Introduction: “Burr holes” are made to insert surgical tools during a craniotomy[1]. Calcium phosphate cement (CPC) is currently used for filling the holes[2]. However, the setting time of CPC is delayed if contact is made with cerebrospinal fluid or blood, they sometimes leak into the brain and also have a low degradation rate. Moreover, CPCs are fragile and may be broken as new bone regenerates.
Shirosaki et al., prepared the chitosan- siloxane-3-glycidoxypropyltrimethoxysilane (GPTMS) porous hybrids via sol-gel and freeze-drying method[3]. Moreover, chitosan-siloxane hybrids incorporated with calcium chloride were soaked in alkaline phosphate solution to deposit the apatite on their surfaces[4]. In previous study[5], Incorporation of calcium ions and hydroxyapatite coating accelerated the calcification. In this study, bone regenerations after 2 and 3 years implantation were observed histologically.
Materials and Methods: Preparation of the porous hybrids: Chitosan (0.5 g, high molecular weight, deacetylation: 79.0%, Aldrich®, USA) was dissolved in aqueous acetic acid (0.25 M, 25 mL). GPTMS (Lancaster, Lancashire, UK) and calcium chloride (Nacalai Tesque, Kyoto, Japan) were added to give a molar ratio of chitosan to GPTMS (ChG) of 1:0.5 or chitosan to GPTMS :to CaCl2 (ChGCa) of 1.0 : 0.5 : 1.0. The mixtures were stirred for 1 h at room temperature and a fraction of each resultant sol was poured into a polystyrene container and frozen at -20°C for 24 h. The frozen hybrids were then transferred to a freeze dryer (FDU-506, EYELA, Tokyo, Japan) for 12 h until dry. The resultant porous ChG and ChGCa hybrids were then washed with NaOH (0.25 M) and distilled water to neutralize remaining acetic acid and were again lyophilized. Some ChGCa hybrids were instead soaked in aqueous Na2HPO4 (0.01 M, pH8.8) at 80°C for 3 days (ChGCa_HAp). These hybrids were then washed with distilled water and lyophilized.
In vivo animal test: The hybrids were sterilized with γ-ray irradiation. All surgical procedures were performed with the approval of the animal care and use committee of Osaka Medical College (No. 26044). The specimens were decalcified and thin sections were prepared by microtome and stained with hematoxyline-eosine (HE) and azan-mallory (AM).
Results and Discussions: The hybrids with HAp (ChGCa_HAp) showed bone formation. Fibrous tissues were observed at the implant site and new bone formed from the interface with the pre-existing skull bone to center of the implant site. Osteoblasts was not observed but a lot of blood vessels with harves canals were observed. The osteoid tissue and calcification were formed along the fibrous tissue. In case of ChGCa and ChG, the regenerated tissues were very similar to ChGCa_HAp, but the osteoid tissue and calcification areas were smaller than ChGCa_HAp. Engler et al[6]., reported that matrix stiffness can have an influence on the cell local-adhesion structure and the cytoskeleton and also differentiation. ChGCa_HAp and ChGCa were harder than ChG because of the accelerated polycondensation of Si-O-Si networks by calcium ions. It indicates that the release of calcium ions, the existence of hydroxyapatite and stiffness of matrix improve bone regeneration in craniotomy.
Conclusion: The chitosan-siloxane porous hybrids coated with hydroxyapatite accelerated the calcification. The chitosan-siloxane porous hybrids could be used to repair burr holes after cranioplasty.
Ministry of Education Culture, Sports, Science and Technology (MEXT); the Foundation for the Promotion Ion Engineering
References:
[1] Kilincer et al., Clin. Neurol. Neurosurg., 107, 5, 412-416, 2005
[2] Provenzano et al., Am. J. Neuroradiol., 25, 7, 1286-1290, 2004
[3] Shirosaki et al., Chem. Eng. J., 137, 1, 122-128, 2008
[4] Shirosaki et al., BioMed. Res. Int., Article ID 392940, 6 pages, 2014
[5] Shirosaki et al., Letters Appl. NanoBioSci., be accepted.
[6] Engler et al., Cell, 126, 677-689, 2006