Effects of serial expansion of septal chondrocytes on tissue-engineered neocartilage composition.

OBJECTIVES: Cartilage grafts for reconstructive surgery may someday be created from harvested autologous chondrocytes that are expanded and seeded onto biodegradable scaffolds in vitro. This study sought to quantify the biochemical composition of neocartilage engineered from human septal chondrocytes and to examine the effects of cell multiplication in monolayer culture on the ultimate composition of the neocartilage. METHODS: Human septal chondrocytes from 10 donors were either seeded immediately after harvest (passage 0 [P(0)]) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P(1)) and passage 2 (P(2)). Cell/scaffold constructs were grown in vitro for 7, 14, and 28 days. Neocartilage constructs underwent histologic analysis for matrix sulfated glycosaminoglycan (S-GAG) and type II collagen as well as quantitative assessment of cellularity (Hoescht 33258 assay), S-GAG content (dimethylmethylene blue assay), and collagen content (hydroxyproline assay). RESULTS: Histologic sections of constructs seeded with P(0) cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P(1) and P(2) cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P(0) constructs was higher than in either P(1) (P < 0.05) or P(2) (P < 0.01) constructs, whereas cellularity and total collagen content showed no difference between passages. CONCLUSION: Neocartilage created from chondrocytes that have undergone serial passages in monolayer culture exhibited decreased matrix S-GAG and type II collagen, indicative of cellular dedifferentiation. SIGNIFICANCE: The alterations of matrix composition produced by dedifferentiated chondrocytes may limit the mechanical stability of neocartilage constructs.     

软骨细胞与静电纺丝聚已内酯支架灌流培养构建组织工程软骨的实验研究

目的 采用静电纺丝聚已内酯(polycaprolactone,PCL)支架与软骨细胞复合培养,比较静态和灌流生物反应器培养条件下对细胞增殖及基质分泌的影响.方法 构建PCL支架,自制灌流生物反应器,分离兔软骨细胞,培养后接种于PCL支架,分为灌流培养组和静态培养组.在培养第3、7、14天对支架-细胞复合体行扫描电镜观察,DNA、糖胺聚糖和总胶原定量检测;在培养第14天分析软骨特异性基因表达并观察软骨基质分泌情况.结果 电镜观察PCL支架纤维直径(1.67±0.76) μm,孔径(17.65土7.11)μm,可见支架中软骨细胞黏附生长良好,灌流培养条件下细胞增殖快,且较好地保持了软骨细胞特征形态.在培养第7天,灌流培养组DNA定量高于静态培养组;在培养第3、7和14天,灌流培养组糖胺聚糖定量均高于静态培养组,灌流培养组糖胺聚糖/DNA比值均高于静态培养组.在培养第14天,灌流培养组Ⅱ型胶原、蛋白聚糖基因表达增加;软骨分化指数高于静态培养组.在培养第14天,组织学染色可见灌流培养促进细胞的增殖和渗透生长,提高了软骨基质的分泌,并见软骨陷窝样结构.结论 在灌流生物反应器培养条件下,静电纺丝PCL支架与软骨细胞复合培养可促进软骨细胞的增殖和基质的分泌,提高了组织工程软骨的质量.     

Fate of a chimeric joint construct in an ectopic site in SCID mice

This study examines the use of a devitalized biological knee as a scaffold for repopulation with chondrocytes and tests the hypothesis that the devitalized scaffold would become repopulated with the foreign chondrocytes when placed in a suitable environment. Chimeric knee constructs were engineered in vitro and their ectopic in vivo fate was examined in SCID mice. The constructs were made by applying porous collagen sponges that contained viable bovine articular chondrocytes to shaved articular surfaces of devitalized embryonic chick knees. The chimeric joints were cultured for 1 week and were subsequently transplanted into dorsal subcutaneous pouches of 5-week-old mice. Specimens were prepared for histological analysis at 1, 3, 6, or 8 weeks after transplantation. Controls included empty collagen sponges, collagen sponges seeded with viable bovine chondrocytes, and devitalized chick knees without collagen sponge inserts. One week after in vitro incubation of the constructs, the porous collagen sponges with viable bovine chondrocytes were adherent to the shaved articular surfaces of the devitalized chick joints. There was abundant metachromatic neomatrix around the chondrocytes in the collagen sponges. During maintenance of the constructs in vivo, the chimeric joints exhibited dramatic changes. Bovine chondrocytes proliferated in the collagen sponges and formed abundant new matrix. Bovine chondrocytes migrated into preexisting chick cartilage canals at 1 week. Subsequently, bovine chondrocytes invaded the matrix of the devitalized chick knees. Bovine neocartilage obliterated the interface between the collagen sponge and the devitalized chick cartilage. With time in vivo, the bovine neocartilage expanded and replaced the chick matrix. The devitalized cartilage appears to provide a framework for supporting chondrogenesis in a chimeric joint.     

