Injectable Liposome-Loaded Hydrogel Formulations with Controlled Release of Curcumin and α-Tocopherol for Dental Tissue Engineering.

An injectable hydrogel formulation is developed utilizing low- and high-molecular-weight chitosan (LCH and HCH) incorporated with curcumin and α-tocopherol-loaded liposomes (Lip/Cur+Toc). Cur and Toc releases are delayed within the hydrogels. The injectability of hydrogels is proved via rheological analyses. In vitro studies are conducted using human dental pulp stem cells (hDPSCs) and human gingival fibroblasts (hGFs) to examine the biological performance of the hydrogels toward endodontics and periodontics, respectively. The viability of hDPSCs treated with the hydrogels with Lip/Cur+Toc is the highest till day 14, compared to the neat hydrogels. During odontogenic differentiation tests, alkaline phosphatase (ALP) enzyme activity of hDPSCs is induced in the Cur-containing groups. Biomineralization is enhanced mostly with Lip/Cur+Toc incorporation. The viability of hGFs is the highest in HCH combined with Lip/Cur+Toc while wound healing occurs almost 100% in both (Lip/Cur+Toc@LCH and Lip/Cur+Toc@HCH) after 2 days. Antioxidant activity of Lip/Cur+Toc@LCH on hGFs is significantly the highest among the groups. Antimicrobial tests demonstrate that Lip/Cur+Toc@LCH is more effective against Escherichia coli whereas so is Lip/Cur+Toc@HCH against Staphylococcus aureus. The antimicrobial mechanism of the hydrogels is investigated for the first time through various computational models. LCH and HCH loaded with Lip/Cur+Toc are promising candidates with multi-functional features for endodontics and periodontics.

Natural origin bilayer pullulan-PHBV scaffold for wound healing applications.

Skin tissue loss that occurs by injury and diseases can turn into chronic wounds as a result of complications alongside infection. Chronic wounds fail to heal by themselves and need advanced treatments like engineered wound dressings and regenerative scaffolds. In this study, a novel, natural origin, bilayer electrospun scaffold was produced from pullulan (PUL) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) biopolymers. PHBV production by Cupriavidus necator bacterial strain was optimized and produced polymer was characterized. Characteristic peaks and bands of PHBV were observed by H-NMR and FTIR analyses. Valerate mol percent of produced PHBV copolymer was determined by H-NMR. Average molecular weight of the polymer was determined by SLS technique and crystallinity of PHBV was calculated from DSC curve. Bilayer scaffold was produced by electrospinning of hydrophilic PUL fibrous membrane onto wet-electrospun hydrophobic PHBV 3D fibrous mat. Bilayer scaffold was designed to involve regenerative and barrier fibrous layers. Nano fibrous PUL membrane with smaller pore size was efficient as a barrier against bacterial transmission while enabling optimum oxygen and water vapor transmission. Water retention and degradation properties were found to be optimum for a skin tissue scaffold. In vitro studies showed that PUL membrane sustained L929 cell proliferation while preventing cells from migrating inside the barrier phase while PHBV layer supported cell viability, proliferation, and migration, creating a regenerative 3D structure. Results showed that, novel natural origin PUL/PHBV bilayer scaffold is a promising candidate for wound healing applications.

Investigation of the effect of ghrelin on bone fracture healing in rats.

Ghrelin is known to have effects on proliferation and differentiation of osteoblasts and improvement of bone mineral density in rats. However, no experimental research on ghrelin's effects on fracture healing has been reported. In this context, the effect of ghrelin on the union of femoral shaft fractures was examined in this study by evaluating whether ghrelin will directly contribute to fracture healing. Forty male Wistar-Albino rats were divided into two groups as control and experimental (ghrelin treated) and standard closed shaft fractures were created in the left femurs of all rats. Daily ghrelin injections were applied to the experimental groups and equal numbers of rats were killed after 14 and 28 days following fracture formation. Tissue samples were examined with radiological, biomechanical, biochemical and histological analyses. Densitometry study showed that bone mineral density was improved after 28 days of ghrelin treatment compared to control. On histological examination, at the end of the 14 and 28 days of recovery, significant union was observed in the ghrelin-treated group. The ghrelin-treated group had higher breaking strength and stiffness at the end of 28 days of recovery. Biochemically, ALP levels were found to be higher in the ghrelin-treated group at the end of 28 days of recovery. Results showed that ghrelin directly contributes to fracture healing and it is promising to consider the effect of ghrelin on fracture healing in human studies with pharmacological applications.

A dual-phase scaffold produced by rotary jet spinning and electrospinning for tendon tissue engineering.

