Electrospun Nanofibrous Scaffolds of Polycaprolactone/Gelatin Reinforced with Layered Double Hydroxide Nanoclay for Nerve Tissue Engineering Applications.

Nerve tissue engineering (NTE) is an effective approach for repairing damaged nerve tissue. In this regard, nanoparticle-incorporated electrospun scaffolds have aroused a great deal of interest in NTE applications. In this study, layered double hydroxide (LDH)-incorporated polycaprolactone (PCL)/gelatin (Gel) nanofibrous scaffolds were fabricated by an electrospinning technique. The physicochemical, mechanical, and biological properties of the scaffolds were examined. Also, the phase identification, morphology, and elemental composition were studied using X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy, respectively. The results revealed that the inclusion of LDH nanoparticles into the PCL/Gel scaffold has improved its mechanical strength and elongation at the break, while the degradation rate was enhanced in comparison with the pure PCL/Gel mat. The LDH-enriched electrospun PCL/Gel scaffolds exhibited a considerable impact on cell attachment and proliferation. The gene expression results showed that the neuron-specific (γγ) enolase (NSE) gene expression was significantly decreased in the scaffolds containing 1 and 10 wt % LDH compared to the scaffold without LDH, whereas in the scaffold with 0.1 wt % LDH, a slight increase in expression was observed. It can be deduced that electrospun PCL/Gel scaffolds containing LDH with optimum concentration can be a promising candidate for nerve tissue engineering applications.

Evaluation of Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cell on Highly Porous Polycaprolactone Scaffold Reinforced With Layered Double Hydroxides Nanoclay.

In recent decades, bone tissue engineering has had an effective role in introducing orthopedic implants. In this regard, polymeric scaffolds reinforced with bioactive nanomaterials can offer great potential in tissue engineering implants for replacing bone loss in patients. In this study, the thermally induced phase separation method was used to fabricate three-dimensional highly porous scaffolds made of layered double hydroxide (LDH)/polycaprolactone (PCL) nanocomposites with varied LDH contents ranging from 0.1 wt.% to 10 wt.%. The Phase identification, morphology, and elemental composition were studied using X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray spectroscopy, respectively. Interconnected pores ranging from 5 to 150 µm were detected in all samples. The results revealed that the inclusion of LDH to PCL scaffold reinforced mechanical strength and compressive modulus increased from 0.6418 to 1.3251 for the pure PCL and PCL + LDH (1 Wt.%) scaffolds, respectively. Also, thermal stability, degradation rate, and biomineralization especially in comparison with the pure PCL were enhanced. Adhesion, viability, and proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) seeded on PCL + LDH scaffolds were improved as compared to the pure PCL. Furthermore, the addition of LDH resulted in the increased mineral deposition as well as expression of ALP and RUNX2 osteogenic genes in terms of differentiation. All in all, our findings revealed that PCL + LDH (1 Wt.%) scaffold might be an ideal choice for 3D scaffold design in bone tissue engineering approaches.

Preparation and characterization of electrospun polycaprolactone/brushite scaffolds to promote osteogenic differentiation of mesenchymal stem cells.

Bone tissue engineering aims to develop effective strategies for repairing or replacing damaged bone tissue. In this study, composite scaffolds consisting of dicalcium phosphate dihydrate (DCDP, brushite) as a bone phase mineral precursor with different weight percentages (0%, 1%, 3%, 5%, and 10%) in combination with polycaprolactone (PCL) were fabricated by electrospinning technique. The morphology and mechanical behavior of scaffolds were characterized using scanning electron microscopy and tensile strength test, respectively. The bioactivity of scaffolds was assessed in simulated body fluid. Adhesion, viability, proliferation, and differentiation of mesenchymal stem cells derived from the human bone marrow on scaffolds were investigated using electron microscopy, MTT assay, live-dead assay, alizarin red staining, alkaline phosphatase activity and, gene expression analysis by real-time PCR. The results showed that the scaffold containing 3 wt. % of DCDP had the highest tensile strength (15.35 MPa). Furthermore, cells seeded on scaffolds showed over 80% viability after 1, 3, 7 days of incubation. Also, the results showed that the addition of DCDP to the PCL significantly increased the alkaline phosphatase activity. The osteocalcin gene expression in the composite scaffold showed a 6.1-fold increase compared to the pure PCL scaffold. It is concluded that electrospun PCL scaffolds containing DCDP with optimum concentration can be a proper candidate for bone tissue engineering applications.

In vivo evaluation of bone regeneration behavior of novel ß-tricalcium phosphate/layered double hydroxide nanocomposite granule as bone graft substitutes.

This study was based on in vivo assessment of bone regeneration capacity of synthesized porous ß-tricalcium phosphate (ß-TCP) nanocomposite granules and aimed to explore the effects of fabricated ß-TCP granules reinforced with layered double hydroxides (LDH) nanoclay compared to ß-TCP granules, in terms of osteoconductivity and biodegradability. Granules with diameters of 2-3 mm were implanted into cavities drilled in rabbit distal femur and were left in situ for up to 3 months. The mechanical study demonstrated that the presence of LDH nanoparticles in ß-TCP granules resulted in a significant increase in compressive modulus from 174.4 to 231.4 MPa, while the porosity was constant at 76%-80%. The results revealed that the obtained granules showed no cytotoxicity. In this study, x-ray radiographic, micro-computed tomography, and histological staining analysis were taken to evaluate the percentage of bone ingrowth and biodegradability of the porous granules. The results exhibited that both granules support bone regeneration and also the amount of new bone formation in the bone defect filled with both granules was almost six times higher than the empty defects. Although no significant difference in bone formation for two different granules was observed, a higher biodegradability was detected in ß-TCP granules in comparison to ß-TCP/LDH granules. Overall, the addition of LDH nanoclay (10%) enhanced the physicochemical and mechanical properties of ß-TCP granules while it is biological and osteoconductity properties have been maintained and its biodegradation rate has been decreased.