The culture of articular chondrocytes in hydrogel constructs within a bioreactor enhances cell proliferation and matrix synthesis

Bovine and human articular chondrocytes were seeded in 2% alginate constructs and cultured for up to 19 days in a rotating-wall-vessel (RWV) and under static conditions. Culture within the RWV enhanced DNA levels for bovine chondrocyte-seeded constructs when compared with static conditions but did not produce enhancement for human cells. There was a significant enhancement of glycosaminoglycans and hydroxyproline synthesis for both bovine and human chondrocytes. In all cases, histological analysis revealed enhanced Safranin-O staining in the peripheral regions of the constructs compared with the central region. There was an overall increase in staining intensity after culture within the RWV compared with static conditions. Type-II collagen was produced by both bovine and human chondrocytes in the peripheral and central regions of the constructs and the staining intensity was enhanced by culture within the RWV. A capsule of flattened cells containing type-I collagen developed around the constructs maintained under static conditions when seeded with either bovine or human chondrocytes, but not when cultured within the RWV bioreactor.     

Culture of human septal chondrocytes in a rotary bioreactor

Objectives (1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture. Study Design Prospective, basic science. Setting Research laboratory. Methods Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured. Results Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P < .01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0. Conclusion Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.     

Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as tissue engineering scaffolding materials for cartilage regeneration

Synthetic biodegradable polyesters poly(L-lactide) (PLLA) and poly(D,L-lactide-coglycolide) (PLGA) (50:50) modified by porcine type II collagen and an Arg-Gly-Asp (RGD)-containing protein were evaluated as scaffolds for cartilage regeneration in this study. Cytocompatibility of the polymer films was tested using immortalized chondrocytes. Neocartilage formation in vitro on cell-seeded scaffolds was further examined using primary porcine chondrocytes. The inflammatory response of the scaffolds was evaluated subcutaneously in rats. A pilot animal study was conducted, in which rabbit allogeneic chondrocyte-seeded scaffolds were implanted to repair the defected rabbit knee cartilage. The results demonstrated that PLGA as well as its blends with PLLA had better cell growth than pure PLLA, and that type II collagen enhanced, but RGD inhibited cell proliferation. Scaffolds made of blended PLLA/PLGA had larger dynamic compressive modulus compared to scaffolds made of PLLA or PLGA single polymer. Chondrocyte-seeded scaffolds modified by type II collagen without RGD had the greater number of cells as well as higher glycosaminoglycan (GAG) and collagen contents compared to scaffolds without type II collagen modification or scaffolds further modified with RDG. Type II collagen modification prevented infiltration by host tissue and capsule formation. Unmodified PLLA and PLLA/PLGA constructs demonstrated persisting inflammatory response after 6 months, while all type II collagen-modified PLLA/PLGA constructs showed complete repair and no inflammation. Partial or full repair was observed after 2 months of postimplantation in type II collagen-modified PLLA/PLGA constructs, with equal cellularity and 75-80% matrix contents of a normal rabbit articular cartilage. It was concluded that PLLA/PLGA blended scaffolds modified by type II collagen were a potential tissue engineering scaffold for cartilage regeneration.     

利用组织工程技术再生软骨组织的实验研究

目的 在有免疫力的动物体兔体内探索组织工程化软骨生成的影响因素。 方法 经不同物质修猸的聚羟基乙酸支架与软骨细胞体外培养,观察基质产生情况,并将细胞支架复合物体内回植,观察软骨的生成,并进行组织学及超微结构评价。结果 以孵磷脂、多聚赖氨酸及聚乳酸共同修饰的聚羟基乙酸与软骨细胞体外培养,结果基质产生旺盛,体内回植后软骨生成良好。 结论 细胞支架复合物体外培养期间有基质产生,是组织工程化软骨生成的前提     

Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage.

