In vitro cartilage tissue formation by Co-culture of primary and passaged chondrocytes

Passaging chondrocytes to increase cell number is one way to overcome the major limitation to cartilage tissue engineering, which is obtaining sufficient numbers of chondrocytes to form large amounts of tissue. Because neighboring cells can influence cell phenotype and because passaging induces dedifferentiation, we examined whether coculture of primary and passaged bovine articular chondrocytes in 3-dimensional culture would form cartilage tissue in vitro. Chondrocytes passaged in monolayer culture up to 4 times were mixed with primary (nonpassaged) chondrocytes (5-40% of total cell number) and grown on filter inserts for up to 4 weeks. Passaged cells alone did not form cartilage, but with the addition of increasing numbers of primary chondrocytes, up to 20%, there was an increase in cartilage tissue formation as determined histologically and biochemically and demonstrated by increasing proteoglycan and collagen accumulation. The passaged cells appeared to be undergoing redifferentiation, as indicated by up-regulation of aggrecan, type II collagen, and SOX9 gene expression and decreased type I collagen expression. This switch in collagen type was confirmed using Western blots. Confocal microscopy showed that fluorescently labeled primary cells were distributed throughout the tissue. This coculture approach could provide a new way to solve the problem of limited cell number for cartilage tissue engineering.     

Cartilage tissue engineering using human auricular chondrocytes embedded in different hydrogel materials

To seek a suitable scaffold for cartilage tissue engineering, we compared various hydrogel materials originating from animals, plants, or synthetic peptides. Human auricular chondrocytes were embedded in atelopeptide collagen, alginate, or PuraMatrix, all of which are or will soon be clinically available. The chondrocytes in the atelopeptide collagen proliferated well, while the others showed no proliferation. A high-cell density culture within each hydrogel enhanced the expression of collagen type II mRNA, when compared with that without hydrogel. By stimulation with insulin and BMP-2, collagen type II and glycosaminoglycan were significantly accumulated within all hydrogels. Chondrocytes in the atelopeptide collagen showed high expression of beta1 integrin, seemingly promoting cell-matrix signaling. The N-cadherin expression was inhibited in the alginate, implying that decrease in cell-to-cell contacts may maintain chondrocyte activity. The matrix synthesis in PuraMatrix was less than that in others, while its Young's modulus was the lowest, suggesting a weakness in gelling ability and storage of cells and matrices. Considering biological effects and clinical availability, atelopeptide collagen may be accessible for clinical use. However, because synthetic peptides can control the risk of disease transmission and immunoreactivities, some improvement in gelling ability would provide a more useful hydrogel for ideal cartilage regeneration.     

Maintenance of cartilaginous gene expression on extracellular matrix derived from serially passaged chondrocytes during in vitro chondrocyte expansion

The loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier for the application of cartilage tissue engineering. The use of matrices mimicking the in vivo extracellular matrix (ECM) microenvironment is anticipated to be an efficient method to suppress chondrocyte phenotype loss. In this study, we developed several types of ECM derived from serially passaged chondrocytes for use as cell-culture substrata and compared their effects on chondrocyte functions. Primary bovine chondrocytes and serially passaged chondrocytes (at passages 2 and 6) were cultured on tissue-culture polystyrene. After culture, the cellular components were selectively removed from the ECM deposited by the cells. The remaining ECM proteins were used as cell-culture substrata. The composition of the deposited ECM depended on the culture stage of the serially passaged chondrocytes used for the ECM production. The deposited ECM supported the adhesion and proliferation of chondrocytes. The effects of the ECM on the chondrocyte dedifferentiation during in vitro passage culture differed dramatically depending on the phenotype of the chondrocytes used to produce the ECM. The primary chondrocyte-derived ECM delayed the chondrocyte dedifferentiation during in vitro passage culture and is a good candidate for chondrocyte subculture for tissue engineering.     

Cartilage tissue engineering for laryngotracheal reconstruction: comparison of chondrocytes from three anatomic locations in the rabbit

Tissue engineering may provide a technique to generate cartilage grafts for laryngotracheal reconstruction in children. The present study used a rabbit model to characterize cartilage generated by a candidate tissue engineering approach to determine, under baseline conditions, which chondrocytes in the rabbit produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. We characterized tissue-engineered cartilage generated in perfused bioreactor chambers from three sources of rabbit chondrocytes: articular, auricular, and nasal cartilage. Biomechanical testing and histological, immunohistochemical, and biochemical assays were performed to determine equilibrium unconfined compression (Young's) modulus, and biochemical composition and structure. We found that cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness necessary for completion of the biomechanical testing, but five of six auricular samples completed the biomechanical testing (moduli of 210 +/- 93 kPa in two samples at 3 weeks and 100 +/- 65 kPa in three samples at 6 weeks). Auricular samples showed more consistent staining for proteoglycans and collagen II and had significantly higher glycosaminoglycan (GAG) content and concentration and higher collagen content than articular or nasal samples. In addition, the delayed gadolinium enhanced MRI of cartilage (dGEMRIC) method revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples. The results indicate that, for the candidate tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. The results also suggest that this and similar tissue engineering approaches must be optimized for each potential source of chondrocytes.     

