Search

The main goal of this study was the formation of bone tissue using dexamethasone (DEX)-loaded [COCH3]-RADARADARADARADA-[CONH2] (RADA 16-I) scaffold that has the ability to release optimal DEX concentration under perfusion force. Bone-marrow samples were collected from three patients during a hip arthroplasty. Human mesenchymal stem cells (hMSCs) were isolated and propagated in vitro in order to be seeded on scaffolds made of DEX-loaded RADA 16-I hydrogel in a perfusion bioreactor. DEX concentrations were as follows: 4 &times, 10&minus, 3, 4 &times, 4 and 4 &times, 5 M. After 21 days in a perfusion bioreactor, tissue was analyzed by scanning electron microscopy (SEM) and histology. Markers of osteogenic differentiation were quantified by real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. Minerals were quantified and detected by the von Kossa method. In addition, DEX release from the scaffold in a perfusion bioreactor was assessed. The osteoblast differentiation was confirmed by the expression analysis of osteoblast-related genes (alkaline phosphatase (ALP), collagen I (COL1A1) and osteocalcin (OC). The hematoxylin/eosin staining confirmed the presence of cells and connective tissue, while SEM revealed morphological characteristics of cells, extracellular matrix and minerals&mdash, three main components of mature bone tissue. Immunocytochemical detection of collagen I is in concordance with given results, supporting the conclusion that scaffold with DEX concentration of 4 &times, 4 M has the optimal engineered tissue morphology. The best-engineered bone tissue is produced on scaffold loaded with 4 &times, 4 M DEX with a perfusion rate of 0.1 mL/min for 21 days. Differentiation of hMSCs on DEX-loaded RADA 16-I scaffold under perfusion force has a high potential for application in regenerative orthopedics.

[EN] The extensive need for hard tissue substituent greatly motivates development of suitable allogeneic grafts for therapeutic recreation. Different calcium phosphate phases have been accepted as scaffold's components with positive influence on osteoinduction and differentiation of human mesenchymal stem cells, in terms of their higher fraction within the graft. Nevertheless, the creation of unlimited nutrients diffusion through newly formed grafts is of great importance. The media flow accomplished by perfusion forces can provide physicochemical, and also, biomechanical stimuli for three-dimensional bone-construct growth. In the present study, the influence of a different scaffold's composition on the human mesenchymal stem cells (hMSCs) differentiation performed in a U-CUP bioreactor under perfusion conditioning was investigated. The histological and immunohistochemical analysis of cultured bony tissues, and the evaluation of osteogenic genes' expression indicate that the lower fraction of in situ formed hydroxyapatite in the range of 10-30% within chitosan scaffold could be preferable for bone-construct development., BICRO PoC5_1_82, the Croatian Science Foundation under the project IP-2014-09-3752 and L'Oreal-UNESCO Foundation 'ForWomen in Science' are gratefully acknowledged.

[EN] Poor mechanical performance of porous chitosan-hydroxyapatite systems is the main limitation in bone tissue engineering. If we merge good mechanical performance of poly(lactic acid) construct with osteoinductive and bioresorbable properties of chitosan-hydroxyapatite porous hydrogel, we can obtain a material that meets necessary requirement for bone tissue substituent. With this in mind, we propose the combination of 3D printing technique and the thermally-induced phase separation method for simultaneous modification of biological properties of poly(lactic acid) and load-bearing properties of chitosan-hydroxyapatite porous hydrogel. 3D printed poly(lactic acid), PLA, construct has been used as a mechanical support with very large pore diameter of 960 +/- 50 mu m allowing enough free space (similar to 60% of porosity) to form porous composite hydrogel by freeze gelation. In situ formation of hydroxyapatite within chitosan hydrogel has ensured higher human mesenchymal stem cell osteogenesis during 21 days of culture. Positive modification of poly(lactic acid) has been simultaneously utilized to improve the compressive strength of composite hydrogel which has been confirmed by Young's modulus ranging from lower values reported for cancellous bone in dry state. Considering positive osteogenic signal accompanied with suitable mechanical properties, our scaffolds have shown good potential as bone tissue substituent. (C) 2016 Elsevier Ltd. All rights reserved., MINECO MAT2013-46467-C4-1-R project and CIBER-BBN-Instituto de Salud Carlos III, HATEA project financed by the Croatian Science Foundation, BICRO PoC5_1_82 and L’Oréal-UNESCO Foundation ‘For Women in Science’ are gratefully acknowledged.

There have been numerous investigations based on the synthesis of new biodegradable scaffolds for bone tissue replacement. To achieve required features of potential biomaterial substituent, wide range of synthetic and natural polymers along with calcium phosphate phases were used. The main goal is to produce a material that can mimic extracellular matrix of natural bone. Chitosan has shown to be a good candidate for tissue engineering materials due to its chemical similarity to biological molecules and short-time biodegradability in vivo through specific enzymatic reactions. Likewise, chitosan can be processed by different techniques for scaffold production (lyophilization, electrospinning, thermally induced phase separation, particulate leaching, microsphere sintering, etc). Scaffold’s topography (surface roughness), charge and wettability, microstructure and interconnected porosity are the main factors influencing the cell adhesion and activity, neovascularization and angiogenesis. Besides surface properties, the alteration of scaffold’s composition during neotissue formation affects further cell proliferation and growth. Calcium phosphate salts have shown good resorption ability in physiological conditions, and depending on its composition, higher solubility. Even though chitosan/hydroxyapatite scaffolds show favourable properties for bone tissue engineering, biomechanical properties of those systems can not meet applications in vivo. To overcome the mechanical drawback, additional synthetic polymers have been used. Up till now, we have demonstrated the importance of scaffold’s composition and microstructure in cell proliferation and differentiation on chitosan/hydroxyapatite three-dimensional structures. In vitro cell cultures in static and dynamical conditions with different cell lines have indicated suitable amount of in situ formed HA within chitosan porous matrix. Since chitosan/hydroxyapatite porous structures form hydrogels in physiological conditions, their mechanical properties do not meet the conditions as bone implants. To overcome those limitations, 3D-printed PLA structure have been applied as a supporting construct for porous chitosan/hydroxyapatite porous hydrogel. Osteogenic evaluation with mesenchymal stem cells after 21 day of culture confirmed good osteogenic properties of three-component porous structure and subsequently indicated its potential application as bone substituent.