Preparation of a biomimetic nanocomposite scaffold for bone tissue engineering

Mahmoud Azami, Nafiseh Baheiraei, Jafar Ai

Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Introduction
Three-dimensional porous scaffolds are mandatory components of bone tissue engineering systems. Biomimetic techniques have been used to reproduce natural bone structure and its chemical composition to make bone scaffolds[1-3].
Dual diffusion of calcium and phosphate ions into hydrogel especially natural polymers such as collagen, gelatin has been considered as a biomimetic method.
The objectives of this study were to use the double diffusion method in a physiologically relevant environment to prepare a biomimetic GEL-ACP nanocomposite scaffold and to investigate ACP’s phase conversion to HA during incubation in a simulated body environment.

Materials and Methods
A double diffusion method was used for biomineralisation of a gelatin hydrogel leading to form a nanocomposite scaffold. Fig 1 shows the details of this setup. Following diffusion of calcium and phosphate ions into the gel, a white precipitate formed within the gel and thickened After 48 h reaching 1 cm. The resulting nanocomposite was extracted and cut into 2 mm layers and freeze-dried to create a porous and finally cross-linked a 1% glutaraldehyde solution for 24 h. To study the possible precipitate phase conversion, nanocomposites samples were soaked in a simulated body fluid (SBF) (kokubo buffer solution) (pH 7.4) and incubated at 37ْC for 5 h. 
Precipitated mineral within gelatin hydrogel and prepared nanocomposite scaffold and were analyzed using scanning electron microscopy, X-ray diffraction (XRD), fourier transform infrared spectroscopy(FTIR), transmission electron microscopy(TEM) and mechanical testing.

Figure1- Schematic view of reactor used for biomineralization of apatite within GEL hydrogel via double diffusion

Results and Discussion
The results showed that prepared nanocomposite scaffolds were porous with three-dimensionally interconnected microstructure, pore size ranging from 150 to 350 µm(Fig2). Porosity was about 82% and nanocrystalline precipitated minerals were dispersed evenly among gelatin fibers. A mineral containing amorphous calcium phosphate and brushite precipitate was formed within the gelatin matrix at 4°C. After incubation in SBF solution at 37°C for 5 days, the mineral phase was transformed to nanocrystalline hydroxyapatite(Fig3).


Figure 2- SEM micrographs obtained from surface of the synthesized biomimetic nanocomposite scaffold

Figure3. XRD diffractogram for the precipitated minerals formed at 4°C (up) and after incubation in SBF (down).

Conclusion
In this study, in situ formed nanocomposite scaffolds were designed and fabricated using a biomimetic approach. A mineral containing ACP and DCPD precipitate was formed within the GEL matrix at 4°C.

After incubation in SBF solution at 37°C for 5 days, the mineral phase was transformed to nanocrystalline HA.

It should be noted that precursor phases inside a scaffold implanted into the body can result in biomimetic conversion of precursors to HA that is very similar to the bone mineral.

This HA has a profound level of biocompatibility.

Thus, our results highlight the potential use of engineered biomimetic bone tissue scaffolds in the bone tissue repair process.

References
1-Manjubala et.al, Acta Biomaterialia 2006; 2: 75–84.
2- Ehrlich et.al, Journal of Membrane Science 2006; 273: 124-128.
3-Gomes et.al, Materials Science Forum 2008; 587/588: 77-81.