Interplay between cytoskeletal polymerization and the chondrogenic phenotype in chondrocytes passaged in monolayer culture

Tubulin and actin exist as monomeric units that polymerize to form either microtubules or filamentous actin. As the polymerization status (monomeric/polymeric ratio) of tubulin and/or actin have been shown to be important in regulating gene expression and phenotype in non‐chondrocyte cells, the objective of this study was to examine the role of cytoskeletal polymerization on the chondrocyte phenotype. We hypothesized that actin and/or tubulin polymerization status modulates the chondrocyte phenotype during monolayer culture as well as in 3D culture during redifferentiation. To test this hypothesis, articular chondrocytes were grown and passaged in 2D monolayer culture. Cell phenotype was investigated by assessing cell morphology (area and circularity), actin/tubulin content, organization and polymerization status, as well as by determination of proliferation, fibroblast and cartilage matrix gene expression with passage number. Bovine chondrocytes became larger, more elongated, and had significantly (P < 0.05) increased gene expression of proliferation‐associated molecules (cyclin D1 and ki67), as well as significantly (P < 0.05) decreased cartilage matrix (type II collagen and aggrecan) and increased fibroblast‐like matrix, type I collagen (COL1), gene expression by passage 2 (P2). Although tubulin polymerization status was not significantly (P > 0.05) modulated, actin polymerization was increased in bovine P2 cells. Actin depolymerization, but not tubulin depolymerization, promoted the chondrocyte phenotype by inducing cell rounding, increasing aggrecan and reducing COL1 expression. Knockdown of actin depolymerization factor, cofilin, in these cells induced further P2 cell actin polymerization and increased COL1 gene expression. To confirm that actin status regulated COL1 gene expression in human P2 chondrocytes, human P2 chondrocytes were exposed to cytochalasin D. Cytochalasin D decreased COL1 gene expression in human passaged chondrocytes. Furthermore, culture of bovine P2 chondrocytes in 3D culture on porous bone substitute resulted in actin depolymerization, which correlated with decreased expression of COL1 and proliferation molecules. In 3D cultures, aggrecan gene expression was increased by cytochalasin D treatment and COL1 was further decreased. These results reveal that actin polymerization status regulates chondrocyte dedifferentiation. Reorganization of the cytoskeleton by actin depolymerization appears to be an active regulatory mechanism for redifferentiation of passaged chondrocytes.     

Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression

To investigate the potential utility of mechanical loading in articular cartilage tissue engineering, porous type II collagen scaffolds seeded with adult canine passaged chondrocytes were subjected to static and dynamic compressions of varying magnitudes (0-50% static strain) and durations (1-24 h), and at different times during culture (2-30 days postseeding). The effects of mechanical compression on the biosynthetic activity of the chondrocytes were evaluated by measuring the amount of (3)H-proline-labeled proteins and (35)S-sulfate-labeled proteoglycans that accumulated in the cell-scaffold construct and was released to the medium during the loading period. Similar to published results on loading of articular cartilage explants, static compression decreased protein and proteoglycan biosynthesis in a time- and dose-dependent manner (each p < 0.005), and selected dynamic compression protocols were able to increase rates of biosynthesis (p < 0.05). The main difference between the results seen for this tissue engineering system and cartilage explants was in the amount of newly synthesized matrix molecules that accumulated within the construct under dynamic loading, with less accumulating in the type II collagen scaffold. In summary, the general biosynthetic response of passaged chondrocytes in the porous type II collagen scaffolds is similar to that seen for chondrocytes in their native environment. Future work needs to be directed to modifications of the cell-seeded construct to allow for the capture of the newly synthesized matrix molecules by the scaffold.     

Centrifugation assay for measuring adhesion of serially passaged bovine chondrocytes to polystyrene surfaces

A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.     

Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering

Cartilage tissue engineering plays an important role in the generation of grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality, and the molecular basis is still not well understood. Therefore, the aim of our study was to investigate the expression of matrix modulators in human chondrocytes during expansion. Human chondrocytes were isolated from septal cartilage (n=32) and held in primary cell culture. Cells were harvested after 1, 6 and 21 days. The differentiation of cells using light microscopy, the expression patterns of various proteins (MMPs, BMPs, and TIMPs) using immunohistochemistry, and the expression of distinct genes using microarray technique, were investigated. The chondrocytes showed strong in vitro proliferation. After 6 and 21 days, BMP-5 and -8 were up-regulated, BMP-2 was down-regulated and BMP-6 was inactivated. Other BMPs were not expressed. The expression of MMP-2, -3 and -13 was up-regulated from day 1 to 21, and MMP-12 and -20 were down-regulated. Other MMPs were not expressed. TIMP-1 was up-regulated and TIMP-3 was down-regulated during expansion. Differential expression of matrix modulators might influence the matrix composition of engineered cartilage. Improving the basic knowledge in this area may ultimately help clinicians to identify and proactively intervene in an attempt to prevent bioartificial cartilage from losing stability.     

表皮生长因子对传代培养的大鼠生长板软骨细胞增殖与胶原合成的影响

目的：探讨不同浓度的表皮生长因子（EGF）对传代培养的大鼠肋生长板软骨细胞（RGC）增殖和胶原合成的影响。 方法： 分离、培养RGC，分别用Western blot和阿尔新蓝（Alcine blue）染色检测各代RGC中Ⅱ型胶原和蛋白多糖的表达；用［³H］-TdR和［³H］-proline掺入分别检测1、10和100 μg/L EGF对第1、3和5代RGC增殖和胶原合成的影响。 结果： 原代培养的RGC表达Ⅱ型胶原和蛋白多糖，从第4代起Ⅱ型胶原和蛋白多糖的表达迅速降低，RGC发生了去分化。［³H］-TdR掺入表明，EGF促进第1代RGC的增殖（P<0.01），3个浓度的作用依次为：1 μg/L>10 μg/L>100 μg/L，差异显著（P<0.01）；3个浓度的EGF对第3代RGC保持了相近的促增殖作用（P<0.01）；对第5代RGC均无促增殖作用(P>0.05)。与促增殖作用不同，不同浓度EGF促不同传代的RGC的［³H］-proline掺入率比对照组均高20%左右（P<0.01）。 结论： EGF具有促进培养RGC增殖和胶原合成的作用，连续传代引起的去分化降低了RGC对EGF促增殖作用的反应，但对EGF的促胶原合成作用没有产生影响。     

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.     

Effect of material geometry on cartilagenous tissue formation in vitro

The effect of material geometry, as defined by average pore size, on chondrocyte phenotype and cartilagenous tissue formation in vitro was examined. Bovine articular chondrocytes were plated on porous titanium alloy (Ti6Al4V) discs of different average pore sizes (13, 43, and 68 microm) and grown in culture for 4 weeks. Chondrocyte phenotype was maintained as indicated by the synthesis of large proteoglycans (Kav +/- SD: 13 microm = 0.28 +/- 0.01; 43 microm = 0.29 +/- 0.01; 68 microm = 0.27 +/- 0.02) and type II collagen. Light microscopical examination of histological sections of the composites showed that cartilagenous tissue had formed on all discs. The cartilagenous tissue on the discs of the smallest average pore size (13 microm) was significantly thicker than the tissue on the discs of larger average pore sizes and also had greater amounts of proteoglycan [mean glycosaminoglycan content +/- SD microg/disc): 13 microm = 246.9 +/- 7.8; 43 microm = 190.4 +/- 10.2; 68 microm = 156.6 +/- 25.8, p = 0.002] and DNA [mean DNA content +/- SD microg/disc): 13 microm = 12.5 +/- 0.6; 43 microm = 8.3 +/- 0.2; 68 microm = 9.3 +/- 0.9, p = 0.0008]. However, the amount of proteoglycan accumulated per cell was similar in the tissues generated on the discs of different average pore sizes. In contrast, the amount of collagen in the cartilagenous tissues showed no significant differences between the different pore sizes, but the amount of collagen accumulated per cell was less in the tissue formed on the smallest pore size disc (13 microm) as compared with the tissue formed on the discs of the larger pore sizes [mean hydroxyproline content/DNA (microg/microg) +/- SD: 13 microm = 1.56 +/- 0.2; 43 microm = 2.19 +/- 0.2; 68 microm = 2.3 +/- 0.3]. These results suggest that material geometry, as defined by pore size, can affect the amount and composition of the cartilagenous tissue that forms.     

