Improvement of Decellularization Efficiency of Porcine Aorta Using Dimethyl Sulfoxide as a Penetration Enhancer.

Decellularization of tissues and organs enables researchers to obtain extracellular matrix (ECM) with the natural conformation and chemical composition of specific tissues. However, drawbacks exist such as the structural alteration of ECM or loss of some important components in ECM due to overexposure to chemicals during the decellularization process. In this study, porcine aorta was decellularized by sodium dodecyl sulfate (SDS). Dimethyl sulfoxide (DMSO) was used as a penetration enhancer in the decellularization process to enhance the penetration of SDS, consequently reducing the exposure time of SDS to treated tissues. It is revealed that by addition of DMSO to the decellularization process 64.4% more DNA was removed when compared with just SDS exposure within a 3 h reaction. Cross-validation by DAPI staining showed that, in the presence of DMSO, the penetration of SDS was improved and almost all cells were removed from the aorta within the 3 h exposure time. Collagen staining revealed that just SDS treatment showed less polarized collagen fibers, while the DMSO addition groups revealed denser and organized collagen fibers. Moreover 77% glycosaminoglycan content was preserved by addition of DMSO in resultant tissues. Scanning electron microscopy analysis of decellularized aortic matrix showed that ECM components remained in the adventitia layer with the addition of DMSO treatment, while the layer was removed with just SDS treatment. Biocompatibility assays proved that after washing the decellularized samples with media supplemented with 3% antibiotic and antimycotic solution for 2 days there was no cytotoxic effect related to the SDS + DMSO decellularization protocol. This study demonstrates that the new decellularization protocol not only improves the removal efficiency of cellular components but also protects the crucial ECM components.

Fabrication of Plasmonic Nanorod-Embedded Dipeptide Microspheres via the Freeze-Quenching Method for Near-Infrared Laser-Triggered Drug-Delivery Applications.

Control of drug release by an external stimulus may provide remote controllability, low toxicity, and reduced side effects. In this context, varying physical external stimuli, including magnetic and electric fields, ultrasound, light, and pharmacological stimuli, have been employed to control the release rate of drug molecules in a diseased region. However, the design and development of alternative on-demand drug-delivery systems that permit control of the dosage of drug released via an external stimulus are still required. Here, we developed near-infrared laser-activatable microspheres based on Fmoc-diphenylalanine (Phe-Phe) dipeptides and plasmonic gold nanorods (AuNRs) via a simple freeze-quenching approach. These plasmonic nanoparticle-embedded microspheres were then employed as a smart drug-delivery platform for native, continuous, and pulsatile doxorubicin (DOX) release. Remarkable sustained, burst, and on-demand DOX release from the fabricated microspheres were achieved by manipulating the laser exposure time. Our results demonstrate that AuNR-embedded dipeptide microspheres have great potential for controlled drug-delivery systems.

A facile in vitro model to study rapid mineralization in bone tissues.

BACKGROUND: Mineralization in bone tissue involves stepwise cell-cell and cell-ECM interaction. Regulation of osteoblast culture microenvironments can tailor osteoblast proliferation and mineralization rate, and the quality and/or quantity of the final calcified tissue. An in vitro model to investigate the influencing factors is highly required. METHODS: We developed a facile in vitro model in which an osteoblast cell line and aggregate culture (through the modification of culture well surfaces) were used to mimic intramembranous bone mineralization. The effect of culture environments including culture duration (up to 72 hours for rapid mineralization study) and aggregates size (monolayer culture as control) on mineralization rate and mineral quantity/quality were examined by osteogenic gene expression (PCR) and mineral markers (histological staining, SEM-EDX and micro-CT). RESULTS: Two size aggregates (on average, large aggregates were 745 µm and small 79 µm) were obtained by the facile technique with high yield. Cells in aggregate culture generated visible and quantifiable mineralized matrix within 24 hours, whereas cells in monolayer failed to do so by 72 hours. The gene expression of important ECM molecules for bone formation including collagen type I, alkaline phosphatase, osteopontin and osteocalcin, varied temporally, differed between monolayer and aggregate cultures, and depended on aggregate size. Monolayer specimens stayed in a proliferation phase for the first 24 hours, and remained in matrix synthesis up to 72 hours; whereas the small aggregates were in the maturation phase for the first 24 and 48 hour cultures and then jumped to a mineralization phase at 72 hours. Large aggregates were in a mineralization phase at all these three time points and produced 36% larger bone nodules with a higher calcium content than those in the small aggregates after just 72 hours in culture. CONCLUSIONS: This study confirms that aggregate culture is sufficient to induce rapid mineralization and that aggregate size determines the mineralization rate. Mineral content depended on aggregate size and culture duration. Thus, our culture system may provide a good model to study regulation factors at different development phases of the osteoblastic lineage.

