Development of 3D PCL microsphere/TiO\u3csub\u3e2\u3c/sub\u3e nanotube composite scaffolds for bone tissue engineering

In this research, the three dimensional porous scaffolds made of a polycaprolactone (PCL) microsphere/TiO2 nanotube (TNT) composite was fabricated and evaluated for potential bone substitute applications. We used a microsphere sintering method to produce three dimensional PCL microsphere/TNT composite scaffolds. The mechanical properties of composite scaffolds were regulated by varying parameters, such as sintering time, microsphere diameter range size and PCL/TNT ratio. The obtained results ascertained that the PCL/TNT (0.5 wt%) scaffold sintered at 60 °C for 90 min had the most optimal mechanical properties and an appropriate pore structure for bone tissue engineering applications. The average pore size and total porosity percentage increased after increasing the microsphere diameter range for PCL and PCL/TNT (0.5 wt%) scaffolds. The degradation rate was relatively high in PCL/TNT (0.5 wt%) composites compared to pure PCL when the samples were placed in the simulated body fluid (SBF) for 6 weeks. Also, the compressive strength and modulus of PCL and PCL/TNT (0.5 wt%) composite scaffolds decreased during the 6 weeks of storage in SBF. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and alkaline phosphates (ALP) activity results demonstrated that a generally increasing trend in cell viability was observed for PCL/TNT (0.5 wt%) scaffold sintered at 60 °C for 90 min compared to the control group. Eventually, the quantitative RT-PCR data provided the evidence that the PCL scaffold containing TiO2 nanotube constitutes a good substrate for cell differentiation leading to ECM mineralization

Collagenous Matrix Supported by A 3D-Printed Scaffold for Osteogenic Differentiation of Dental Pulp Cells

Objective A systematic characterization of hybrid scaffolds, fabricated based on combinatorial additive manufacturing technique and freeze-drying method, is presented as a new platform for osteoblastic differentiation of dental pulp cells (DPCs). Methods The scaffolds were consisted of a collagenous matrix embedded in a 3D-printed beta-tricalcium phosphate (β-TCP) as the mineral phase. The developed construct design was intended to achieve mechanical robustness owing to 3D-printed β-TCP scaffold, and biologically active 3D cell culture matrix pertaining to the Collagen extracellular matrix. The β-TCP precursor formulations were investigated for their flow-ability at various temperatures, which optimized for fabrication of 3D printed scaffolds with interconnected porosity. The hybrid constructs were characterized by 3D laser scanning microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and compressive strength testing. Results The in vitro characterization of scaffolds revealed that the hybrid β-TCP/Collagen constructs offer superior DPCs proliferation and alkaline phosphatase (ALP) activity compared to the 3D-printed β-TCP scaffold over three weeks. Moreover, it was found that the incorporation of TCP into the Collagen matrix improves the ALP activity. Significance The presented results converge to suggest the developed 3D-printed β-TCP/Collagen hybrid constructs as a new platform for osteoblastic differentiation of DPCs for craniomaxillofacial bone regeneration

Preparation, Characterization and Evaluation of Drug Release Properties of Simvastatin-loaded PLGA Microspheres

Abstract Microspheres formulated from poly (D, L-lactic-co-glycolide) (PLGA), a biodegradable polymer, have been extensively evaluated as a drug delivery system. In this study, the preparation, characterization and drug release properties of the PLGA microspheres were evaluated. Simvastatin (SIM)-loaded PLGA microspheres were prepared by oil-in-water emulsion/solvent evaporation method. The microspheres were then frozen to −80 °C, they were freeze dried for 24 h. Characterization of SIM-loaded PLGA microspheres was evaluated by X-ray diffraction analysis, Fourier transform infrared spectroscopy analysis, and scanning electron microscopy (SEM). Drug release potential was evaluated by UV-spectrophotometry. The experimental results revealed that SIM-loaded PLGA microspheres can be successfully obtained through solvent evaporation method with appropriate morphologic characteristics and high encapsulation efficiency. The drug release pattern from polymeric microspheres in the phosphate buffered saline medium was measured during a 21-day period using UV-spectrophotometry. The correlation coefficient value (r 2 = 0.9878) of the trend lines of the graph showed that the SIM-loaded PLGA microspheres best fit with zero order release pattern. No burst release was observed with polymeric matrix. The drug release characteristic of the microspheres ascertained that the release was about 27% for SIM-loaded microspheres, which occurred within the first 6 days after maintaining the microspheres in phosphate buffer saline. Also, the microspheres successfully presented a slow release and the duration of the release lasted for more than 21 days. It can be concluded that SIM-loaded PLGA microspheres hold great promise for using as a drug-delivery system in biomedical applications, especially in drug delivery systems and tissue engineering

