Non-Cytotoxic Agarose/Hydroxyapatite Composite Scaffolds for Drug Release

Abstract

Healing of large bone defects requires implants or scaffolds that provide structural guidance for cell growth, differentiation, and vascularization. In the present work, an agarose-hydroxyapatite composite scaffold was developed that acts not only as a 3D matrix, but also as a release system. Hydroxyapatite (HA) was incorporated into the agarose gels in situ in various ratios by a simple procedure consisting of precipitation, cooling, washing, and drying. The resulting gels were characterized regarding composition, porosity, mechanical properties, and biocompatibility. A pure phase of carbonated HA was identified in the scaffolds, which had pore sizes of up to several hundred micrometers. Mechanical testing revealed elastic moduli of up to 2.8 MPa for lyophilized composites. MTT testing on Lw35human mesenchymal stem cells (hMSCs) and osteosarcoma MG-63 cells proved the biocompatibility of the scaffolds. Furthermore, scaffolds were loaded with model drug compounds for guided hMSC differentiation. Different release kinetic models were evaluated for adenosine 5'-triphosphate (ATP) and suramin, and data showed a sustained release behavior over four days.

Keywords: agarose; biocomposite; bone tissue engineering; drug release; hydrogel; hydroxyapatite.

Figure A1

EDX mapping of a supercritically-dried AG33HA67 scaffold. (a) SEM picture; (b) mapping overlay of carbon (blue), calcium (red), phosphorus (green), oxygen (yellow), and sodium (white); (c) mapping overlay of calcium (red) and phosphorus (green); (d) mapping overlay of carbon (blue) and oxygen (yellow); (e) EDX spectrum of mapping data. The scale bar in mappings is 5 $\mathsf{\mu }$$\mathrm{m}$; voltage 15 $\mathrm{k}$$\mathrm{V}$; working distance $4.3$ $\mathrm{m}$$\mathrm{m}$.

Figure A2

Complete dataset of swelling by volume and water uptake experiments. All three scaffolds were tested at different pH (5.0, 7.4, and 9.0) over 48 $\mathrm{h}$ after different drying methods (vacuum drying, lyophilization, and as native hydrogel).

Figure 1

X-ray diffraction of hydroxyapatite (HA) dried at 500 ${}^{\circ }\mathrm{C}$ and lyophilized agarose/HA composite (AG33HA67).

Figure 2

FT-IR spectra of agarose (black), hydroxyapatite (red), and agarose/hydroxyapatite composite (AG33HA67; blue).

Figure 3

SEM images of agarose lyophilized (LYO; (a–c)) and supercritically-dried (SCD; (d–f)) and AG33HA67 composite LYO (g–i) and SCD (k–m) at three different magnifications. The scale bar is 10 $\mathsf{\mu }$$\mathrm{m}$ (left), 1 $\mathsf{\mu }$$\mathrm{m}$ (middle), and $0.2$ $\mathsf{\mu }$$\mathrm{m}$ (right), respectively.

Figure 4

Images of (a) agarose hydrogel (AG100HA0) and (b) composite hydrogel (AG33HA67) in the native state, lyophilized (LYO), or vacuum dried (VD) and after reswelling of the dried gels. Comparison of (c) swelling, (d) water uptake, and (e) volume and mass loss for dried and native gels.

Figure 5

Compressive properties of native hydrogels, lyophilized, and rehydrated gels. (a) Compressive strength, elastic modulus E, and strain at compressive strength, derived from uni-axial unconfined compression ($n=6$). (b) Typical stress-strain and stress ($\lambda -1/{\lambda }^2$) curves for AG33HA67 gels.

Figure 6

Cell viability of hMSCs Lw35and MG-63 cell lines on pure agarose (AG100HA0) and AG33HA67 composites. Scaffolds washed with PBS prior to MTT assay display higher cell viability. One hundred percent viability (dotted line) and 75% viability (solid line) are marked for clarification.

Figure 7

Release data of (a) ATP and (b) suramin from AG100HA0 (black), AG50HA50 (orange), and AG33HA67 (blue) scaffolds. Data fit: Weibull equation $M=M_f·(1-e^{\frac{-{(t-T)}^b}{a}})$.