repair of cartilage damage. To date, however, no engineered cartilage has proven to be equivalent to native cartilage in terms of biochemical and compression properties, as well as histological features. An alternative strategy for cartilage engineering is to focus on the in vivo regeneration potential of immature engineered cartilage. Here, we used a rabbit model to evaluate the extent to which the maturity of engineered cartilage influenced the remodeling and integration of implanted extracellular matrix scaffolds containing allogenous chondrocytes. Full-thickness osteochondral defects were created in the trochlear groove of New Zealand white rabbits. Left knee defects were left untreated as a control (group 1), and right knee defects were implanted with tissue-engineered cartilage cultured in vitro for 2 days (group 2), 2 weeks (group 3), or 4 weeks (group 4). Histological, chemical, and compression assays of engineered cartilage in vitro showed that biochemical composition became more cartilagenous, and biomechanical property for compression gradually increased with culture time. In an in vivo study, gross imaging and histological observation at 1 and 3 months after implanting in vitro-cultured engineered cartilage showed that defects in groups 3 and 4 were repaired with hyaline cartilage-like tissue, whereas defects were only partially filled with fibrocartilage after 1 month in groups 1 and 2. At 3 months, group 4 showed striking features of hyaline cartilage tissue, with a mature matrix and a columnar arrangement of chondrocytes. Zonal distribution of type II collagen was most prominent, and the International Cartilage Repair Society score was also highest at this time. In addition, the subchondral bone was well ossified. In conclusion, in vivo engineered cartilage was remodeled when implanted; however, its extent to maturity varied with cultivation period. Our results showed that the more matured the engineered cartilage was, the better repaired the osteochondral defect was, highlighting the importance of the in vitro cultivation period.     

Potential of 3-D tissue constructs engineered from bovine chondrocytes/silk fibroin-chitosan for in vitro cartilage tissue engineering

The use of cell-scaffold constructs is a promising tissue engineering approach to repair cartilage defects and to study cartilaginous tissue formation. In this study, silk fibroin/chitosan blended scaffolds were fabricated and studied for cartilage tissue engineering. Silk fibroin served as a substrate for cell adhesion and proliferation while chitosan has a structure similar to that of glycosaminoglycans, and shows promise for cartilage repair. We compared the formation of cartilaginous tissue in silk fibroin/chitosan blended scaffolds seeded with bovine chondrocytes and cultured in vitro for 2 weeks. The constructs were analyzed for cell viability, histology, extracellular matrix components glycosaminoglycan and collagen types I and II, and biomechanical properties. Silk fibroin/chitosan scaffolds supported cell attachment and growth, and chondrogenic phenotype as indicated by Alcian Blue histochemistry and relative expression of type II versus type I collagen. Glycosaminoglycan and collagen accumulated in all the scaffolds and was highest in the silk fibroin/chitosan (1:1) blended scaffolds. Static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls. The results suggest that silk/chitosan scaffolds may be a useful alternative to synthetic cell scaffolds for cartilage tissue engineering.     

Development of mature cartilage constructs using novel three-dimensional porous scaffolds for enhanced repair of osteochondral defects

In this study, we successfully developed two types of volume-reduced three-dimensional scaffolds, including cushion- and cylinder-shape scaffolds, fabricated from chitosan-based hyaluronic acid hybrid polymer fibers. Using these scaffolds combined with a bioreactor system, we regenerated histologically and mechanically mature cartilage constructs. The final goal of this study was to clarify the ability of this engineered cartilage construct to induce cartilage repair in osteochondral defects. The mature cartilage constructs regenerated with two types of scaffolds were implanted into 5-mm diameter osteochondral defects in the patellar groove of rabbits. At 12 weeks after implantation, the reparative tissues consisted of hyaline-like cartilage with evidence of stable fusion to adjacent native cartilage and normal reconstitution of subchondral bone. The histological score of these tissues significantly outranked the value of untreated tissue. Biomechanically, compression modulus of reparative tissue at 12 weeks postoperatively was comparative to that of normal articular cartilage. Our results indicate that the implantation of constructs with mature cartilage have potential as a better approach for joint resurfacing.     

Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.     

