Effects of multiple chondroitinase ABC applications on tissue engineered articular cartilage

Abstract

Increasing tensile properties and collagen content is a recognized need in articular cartilage tissue engineering. This study tested the hypothesis that multiple applications of chondroitinase ABC (C-ABC), a glycosaminoglycan (GAG) degrading enzyme, could increase construct tensile properties in a scaffold-less approach for articular cartilage tissue engineering. Developing constructs were treated with C-ABC at 2 weeks, 4 weeks, or both 2 and 4 weeks. At 4 and 6 weeks, construct sulfated GAG composition, collagen composition, and compressive and tensile biomechanical properties were assessed, along with immunohistochemistry (IHC) for collagens type I, II, and VI, and the proteoglycan decorin. At 6 weeks, the tensile modulus and ultimate tensile strength of the group treated at both 2 and 4 weeks were significantly increased over controls by 78% and 64%, reaching values of 3.4 and 1.4 MPa, respectively. Collagen concentration also increased 43%. Further, groups treated at either 2 weeks or 4 weeks alone also had increased tensile stiffness compared to controls. Surprisingly, though GAG was depleted in the treated groups, by 6 weeks there were no significant differences in compressive stiffness. IHC showed abundant collagen type II and VI in all groups, with no collagen type I. Further, decorin staining was reduced following C-ABC treatment, but returned during subsequent culture. The results support the use of C-ABC in cartilage tissue engineering for increasing tensile properties.

Figure 1. Histology and IHC

A-H) Safranin-O/fast green stain for GAGs, I-P) IHC for decorin, a-h) IHC for collagen type I, and i-p) IHC for collagen type II. At 4 wks, note the absence of GAG and substantially decreased decorin staining immediately following C-ABC treatment in the group treated at 4 wks and group treated at both 2 and 4 wks (C, D, K, and L, respectively). GAG and decorin staining returned in these groups by 6 wks (G, H, O, and P, respectively). Scale bar in P is 200µm.

Figure 2. 4 week biomechanical properties

A) Compressive stiffness and B) ultimate tensile strength. Bars represent mean ± S.D. Groups not connected by the same letter are significantly different. The aggregate modulus of all treated groups was significantly less than control. The ultimate tensile strength of all treated groups was significantly greater than control, reaching 931 ± 155 kPa in the combined group. The group treated at both 2 and 4 wks was significantly greater than the groups treated at only 2 or 4 wks.

Figure 3. 6 week biomechanical properties

A) Compressive stiffness and B) tensile stiffness. Bars represent mean ± S.D. Groups not connected by the same letter are significantly different. There were no significant differences in aggregate modulus. Young’s modulus of all treated groups was significantly greater than control, reaching 3.4 ± 0.5 MPa in the combined group. Further, the group treated at both 2 and 4 wks was significantly greater than the groups treated at only 2 or 4 wks, which measured 2.4 ± 0.4 and 2.6 ± 0.5 MPa, respectively.