Cylindrical constructs with parallel aligned pores were prepared by using ionotropic gelation of alginate/calcium phosphate hydroxyapatite (HAP) mixtures in regard to applications as scaffold for bone regeneration. The starting powder and stabilizing agents were characterized by measurement of electrosonic amplitude, particle size distribution, and specific surface. The shrinkage of the gels was investigated in dependence on the drying methods. The pore size relied on preparation conditions such as amount of HAP and concentrations of gelling agent or alginate sol. A wide field of pore sizes could be fabricated by varying the kind and concentration of additives. Micro computer tomography-investigations of freeze dried scaffolds demonstrated the pore progression over a length of 4 mm. The pore dimension and structure were adequate for cell seeding and blood capillary ingrowth. Biocompatibility was proven by in vitro experiments with human mesenchymal stem cells by fluorescence microscopy. A high stability of the wet gels was maintained under cell culture conditions for a period of 3 weeks.
Porous and mineralised scaffolds are required for various applications in hard tissue engineering. Scaffolds with oriented tube-like pores facilitate homogenous cell seeding, a sufficient nutrient supply during cell culture (even in big constructs) and a fast vascularisation after implantation. The phenomenon of ionotropic gelation has been known since more than 30 years which describes that alginate forms gels with capillary-like pores when covered with solutions of di-or trivalent cations [1]. This technique has been used here to develop scaffolds with tube-like and regular pores from alginate/calcium phosphate composites and to stabilise them by mineralisation with hydroxyapatite from solution.
Alginates form gels with tube-like pores when covered with solutions of di-or trivalent cations. This phenomenon also referred to as ionotropic gelation has been known for more than 30 years.[1,2] By mixing a calcium phosphate powder and an alginate as the starting material, the mineral phase of bone is incorporated. Such porous structures can be used for scaffolds in hard tissue engineering. The starting materials and stabilizing additives are dispersed in an aqueous solution. Then a solution of Ca-ions is deposited onto the surface of the slurry. The slurry can be gelled by ion exchange of Na-ions in the alginate with Ca-ions. A primary thin gel layer with the function of a membrane is immediately formed. By diffusional control of cation transport through the membrane, the slurry gradually transforms to the gel forming tube-like pores in direction of cation diffusion. Like the gelation of pure alginate the concentration of electrolyte and the kind of cations and anions influence the size (diameter and length) and size growth of the pores, but the tolerance in the preparation conditions is much smaller. The diameters of the pores can be adjusted between 50 and 500 µm which fits the optimum size for cell seeding and blood capillary ingrowth very well. By selecting the proper drying method the inherent shrinkage can be controlled. Hydroxyapatite sintered at high temperatures loses the ability to be resorbed by osteoclasts in vivo. Therefore, we have developed scaffolds with channel-like pores from alginate/calcium phosphate composites without the necessity for heating them to higher temperatures.
Scaffolds for bone regeneration are mostly prepared with an isotropic, sponge-like structure mimicking the architecture of trabecular bone. We have developed an anisotropic bioceramic with parallel aligned pores resembling the honeycomb arrangement of Haversian canals of cortical bone and investigated its potential as a scaffold for tissue engineering. Parallel channel-like pores were generated by ionotropic gelation of an alginate-hydroxyapatite (HA) slurry, followed by ceramic processing. Organic components were thermally removed at 650 °C, whereas the pore system was preserved in the obtained HA bioceramic in the processing stage of a bisque. Even without further sintering at higher temperatures, the anisotropic HA bisque (AHAB) became mechanically stable with a compressive strength (4.3 MPa) comparable to that of native trabecular bone. Owing to the low-temperature treatment, a nanocrystalline microstructure with high porosity (82%) and surface area (24.9 m(2)/g) was achieved that kept the material dissolvable in acidic conditions, similar to osteoclastic degradation of bone. Human mesenchymal stem cells (hMSCs) adhered, proliferated and differentiated into osteoblasts when osteogenically induced, indicating the cytocompatibility of the bisque scaffold. Furthermore, we demonstrated fusion of human monocytes to osteoclast-like cells in vitro on this substrate, similar to the natural pathway. Biocompatibility was demonstrated in vivo by implantation of the bisque ceramic into cortical rabbit femur defects, followed by histological analysis, where new bone formation inside the channel-like pores and generation of an osteon-like tissue morphology was observed.
Conventionally sintered hydroxyapatite-based materials for bone repair show poor resorbability due to the loss of nanocrystallinity. The present study describes a method to establish nanocrystalline hydroxyapatite granules. The material was prepared by ionotropic gelation of an alginate sol containing hydroxyapatite (HA) powder. Subsequent thermal elimination of alginate at 650 °C yielded non-sintered, but unexpectedly stable hydroxyapatite granules. By adding stearic acid as an organic filler to the alginate/HA suspension, the granules exhibited macropores after thermal treatment. A third type of material was achieved by additional coating of the granules with silica particles. Microstructure and specific surface area of the different materials were characterized in comparison to the already established granular calcium phosphate material Cerasorb M(®). Cytocompatibility and potential for bone regeneration of the materials was evaluated by in vitro examinations with osteosarcoma cells and osteoclasts. Osteoblast-like SaOS-2 cells proliferated on all examined materials and showed the typical increase of alkaline phosphatase (ALP) activity during cultivation. Expression of bone-related genes coding for ALP, osteonectin, osteopontin, osteocalcin and bone sialoprotein II on the materials was proven by RT-PCR. Human monocytes were seeded onto the different granules and osteoclastogenesis was examined by activity measurement of tartrate-specific acid phosphatase (TRAP). Gene expression analysis after 23 days of cultivation revealed an increased expression of osteoclast-related genes TRAP, vitronectin receptor and cathepsin K, which was on the same level for all examined materials. These results indicate, that the nanocrystalline granular materials are of clinical interest, especially for bone regeneration.
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