Application of sodium triple‐quantum coherence NMR spectroscopy for the study of growth dynamics in cartilage tissue engineering

We studied the tissue growth dynamics of tissue‐engineered cartilage at an early growth stage after cell seeding for four weeks using sodium triple‐quantum coherence NMR spectroscopy. The following tissue‐engineering constructs were studied: 1) bovine chondrocytes cultured in alginate beads; 2) bovine chondrocytes cultured as pellets (scaffold‐free chondrocyte pellets); and 3) human marrow stromal cells (HMSCs) seeded in collagen/chitosan based biomimetic scaffolds. We found that the sodium triple‐quantum coherence spectroscopy could differentiate between different tissue‐engineered constructs and native tissues based on the fast and slow components of relaxation rate as well as on the average quadrupolar coupling. Both fast (T_f) and slow (T_s) relaxation times were found to be longer in chondrocyte pellets and biomimetic scaffolds compared to chondrocytes suspended in alginate beads and human articular cartilage tissues. In all cases, it was found that relaxation rates and motion of sodium ions measured from correlation times were dependent on the amount of macromolecules, high cell density and anisotropy of the cartilage tissue‐engineered constructs. Average quadrupolar couplings were found to be lower in the engineered tissue compared to native tissue, presumably due to the lack of order in collagen accumulated in the engineered tissue. These results support the use of sodium triple‐quantum coherence spectroscopy as a tool to investigate anisotropy and growth dynamics of cartilage tissue‐engineered constructs in a simple and reliable way. Copyright © 2013 John Wiley & Sons, Ltd.     

Poly(hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of articular cartilage chondrocytes in vitro

The present investigation describes the production of extracellular matrix of rabbit articular cartilage chondrocytes grown on scaffolds of polyhydroxybutyrate (PHB) blended with poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for up to 7 days. The mRNA level of type II collagen of chondrocytes seeded on all scaffolds consisting of PHBHHx were obviously higher than that of PHB-only scaffold throughout the culture period, suggesting the positive effect of PHBHHx on extracellular matrix production. Second-harmonic generation (SHG) imaging technique, combined with confocal fluorescence microscopy (CFM) revealed that PHBHHx in PHB scaffold provided better surface properties for anchoring type II collagen filaments and their penetration into internal layers of the scaffolds. Glycosaminoglycan (GAG), a major composition of extracellular matrix, showed a sharp increase in construct of 1:2 PHB/PHBHHx scaffold after 7 day cultivation, while only a small increase was observed in all other tested scaffolds. At the same time, total collagen contents in all scaffolds containing PHBHHx increased with time, with the maximum collagen production of 742.1+/-99.2mg/g dry weight observed in construct of 1:2 PHB/PHBHHx scaffold inoculated for 7 days, this was almost 4-fold higher than that in scaffold of PHB only. It appears that the presence of right proportion of PHBHHx in the composite system of PHB/PHBHHx highly favored the production of extracellular matrix of articular cartilage chondrocytes.     

In vitro generation of osteochondral composites

Osteochondral repair involves the regeneration of articular cartilage and underlying bone, and the development of a well-defined tissue-to-tissue interface. We investigated tissue engineering of three-dimensional cartilage/bone composites based on biodegradable polymer scaffolds, chondrogenic and osteogenic cells. Cartilage constructs were created by cultivating primary bovine calf articular chondrocytes on polyglycolic acid meshes; bone-like constructs were created by cultivating expanded bovine calf periosteal cells on foams made of a blend of poly-lactic-co-glycolic acid and polyethylene glycol. Pairs of constructs were sutured together after 1 or 4 weeks of isolated culture, and the resulting composites were cultured for an additional 4 weeks. All composites were structurally stable and consisted of well-defined cartilaginous and bone-like tissues. The fraction of glycosaminoglycan in the cartilaginous regions increased with time, both in isolated and composite cultures. In contrast, the mineralization in bone-like regions increased during isolated culture, but remained approximately constant during the subsequent composite culture. The integration at the cartilage/bone interface was generally better for composites consisting of immature (1-week) than mature (4-week) constructs. This study demonstrates that osteochondral tissue composites for potential use in osteochondral repair can be engineered in vitro by culturing mammalian chondrocytes and periosteal cells on appropriate polymer scaffolds.     

