组织工程软骨体外构建的相关实验研究

目的 探索组织工程软骨体外构建技术体系可行性.方法 种子细胞选用胎儿软骨细胞(口服药物流产胎儿,胎龄3～6个月).酶消化法获得第1代细胞,以50×106/ml浓度均匀接种于经聚乳酸(PLA)包埋聚乙醇酸(PGA)高分子聚合物支架,形成细胞-支架复合体,在体外静态培养.分别于2周、4周、8周进行大体观察、扫描电镜及组织学检测.结果 体外构建的组织工程软骨,随培养时间延长,色泽由2周时的乳白色逐渐呈现半透明,8周时接近正常软骨外观.扫描电镜显示软骨细胞与材料具有良好相容性,培养7天PGA纤维之间有基质沉积.HE染色示2周有大量软骨陷窝形成和均匀嗜碱性基质分泌,Safranin'O染色示基质有酸性蛋白多糖分布,Massons's trichome染色示基质有胶原成分,但含量较少,经免疫组织化学检测为特异Ⅱ型胶原.培养4周胶原成分开始明显增多,软骨陷窝形态接近成熟,8周细胞外基质蛋白多糖和Ⅱ型胶原含量丰富且分布均匀.结论 以成熟软骨细胞为种子细胞,运用组织工程技术在体外能构建出具有正常软骨组织结构特征的人组织工程软骨.     

Feasibility of Autologous Bone Marrow Mesenchymal Stem Cell–Derived Extracellular Matrix Scaffold for Cartilage Tissue Engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell–derived ECM (aBMSC‐dECM) membrane was collected and fabricated into a three‐dimensional porous scaffold via cross‐linking and freeze‐drying techniques. Articular chondrocytes were seeded into the aBMSC‐dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC‐dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC‐dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC‐dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC‐dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.     

PLGA–PTMC–Cultured Bone Mesenchymal Stem Cell Scaffold Enhances Cartilage Regeneration in Tissue‐Engineered Tracheal Transplantation

The treatment of long‐segment tracheal defect requires the transplantation of effective tracheal substitute, and the tissue‐engineered trachea (TET) has been proposed as an ideal tracheal substitute. The major cause of the failure of segmental tracheal defect reconstruction by TET is airway collapse caused by the chondromalacia of TET cartilage. The key to maintain the TET structure is the regeneration of chondrocytes in cartilage, which can secrete plenty of cartilage matrices. To address the problem of the chondromalacia of TET cartilage, this study proposed an improved strategy. We designed a new cell sheet scaffold using the poly(lactic‐co‐glycolic acid) (PLGA) and poly(trimethylene carbonate) (PTMC) to make a porous membrane for seeding cells, and used the PLGA–PTMC cell‐scaffold to pack the decellularized allogeneic trachea to construct a new type of TET. The TET was then implanted in the subcutaneous tissue for vascularization for 2 weeks. Orthotopic transplantation was then performed after implantation. The efficiency of the TET we designed was analyzed by histological examination and biomechanical analyses 4 weeks after surgery. Four weeks after surgery, both the number of chondrocytes and the amount of cartilage matrix were significantly higher than those contained in the traditional stem‐cell–based TET. Besides, the coefficient of stiffness of TET was significantly larger than the traditional TET. This study provided a promising approach for the long‐term functional reconstruction of long‐segment tracheal defect, and the TET we designed had potential application prospects in the field of TET reconstruction.     

培养软骨移植修复关节软骨缺损的实验研究

目的：为探讨一种新的关节软骨缺损修复方法。方法：将体外培养２周形成软骨样组织,移植修复兔关节软骨全层缺损。于移植术后２、４、８周分别行功能评价、大体形态及组织学检查。结果：全部实验兔于术后２周内恢复正常活动。２周时移植修复组织由非成熟透明软骨组成。４周时部分移植组出现成熟透明软骨。８周时移植组关节软骨缺损全部由成熟透明软骨充填修复,修复组织与邻近关节软骨融合。培养软骨移植修复关节软骨全层缺损明显优于自身修复（Ｐ＜００１）。结论：本实验提示使用具有高有丝分裂率的软骨细胞,经离心管培养形成骺软骨样组织,植入关节软骨全层缺损后,软骨细胞生长良好,逐渐成熟和转化,能发挥良好的修复作用。     