Cartilaginous constructs have been grown in vitro with use of isolated cells, biodegradable polymer scaffolds, and bioreactors. In the present work, the relationships between the composition and mechanical properties of engineered cartilage constructs were studied by culturing bovine calf articular chondrocytes on fibrous polyglycolic acid scaffolds (5 mm in diameter, 2-mm thick, and 97% porous) in three different environments: static flasks, mixed flasks, and rotating vessels. After 6 weeks of cultivation, the composition, morphology, and mechanical function of the constructs in radially confined static and dynamic compression all depended on the conditions of in vitro cultivation. Static culture yielded small and fragile constructs, while turbulent flow in mixed flasks yielded constructs with fibrous outer capsules; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels were the largest, contained continuous cartilage-like extracellular matrices with the highest fractions of glycosaminoglycan and collagen, and had the best mechanical properties. The equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential correlated with the wet-weight fractions of glycosaminoglycan, collagen, and water. These findings suggest that the hydrodynamic conditions in tissue-culture bioreactors can modulate the composition, morphology, mechanical properties, and electromechanical function of engineered cartilage.     

Growth and integration of neocartilage with native cartilage in vitro.

Poor integration of neocartilage with recipient has been a major obstacle to articular cartilage restoration. An in vitro study was designed to provide insights regarding the integration process. Cartilage explants and chondrocytes were harvested from the distal sternum of 16-day-old chick embryos. Four million chondrocytes and one 1mm(3) explant were centrifuged together in a 0.75ml tube. In the constructs, consisting of cartilage explant and chondrocyte pellet, isolated chondrocytes attached to the surface of the explant at the beginning of the culture, followed by significant chondrocyte death at the interface between chondrocyte pellet and explant. Chondrocyte apoptosis was seen almost exclusively at this interface. Meanwhile, the interface was a zone with active extracellular matrix deposition as demonstrated by immunohistochemistry. By two weeks, the junction of neocartilage and native cartilage explant had formed an acellular zone with collagen fibrils orientated parallel with the surface of the cartilage explant. In conclusion, chondrocyte death leads to acellularity and fibril network reorganization at the neocartilage/explant interface, and impacts the quality of cartilage repair as abnormal matrix remodeling implies.     

Effects of Pulsatile Bioreactor Culture on Vascular Smooth Muscle Cells Seeded on Electrospun Poly (lactide‐co‐ε‐caprolactone) Scaffold

Electrospun nanofibrous scaffolds have several advantages, such as an extremely high surface‐to‐volume ratio, tunable porosity, and malleability to conform over a wide variety of sizes and shapes. However, there are limitations to culturing the cells on the scaffold, including the inability of the cells to infiltrate because of the scaffold's nano‐sized pores. To overcome the limitations, we developed a controlled pulsatile bioreactor that produces static and dynamic flow, which improves transfer of such nutrients and oxygen, and a tubular‐shaped vascular graft using cell matrix engineering. Electrospun scaffolds were seeded with smooth muscle cells (SMCs), cultured under dynamic or static conditions for 14 days, and analyzed. Mechanical examination revealed higher burst strength in the vascular grafts cultured under dynamic conditions than under static conditions. Also, immunohistology stain for alpa smooth muscle actin showed the difference of SMC distribution and existence on the scaffold between the static and dynamic culture conditions. The higher proliferation rate of SMCs in dynamic culture rather than static culture could be explained by the design of the bioreactor which mimics the physical environment such as media flow and pressure through the lumen of the construct. This supports regulation of collagen and leads to a significant increase in tensile strength of the engineered tissues. These results showed that the SMCs/electrospinning poly (lactide‐co‐ε‐caprolactone) scaffold constructs formed tubular‐shaped vascular grafts and could be useful in vascular tissue engineering.     

The effect of a cyclic uniaxial strain on urinary bladder cells

Purpose

Pre-conditioning of a cell seeded construct may improve the functional outcome of a tissue engineered construct for augmentation cystoplasty. The precise effects of mechanical stimulation on urinary bladder cells in vitro are not clear. In this study we investigate the effect of a cyclic uniaxial strain culture on urinary bladder cells which were seeded on a type I collagen scaffold.

