Construction of tissue engineering cartilage with collagen/hydroxyapatite composite scaffolds loaded chondrocytes in vitro

Huading LU; Daozhang CAI; Gang WU; Chun ZENG

ABSTRACT

BACKGROUND:

A new composite scaffold for cartilage tissue engineer ing has been employed to culture chondrocytes and overcome many limits related to traditional scaffolds, such as poor biocompatibility, inferior mechanical property, inappropriate biodegradability, and simplex structure which can not match layered structure of articular cartilage, etc. The new composite scaffolds provided a new approach for the research of cartilage tissue engineering.

OBJECTIVE:

To evaluate the feasibility and value of layered cylindrical collagen/hydroxyapatite (HA) composite for cartilage tissue engineering by observing how it absorbs chondrocytes and affects its cellular characteristics.

DESIGN:

Completely randomized design and controlled experimental study.

SETTING:

Department of Orthopaedics, Third Hospital Affiliated to Sun Yat-sen University, and College of Material Science, South China University of Technology.MATERIALS The experiment was conducted at the central experimental laboratory of the Third Hospital Affiliated to Sun Yat-sen University from June to November 2004. One two-week-old male healthy New Zealand rabbit,which was bred in 20 ℃ and 40% humidity, was used in this experiment.

METHODS:

①Right amount of deionized water was added into HA, collagen I solution was added to disperse HA, then carbodiimide was added in the mixture at a proportion for getting the collagen/HA composite at different ratios. Pour to the certain mould in successive layers. The upper layer was pure collagen and the bottom was pure HA. The prepared layered cylindrical collagen/HA composite was put into the ultra low temperature freezer, lyophilized, and sterilized by ethylene oxide for the following procedures. ② Chondrocytes of juvenal rabbit were isolated and multiplied in vitro, then chondrocytes were seeded onto porous collagen/HA composite scaffold and cultured. The effects of composite scaffold on chondrocytes'morphological changes, proliferation, and function were evaluated through a series of examinations such as inverted phase-contrast microscopy, histological, scanning electron microscopy and immunohistochemistry.MAIN OUTCOME

MEASURES:

①Morphology of collagen/HA porousscaffold. ②The characteristics of chondrocytes cultured in vitro. ③ The chondrocytes' compatibility with the collagen/HA porous scaffold.

RESULTS:

①Morphology of collagen/HA porous scaffold Collagen/ HA was a cylindrical three-dimensional porous scaffold in layer structure with certain mechanical strength. The top layer was pure collagen, the bottom was pure HA, and the intermediate layer was collagen and HA composite.Under scanning electron microscope, the scaffold had irregular blowhole structure, the apertures were even and pores were communicated each other. The average pore diameter was about 147 μm. ②The characteristics of chondrocytes cultured in vitro The chodrocytes that had just been isolated were spherical; rate of living cells was 95%. 24 hours later, the cells were going to attach to the wall, extend to be triangle or polygon. The cytoplasms were abundant with secretary granules inside. The cells kept on growing in monolayer and the multiply rate accelerated after passage, and chondrocytes overspreaded the culture flask in 4 days. After passaged for several times, the multiply rate decreased. In passage Ⅵ, the ability of cell division was decreased obviously, the cytomorphosis was clear. Cellular sizes were larger. Most of cells were shuttle-shape, less refracted with blur edges, which trended to age and dedifferentiate to fibroblast. ③The chondrocytes' compatibility with the collagen/HA porous scaffold Cylindrical collagen/ HA composite scaffold in layer structure had good hydrophilicity. The chondrocytes adhered to the surface of the scaffold, proliferated and migrated toward the scaffold through the pore. Chondrocytes attached to wall of microholes of the scaffold had largely maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold.

CONCLUSION:

Cylindrical collagen/ HA composite scaffold in layer structure has good cellular compatibility.It is stronger in mechanical property than pure collagen, is anideal scaffold for cartilage tissue engineering.