软骨细胞膜片复合3D打印内支撑构建管状软骨的初步研究

目的 利用猪耳廓软骨细胞制备软骨细胞膜片,与3D打印聚酰胺带孔管状支架结合,构建管状软骨组织,用于气管软骨的缺损重建。方法 用酶消化法分离获得原代猪耳廓软骨细胞,以5×105cells/m L的密度将第2代软骨细胞接种于6 cm细胞培养皿上,在软骨细胞膜片培养环境中培养约14 d,获得完整软骨细胞膜片;4层软骨细胞膜片依次卷叠于3D打印的聚酰胺(PA12)带孔管状内支架上,以构建管状软骨组织复合物;体外静态培养12周或裸鼠皮下培养8周,对形成的管状软骨组织行大体观察及石蜡切片组织学染色鉴定。结果 体外静态培养12周后,半透明管状软骨组织形成,组织学染色可见少量分泌的软骨细胞外基质,质软,弹性差;体内培养8周后,白色管状软骨组织形成,其组织学染色可见大量软骨细胞外基质分泌,质较硬,有一定弹性。结论 利用软骨细胞膜片复合3D打印聚酰胺支架,可构建组织工程管状软骨,有望将其用于气管软骨缺损的修复。     

藻酸钙载体复合软骨细胞成软骨的实验观察

目的：通过软骨细胞复合于载体后在同种异体动物体内异位成软骨情况，为组织工程方法修复关节软骨缺损的研究提供实验依据。方法：用一定浓度的海灌酸钠溶液与葡萄糖酸钙溶液按比例混合均匀，置特殊柱形模具中经冷冻、脱水得到具有一定弹性和韧性的柱形灌酸钙载体，将其复合软骨细胞植入同种动物体内，分别于术后2、4周取材进行组织学观察。结果：软骨细胞在体内生长增殖良好，4周时可见新生软骨组织形成。结论：灌酸钙可作为载体复合细胞进行软骨组织工程的研究。     

Novel Strategy to Engineer Trachea Cartilage Graft With Marrow Mesenchymal Stem Cell Macroaggregate and Hydrolyzable Scaffold

Limited donor sites of cartilage and dedifferentiation of chondrocytes during expansion, low tissue reconstruction efficiency, and uncontrollable immune reactions to foreign materials are the main obstacles to overcome before cartilage tissue engineering can be widely used in the clinic. In the current study, we developed a novel strategy to fabricate tissue‐engineered trachea cartilage grafts using marrow mesenchymal stem cell (MSC) macroaggregates and hydrolyzable scaffold of polylactic acid–polyglycolic acid copolymer (PLGA). Rabbit MSCs were continuously cultured to prepare macroaggregates in sheet form. The macroaggregates were studied for their potential for chondrogenesis. The macroaggregates were wrapped against the PLGA scaffold to make a tubular composite. The composites were incubated in spinner flasks for 4 weeks to fabricate trachea cartilage grafts. Histological observation and polymerase chain reaction array showed that MSC macroaggregates could obtain the optimal chondrogenic capacity under the induction of transforming growth factor‐β. Engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans. PLGA scaffold degraded totally during in vitro incubation and the engineered cartilage graft was composed of autologous tissue. Based on this novel, MSC macroaggregate and hydrolyzable scaffold composite strategy, ready‐to‐implant autologous trachea cartilage grafts could be successfully fabricated. The strategy also had the advantages of high efficiency in cell seeding and tissue regeneration, and could possibly be used in future in vivo experiments.     

