A novel pathway for in situ synthesis of modified gelatin microspheres by silane coupling agents as a bioactive platform Farnaz Ghorbani, 1 Ali Zamanian , 2 Aliasghar Behnamghader, 2 Morteza Daliri Joupari 3 1 Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, P.O. Box 4515-775, Tehran, Iran 2 Department of Nanotechnology and Advanced Materials, Materials and Energy Research Center, P.O. Box 14155-4777, Karaj, Iran 3 Department of Animal, Avian and Marine Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965-161, Tehran, Iran Correspondence to: A. Zamanian (E - mail: a-zamanian@merc.ac.ir) ABSTRACT: This study investigated the fabrication and modification of gelatin microspheres via single emulsion method and silane cou- pling agents, respectively. Herein, the influence of oil phase and washing procedure on morphology and uniformity of microspheres was evaluated. The results of field emission scanning electron microscopy indicated a positive effect of olive oil on the production of uniform and approximately mono-size spheres while monodispersity increased after samples were washed under ultrasonication. Investigation of crosslinking degree demonstrated that GPTMS could not show efficiency as high as glutaraldehyde, but the lack of toxicity and inducing bioactive behavior to the microspheres has made them appropriate for bone applications. Moreover, the stability of spheres after 14 days in phosphate buffered saline proved the ability of (3-glycidyloxypropyl)trimethoxysilane to crosslink gelatin. The presence of silane cou- pling agents in prepared spheres caused the biomimetic formation of hydroxyapatite after a 14-day immersion in simulated body fluid as observed by field emission scanning electron microscopy-energy-dispersive X-ray spectroscopy, changes in pH, Fourier transform infrared spectroscopy, X-ray diffraction, and atomic force microscopy. Adhesion, spreading, and proliferation of L929 cells on the hybrid microspheres corroborated the potential of gelatin spheres for biomedical application. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018, 00, 46739. KEYWORDS: biomedical applications; biomimetics; biomineralization; drug delivery systems; gels; hybrid materials; polymeric materials; rheology; structure–property relationships Received 26 January 2018; accepted 18 May 2018 DOI: 10.1002/app.46739 INTRODUCTION Natural bone is a composite of approximately 22 wt % longitudinal collagen as a matrix phase reinforced with 69 wt % nonstoichio- metric carbonate hydroxyapatite [HA, (Ca 10 (PO 4 ) 6 (OH) 2 ]. 1 Bone structure is exposed to many dangers that originate from trauma, arthritis, fracture, etc., and requires replacement or regeneration to recover its function. 2 There exist many strategies to regenerate damaged bones and restore functionality, but in recent decades reconstruction via degradable and bioengineered structures have been given considerable attention owing to biocompatibility and nontoxicity, rare immune reaction, possibility of producing con- structs with desired properties and high degree of compliance, stim- ulating natural tissue/organ regeneration, access to abundant resources, 3–5 etc. Lack of stress-shielding through the use of poly- meric biomaterials has led to the versatile application in the recon- struction of defect. 6 But in hard tissue regeneration, there is the need to mix with bioactive materials in order to increase their potential for simulation of bone composition and chemical bonding ability with natural bone, 7,8 mechanical stability, 9 etc. The blending of polymers and ceramics necessitates the development of hybrid biomaterials for simulating multiphase tissue such as bone. A variety of constructs has been used in the regeneration process. One of the most common types is sphere-like shaped structures in micron-sized or nanoscale distribution. Microspheres can reduce side effects and immune rejection, produce efficiently, increase duration and stability of the structure, and are afford- able, injectable, and sterilizable. 10 They are numerously used indi- vidually in bone reconstruction, 11 or in three-dimensional scaffolds; 12 moreover, their ability to encapsulate drugs, genes, hormone, etc., increases their effectiveness. Despite the introduc- tion of a variety of techniques for fabrication of these biocompat- ible vehicles such as spraying, 13 phase separation, 14 emulsion- based methods, 15,16 suspension or dispersion polymerization, 17 extrusion methods, 18 etc., single emulsion technique have been © 2018 Wiley Periodicals, Inc. 46739 (1 of 10) J. APPL. POLYM. SCI. 2018, DOI: 10.1002/APP.46739