Cartilage regeneration using improved surface electrospun bilayer polycaprolactone scaffolds loaded with transforming growth factor-beta 3 and rabbit muscle-derived stem cells.

Polycaprolactone (PCL) has recently received significant attention due to its mechanical strength, low immunogenicity, elasticity, and biodegradability. Therefore, it is perfectly suitable for cartilage tissue engineering. PCL is relatively hydrophobic in nature, so its hydrophilicity needs to be enhanced before its use in scaffolding. In our study, first, we aimed to improve the hydrophilicity properties after the network of the bilayer scaffold was formed by electrospinning. Electrospun bilayer PCL scaffolds were treated with ozone and further loaded with transforming growth factor-beta 3 (TGFß3). In vitro studies were performed to determine the rabbit muscle-derived stem cells' (rMDSCs) potential to differentiate into chondrocytes after the cells were seeded onto the scaffolds. Statistically significant results indicated that ozonated (O) scaffolds create a better environment for rMDSCs because collagen-II (Coll2) concentrations at day 21 were higher than non-ozonated (NO) scaffolds. In in vivo studies, we aimed to determine the cartilage regeneration outcomes by macroscopical and microscopical/histological evaluations at 3- and 6-month time-points. The Oswestry Arthroscopy Score (OAS) was the highest at both mentioned time-points using the scaffold loaded with TGFß3 and rMDSCs. Evaluation of cartilage electromechanical quantitative parameters (QPs) showed significantly better results in cell-treated scaffolds at both 3 and 6 months. Safranin O staining indicated similar results as in macroscopical evaluations-cell-treated scaffolds revealed greater staining with safranin, although an empty defect also showed better results than non-cell-treated scaffolds. The scaffold with chondrocytes represented the best score when the scaffolds were evaluated with the Mankin histological grading scale. However, as in previous in vivo evaluations, cell-treated scaffolds showed better results than non-cell-treated scaffolds. In conclusion, we have investigated that an ozone-treated scaffold containing TGFß3 with rMDSC is a proper combination and could be a promising scaffold for cartilage regeneration.

Functionalized Electrospun Scaffold-Human-Muscle-Derived Stem Cell Construct Promotes In Vivo Neocartilage Formation.

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-ß3. 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-ß3 and hMDSC compared to NO and scaffolds without TGF-ß3 in in vivo animal experiments. Coll2-positive extracellular matrix was observed within O and NO scaffolds containing TGF-ß3 and hMDSC for up to 8 weeks after implantation. These findings suggest that ozone-treated, TGF-ß3-loaded scaffold with hMDSC is a promising tool in neocartilage formation.

The Effect of Ozone Treatment on the Physicochemical Properties and Biocompatibility of Electrospun Poly(ε)caprolactone Scaffolds.

Ozonation has been proved as a viable surface modification technique providing certain properties to the scaffolds that are essential in tissue engineering. However, the ozone (O3) treatment of PCL scaffolds in aqueous environments has not yet been presented. O3 treatment performed in aqueous environments is more effective compared with traditional, executed in ambient air treatment due to more abundant production of hydroxyl radicals (•OH) within the O3 reaction with water molecules. During interaction with •OH, the scaffold acquires functional groups which improve wettability properties and encapsulate growth factors. In this study, a poly(ε)caprolactone (PCL) scaffold was fabricated using solution electrospinning and was subsequently ozonated in a water reactor. The O3 treatment resulted in the expected occurrence of oxygen-containing functional groups, which improved scaffold wettability by almost 27% and enhanced cell proliferation for up to 14 days. The PCL scaffold was able to withhold 120 min of O3 treatment, maintaining fibrous morphology and mechanical properties.

The effect of biomimetic coating and cuttlebone microparticle reinforcement on the osteoconductive properties of cellulose-based scaffolds.

Polymer-based scaffolds have already gained popularity in many biomedical applications due to convenient routes for fabrication and favourable structural, physicochemical and functional characteristics. However, polymeric scaffolds lack osteoconductivity and some synthetic polymers carry the risk of inflammatory response caused by degradation by-products. Those facts limit their practical use in bone tissue engineering. In this study, three-dimensional (3D) porous scaffolds from naturally derived polymer, namely regenerated cellulose, were prepared using a non-hydrolytic sol-gel and lyophilization techniques. To induce osteoconductive properties of the polymeric scaffolds, cuttlebone microparticles were immobilized and the surface coating was achieved via in vitro mineralization using 10-fold concentrated simulated body fluid (10x SBF). Biogenic activity of cuttlebone is explained by its chemical composition, which includes polysaccharide ß-chitin and macro-, micro- and trace elements favourable for mineralization. Parallel the scaffolds were examined during long-term (24 weeks) in vitro mineralization in 1x SBF for the purpose to investigate apatite-forming ability of the scaffolds. A nice cauliflower-like structures and needle-like dents of the spherical aggregates, which are characteristic to hydroxyapatite precursors, were observed on the surface of cellulose/cuttlebone scaffolds by SEM. 10x SBF coating enhanced cell attachment to the scaffolds because SBF elements are known to increase bioactivity by inducing re-deposition of carbonate apatite crystallites on scaffold surface. Additionally, calcium and phosphate depositions were clearly observed on the developed scaffolds using von Kossa and Alizarin Red S staining. Proliferative and osteoconductive effects on the osteoblast-like MG-63 cells demonstrate the cellulose/cuttlebone scaffolds soaked in 10x SBF as a favourable material for bone tissue engineering.

Osteoconductive 3D porous composite scaffold from regenerated cellulose and cuttlebone-derived hydroxyapatite.

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 cellulose-based composite scaffolds. Interconnected highly porous structure of the scaffolds was confirmed by micro-computed tomography. The mean pore size of the scaffolds was 510 µm 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.

Novel cellulose/hydroxyapatite scaffolds for bone tissue regeneration: In vitro and in vivo study.

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.