以壳聚糖-胶原共混膜为三维支架同种异体工程化软骨的构建

目的探讨以壳聚糖-胶原共混膜为三维支架材料的同种异体软骨细胞构建组织工程化软骨的能力。方法将分离、培养、扩传兔软骨细胞，接种在壳聚糖-胶原共混膜上，倒置显微镜下观察细胞在共混膜上的生长情况。体外培养7d后，将细胞-材料复合物种植在新西兰兔皮下，6周取材，对获得的同种异体工程化软骨进行组织学评价。结果兔软骨细胞接种于壳聚糖-胶原共混膜上4h后有贴壁现象出现，细胞呈梭形。培养48h后，软骨细胞分裂增殖越来越多并向周围延伸，培养第7天取材，HE染色示细胞生长良好，呈梭形。体内培养6周取材，HE染色、Masson染色为均一的成熟软骨组织，且共混膜已降解。结论以壳聚糖-胶原共混膜为支架材料同种异体软骨细胞在有免疫力的动物体内可形成工程化软骨。     

Cartilage reshaping via in vitro mechanical loading

Shaped cartilage grafts can be used in the restoration of injured joints and the reconstruction of deformities of the head and neck. This study describes a novel method for altering cartilage shape, based on the hypothesis that mechanical loading coupled with in vitro tissue growth and remodeling facilitates tissue reshaping. Static bending deformations were imposed on strips of immature articular cartilage, and retention of the imposed shape and structural and biochemical measures of growth were assessed after 2, 4, and 6 days of incubation. The results show that mechanical reshaping of tissue is feasible, because shape retention was greater than 86% after 6 days of culture. The imposed mechanical deformations had little effect on measures of tissue viability or growth within the 6-day culture period. The addition of cycloheximide to the culture medium only slightly reduced the ability to reshape these tissues, but cycloheximide plus a lower culture temperature of 4 degrees C markedly inhibited the reshaping response. These results suggest a limited role for chondrocyte biosynthesis but a potentially important role for metabolic reactions in the cartilage matrix in the reshaping process. The ability to modulate cartilage shape in vitro may prove useful for tissue engineering of shaped cartilage grafts.     

Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes.

The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.     

Aragonite crystalline biomatrices support astrocytic tissue formation in vitro and in vivo

Astrocytes play a pivotal role in the development and function of the central nervous system by regulating synaptic activity and supporting and guiding growing axons. It is therefore a central therapeutic and scientific challenge to develop means to control astrocytic survival and growth. We cultured primary hippocampal astrocytes on a crystalline three-dimensional (3D) aragonite biomatrix prepared from the exoskeleton of the coral Porites lutea. Such culturing led to the formation of astrocytic tissue-like 3D structures in which the cells had a higher survival rate than astrocytes grown in conventional cell culture. Within the pore void areas, multiple layers of astrocytic processes formed concave sheet structures that had no physical contact with the surface. The astrocytes attached to the crystalline perpendicular edges of the crystalline template surface extended processes in 3D and expressed glial fibrillary acidic protein. The astrocytes also expressed gap junctions and developed partly synchronized cytosolic Ca2+ oscillations. Preliminary in vivo models showed that astrocytic networks were also developed when the matrices were implanted into cortical areas of postnatal rat brains. Hence, we suggest that the biomatrix is a biocompatible supportive scaffold for astrocytes and may be exploited in applications for neuronal tissue restoration in injured or diseased central nervous system.     

碱性成纤维细胞生长因子对离心管内培养骺板细胞生成软骨组织的作用

背景：采用离心管技术体外培养骺板细胞的报道已很多。 目的：观察碱性成纤维细胞生长因子对离心管内培养的骺板细胞生成类骺板组织的影响。 方法：获取3周龄新西兰白兔股骨远端的骺板组织,利用组织块纱巾培养法获得骺板细胞,加入含有10 μg/L碱性成纤维细胞生长因子的DMEM培养液,连续培养4周。 结果与结论：离心管内聚集的骺板细胞在含有碱性成纤维细胞生长因子的DMEM培养液中形成类骺板样组织块。在组织块外周形成类似骺软骨的生发层,中心的骺板细增殖情况良好,向肥大细胞方向分化。类骺板样组织甲苯胺蓝及番红“O”染色阳性,Ⅱ型胶原免疫组织化学染色呈强阳性。说明碱性成纤维细胞生长因子能够促进离心管中的骺板细胞形成富含蛋白聚糖及Ⅱ型胶原等细胞外基质的类骺板样软骨组织。