(180bw) Self-Assembled Rosette Nanotube Composites Improve Chondrocyte Functions

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 BCA^TM 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 37°C 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:

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2.      Zhang, L.;
Hemraz, U. D.; Fenniri, H.; Webster, T. J., Tuning Cell Adhesion on Titanium
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2010.

3.      Chen, Y.;
Pareta, R. A.; Bilgen, B.; Myles, A. J.; Fenniri, H.; Ciombor D. M.; Aaron, R.
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