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Bone tissue engineering using macroporous PHA-PLA and PHBV scaffolds produced by additive manufacturing and wet spinning

Alagöz, Ayşe Selcen
Bone supports and protects organs of body, stores minerals, produces blood cells and enables the movement of body. In addition, bone regulates homeostasis by controlling the concentration of key electrolytes in the blood and in the storage of Ca+2 and PO43- ions. Trauma, tumor, nonunion fractures and diseases like osteoporosis lead to bone loss that affects millions of people. Current clinical treatments such as application of autograft and allograft for treatment of these problems are limited due to donor scarcity, donor site morbidity, disease transmission and rejection. Bone tissue engineering uses life science and engineering principles and presents a promising approach to treat bone defects. Scaffolds, signaling molecules, and cells are essential components of any tissue engineering application. The aim of this study was to develop three dimensional structures which have suitable architecture for the treatment of bone defects. For this purpose, two different polymers, PHBV and PHA-PLA, were used to produce scaffolds by using two different techniques, rapid prototyping (fused deposition modelling, FDM) and wet spinning. With FDM the pore size, pore distribution within the 3D structure of scaffolds can be controlled. Wet spinning produces scaffolds with pores that are random and nonhomogeneous in size and distribution. Thus, the properties of the FDM products are predetermined. PHA-PLA was used to make scaffolds using both methods while PHBVwas only wet spun. Results showed that wet spun PHA-PLA and PHBV scaffolds had similar porosity (77% and 75%), and pore size (300 μm and 250 μm). On the other hand, FDM PHA-PLA scaffolds have higher compressive property than wet spun scaffolds because fibers in a layer contact with fibers at the subsequent layer. Oxygen plasma treatment is known to improve the hydrophilicity of polymers and also increase surface reactivity to coat ELP-REDV on the surface of the polymer to promote endothelial cell attachment and increase proliferation of cells around the defect site. Optimum oxygen plasma treatment times and powers were determined as 4 min for PHBV scaffolds and 2 min for PHA-PLA scaffolds at 50W. The effect of oxygen plasma treatment and surface coating with ELP-REDV were shown by goniometer for contact angle, atomic force microscope for surface topography, FTIR-ATR, and Toluidine Blue staining for binding. It was seen that hydrophilicity of all scaffolds increased and moderately hydrophilic surfaces were obtained. FTIR-ATR analysis showed that surfaces of scaffolds were coated with ELP-REDV resulting in formation of amide I and amide II bands. Besides, oxygen plasma treatment and ELP-REDV attachment resulted in the increase of roughness (formation of valley and peaks) on the surfaces of samples and changed the surface roughness. Isolated rabbit bone marrow stem cells were seeded on scaffolds and cell behavior (attachment, proliferation and differentiation) were studied. High cell proliferation on FDM scaffolds was observed compared with wet spun scaffolds. This shows that FDM scaffolds can provide surfaces suitable for cell proliferation. Presence of ELP-REDV sequences enhanced cell attachment and proliferation on the scaffolds. Alkaline phosphatase activity on FDM scaffolds was higher than on wet spun scaffolds because of more cell proliferation on FDM scaffolds. Osteopontin staining showed that after culturing for 3 weeks in the differentiation medium, cells secreted osteopontin which show osteogenic differentiation because this protein is secreted by mature osteoblasts at the later stages of osteoblastic differentiation. SEM images showed that cells cultured on the scaffolds proliferated and penetrated into the scaffolds and deposited calcium containing minerals. Ca+2 deposition was observed on all types of scaffolds by Alizarin Red staining.It was concluded that FDM PHA-PLA and wet spun PHBV and PHA-PLA scaffolds have a significant potential for using bone tissue engineering.