Tendon is a highly hierarchical and oriented tissue that provides high mechanical strength. Tendon injuries lead to loss of function, disability, and a decrease in quality of life. The limited healing capacity of tendon tissue leads to scar tissue formation, which can affect mechanical strength and cause a re-tear. Tissue engineering can be the solution to achieving complete and proper healing of tendon. The developed constructs should be mechanically strong while maintaining a suitable environment for cell proliferation. In this study, a dual-phase fibrous scaffold was produced by combining fibrous mats produced by rotary jet spinning (RJS) and wet electrospinning (WES), with the intent of improving the healing capacity of the construct. Dual-phase scaffolds were formed from aligned poly(ϵ-caprolactone) (PCL) fibers (Shell) produced by RJS and randomly oriented PCL or PCL/gelatin fibers (Core) produced by WES systems. The scaffolds mimicked i) the repair phase of tendon healing, in which randomly-oriented collagen type III is deposited by randomly-oriented WES fibers and ii) the remodeling stage, in which aligned collagen type I fibers are deposited by aligned RJS fibers. In vitro studies showed that the presence of randomly-oriented core fibers inside the aligned PCL fiber shell of the dual-phase scaffold increased the initial attachment and viability of cells. Scanning electron microscopy and confocal microscopy analysis showed that the presence of aligned RJS fibers supported the elongation of cells through aligned fibers which improves tendon tissue healing by guiding oriented cell proliferation and extracellular matrix deposition. Tenogenic differentiation of human adipose-derived mesenchymal stem cells on scaffolds was studied when supplemented with growth differentiation factor 5 (GDF-5). GDF-5 treatment improved the viability, collagen type III deposition and scaffold penetration of human adipose derived stem cells. The developed FSPCL/ESPCL-Gel 3:1 scaffold (FS = centrifugal force spinning/RJS, ES = wet electrospinning, Gel = gelatin) sustained high mechanical strength, and improved cell viability and orientation while supporting tenogenic differentiation.

Nanocrystalline Zn2+ and SO42- binary doped fluorohydroxyapatite: A novel biomaterial with enhanced osteoconductive and osteoinconductive properties.

In this study, we have successfully doped hydroxyapatite (HA) with zinc (Zn2+), sulphate (SO42-) and fluoride (F-) ions to develop a new composition of bioceramic, Ca10-x Znx(PO4)6-y(SO4)y(OH)2-z-yFz(SO4)y, (x = 0, 0.2, 0.6, 1.0, y = 0, 0.5 and z = 0,1.0 mol), using wet precipitation method. The obtained materials were analysed using XRD, FTIR, FESEM, and XPS techniques to investigate the phase purity, particle morphology and elemental composition, respectively. A model anticancer drug (Doxorubicin, DOX) was loaded onto the surface of the Zn/SO4-FHA materials. About 100% loading of DOX with a controlled release profile was obtained. Degradation of materials in Simulated body fluid (SBF) was greatly improved with the incorporation of Zn2+/SO42- ions in comparison to HA/FHA, which makes it highly bioactive materials. In vitro cell viability and adhesion of Human fetal osteoblast (hFOB) cell were investigated. Cell viability has demonstrated that the hFOB cells proliferated at a high rate on Zn/SO4-FHA materials, confirming the in vitro biocompatibility of the materials. Alkaline phosphatase (ALP) activity and intracellular calcium deposition of hFOB cells seeded on 1ZnSO4-FHA disc surface was statistically higher than observed on pure HA and FHA discs, indicating that hFOB cells differentiated into mature osteoblasts on 1Zn/SO4-FHA disc surfaces. Taken together, our results suggest that HA substituted by (Zn2+, 0.2 mol), (SO42-, 0.5 mol) and (F-, 1 mol) (1Zn/SO4-FHA) material was a promising material for hard tissue scaffolds.

Diatom shell incorporated PHBV/PCL-pullulan co-electrospun scaffold for bone tissue engineering.

Tissue engineering can benefit from wide variety of materials produced by microorganisms. Natural origin materials often possess good biocompatibility, biodegradability with sustainable production by microorganisms. A phytoplankton, diatom, produces an amorphous silica shell that can be obtained by a cost efficient production process. Diatom shells (DS) are promising for bone tissue engineering since silicon enhances bone regeneration. Biocompatible and biodegradable biopolymers with microorganism origin can be combined with DS to produce tissue engineering constructs. In this study, a novel multifunctional 3D fibrous scaffold for bone tissue engineering was produced by co-electrospinning system; antibiotic loaded poly(hydroxybutyrate-co-hydroxyvalerate)/poly(ε-caprolactone) (PHBV/PCL) fibers and DS incorporated pullulan (PUL) fibers. Controlled release of cefuroxime axetil (CA) from DS and scaffolds were investigated upon loading CA into DS or PHBV/PCL fibers. Purified DS were characterized with ESCA, SEM, and EDX analyses while scaffolds were evaluated in terms of morphology, porosity, degradation, calcium deposition, water retention and mechanical properties. In vitro studies showed that scaffolds bearing DS have improved human osteosarcoma (Saos-2) cell viability. Developed co-electrospun scaffold showed higher osteocompatibility with better cell spreading and cell distribution. Results showed that DS loaded, co-electrospun scaffold having both hydrophobic and hydrophilic characteristics can be a promising biomaterial for bone tissue engineering.