Combination of baculovirus-mediated gene transfer and rotating-shaft bioreactor for cartilage tissue engineering

We have previously demonstrated efficient baculovirus transduction of rat chondrocytes in 6-well plates. To further explore the potential of baculovirus in cartilage tissue engineering, the baculovirus-transduced chondrocytes were seeded into porous scaffolds and cultivated in a rotating-shaft bioreactor (RSB) which was developed for two-phase cultivation of tissue engineered cartilage. The baculovirus transduction resulted in efficiencies up to 90%, and affected neither cell adhesion to the scaffolds nor cell survival in the RSB. After 4-week RSB cultivation, the transduced cells remained highly differentiated and grew into constructs that resembled the untransduced constructs with regard to gross appearance, construct size, cell morphology, cell spatial distribution, glycosaminoglycan and collagen production and deposition. Importantly, baculovirus transduction did not alter the expression of chondrocytic genes. These data confirmed that baculovirus transduction neither harms chondrocytes nor retards the formation of cartilage-like tissues in the RSB, thus implicating the potentials of combining baculovirus-mediated gene transfer with RSB cultivation in in vitro cartilage tissue engineering.     

Study on the three-dimensional proliferation of rabbit articular cartilage-derived chondrocytes on polyhydroxyalkanoate scaffolds

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx)/polyhydroxybutyrate (PHB) (PHBHHx/PHB) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture. Blend polymers of PHBHHx/PHB were fabricated into three-dimensional porous scaffolds by the salt-leaching method. Chondrocytes isolated from rabbit articular cartilage (RAC) were seeded on the scaffolds and incubated over 28 days, with change of the culture medium every 4 days. PHB scaffold was taken as a control. Methylthiazol tetrazolium (MTT) (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zolium bromide) assay was used to quantitatively examine the proliferation of chondrocytes. Results showed that chondrocytes proliferated better on the PHBHHx/PHB scaffolds than on PHB one. The maximal cell densities were all observed after 7 days of incubation. As for the blend polymers, cells grew better on scaffolds consisting of PHBHHx/PHB in ratios of 2:1 and 1:2 than they did on PHBHHx/PHB of 1:1. Scanning electron microscopy (SEM) also showed that large quantities of chondrocytes grew initially on the surface of the scaffold. After 7 days, they further grew into the open pores of the blend polymer scaffolds. Morphologically, cells found on the surface of the scaffold exhibited a flat appearance and slowly form confluent cell multilayers starting from 14 to 28 days of the growth. In contrast, cells showed rounded morphology, formed aggregates and islets inside the scaffolds. In addition, chondrocytes proliferated on the scaffold and preserved their phenotype for up to 28 days.     

The influence of scaffold material on chondrocytes under inflammatory conditions

Cartilage tissue engineering aims to repair damaged cartilage tissue in arthritic joints. As arthritic joints have significantly higher levels of pro-inflammatory cytokines (such as IL-1β and TNFα that cause cartilage destruction, it is critical to engineer stable cartilage in an inflammatory environment. Biomaterial scaffolds constitute an important component of the microenvironment for chondrocytes in engineered cartilage. However, it remains unclear how the scaffold material influences the response of chondrocytes seeded in these scaffolds under inflammatory stimuli. Here we have compared the responses of articular chondrocytes seeded within three different polymeric scaffolding materials (silk, collagen and polylactic acid (PLA)) to IL-1β and TNFα. These scaffolds have different physical characteristics and yielded significant differences in the expression of genes associated with cartilage matrix production and degradation, cell adhesion and cell death. The silk and collagen scaffolds released pro-inflammatory cytokines faster and had higher uptake water abilities than PLA scaffolds. Correspondingly, chondrocytes cultured in silk and collagen scaffolds maintained higher levels of cartilage matrix than those in PLA, suggesting that these biophysical properties of scaffolds may regulate gene expression and the response to inflammatory stimuli in chondrocytes. Based on this study we conclude that selecting the proper scaffold material will aid in the engineering of more stable cartilage tissues for cartilage repair, and that silk and collagen are better scaffolds in terms of supporting the stability of three-dimensional cartilage under inflammatory conditions.     