应用细胞膜片复合硅胶管内支撑构建组织工程管状软骨

目的 研究利用羊耳软骨细胞形成细胞膜片,构建无细胞支架组织工程管状软骨的可行性.方法 体外培养扩增羊耳软骨细胞至第2代,以2.0×106 cells/mL的高密度,培养7d,形成直径为35 mm的圆形细胞膜片,将细胞膜片均匀包裹于硅胶管,植入裸鼠背部皮下,4周后取材检测.以大体观察、组织学、免疫组织化学等方法,对形成的软骨进行评价.结果 大体观察可见形成良好的管状软骨,HE染色见软骨陷窝形态规则,番红O染色及Ⅱ型胶原免疫组化均呈阳性表达.结论 细胞膜片复合硅胶管内支撑的方法能形成管状软骨,为气管软骨的再造提供了新的方法.     

利用软骨细胞膜片技术在山羊皮下构建软骨样组织的研究

目的 探讨软骨细胞膜片技术在大型哺乳动物体内构建软骨的优越性.方法 以山羊耳廓软骨细胞为种子细胞,分别利用细胞膜片技术(实验组)和细胞复合PGA/PLA支架材料的方法(对照组)构建软骨组织.体外培养4周回植到动物腹部皮下,分别于回植前、回植后4周及回植后8周时采集标本,观察比较两组软骨形成情况.结果 体外培养4周后,两组均形成一定量的软骨样组织,但对照组可见未降解的材料纤维;体内构建4周后,实验组形成成熟软骨样组织,对照组为纤维结缔组织替代,伴有大量炎性细胞浸润;体内培养8周时,实验组较4周前无明显变化,对照组体积明显缩小,仍为纤维结缔组织.结论 软骨细胞膜片技术与细胞复合PGA/PLA支架材料构建组织工程软骨方法相比,原代细胞用量少,体外培养时间短,在大动物体内能形成成熟软骨,具有更广泛的临床应用前景.     

软骨细胞和骨髓间质干细胞混合培养构建组织工程软骨的实验研究

目的 探讨软骨细胞和骨髓间质干细胞(bone mesenchymal stem cells,BMSCs)混合培养对构建组织工程软骨的影响,并确定两者的最佳比例.方法 体外分离培养兔(1月龄)关节软骨细胞和BMSCs,按不同比例(软骨细胞和BMSCs的浓度比为:4:1、2:1、1:1、1:2、1:4及单纯软骨细胞)混合培养一代.以4×107/ml的细胞终浓度接种40μl于聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA:4mm×4mm×2mm),静态培养2 d,然后移至周期性压力场(压力0～200 kPa,频率:0.1 Hz,时间:8 h/d)培养3周.收获组织工程软骨行大体观察,组织切片,定量检测糖胺聚糖(glyeosaminoglycans,GAGs)含量、DNA含量及Ⅱ型胶原染色面积.结果 软骨细胞和BMSCs混合培养组与单纯软骨细胞组相比,体积较大,表面光滑,有弹性,有光泽.组织学检查显示混合培养组结构致密,细胞外基质分布更均匀,其中软骨细胞和BMSCs的浓度比为2:1组可见软骨陷窝.混合培养组的Ⅱ型胶原染色面积、GAGs含量、DNA含量高于单纯软骨细胞组,其中软骨细胞和BMSCs的浓度比为2:1组含量最高,与其他比例混合组比较,差异有统计学意义.结论 软骨细胞和BMSCs混合培养能提高组织工程软骨的质量,其中以软骨细胞和BMSCs的浓度比为2:1最佳.     

体内外环境对组织工程软骨组织结构与力学性能的影响

目的 探讨体外构建组织工程化软骨在体、内外环境中力学性能及组织结构的变化,以及微环境对组织形成的影响,为工程化软骨构建提供适当参数。方法 体外培养扩增人胎儿关节软骨细胞,取第2代细胞以6×107个细胞/ml密度接种到聚羟基乙酸/聚乳酸(Polyglycolic acid/Polylactic acid,PGA/PLA)材料制成的圆柱形三维支架上,常规体外培养4周后,分为体内组(C、D组)和体外组（A、B组）,C、D组植于裸鼠皮下,A、B组继续常规培养液培养,于6、12周后取材,以正常软骨作为对照,行大体观察,以及组织学、组织化学、生物力学、超微结构等检测。结果 A、B、C、D4组均形成大体形态良好的透明软骨样组织。C、D组软骨呈乳白色,表面光滑,超微结构上胶原纤维排列致密而有规则,可形成有横纹的粗大胶原纤维,类似正常成人软骨;A、B组颜色偏黄,表面略粗糙,外观及超微结构近似半透明的胎儿关节软骨。工程化软骨植入体内12周后压缩弹性模量及胶原直径分别为（38.28±3.95）MPa和（41.58 ±2.78）nm,明显优于体外同时期组的(4.12±0.63) MPa和（15.83±1.70）nm(P ＜0.01)。结论 组织工程软骨的结构和功能在体内环境逐步成熟,体内组软骨超微结构上能形成粗大胶原纤维网络,胶原的交联增强可能是其力学性能较体外组明显提高的重要原因。     

