Biomaterials 23 (2002) 4557–4563 Variation of nanomechanical properties of bone by gene mutation in the zebrafish X.M. Wang, F.Z. Cui*, J. Ge, Y. Zhang, C. Ma Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Received 5 March 2002; accepted 16 May 2002 Abstract Significant variations of nanomechanical properties and fracture morphology between gene-mutated liliput dtc232 (lil/lil) zebrafish skeletal bone and wild-type bone have been observed. Nanoindentation measurement disclosed that lil/lil bone has 36% lower nanohardness and 32% lower elastic modulus. The standard deviations of hardness and elastic modulus of lil/lil bone were both much higher than those of wild-type bone. SEM morphology of fracture surfaces further revealed that in bones after gene mutation, formative microcracks make the performance reduction and the increasing of brittleness. What is more, the plywood-like structure of the normal bone does not exist in the lil/lil bone. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Gene mutation; Nanoindentation; Bone; Zebrafish 1. Introduction The application of gene technique to materials is an emerging field [1–4]. Currently, this field mainly focuses on biosynthesis and modifications of fibers and bio- composites through integration of polymer synthesis, mineralization and self-assembly. Bone material is a particularly complex example of such composites, because it contains multiple hierarchical levels of organization [5]. Recently, the alteration of bone development by transgene or gene mutation has been widely reported [6–9]. However, to the best of our knowledge, variations on the mechanical properties and microstructure of gene-mutated bone have never been revealed. Whereas the information on these variations has considerable significance in many areas, such as understanding the roles of genes associated with bone materials, studying the relationship among genes, microstructure and mechanical properties of materials, beating out the mechanism of biomineralization essen- tially and providing novel ideals to study bone-materials fabrication via gene engineering and human heritable bone disease. The zebrafish (Danio rerio) has been a powerful model organism for the study of vertebrate biology, being well suited to both developmental and genetic analysis [10]. The gene-mutated liliput dtc232 zebrafish was found to have a dominant reduction of the overall body length in the adult, which also indicated the reduction in length of the vertebra [9]. However, it is unclear whether there is any variation of tissue mechanical properties and microstructure. We have examined bone mineral and microstructure of lil/lil mutants, which will be discussed in detail in another paper. The diversity of structures reflects the fine-tuning of the structure of its function. As a significant variation of microstructure was found in lil/ lil bone, further studies on mechanical behaviors in lil/lil bone are therefore needed. Investigation of mechanical behavior of bone is of particular challenge, both because of its dependence on the complex hierarchical structures of bone [11,12] and because of the technical difficulties involved in measur- ing the mechanical properties of very small samples [13]. Nanoindentation is now used widely for probing the mechanical properties of small volumes, thin films, and small microstructural features [14,15]. Nanoindentation was therefore thought to be a good choice to evaluate *Corresponding author. Tel.: +86-10-62772850; fax: +86-10- 62771160. E-mail address: cuifz@mail.tsinghua.edu.cn (F.Z. Cui). 0142-9612/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII:S0142-9612(02)00201-6