Synthesis of Conductive Carbon Aerogels Decorated with ß-Tricalcium Phosphate Nanocrystallites.

There has been substantial interest in research aimed at conductive carbon-based supports since the discovery that the electrical stimulus can have dramatic effect on cell behavior. Among these carbon-aerogels decorated with biocompatible polymers were suggested as future materials for tissue engineering. However, high reaction temperatures required for the synthesis of the aerogels tend to impair the stability of the polymeric networks. Herein, we report a synthetic route towards carbon-aerogel scaffolds decorated with biocompatible ceramic nanoparticles of tricalcium phosphate. The composites can be prepared at temperature as high as 1100 °C without significant effect on the morphology of the composite which is comparable with the original aerogel framework. Although the conductivity of the composites tends to decrease with the increasing ceramic content the measured conductivity values are similar to those previously reported on polymer-functionalized carbon-aerogels. The cell culture study revealed that the developed constructs support cell proliferation and provide good cell attachment suggesting them as potentially good candidates for tissue-engineering applications.

Critical-size alveolar defect treatment via TGF-ß3 and BMP-2 releasing hybrid constructs.

In the present study a combination of Transforming Growth Factor Beta 3 (TGF-ß3) and Bone Morphogenetic Protein-2 (BMP-2) loaded gelatin films sandwiched between poly (L-lactide) (PLLA)/poly (ε-caprolactone) (PCL) matrices were produced to enhance bone formation in alveolar bone defects. Osteogenic properties of tissue constructs were tested in alveolar bone defect model in rats. Bone healing was assessed by osteogenic gene expression levels of bone sialoprotein (BSP), alkaline phosphatase (ALP), osteonectin (ON, SPARC), osteocalcin (OC), runt-related transcription factor 2 (RUNX2), bone specific alkaline phosphatase (BALP) activity, histomorphometry and microtomography. Increase in osteogenic gene expression levels and BALP activity results showed that new bone formation was significantly accelerated in TGF-ß3 + BMP-2 loaded scaffold group compared to growth factor free and only BMP-2 loaded groups. The micro-computed tomography (µ-CT) data from the 4th months revealed that (TGF-ß3+ BMP-2) loaded scaffolds displayed increased bone formation and was able to fulfill 84% of the defect area (p < 0.05). Accelerated bone formation in the S-GF-B-T group compared to that of the S-GF group at the end of the 4th month was further verified via histomorphometric analysis (p = 0.008). Gene expression, BALP activity, microtomography and histomorphometry analysis indicated that (TGF-ß3 + BMP-2) loaded PLLA/PCL scaffolds increased the new bone formation. BMP-2 loaded scaffolds were less effective than combination of TGF-ß3 and BMP-2 loaded scaffolds. These findings demonstrated that focusing on the PLLA/PCL hybrid scaffolds combined with (TGF-ß3 + BMP-2) may lay the groundwork for future therapy-oriented efforts to enhance bone formation in alveolar defects.