Fabrication and Evaluation of Layered Double Hydroxide-Enriched ß-Tricalcium Phosphate Nanocomposite Granules for Bone Regeneration: In Vitro Study.

One of the most important challenges facing tissue engineering researches is the scaffold design with optimum physical and mechanical properties for growth and proliferation of cells, and tissue formation. The aim of this study was to produce a novel nanocomposite containing ß-tricalcium phosphate and layered double hydroxide (ß-TCP-LDH) and analyzing the capacity of its osteogenic activity in vitro. In this paper, ß-tricalcium phosphate and layered double hydroxide powders were synthesized by co-precipitation processes. Then, the porous nanocomposite granules were prepared by the polyurethane sponge replication method. In this study, four kinds of ß-TCP granules containing LDHs nanoparticles (ranging from 0.1 to 10 wt%) have been prepared. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) analyses were selected to study the phase structure, morphology, and phase distribution, respectively. Physicochemical characterizations demonstrated that the granules were synthesized successfully. Interconnected macro pores ranging over 200-500 µm were observed for all kinds of granules. SEM micrographs showed that human mesenchymal stem cells (hMSCs) were attached to the surfaces of the granules and proliferated in good shape. The results warranted that the synthesized granules exhibited good biocompatibility and mineralization. Based on the results of compressive strength and porosity tests, the most suitable type of granule is ß-TCP/LDH 10 wt% with 77% porosity and compressive modulus of 231.4 MPa, which can be utilized in bone tissue engineering. To our knowledge, layered double hydroxides have not previously been incorporated into tricalcium phosphate granules for bone grafting. Also, this study is the first report on the effects of LDH on the mechanical properties and porosity of ß-TCP granules. Our results demonstrated that ß-TCP/LDH nanocomposite granule has a great potential for bone defects regeneration and tissue engineering applications.

Structure, Properties, and In Vitro Behavior of Heat-Treated Calcium Sulfate Scaffolds Fabricated by 3D Printing.

The ability of inkjet-based 3D printing (3DP) to fabricate biocompatible ceramics has made it one of the most favorable techniques to generate bone tissue engineering (BTE) scaffolds. Calcium sulfates exhibit various beneficial characteristics, and they can be used as a promising biomaterial in BTE. However, low mechanical performance caused by the brittle character of ceramic materials is the main weakness of 3DP calcium sulfate scaffolds. Moreover, the presence of certain organic matters in the starting powder and binder solution causes products to have high toxicity levels. A post-processing treatment is usually employed to improve the physical, chemical, and biological behaviors of the printed scaffolds. In this study, the effects of heat treatment on the structural, mechanical, and physical characteristics of 3DP calcium sulfate prototypes were investigated. Different microscopy and spectroscopy methods were employed to characterize the printed prototypes. The in vitro cytotoxicity of the specimens was also evaluated before and after heat treatment. Results showed that the as-printed scaffolds and specimens heat treated at 300°C exhibited severe toxicity in vitro but had almost adequate strength. By contrast, the specimens heat treated in the 500°C-1000°C temperature range, although non-toxic, had insufficient mechanical strength, which was mainly attributed to the exit of the organic binder before 500°C and the absence of sufficient densification below 1000°C. The sintering process was accelerated at temperatures higher than 1000°C, resulting in higher compressive strength and less cytotoxicity. An anhydrous form of calcium sulfate was the only crystalline phase existing in the samples heated at 500°C-1150°C. The formation of calcium oxide caused by partial decomposition of calcium sulfate was observed in the specimens heat treated at temperatures higher than 1200°C. Although considerable improvements in cell viability of heat-treated scaffolds were observed in this study, the mechanical properties were not significantly improved, requiring further investigations. However, the findings of this study give a better insight into the complex nature of the problem in the fabrication of synthetic bone grafts and scaffolds via post-fabrication treatment of 3DP calcium sulfate prototypes.

Epigallocatechin Gallate/Layered Double Hydroxide Nanohybrids: Preparation, Characterization, and In Vitro Anti-Tumor Study.

In recent years, nanotechnology in merging with biotechnology has been employed in the area of cancer management to overcome the challenges of chemopreventive strategies in order to gain promising results. Since most biological processes occur in nano scale, nanoparticles can act as carriers of certain drugs or agents to deliver it to specific cells or targets. In this study, we intercalated Epigallocatechin-3-Gallate (EGCG), the most abundant polyphenol in green tea, into Ca/Al-NO3 Layered double hydroxide (LDH) nanoparticles, and evaluated its efficacy compared to EGCG alone on PC3 cell line. The EGCG loaded LDH nanohybrids were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy (TEM) and nanosizer analyses. The anticancer activity of the EGCG-loaded LDH was investigated in prostate cancer cell line (PC3) while the release behavior of EGCG from LDH was observed at pH 7.45 and 4.25. Besides enhancing of apoptotic activity of EGCG, the results showed that intercalation of EGCG into LDH can improve the anti- tumor activity of EGCG over 5-fold dose advantages in in-vitro system. Subsequently, the in-vitro release data showed that EGCG-loaded LDH had longer release duration compared to physical mixture, and the mechanism of diffusion through the particle was rate-limiting step. Acidic attack was responsible for faster release of EGCG molecules from LDH at pH of 4.25 compared to pH of 7.4. The results showed that Ca/Al-LDH nanoparticles could be considered as an effective inorganic host matrix for the delivery of EGCG to PC3 cells with controlled release properties.