聚羟基烷酸酯聚合物负载软骨细胞修复同种异体喉软骨缺损

背景：软骨组织工程基础研究相当深入,但在耳鼻咽喉科实际应用研究颇少,探索组织工程技术简便实用的喉软骨修复方法是值得研究的课题。 目的：比较多孔海绵状聚羟基丁酸酯与聚羟基己酸酯共聚物生物材料负载软骨细胞体外培养形成的初期组织工程软骨组织与体内植入一定时期形成的较成熟组织工程软骨组织修复同种异体甲状软骨缺损的效果。 方法：收集体外培养第3代乳兔(3 d龄)软骨细胞,以多孔海绵状聚羟基丁酸酯与聚羟基己酸酯共聚物生物材料为细胞外基质,采用组织工程技术制备细胞-材料复合物,共同体外培养形成初级组织工程软骨组织后直接应用于成兔甲状软骨缺损的修复(实验组A,n=5)或将初级组织工程软骨组织体内植入一定时期形成较成熟组织工程软骨再应用于甲状软骨缺损的修复(实验组B,n=5)。设单纯聚羟基丁酸酯与聚羟基己酸酯共聚物材料修复组(对照A组,n=4)和单纯软骨细胞修复组(对照B组,n=4)作为对照。分别于术后4周(实验B组)和8周(实验A组、对照A组、对照B组)取材,对甲状软骨缺损修复效果进行大体和组织学评价。 结果与结论：两者大体支架形态基本一致,修复区与原有软骨均相续平坦,无凹陷及缺损。但实验A组存在界面无细胞区,修复区基质分泌不丰富;实验B组界面区有细胞生长,基质分泌良好。两者炎细胞浸润均不明显。对照组修复区凹陷,呈暗红色软组织充填,组织学及特殊染色检查未发现软骨样结构及其分泌的基质成分。结果表明在有免疫力的动物体内,初级组织工程软骨组织直接应用与体内植入后再应用均能有效修复同种异体甲状软骨缺损,无明显免疫反应;相同时期内,应用较成熟组织工程软骨组织修复效果优于应用初级组织工程软骨组织。然而,直接应用初级组织工程软骨组织可节省时间、成本、工作量及操作环节,避免二次皮下手术的痛苦,是比较实用的方法之一。     

The Effect of Hybridization of Hydrogels and Poly(L-lactide-co-ε-caprolactone) Scaffolds on Cartilage Tissue Engineering

For repairing cartilage defects by cartilage tissue engineering, it is important that engineered cartilage that is fabricated with scaffolds and cells can maintain the biological and physiological functions of cartilage, and also can induce three-dimensional spatial organization of chondrocytes. In this sense, hydrogels such as fibrin gels (FG) and hyaluronan (HA) are widely used for application in cartilage treatment. However, the use of hydrogels alone as a scaffold has a physical weakness; the mechanical properties of hydrogels are too weak to endure complex loading in the body. In this study, for mimicking a native cartilage microenvironment, we made cell–hybrid scaffold constructs with poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds and hydrogels to guide three-dimensional spatial organization of cells and extracellular matrix. A highly elastic scaffold was fabricated from PLCL with 85% porosity and 300–500 μm pore size using a gel-pressing method. The mixture of rabbit chondrocytes and hydrogels was seeded on PLCL scaffolds, and was subcutaneously implanted into nude mice for up to eight weeks. The cell seeding efficiency of the hybrid scaffolds with FG or HA was higher than that of the PLCL scaffolds. From in vivo studies, the accumulation of cartilaginous extracellular matrices of constructs, which was increased by hybridization of hydrogels and PLCL scaffolds, showed that the cell–hybrid scaffold constructs formed mature and well-developed cartilaginous tissue. In conclusion, the hybridization of hydrogels and PLCL scaffold for three-dimensional spatial organization of cells would provide a biomimetic environment where cartilage tissue growth is enhanced and facilitated. It can enhance the production of cartilaginous extracellular matrices and, consequently, improve the quality of the cartilaginous tissue formed.     