Methods

Isolated porcine smooth muscle cells or urothelial cells were seeded on a type I collagen scaffolds and cultured under static and dynamic conditions. A uniform cyclic uniaxial strain was applied to the seeded scaffold using a Bose Electroforce Bio-Dynamic bioreactor. Cell proliferation rate and phenotype were investigated, including SEM analysis, RT-PCR and immunohistochemistry for α-Smooth muscle actin, calponin-1, desmin and RCK103 expression to determine the effects of mechanical stimulation on both cell types.

Results

Dynamic stimulation of smooth muscle cell seeded constructs resulted in cell alignment and enhanced proliferation rate. Additionally, expression of α-Smooth muscle actin and calponin-1 was increased suggesting differentiation of smooth muscle cells to a more mature phenotype.

Conclusions

Mechanical stimuli did not enhance the proliferation and differentiation of urothelial cells. Mechanical stimulation, i.e., preconditioning may improve the functional in vivo outcome of smooth muscle cell seeded constructs for flexible organs such as the bladder.

     

A peek into the possible future of management of articular cartilage injuries: gene therapy and scaffolds for cartilage repair

Two rapidly progressing areas of research will likely contribute to cartilage repair procedures in the foreseeable future: gene therapy and synthetic scaffolds. Gene therapy refers to the transfer of new genetic information to cells that contribute to the cartilage repair process. This approach allows for manipulation of cartilage repair at the cellular and molecular level. Scaffolds are the core technology for the next generation of autologous cartilage implantation procedures in which synthetic matrices are used in conjunction with chondrocytes. This approach can be improved further using bioreactor technologies to enhance the production of extracellular matrix proteins by chondrocytes seeded onto a scaffold. The resulting "neo-cartilage implant" matures within the bioreactor, and can then be used to fill cartilage defects.     

纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞修复兔膝关节软骨缺损的实验研究

目的：探讨纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞作为软骨组织工程支架的可行性及有效性,并为后续研究可注射性材料做基础。方法：体外分离培养软骨细胞后接种到纤维蛋白凝胶和脱钙骨基质支架材料体外培养4周,然后植入兔膝关节软骨缺损区继续培养4、8、12周后取材,分别行大体、组织学、Ⅱ型胶原免疫组织化学观察。并进行Wakitani评分,观察其体内修复关节缺损效果。结果：大体观察4周后,实验组软骨缺损区可有乳白色组织修复,12周可修复完全,并无明显凹凸感。光镜下8周可见大量软骨细胞修复,并在TB染色下见Ⅱ型胶原比4周时明显增多。12周时软骨陷窝结构形成,细胞形态排列及Ⅱ型胶原与正常软骨组织相近。结论：纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞可以作为软骨组织工程支架材料,能够用于再生修复软骨的缺损。并为构建可注射性修复材料提供途径。     

Engineered fetal cartilage: structural and functional analysis in vitro

Background/Purpose: This study was aimed at characterizing the structure and function of engineered fetal cartilage in vitro. Methods: Chondrocytes from ovine specimens of fetal elastic, fetal hyaline, and adult elastic cartilage were expanded in culture and their growth rates determined. Cells were seeded onto synthetic scaffolds, which were then maintained in a bioreactor. Matrix deposition was determined by specific staining and quantitative assays for glycosaminoglycans (GAG), type II collagen (CII), and elastin, as well as compared with native tissue. Statistical analysis was by analysis of variance (ANOVA) and Students' t test, with significance set at P less than .01. Results: Fetal elastic chondrocytes grew significantly faster than all other cell types. All fetal constructs resembled hyaline cartilage, regardless of the cell source. There were significantly higher levels of GAG and CII in fetal versus adult constructs, but no significant difference between fetal constructs from different sources. Unlike their adult counterparts, fetal constructs had GAG and CII levels similar to native tissues. Conclusions: Fetal chondrocytes can be rapidly expanded in culture. Compared with adult constructs, matrix deposition is enhanced in engineered fetal cartilage, which closely resembles native tissue, regardless of the cell source. Engineered fetal cartilage may be a preferable option during surgical reconstruction of select congenital anomalies.     

