Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels.

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.

Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system.

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.

Subculture of chondrocytes on a collagen type I-coated substrate with suppressed cellular dedifferentiation.

To evaluate the degree of cellular dedifferentiation, subculture of chondrocytes was conducted on a surface coated with collagen type I at a density of 1.05 mg/cm(2). In the primary culture, most of the cells were round in shape on the collagen (CL) substrate, whereas fibroblastic and partially extended cells were dominant on the polystyrene plastic (PS) substrate. Stereoscopic observation revealed that the round-shaped cells on the CL substrate were hemispherical with nebulous and punctuated F-actin filaments, whereas the fibroblastic cells on the PS substrate were flattened with fully developed stress fibers. This suggested that cell polarization was suppressed during culture on the former substrate. Although serial passages of chondrocytes through subcultures on the CL and PS substrates caused a decrease in the number of round-shaped cells, the morphological change was appreciably suppressed on the CL substrate, as compared with that on the PS substrate. It was found that only round-shaped cells formed collagen type II, which supports the view that cellular dedifferentiation can be suppressed to some extent on the CL substrate. Three-dimensional cultures in collagen gel were performed with cells isolated freshly and passaged on the CL or PS substrate. Cell density at 21 days in the culture of cells passaged on the CL substrate was comparable to that in the culture of freshly isolated cells, in spite of a significant reduction in cell density observed in the culture of cells passaged on the PS substrate. In addition, histological analysis revealed that the expression of glycosaminoglycans and collagen type II was of significance in the collagen gel with cells passaged on the CL substrate, and likewise in the gel with freshly isolated cells. This indicated that the CL substrate could offer a monolayer culture system for expanding chondrocyte cells.

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

Process design of chondrocyte cultures with monolayer growth for cell expansion and subsequent three-dimensional growth for production of cultured cartilage.

The subculture of rabbit chondrocytes with serial passaging was carried out for cell expansion on a collagen-coated surface, and the morphological transition of round-shaped cells to spindle-shaped ones was examined. The observation of cytoskeletal formation by staining F-actin and vinculin supported the view that the round-shaped cells were in the process of differentiation with immature stress fibers relating to less cellular polarity. The cellular morphology was estimated in terms of the distribution of roundness, R(C), during the subculturing on the collagen substrate. The frequency of the number of round-shaped cells, which was defined as the ratio of the number of cells with R(C) >0.9 against the total cell number, was correlated in a logarithmic formula with the number of population doublings during the subcultures. Kinetic models were adopted for the process design of the combined culture of chondrocytes with monolayer growth on the collagen substrate and subsequent three-dimensional growth in Atelocollagen gel, employing the boundary conditions based on the population balance between differentiated and dedifferentiated cells. The combined culture was performed successfully according to the process design scheduled as monolayer growth for 240 h and three-dimensional growth for 264 h, the number of seed cells being 68% of that in the conventional culture for 504 h where monolayer growth for cell expansion was not included.

Effect of glycosaminoglycan production on hardness of cultured cartilage fabricated by the collagen-gel embedding method.

To assess applicability of the tactile sensor in hardness measurement of cultured cartilage and to clarify the relationship between hardness and tissue structure of cultured cartilage fabricated by the collagen-gel embedding method, we studied the effect of glycosaminoglycans on hardness of such cultured cartilage using a tactile sensor and electron probe x-ray microanalyzer (EPMA). Hardness measured by the tactile sensor, that is, change in frequency of naturally oscillating piezoelectric elements caused by contact with a testing material, increased with the number of days of culture or seeding cell density. Analysis of the sulfur component in EPMA results mainly reflected glycosaminoglycans produced by chondrocytes. Sulfur mapping indicated that tissue of the cultured cartilage differed between its surface and the inside; layers rich in glycosaminoglycans and cells had formed in the surface. Changes in frequency showed close correlation with the amount of glycosaminoglycans in the surface and the inside (r = 0.98 and 0.85, respectively) of cultured cartilage measured by EPMA. Thus, the tactile sensor is capable of measuring hardness of cultured cartilage, reflecting the change in tissue structure between the surface and the inside of the cartilage.

Novel poly(ethylene glycol) scaffolds crosslinked by hydrolyzable polyrotaxane for cartilage tissue engineering.

Highly porous poly(ethylene glycol) (PEG) hydrogel scaffolds crosslinked with hydrolyzable polyrotaxane for cartilage tissue engineering were prepared by a solvent casting/salt leaching technique. The resultant scaffolds have well interconnected microporous structures ranging from 87 to 90%. Pore sizes ranging from 115.5-220.9 microm appeared to be dependent on the size of the sieved sodium chloride particulates. Moreover, a dense surface skin layer was not found on either side of the scaffold surfaces. Using microscopic Alcian blue staining of the chondrocyte-seeded scaffolds, well adhered cells and newly produced glycosaminoglycans (GAG) were confirmed. Following the initial chondrocyte seeding onto the hydrogel scaffolds, the cell number was significantly increased, reaching 149, 877, and 1228 cells/mg of tissue at 8, 15, and 21 days in culture, respectively. The micrograph shows well adhered and spread chondrocytes in the interior pores and a cartilaginous extracellular matrix with a GAG fraction produced from the chondrocytes. Results suggest that the PEG hydrogel scaffolds crosslinked with the hydrolyzable polyrotaxane are a promising candidate for chondrocyte culture.

Effect of transforming growth factor-beta1 on morphological characteristics relating to migration and differentiation of rabbit chondrocytes cultured in collagen gels.

The influence of transforming growth factor-beta1 (TGFbeta1) on the behavior of rabbit chondrocytes embedded in collagen gels was examined in terms of cell migration and consequent architecture of cell aggregation. In a low-seeding density culture (X(0)=2.0 x 10(5) cells/cm(3)) TGFbeta1 (0-10.0 ng/ml) was added and observed during a 14-d culture period. Stereoscopic observation was performed on 5 d employing the morphology-related parameter of sphericity (S(c)) for individual cells in the gels. The frequency of migrating cells with S(c) less than 0.95 increased in a dose-dependent manner in response to TGFbeta1. Moreover, the frequency of migrating cells in the culture with 10.0 ng/ml TGFbeta1 was 0.32, two times higher than that in the reference culture without TGFbeta1, while the frequency of dividing cells in the same culture was less than half of that in the reference culture. The histological observation of cultured gels on 14 d revealed that the starburst and loose aggregates with the spindle-shaped cells emerged in the TGFbeta1-free culture, accompanying the poor production of collagen type II by the cells. On the other hand, the spherical-shaped cells were observed in the starburst aggregates with rich excretion of collagen type II in the culture with 5.0 ng/ml TGFbeta1. Moreover, the mRNA levels of differentiation-marker genes (collagen types I and II) were regulated in accordance with the morphological analyses concerning the cell migration and aggregation in the cultures with and without TGFbeta1. From these results, it was concluded that TGFbeta1 had a culture time-dependent effect on the morphological characteristics relating to the migration and differentiation of the chondrocytes in the collagen gel-embedded cultures seeded at low density, that is, the growth factor promotes cell migration with deteriorated proliferation in the early culture phase, and accelerates the transformation of spindle-shaped cells to spherical-shaped ones in the prolonged culture.