Cellulose scaffolds containing nano- or micro-hydroxyapatite (nHA or μHA) were prepared by the regeneration of cellulose from its acetylated derivative and the mechanical immobilization of inorganic particles, followed by freeze-drying. Microtomographic (micro-computed tomography) evaluation revealed that both scaffolds presented a highly interconnected porous structure, with a mean pore diameter of 490 ± 94 and 540 ± 132 μm for cellulose/nHA and cellulose/μHA, respectively. In vitro and in vivo characterizations of the developed scaffolds were investigated. Commercially available bone allograft was used as a control material. For the in vitro characterization, osteoblastic cell cultures were used and characterized over time to evaluate cell adhesion, metabolic activity, and functional output (alkaline phosphatase activity and osteoblastic gene expression). The results revealed greater spreading cell distribution alongside an increased number of filopodia, higher MTT values, and significantly increased expression of osteoblastic genes (Runx-2, alkaline phosphatase, and BMP-2) for cellulose/nHA, compared with cellulose/μHA and the control. The in vivo biocompatibility was evaluated in a rabbit calvarial defect model. The investigated scaffolds were implanted in circular rabbit calvaria defects. Four- and 12-week bone biopsies were investigated using micro-computed tomography and histological analysis. Although both cellulose/HA scaffolds outperformed the assayed control, a significantly higher amount of newly formed mineralized tissue was found within the defects loaded with cellulose/nHA. Within the limitations of this study, the developed cellulose/HA scaffolds showed promising results for bone regeneration applications. The biological response to the scaffold seems to be greatly dependent on the HA particles' characteristics, with cellulose scaffolds loaded with nHA eliciting an enhanced bone response.

Recently, usage of marine-derived materials in biomedical field has come into prominence due to their promising characteristics such as biocompatibility, low immunogenicity and wide accessibility. Among these marine sources, cuttlebone has been used as a valuable component with its trace elemental composition in traditional medicine. Recent studies have focused on the use of cuttlebone as a bioactive agent for tissue engineering applications. In this study, hydroxyapatite particles were obtained by hydrothermal synthesis of cuttlebone and incorporated to cellulose scaffolds to fabricate an osteoconductive composite scaffold for bone regeneration. Elemental analysis of raw cuttlebone material from different coastal zones and cuttlebone-derived HAp showed that various macro-, micro-and trace elements-Ca, P, Na, Mg, Cu, Sr, Cl, K, S, Br, Fe and Zn were found in a very similar amount. Moreover, biologically unfavorable heavy metals, such as Ag, Cd, Pb or V, were not detected in any cuttlebone specimen. Carbonated hydroxyapatite particle was further synthesized from cuttlebone microparticles via hydrothermal treatment and used as a mineral filler for the preparation of cellulosebased composite scaffolds. Interconnected highly porous structure of the scaffolds was confirmed by micro-computed tomography. The mean pore size of the scaffolds was 510 mm with a porosity of 85%. The scaffolds were mechanically characterized with a compression test and cuttlebone-derived HAp incorporation enhanced the mechanical properties of cellulose scaffolds. In vitro cell culture studies indicated that MG-63 cells proliferated well on scaffolds. In addition, cuttlebone-derived hydroxyapatite significantly induced the ALP activity and osteocalcin secretion. Besides, HAp incorporation increased the surface mineralization which is the major step for bone tissue regeneration.

Fibrous matrix of cellulose was obtained by wet-type electrospinning of cellulose in ionic liquid, 1-butyl-3-methylimidazolium acetate (BMIMAc). The experiments were designed to determine the effects of electric field intensity and the amount of dissolved cellulose on cellulose fiber morphology and diameter. Taking to account practical implications, that is, fiber size and the effectiveness of production, the most effective production of fibers took place using 3% cellulose/BMIMAc solution at electric field of 4.8 kV cm À1 , feed rate of 2.38 ml h À1 . Analysis has shown that cellulose was fully dissolved and consisted purely of regenerated cellulose (type II), while having porosity of 90% and average fiber width of 1.95 ± 0.9 μm. The scanning electron microscopy and micro-computed tomography analyses revealed a robust structural integrity of the formed fibrous matrix, which featured an area density of 85 ± 8 g/ m 2 . The mechanical properties (strength of 12.03 ± 1.1 MPa; strain at break 2.6 ± 0.3%) indicate that in this study strong fibrous cellulose matrix was formed which could be used for the production of biocomposites or as biocompatible scaffolds.

Polycaprolactone (PCL) is a non-cytotoxic, completely biodegradable biomaterial, ideal for cartilage tissue engineering. Despite drawbacks such as low hydrophilicity and lack of functional groups necessary for incorporating growth factors, it provides a proper environment for different cells, including stem cells. In our study, we aimed to improve properties of scaffolds for better cell adherence and cartilage regeneration. Thus, electrospun PCL–scaffolds were functionalized with ozone and loaded with TGF-β3. Together, human-muscle-derived stem cells (hMDSCs) were isolated and assessed for their phenotype and potential to differentiate into specific lineages. Then, hMDSCs were seeded on ozonated (O) and non-ozonated (“naïve” (NO)) scaffolds with or without protein and submitted for in vitro and in vivo experiments. In vitro studies showed that hMDSC and control cells (human chondrocyte) could be tracked for at least 14 days. We observed better proliferation of hMDSCs in O scaffolds compared to NO scaffolds from day 7 to day 28. Protein analysis revealed slightly higher expression of type II collagen (Coll2) on O scaffolds compared to NO on days 21 and 28. We detected more pronounced formation of glycosaminoglycans in the O scaffolds containing TGF-β3 and hMDSC compared to NO and scaffolds without TGF-β3 in in vivo animal experiments. Coll2-positive extracellular matrix was observed within O and NO scaffolds containing TGF-β3 and hMDSC for up to 8 weeks after implantation. These findings suggest that ozone-treated, TGF-β3-loaded scaffold with hMDSC is a promising tool in neocartilage formation.

No abstract