组织工程化软骨修复兔膝关节软骨缺损的实验研究

目的 评估同种异体组织工程软骨修复兔膝关节全层软骨缺损的有效性。方法分离收集成年新西兰大白兔软骨细胞进行体外培养。建立双侧兔膝关节软骨缺损模型，用去端肽胶原（atelocollagen）凝胶与所培养的异体兔关节软骨细胞共同植入兔膝关节软骨缺损处，并设对照组。分别于手术后4周、8周观察大体标本以及组织学修复结果，并进行Wakitan的评分，评估此方法的有效性。结果大体观察结果表明，与对照组相比，实验组缺损处由软骨组织修复而对照组缺损处由纤维样组织填充。组织学观察可以见到实验组关节软骨缺损处有密集的软骨细胞而对照组关节缺损处只有纤维细胞无软骨细胞。结论通过短期观察表明以同种异体软骨细胞-去端肽胶原复合物修复全层软骨缺损的方法是有效可行的，为其进一步临床应用提供了参考。     

不同比例软骨细胞与间充质干细胞对构建组织工程软骨的影响

目的以不同比例将大鼠软骨细胞及骨髓间充质干细胞（bonemarrowstromalceu,BMSCs）依次体外及体内混合培养,探讨二者构建组织工程软骨的最佳比例。方法分别取1个月及1d龄SD大鼠的BMSCs及关节软骨,原代软骨细胞与第2代BMSCs混合比例为全软骨细胞组、3：1、1：1、1：3、全BMSCs组,共五组。以终浓度4×10^7／mI．种于聚乳酸一聚羟基乙酸支架上,体外培养4周后,植入裸鼠皮下继续培养,8周后对标本进行大体观察、组织学切片、Ⅱ型胶原免疫荧光染色及检测氨基糖胺多糖的含量。结果软骨细胞与BMSCs混合比例3：1组较其他各组所构建出的组织工程软骨体积大,厚度厚,有光泽;组织染色见软骨细胞增殖较旺盛,被大量细胞外基质包围,分布均匀,并可见软骨陷窝;Ⅱ型胶原免疫荧光染色见细胞内和细胞外基质发出的红色荧光较明亮;3：1组的组织块氨基糖胺多糖含量（470．38±29．78）μg高于其他各组（P〈O．05）。结论软骨细胞与BMSCs混合培养有助于节省软骨细胞,二者混合浓度比例以3：1最好。     

胶原/羟基磷灰石支架负载软骨细胞修复兔膝关节骨软骨缺损

目的 探讨胶原复合梯度羟基磷灰石(Col/HA)双相支架负载软骨细胞修复兔膝关节骨软骨缺损的可行性及疗效.方法 构建Col/HA双相支架,将软骨细胞种植于支架培养1周,再将软骨细胞-支架复合体移植修复兔膝关节股骨髁的骨软骨缺损,并对骨软骨缺损的修复进行检测.结果 光镜及扫描电镜观察显示软骨细胞在Col/HA支架中贴附良好,表型维持稳定,分泌胞外基质.大体观察和组织学检测显示,植入体内16周后实验组软骨层呈透明软骨样修复,软骨下骨缺损有新骨构建;对照组骨软骨缺损修复不良,组织学检测以纤维性组织或纤维软骨组织形成.Wakitani评分显示实验组修复组织优于对照组,差异有统计学意义(P＜0.05).结论 双相Col/HA复合支架可作为骨软骨组织工程支架,负载软骨细胞可修复兔膝关节骨软骨缺损,重建关节软骨的结构和功能.     

