Calcium phosphate ceramics, such as hydroxyapatite (HA), play a unique role in the regeneration of damaged bone due to their bioactivity and osteoconductivity.1-4
To accelerate in-growth of bone and osteo-integration, the HA ceramic needs to be prepared in a porous scaffold structure mimicking cancellous bone, the spongy material inside long bones.
Bone regeneration occurs through a remodeling process that results from a dynamic balance between bone forming (osteoblast) and absorbing (osteoclast) cells. The latter break down bone tissue by secreting acid and enzymes that dissolve HA and collagen, respectively.5-7
The liberated calcium and protein prompt osteoblasts to lay down a new matrix, which remineralizes forming HA and collagen. After a bone is damaged, the body first produces woven bone by this process. This type of bone is weak, as it contains only a few, randomly oriented collagen fibers. Through the dynamic equilibrium between osteoclasts and osteoblasts, woven bone is transformed into lamellar bone, which contains ordered collagen and is much stronger. The uncoupling of this equilibrium results in either unwanted calcification or a reduction in bone mineral density, a condition commonly known as osteoporosis.
To speed the healing of damaged bones, we have developed a drug delivery system that mimicks the structure of cancellous (spongy) bone and has a highly controlled pharmaceutical release capability that can be sustained over a long period of time and does not have an initial burst release. This system can act not only as a convenient drug delivery reservoir but also as a scaffold for the regeneration of damaged hard tissue. To create the system, we coated porous HA with hybrid gels of bicontinuous sponge-phased L3 silicate and thermo-responsive poly(N-isopropylacrylamide) (L3-PNIPAm gels) without blocking the material's 3D pore channels. To test its drug-release capability, we introduced indomethacin (IMC), an anti-inflammatory drug for treatment of chronic rheumatism, into the L3-PNIPAm/HA biomimetic scaffolds. The in-vitro drug-release characteristics were demonstrated during step-wise temperature changes, and an in vitro cytotoxicity test was performed with murine (mouse) L-929 cells to demonstrate clinical feasibility.
Before L3-PNIPAm coating, scanning electron microscope images suggested that the synthetic HA skeleton had a 3D interconnected macro-porous structure with pore diameters of 100-200µm. The scaffold struts were well consolidated with fine crystalline grains of 3µm on average. After successful coating with silicified L3-PNIPAm gels, the macropores were still well connected to each other. The coating with silicified L3-PNIPAm created three sizes of channels in the material: tiny ones in the PNIPAm, larger ones in the L3, and the largest ones in the HA skeleton.
The silicified L3-PNIPAm gels regulate drug release by modulating the diameter of the pores. At low temperature, the PNIPAm polymers are swelled, preventing release of the drug. As the temperature rises from 25°C, the polymers shrink, allowing the drug to squeeze into the L3 porous channels. When the temperature increases above the lower critical solution temperature of L3-PNIPAm (34-36°C), the pore structure opens completely. As such, we examined the thermoresponsive release of IMC from L3-PNIPAm/HA composites during stepwise temperature changes between 25°C and 40°C.
The total IMC release rate can be represented as a combination of the individual diffusion rates in each matrix, which are influenced by the diameter and conformational shape of the pores. Consequently, the control of the burst release pattern resulted from the combinational retardation of the overall diffusion rate through the heterogeneous nanoporous channels. IMC molecules gradually squeezed out of the different dimensions of porous channels, first through the sub-nano and nanoscale PNIPAm polymeric network, then the 3D bicontinuous disordered nanoscale L3-channels, and finally the macroporous HA channels.
The in vitro cytotoxicity test was performed only with L3-PNIPAm/HA because PNIPAm and HA are known to be non-cytotoxic. Microscopic observation after seven days revealed that the murine L-929 cells were attached well onto both the matrix between HA and L3-PNIPAm and were in good contact with each other. They exhibited low cytotoxicity grades of 1 (slight) for HA and 2 (mild) for L3-PNIPAm/HA, according to the five grade criteria of the Korean National Standard for Medical Devices and Implants.
We have prepared drug delivery vehicles made of silicified L3-PNIPAm/HA nanocomposites that are free from initial burst release during stepwise temperature changes. Polymer shrinkage opened up the 3D interconnected pores in the material, gradually releasing IMC. The L3-PNIPAm/HA nanocomposites demonstrated a remarkable thermoresponsive on-off regulation. In vitro analysis carried out with the prepared material revealed no cytotoxicity, and the cells grew well on the surface of the matrix. As such, these L3-PNIPAm/HA nanocomposites could be used as highly long term drug release materials in the regeneration of damaged bones.
Jeong Ho Chang, Kyung Ja Kim
Korea Institute of Ceramic Engineering and Technology
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