287897 Self-Assembled Rosette Nanotube Composites Improve Chondrocyte Functions

Monday, October 29, 2012
Hall B (Convention Center )
Linlin Sun1, Lijie Grace Zhang2, Usha Hemraz3, Hicham Fenniri4 and Thomas J. Webster1,5, (1)School of Engineering, Brown University, Providence, RI, (2)Department of Mechanical and Aerospace Engineering and Institute for Biomedical Engineering, The George Washington University, Washington, DC, (3)National Institute for Nanotechnology and Departments of Chemistry and Biomedical Engineering, University of Alberta, Edmonton, AB, Canada, (4)National Institute for Nanotechnology and Department of Chemistry, University of Alberta, Edmonton, AB, Canada, (5)Department of Orthopaedics, Brown University, Providence, RI

Introduction:

Even with intensive studies over the past several decades, cartilage injuries are still one of the most difficult challenges in medicine. The main reason is that cartilage has a limited regenerative capacity due to it non-vascular structure and small number of chondrocytes able to heal cartilage damage. Novel materials for cartilage injury treatment and regeneration require biocompatibility and bioactivity to enhance chondrocyte functions as well as mechanical properties similar to natural cartilage. With unique biological and mechanical properties, rosette nanotubes, which are self-assembled by small molecules composed of DNA bases guanine and cytosine, could serve as novel materials of cartilage implants. In this study, one type of rosette nanotubes (termed TBL) [1-3] and poly(2-hydroxyethyl methacrylate) (pHEMA) were used to generate biocompatible, bioactive, and injectable composites for cartilage applications.

Materials and Methods:

Preparation of TBL/HA/pHEMA composites: TBL building blocks were synthesized according to a previously reported synthetic strategy [1,2] in twelve steps, then it was dissolved in dH2O to a final concentration of 4 mg/mL. This solution was sterilized by filtration through a 0.22 µm syringe filter.

A mixture of 2-hydroxyethyl methacrylate (HEMA) monomer (5 mL, Polysciences, PA), dH2O, TBL (0.01 mg/mL), and initiator 2,2'-azobisisobutyronitrile (AIBN, 3 mg/mL, Sigma-Aldrich) were heated in an oven at 60ºC until the samples solidified completely. After polymerization, the TBL/pHEMA composites were sterilized by soaking in 70% ethanol for 20 min and exposed to ultraviolet (UV) light overnight before cell experiments. 

Chondrocyte adhesion and proliferation studies: To determine the adhesion density and proliferation viability of chondrocytes, the cell proliferation assay (CellTiter 96, Promega) was used. Briefly, pig chondrocyte cells at passage number 4-6 were seeded at 3,500 cells/cm2 in Dulbecco's Modified Eagle's (DMEM, GIBCO)/Ham F-12 media supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (P/S, Hyclone) and incubated for 4 hours, 1 day, and 3 days. The dye solution was added to the cells after the end of the prescribed period for 4 h, then the stop solution was added and incubated overnight. A plate reader was used to test the cell density. 

Total protein synthesis: Total protein content in the cell lysates was measured using a commercial BCATM Protein Assay Reagent Kit (Pierce Biotechnology) and following the manufacturer's instructions. Chondrocytes were seeded at a seeding density of 10,000 cells/cm2 onto the substrates for 3 and 5 days, and then lysated by freeze-thaw cycles for at least three times. Aliquots from the supernatants of the protein-containing cell lysates (150 µl) were mixed with the reagent solutions and incubated at 37C for 2 h. Optical absorbance was measured at 562 nm on a spectrophotometer (SpectraMax 340PC, Molecular Devices).

GAG synthesis: For chondrocyte differentiation studies, chondrocytes were seeded at a seeding density of 10,000 cells/cm2 onto the substrates. Cells were cultured for 3 and 5 days under standard cell culture conditions with chondrogenic medium. Glycosaminoglycan (GAG) concentration was measured spectrophotometrically by a 1- 9- dimethylmethylene blue (DMMB) dye assay.

Statistical analysis. Numerical data were analyzed with Student's t-test to make pair-wise comparisons. Statistical significance was considered at p<0.05.

Results and Discussion:

All of the composites containing TBLs (0.01 mg/ml) enhanced chondrocyte adhesion and proliferation compared to composites without TBL (Figure 1). In particular, TBL /pHEMA/20%H2O composites had the highest chondrocyte adhesion density after 3 days of culturing. The addition of TBLs increased chondrocyte differentiation including total protein and GAG synthesis. The tensile strength of the composites increased with water content. The mechanical properties were closer to those of cartilage tissue with 20% water. Moreover, composite injectability was controlled by varying water concentrations. Therefore, this study showed that the TBL/pHEMA composites are promising for the design of injectable bioactive cartilage implants.

Figure 1. Chondrocyte density on pHEMA composites containing no TBL, TBL with 10%, 20%, or 30% H2O after 1 and 3 day of culturing. All the composites contained TBLs (0.01 mg/ml). Values are mean SEM; n=3. (*) p<0.05 compared to pHEMA without TBL composites after 1 day of culturing. (**) p<0.05 compared to pHEMA with TBL composite. (***) p<0.05 compared to pHEMA with TBL and 10% H2O composite. (#) p<0.05 compared to pHEMA without TBL composites after 3 days.

 

Conclusions:

This study indicated that TBL/pHEMA composites are promising injectable materials for cartilage applications since they possess desirable mechanical and cytocompatible properties.

Acknowledgements:

The authors acknowledge Audax Medical, Inc. for financial assistance.

References:

1.      Moralez, J. G.; Raez, J.; Yamazaki, T.; Kishan, M. R.; Kovalenko, A.; Fenniri, H., Helical Rosette Nanotubes with Tunable Stability and Hierarchy, J. Am. Chem. Soc. 127, 8307C8309, 2005.

2.      Zhang, L.; Hemraz, U. D.; Fenniri, H.; Webster, T. J., Tuning Cell Adhesion on Titanium using Osteogenic Rosette Nanotubes. J. Biomed. Mater. Res. Part A 95A, 550-563, 2010.

3.      Chen, Y.; Pareta, R. A.; Bilgen, B.; Myles, A. J.; Fenniri, H.; Ciombor D. M.; Aaron, R. K.; Webster, T. J., Self-assembled Helical Rosette Nanotubes/Hydrogel Composites for Cartilage Tissue Engineering. Tissue Eng. Part C 16, 1233C1243, 2010.

 

 


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