Effects of a cultured autologous chondrocyte-seeded type II collagen scaffold on the healing of a chondral defect in a canine model.

Using a previously established canine model for repair of articular cartilage defects, this study evaluated the 15-week healing of chondral defects (i.e., to the tidemark) implanted with an autologous articular chondrocyte-seeded type II collagen scaffold that had been cultured in vitro for four weeks prior to implantation. The amount and composition of the reparative tissue were compared to results from our prior studies using the same animal model in which the following groups were analyzed: defects implanted with autologous chondrocyte-seeded collagen scaffolds that had been cultured in vitro for approximately 12 h prior to implantation, defects implanted with autologous chondrocytes alone, and untreated defects. Chondrocytes, isolated from articular cartilage harvested from the left knee joint of six adult canines, were expanded in number in monolayer for three weeks, seeded into porous type II collagen scaffolds, cultured for an additional four weeks in vitro and then implanted into chondral defects in the trochlear groove of the right knee joints. The percentages of specific tissue types filling the defects were evaluated histomorphometrically and certain mechanical properties of the repair tissue were determined. The reparative tissue filled 88+/-6% (mean+/-SEM; range 70-100%) of the cross-sectional area of the original defect, with hyaline cartilage accounting for 42+/-10% (range 7-67%) of defect area. These values were greater than those reported previously for untreated defects and defects implanted with a type II collagen scaffold seeded with autologous chondrocytes within 12 h prior to implantation. Most striking, was the decreased amount of fibrous tissue filling the defects in the current study, 5+/-5% (range 0-26%) as compared to previous treatments. Despite this improvement, indentation testing of the repair tissue formed in this study revealed that the compressive stiffness of the repair tissue was well below (20-fold lower stiffness) that of native articular cartilage.     

传代对残耳软骨细胞体内软骨形成能力的影响

目的：研究传代对残耳软骨细胞体内软骨形成能力的影响。方法分离培养人残耳软骨细胞,将第3-8代细胞分别复合聚羟基乙酸/聚乳酸支架,构建组织工程化软骨;体外培养4周后植入裸鼠体内观察8周。采用组织学染色观察各组标本的软骨形成情况;Real-time PCR检测软骨分化相关基因的表达;生物力学分析新生软骨的弹性模量。结果各代复合物体外培养4周时均不能形成软骨组织,但第3-5代残耳软骨细胞COL 2A1、第3-4代的SOX 9和第3代的DLK 1仍可维持较高的表达水平（P〈0.05）;体内植入8周后,第3-6代复合物均有不同程度的弹性软骨结构形成,并随代次增高而减少,第3-6代复合物的弹性模量明显高于第7、8代。结论残耳软骨细胞传至第4代仍能保持良好的体内软骨形成能力,但扩增传代对残耳软骨细胞软骨表型去分化的影响在第7代后已无法逆转。     

同种异体软骨细胞移植术后关节软骨蛋白多糖的测定

目的 应用Pluronic F-127负载同种异体软骨细胞移植修复兔全厚关节软骨损伤,对于新生的修复组织进行基质蛋白多糖含量测定,以探讨此方法修复全厚关节软骨损伤的可行性.方法 取3个月龄新西兰大白兔关节软骨细胞体外培养扩增,与20%Plurortic F-127凝胶混合.选27只健康同种成年大白兔,人为造成双侧膝关节软骨缺损.实验组软骨缺损处植入培养的软骨细胞/Pluronic F-127混合物,对照组缺损处单纯注入Pluronic F-127凝胶和空白对照.然后,对修复组织进行大体观察及蛋白多糖含量测定.结果 移植的软骨细胞-载体复合物中的软骨细胞能良好地生长,12周时再生组织与周围正常软骨组织外观相似,界限模糊.实验组与对照组各时期蛋白多糖含量均有非常显著性差异,实验组不同时期的蛋白多糖含量之间均有显著性差异,实验组12周时蛋白多糖含量与正常软骨组织无显著性差异.结论 Pluronic F-127负载同种异体软骨细胞移植是治疗关节软骨缺损的有效方法.     