A new therapeutic combination for osteosarcoma: Gemcitabine and Clofazimine co-loaded liposomal formulation.

Osteosarcoma is the most common cancer in bone. Drug resistance is a challenge of current treatments that needs to be improved with novel treatment strategies. In this research, a new dual drug delivery system was developed with Gemcitabine (GEM) and Clofazimine (CLF) co-loaded liposome formulations. GEM is a well-known anticancer agent and CLF is a leprostatic and anti-inflammatory drug recently recognized as effective on cancer. GEM and CLF co-loaded liposomal formulation was achieved with compartmentalization as hydrophilic GEM being in core and lipophilic CLF sequestering in lipid-bilayer. Liposomes had high encapsulation efficiency (above 90%, GEM and above 80%, CLF). CLF release was enhanced while GEM release was slowed down in co-loaded liposomes compared to single cases. GEM/CLF co-loaded liposomes significantly enhanced cytotoxicity than GEM or CLF loaded liposomes on osteosarcoma cell line. CLF and GEM had synergistic effect (CI < 1). Results of flow cytometry showed higher apoptotic cell ratio, caspase-3 activity, mitochondrial membrane depolarized cells' ratio for GEM/CLF co-loaded liposome treatments than other liposomes. Cytotoxicity of CLF on bone cancer cells and also its synergistic effect with GEM on osteosarcoma is reported for the first time with this study. CLF's loading with GEM into liposome was also a new approach for enhancement of anticancer effect on Saos-2 cells. Therefore, GEM/CLF co-loaded liposomal delivery system is proposed as a novel approach for treatment of osteosarcoma.

Silicate-doped nano-hydroxyapatite/graphene oxide composite reinforced fibrous scaffolds for bone tissue engineering.

In this study, novel graphene oxide-incorporated silicate-doped nano-hydroxyapatite composites were prepared and their potential use for bone tissue engineering was investigated by developing an electrospun poly(ε-caprolactone) scaffold. Nanocomposite groups were synthesized to have two different ratios of graphene oxide (2 and 4 wt%) to evaluate the effect of graphene oxide incorporation and groups with different silicate-doped nano-hydroxyapatite content was prepared to investigate optimum concentrations of both silicate-doped nano-hydroxyapatite and graphene oxide. Three-dimensional poly(ε-caprolactone) scaffolds were prepared by wet electrospinning and reinforced with silicate-doped nano-hydroxyapatite/graphene oxide nanocomposite groups to improve bone regeneration potency. Microstructural and chemical characteristics of the scaffolds were investigated by X-ray diffraction, Fourier transform infrared spectroscope and scanning electron microscopy techniques. Protein adsorption and desorption on material surfaces were studied using fetal bovine serum. Presence of graphene oxide in the scaffold, dramatically increased the protein adsorption with decreased desorption. In vitro biocompatibility studies were conducted using human osteosarcoma cell line (Saos-2). Electrospun scaffold group that was prepared with effective concentrations of silicate-doped nano-hydroxyapatite and graphene oxide particles (poly(ε-caprolactone) - 10% silicate-doped nano-hydroxyapatite - 4% graphene oxide) showed improved adhesion, spreading, proliferation and alkaline phosphatase activity compared to other scaffold groups.

Maltodextrin modified liposomes for drug delivery through the blood-brain barrier.

Central nervous system acting drugs, when administered intravenously, cannot show their effect in the brain due to the difficulty in crossing the blood-brain barrier (BBB). Levodopa is one of those drugs that are used to treat Parkinson's disease. In this study, a new liposomal levodopa delivery system that is modified with maltodextrin was developed in order to target and enhance transport through the BBB. An antioxidant, glutathione, was co-loaded in liposomes as a supportive agent and its effect on liposome stability and delivery was investigated. Glutathione co-loading had a positive effect on the viabilities of 3T3 and SH-SY5Y cells. Maltodextrin targeted liposomes showed high in vitro levodopa passage in the parallel artificial membrane permeability assay and had superior binding to MDCK cells. Results suggest that maltodextrin modification of liposomes is an effective way of targeting the BBB and the developed liposomal formulation would improve brain delivery of central nervous system agents.