Development of human articular cartilage derived microcarrier / 中华骨科杂志

ABSTRACT

Objective To develop a method to prepare human articular cartilage derived microcarrier for both rapid propagating chondrocytes and being used as scaffold to support chondrogenesis. Methods Human articular cartilage was crushed into small pieces by muller after lyophilization, and sorted through two different meshes to collect only those specimens measuring 150-200 microns. Then, in turn, the specimens were subjected to 0.25% trypsin at 37 ℃ for 24 hours and 1% Triton X-100 for 72 hours, respectively. The specimens were observed by inverted phase contrast microscopy, and assessed by staining with haematoxylin-eosin, safranin-O (for GAG), as well as by the immunohistochemistry of aggrecan, collagen type Ⅱ. The microcarriers were seeded with human chondrocytes after being irradiated by 60Co. Results Using inverted phasecontrast microscope, the freezing-dry cartilage particles were observed as yellow, different shapes, and their surfaces were uneven, and with many pits. After treating with trypsin and Triton X-100, the microcarriers showed light yellow, without cartilage morphology. The microcarriers became flocculous or like a hairbrush, and the area of contacting surface significant increased. After culture with cartilage cell for 2 hours, lots of spherical chondrocytes adhered to the microcarriers. HE stain of section confirmed that the celluar constituents of the specimens were removed, the specimens stained weakly positive for GAG, negatively for aggrecan, and positively for collagen type Ⅱ, respectively. Conclusion The detergent and trypsin can remove the cellular constituents and knock out the aggrecan from human articular cartilage while maintaining collagen type Ⅱ and GAG, and made the cartilage pieces flocculous or hairbrush-like. The chondrocytes can be well maintained in human articular cartilage derived microcarriers. Human articular cartilage derived microcarriers were prepared successfullly.