以聚羟基丁酸已酯为支架材料的组织工程隆鼻法的实验研究

目的:研究以聚羟基丁酸已酯(PHB)为支架材料形成的组织工程软骨作为隆鼻填充材料的可行性。方法:实验组:取兔的耳软骨细胞,行体外培养扩增,达到一定数量后,再接种于PHB支架上,置于新西兰白兔的鼻背部骨膜下,从大体和组织学方面观察软骨形成情况;对照组:单纯以未接种软骨细胞的支架移植于兔的鼻背部骨膜下,于相同时间点进行检测。结果:实验组:植入4周后该复合物在兔鼻背下支架表层有软骨层形成,并可见软骨细胞突入至支架材料中间。8周后支架材料继续降解,软骨细胞及细胞基质生成明显,伴有少量血管及胶原长入,对照组无相应变化。结论:以聚羟基丁酸已酯(PHB)为支架材料的组织工程软骨可用于隆鼻的填充材料。     

Mechanical properties and structure‐function relationships of human chondrocyte‐seeded cartilage constructs after in vitro culture

Autologous Chondrocyte Implantation (ACI) is a widely recognized method for the repair of focal cartilage defects. Despite the accepted use, problems with this technique still exist, including graft hypertrophy, damage to surrounding tissue by sutures, uneven cell distribution, and delamination. Modified ACI techniques overcome these challenges by seeding autologous chondrocytes onto a 3D scaffold and securing the graft into the defect. Many studies on these tissue engineered grafts have identified the compressive properties, but few have examined frictional and shear properties as suggested by FDA guidance. This study is the first to perform three mechanical tests (compressive, frictional, and shear) on human tissue engineered cartilage. The objective was to understand the complex mechanical behavior, function, and changes that occur with time in these constructs grown in vitro using compression, friction, and shear tests. Safranin‐O histology and a DMMB assay both revealed increased sulfated glycosaminoglycan (sGAG) content in the scaffolds with increased maturity. Similarly, immunohistochemistry revealed increased lubricin localization on the construct surface. Confined compression and friction tests both revealed improved properties with increased construct maturity. Compressive properties correlated with the sGAG content, while improved friction coefficients were attributed to increased lubricin localization on the construct surfaces. In contrast, shear properties did not improve with increased culture time. This study suggests the various mechanical and biological properties of tissue engineered cartilage improve at different rates, indicating thorough mechanical evaluation of tissue engineered cartilage is critical to understanding the performance of repaired cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2298–2306, 2017.

     

Construction of a Tissue‐Engineered Annulus Fibrosus

The intervertebral disc is composed of load‐bearing fibrocartilage that may be subjected to compressive forces up to 10 times the body weight. The multilaminated outer layer, the annulus fibrosus (AF), is vulnerable to damage and its regenerative potential is limited, sometimes leading to nuclear herniation. Scaffold‐based tissue engineering of AF using stem cell technology has enabled the development of bi‐laminate constructs after 10 weeks of culture. It is difficult to know if these constructs are limited by the differentiation state of the stem cells or the culture system. In this study, we have characterized an expandable scaffold‐free neoconstruct using autologous AF cells. The construct was prepared from pellet cultures derived from monolayer cultures of AF cells from mature pigs that became embedded in their own extracellular matrix. The pellet cultures were incubated for 24 h in a standardized conical tube and then carefully transferred intact to a culture flask and incubated for 21 days to allow continued matrix synthesis. Cell viability was maintained above 90% throughout the culture period. The engineered scaffold‐free construct was compared with the native AF tissue by characterization of gene expression of representative markers, histological architecture, and biochemical composition. The morphological and biochemical characteristics of the cultured disc construct are very similar to that of native AF. The cell number per gram of construct was equal to that of native AF. Expression of aggrecan was elevated in the engineered construct compared with RNA extracted from the AF. The glycosaminoglycan content in the engineered construct showed no significant difference to that from native construct. These data indicate that scaffold‐free tissue constructs prepared from AF cells using a pellet‐culture format may be useful for in vitro expansion for transplantation into damaged discs.