不同生物材料复合支架对关节软骨缺损的修复评价

背景：不同生物材料制备的复合软骨支架其修复软骨缺损也各具特点。 目的：探讨不同生物材料制备复合支架的组织工程学特性及其修复关节软骨缺损的性能评价。 方法：以“软骨组织工程,生物材料,工程软骨,复合支架”为中文关键词,以“tissue enginneering,articular cartilage,scaffold material”为英文关键词,采用计算机检索中国期刊全文数据库、PubMed数据库(1993-01/2010-11)相关文章。纳入复合支架材料-细胞复合物修复关节软骨损伤等相关的文章,排除重复研究或Meta分析类文章。 结果与结论：复合支架是当前软骨组织工程中应用较多的支架,它是将具有互补特征的生物相容性可降解支架,按一定比例和方式组合,设计出结构与性能优化的复合支架。较单一支架材料具有更好的生物相容性和一定强度的韧性,较好的孔隙和机械强度。复合支架的制备不仅包括同一类生物材料的复合,还包括不同类别生物材料之间的交叉复合。可分为纯天然支架材料、纯人工支架材料以及天然与人工支架材料的复合等3类。复合支架使生物材料具有互补特性,一定程度上满足了理想生物材料支架应具的综合特点,但目前很多研究仍处于实验阶段,还有一些问题有待于解决,如不同材料的复合比例、复合工艺等。     

Effect of initial cell seeding density on 3D-engineered silk fibroin scaffolds for articular cartilage tissue engineering

The repair of articular cartilage defects poses a continuing challenge. Cartilage tissue engineering through the culture of chondrocytes seeded in 3D porous scaffolds has the potential for generating constructs that repair successfully. It also provides a platform to study scaffold-cell and cell-cell interactions. The scaffold affects the growth and morphology of cells growing on it, and concomitantly, cells affect the properties of the resultant tissue construct. Silk fibroin protein from Antheraea mylitta, a non-mulberry Indian tropical tasar silkworm, is a potential biomaterial for diverse applications due to its widespread versatility as a mechanically robust, biocompatible, tissue engineering material. Analysis of silk fibroin scaffolds seeded with varying initial densities (25, 50 and 100 million cells/ml) and cultured for 2 weeks showed that thickness and wet weight increased by 60-70% for the highest cell density, and DNA, GAG and collagen content of the cartilaginous constructs increased with increasing cell density. Mechanical characterization of the constructs elucidated that the highest density constructs had compressive stiffness and modulus 4-5 times that of cell-free scaffolds. The present results indicate the importance of cell seeding density in the rapid formation of a functional cartilaginous tissue.     

Tissue engineering of articular cartilage using an allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold (ACHMS scaffold)

The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or non-filled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects.     

Nasal chondrocytes and fibrin sealant for cartilage tissue engineering

Hybrid constructs associating a biodegradable matrix and autologous chondrocytes hold promise for the treatment of articular cartilage defects. In this context, our objective was to investigate the potential use of nasal chondrocytes associated with a fibrin sealant for the treatment of articular cartilage defects. The phenotype of primary nasal chondrocytes (NC) from human (HNC) and rabbit (RNC) origin were characterized by RT-PCR. The ability of constructs associating fibrin sealant and NC to form a cartilaginous tissue in vivo was investigated, firstly in a subcutaneous site in nude mice and secondly in an articular cartilage defect in rabbit. HNC express type II collagen and aggrecan, the two major hallmarks of a chondrocytic phenotype. Furthermore, when injected subcutaneously into nude mice within a fibrin sealant, these chondrocytes were able to form a cartilage-like tissue. Our data indicate that RNC also express type II collagen and aggrecan and maintained their phenotype in three-dimensional culture within a fibrin sealant. Moreover, treatment of rabbit articular cartilage defects with autologous RNC embedded in a fibrin sealant led to the formation of a hyalin-like repair tissue. The use of fibrin sealant containing hybrid autologous NC therefore appears as a promising approach for cell-based therapy of articular cartilage.     

