An improved collagen scaffold for skeletal regeneration

Abstract

Bone repair and regeneration is one of the most extensively studied areas in the field of tissue engineering. All of the current tissue engineering approaches to create bone focus on intramembranous ossification, ignoring the other mechanism of bone formation, endochondral ossification. We propose to create a transient cartilage template in vitro, which could serve as an intermediate for bone formation by the endochondral mechanism once implanted in vivo. The goals of the study are (1) to prepare and characterize type I collagen sponges as a scaffold for the cartilage template, and (2) to establish a method of culturing chondrocytes in type I collagen sponges and induce cell maturation. Collagen sponges were generated from a 1% solution of type I collagen using a freeze/dry technique followed by UV light crosslinking. Chondrocytes isolated from two locations in chick embryo sterna were cultured in these sponges and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Material strength testing as well as microscopic and biochemical analyzes were conducted to evaluate the properties of sponges and cell behavior during the culture period. We found that our collagen sponges presented improved stiffness and supported chondrocyte attachment and proliferation. Cells underwent maturation, depositing an abundant extracellular matrix throughout the scaffold, expressing high levels of type X collagen, type I collagen and alkaline phosphatase. These results demonstrate that we have created a transient cartilage template with potential to direct endochondral bone formation after implantation.

Figure 1. Stress vs. strain curves of hydrated collagen type I sponges

Sponges deformation was evaluated in compression tests. Stress and strain was calculated from the recorded values of force and displacement. Image A shows whole compression curve while image B shows the linear equation and the trend-line of a restrict area close to 30% of deformation.

Figure 2. Scanning electron microscopy of collagen type I sponges

Photomicrographs of cross sections of collagen sponges are shown. Image A (50X) shows homogenous size and distribution of pores and image B (150X) shows the good interconnectivity between the pores.

Figure 3. Scanning electron microscopy of collagen sponges cultured with chondrocytes

CP and CD chondrocytes were grown on collagen sponges for 5 days and then treated with retinoic acid for the next 5 days to induce maturation. CP chondrocyte cultures, A (day 5), B and C (day 10). CD chondrocyte cultures, D (day 5), E and F (day 10)

Figure 4. Rapid proliferation of chondrocytes on collagen sponges

CP and CD chondrocytes were grown on collagen sponge for 5 days and then treated with retinoic acid for the next 5 days to induce chondrocyte maturation. DNA was measured after 5 and 10 days in culture. *Significant difference from day 5 for both CP and CD chondrocytes.

Figure 5. Immunohistochemical staining collagen sponges cultured with chondrocytes

Histological cross sections of sponges collected after 10 days in culture were immunostained with antibodies against alkaline phosphatase (A, B), and type X collagen (C,D). A and C are cross sectional views of the sponges with CD chondrocytes, while B and D are sponges with CP chondrocytes. AP and type X collagen are evidenced by the presence of brown color.

Figure 6. Retinoic acid treatment increases AP activity in CP chondrocytes

CP and CD chondrocytes were grown on collagen sponges for 5 days and then treated with retinoic acid for the next 5 days to induce chondrocyte maturation. Graph shows AP activity levels, measured spectrophotometrically, and normalized for total protein content in the samples. *Significant difference from CP chondrocytes at day 5. **Significant difference from CD chondrocytes day10.

Figure 7. Gene expression profile of upper sternal chondrocytes when compared to lower sternal chondrocytes

CP and CD chondrocytes were grown to confluence for 5 days and then treated with retinoic acid for additional 5 days. mRNA was extracted at the end of the culture period (10 days) from CP and CD chondrocytes. RT-PCR was performed using primers specific for chick genes. Expression levels are presented as “fold change” in mRNA levels in CP chondrocytes in relation to CD chondrocytes. *Significantly different from CD chondrocytes.