Effect of different solvents and crosslinkers on cytocompatibility of Type II collagen scaffolds for chondrocyte seeding

Type II collagen extracted from porcine costal cartilage was evaluated as scaffolds for cartilage tissue engineering. Chemical crosslinkers were employed to improve the mechanical properties and the resistance toward degradation. Films and porous scaffolds were prepared from collagen solutions dissolved in 3% acetic acid (designated A) or in deionized water (designated W) and crosslinked by an epoxy (designated E) or by a carbodiimide (designated C). Immortalized rat chondrocytes and rabbit chondrocytes were used to assess cytocompatibility of crosslinked collagen matrices. Cell adhesion rate onto the films made by different preparations ranked in the order of WE > or = WC > AC > or = AE. Cell proliferation ranked in the order of AC > WC > AE > or = WE. Cells maintained round morphology only on AC and WC films. In 3-D seeding, AC scaffolds also were found to be the most cytocompatible. WC scaffolds, however, had better dimensional stability. It was concluded that Type II collagen scaffolds, when prepared by using deionized water as the solvent and carbodiimide as the crosslinker, could promote chondrocyte growth and matrix production.     

残耳软骨细胞种植于聚羟基丁酸酯-聚羟基己酸酯共聚物形成组织工程软骨

目的探讨以聚羟基丁酸酯-聚羟基己酸酯(PHB-PHH)共聚物为细胞外支架、以残耳软骨作为种子细胞形成组织工程软骨的可能性。方法取先天性小耳畸形8例患者的残耳软骨，以胶原酶消化后种植于PHBPHH支架，体外培养1周后种植于8只裸鼠一侧背部皮下为实验组，另一侧只植入支架材料作为对照组。于4周、8周后取出标本，做大体观察及HE染色、Masson三色染色检查。结果4周时实验组镜下显示有新生软骨形成，但仍有部分支架材料残留；8周时实验组标本大体观察及HE染色、Masson三色染色检查新生软骨与人耳软骨相似，支架材料已完全吸收。对照组无软骨形成。结论以残耳软骨作为种子细胞，以PHB-PHH共聚物为细胞外支架可以形成组织工程软骨，新生软骨大体观察、组织学检查与人耳软骨相似。     

力学刺激促进软骨细胞与骨髓基质干细胞共培养体外软骨分化

目的：研究不同的应力刺激对软骨细胞与骨髓基质干细胞（BMSCs）共培养体外构建组织工程化软骨的影响。方法：分离、培养、扩传兔MSCs及软骨细胞,二者按7：3比例混和,以5．0×107／ml的细胞密度接种于聚羟基乙酸（PGA）支架上,一周后根据不同的施加力分为4组：离心组、摇床组、搅拌组,静止培养作为对照组。6周后取材行相关检测。结果：三受力组形成的细胞材料复合物基本保持原来的体积与外形。HE染色结果显示大量成熟软骨陷窝形成,细胞外基质沉积均匀;Safranin-O及甲苯胺兰染色显示有大量的GAG形成,免疫组化检测II型胶原表达强阳性。三受力组标本组织湿重、体积、GAG含量等指标均优于对照组。结论：力学刺激有利于促进少量软骨细胞与BMSCs共培养体外软骨分化;并在三维支架材料上构建组织工程化软骨。     

组织工程肌腱种子细胞的比较研究

目的 探讨猪肌腱细胞、真皮成纤维细胞、骨髓问充质干细胞中何种细胞最适宜作为体外组织工程化肌腱构建的种子细胞.方法 收集猪肌腱细胞、真皮成纤维细胞及骨髓间充质干细胞,以50×106个细胞密度均匀接种于圆柱状聚羟基乙酸(Polyglycolic acids,PGA)上,按细胞种类分为三组,每组n=3,体外培养,并于1、2、6周取材,进行组织学检测、免疫组化检测、胶原定量测定和大体观察.结果 细胞-PGA复合物体外培养时有细胞外基质产生,六周时肌腱细胞组产生胶原量最多,明显优于真皮成纤维细胞和骨髓间充质干细胞(p＜0.01).免疫组化显示形成的主要为Ⅰ型胶原.结论 体外构建组织工程肌腱时肌腱细胞合成胶原能力最强,在现有条件下是体外构建组织工程肌腱的最佳种子细胞.