丝素蛋白-壳聚糖支架复合骨髓间充质干细胞体内构建组织工程化软骨的生物相容性

文题释义： 生物相容性：是指生命体组织对非活性材料产生的一种性能,一般是指材料与宿主之间的相容性,包括组织相容性和血液相容性。 检测相容性的方法：是将支架材料与种子细胞在体外共培养,检测支架毒性、细胞活性、细胞增殖及细胞与支架的黏附情况等指标,该方法具有客观性强、可重复性强、影响因素相对简单及敏感性高等特点。 背景：课题组前期的研究中发现,丝素蛋白-壳聚糖支架材料复合诱导后骨髓间充质干细胞在兔体内能修复缺损的软骨组织,但对于该组织工程化软骨组织的生物相容性还未进一步研究。 目的：研究丝素蛋白-壳聚糖支架材料复合骨髓间充质干细胞在体内构建组织工程化软骨的生物相容性。 方法：使用丝素蛋白-壳聚糖按1∶1比例混合制备三维支架材料,提取兔骨髓间充质干细胞,将诱导后的骨髓间充质干细胞与丝素蛋白-壳聚糖支架构建修复体,再将修复体移植到兔关节软骨缺损模型中修复软骨组织。实验分为3组,实验组植入诱导后骨髓间充质干细胞+丝素蛋白-壳聚糖支架,对照组植入丝素蛋白-壳聚糖支架干预,空白组未植入修复体。 结果与结论：①实验成功制备丝素蛋白-壳聚糖三维支架材料及提取骨髓间充质干细胞,并构建软骨缺损的修复体,将修复体植入兔体内能成功修复缺损的软骨组织;②建模后2,4,8,12周,3组血常规、降钙素原、血沉、C-反应蛋白结果提示无明显的全身感染征象,3组血常规及肝肾功能各时间段比较差异无显著性意义(P > 0.05);③一般观察、苏木精-伊红染色及扫描电镜观察：建模后12周,相比其他两组,实验组软骨缺损已修复,支架材料已吸收,修复组织周围未见炎性细胞,修复组织已正常组织整合良好;④结果证实,丝素蛋白-壳聚糖支架复合骨髓间充质干细胞在体内构建的组织工程化软骨具有良好的生物相容性。 ORCID: 0000-0002-8139-1175(佘荣峰) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro

Augmentative and reconstructive rhinoplasty surgical procedures use autologous tissue grafts or synthetic grafts to repair the nasal defect and aesthetic reconstruction. Donor site trauma and morbidity are common in autologous grafts. The desperate need for the production of grafted 3D cartilage tissues as rhinoplasty grafts without the adverse effect is the need of the hour. In the present study, we developed a bioactive 3D histotypic construct engineered with the various ratio of adipose-derived stem cells (ADSC) and chondrocytes together with decellularized porcine nasal cartilage graft (dPNCG). We decellularized porcine nasal cartilage using supercritical carbon dioxide (SCCO2) extraction technology. dPNCG was characterized by H&E, DAPI, alcian blue staining, scanning electron microscopy and residual DNA content, which demonstrated complete decellularization. 3D histotypic constructs were engineered using dPNCG, rat ADSC and chondrocytes with different percentage of cells and cultured for 21 days. dPNCG together with 100% chondrocytes produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. H&E and alcian blue staining showed an intact mass, with cartilage granules bound to one another by extracellular matrix and proteoglycan, to form a 3D structure. Besides, the expression of chondrogenic markers, type II collagen, aggrecan and SOX-9 were elevated indicating chondrocytes cultured on dPNCG substrate facilitates the synthesis of type II collagen along with extracellular matrix to produce 3D histotypic cartilage. To conclude, dPNCG is an excellent substrate scaffold that might offer a suitable environment for chondrocytes to produce 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty.     

以交联透明质酸钠为支架体外构建组织工程软骨

背景：采用组织工程技术再生和重建软骨是目前修复软骨组织缺损效果最好、最有应用前景的方法。 目的：以体外培养的软骨细胞和交联透明质酸钠为支架材料,开发一套体外构建组织工程软骨的完整方案。 方法：分离新西兰兔膝关节软骨细胞,制成细胞悬液滴加于交联透明质酸钠支架上,体外复合培养21 d,提取RNA进行RT-PCR检测,制备冰冻切片进行显微观察和免疫组织化学观察。 结果与结论：软骨细胞接种于交联透明质酸钠支架材料后,可贴附于支架上生长,并且大量细胞聚集成团,在支架材料的纤维间隙中生长或呈单层细胞附着于支架材料纤维。细胞-支架复合物表达软骨组织特异性蛋白聚糖基因和Ⅱ型胶原α1基因,以及软骨组织特异性蛋白Ⅱ型胶原蛋白,可维持软骨细胞表型。表明培养的细胞-支架复合物在体外培养可形成软骨细胞外基质,有望获得组织工程软骨组织。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：