Enhancing Cell Seeding and Osteogenesis of MSCs on 3D Printed Scaffolds Through Injectable BMP2 Immobilized ECM-Mimetic Gel

Objective Design of bioactive scaffolds with osteogenic capacity is a central challenge in cell-based patient-specific bone tissue engineering. Efficient and spatially uniform seeding of (stem) cells onto such constructs is vital to attain functional tissues. Herein we developed heparin functionalized collagen gels supported by 3D printed bioceramic scaffolds, as bone extracellular matrix (ECM)-mimetic matrices. These matrices were designed to enhance cell seeding efficiency of mesenchymal stem cells (MSCs) as well as improve their osteogenic differentiation through immobilized bone morphogenic protein 2 (BMP2) to be used for personalized bone regeneration. Methods A 3D gel based on heparin-conjugated collagen matrix capable of immobilizing recombinant human bone morphogenic protein 2 (BMP2) was synthesized. Isolated dental pulp Mesenchymal stem cells (MSCs) were then encapsulated into the bone ECM microenvironment to efficiently and uniformly seed a bioactive ceramic-based scaffold fabricated using additive manufacturing technique. The designed 3D cell-laden constructs were comprehensively investigated trough in vitro assays and in vivo study. Results In-depth rheological characterizations of heparin-conjugated collagen gel revealed that elasticity of the matrix is significantly improved compared with freely incorporated heparin. Investigation of the MSCs laden collagen-heparin hydrogels revealed their capability to provide spatiotemporal bioavailability of BMP2 while suppressing the matrix contraction over time. The in vivo histology and real-time polymerase chain reaction (qPCR) analysis showed that the designed construct supported the osteogenic differentiation of MSCs and induced the ectopic bone formation in rat model. Significance The presented hybrid constructs combine bone ECM chemical cues with mechanical function providing an ideal 3D microenvironment for patient-specific bone tissue engineering and cell therapy applications. The implemented methodology in design of ECM-mimetic 3D matrix capable of immobilizing BMP2 to improve seeding efficiency of customized scaffolds can be exploited for other bioactive molecules

Efficacy of the Biomaterials 3 wt%-nanostrontium-hydroxyapatite-enhanced Calcium Phosphate Cement (nanoSr-CPC) and nanoSr-CPC-incorporated Simvastatin-loaded Poly(lactic-co-glycolic-acid) Microspheres in Osteogenesis Improvement

Aims The purpose of this multi-phase explorative in vivo animal/surgical and in vitro multi-test experimental study was to (1) create a 3 wt%-nanostrontium hydroxyapatite-enhanced calcium phosphate cement (Sr-HA/CPC) for increasing bone formation and (2) creating a simvastatin-loaded poly(lactic-co-glycolic acid) (SIM-loaded PLGA) microspheres plus CPC composite (SIM-loaded PLGA + nanostrontium-CPC). The third goal was the extensive assessment of multiple in vitro and in vivo characteristics of the above experimental explorative products in vitro and in vivo (animal and surgical studies). Methods and results pertaining to Sr-HA/CPC Physical and chemical properties of the prepared Sr-HA/CPC were evaluated. MTT assay and alkaline phosphatase activities, and radiological and histological examinations of Sr-HA/CPC, CPC and negative control were compared. X-ray diffraction (XRD) indicated that crystallinity of the prepared cement increased by increasing the powder-to-liquid ratio. Incorporation of Sr-HA into CPC increased MTT assay (biocompatibility) and ALP activity (P \u3c 0.05). Histomorphometry showed greater bone formation after 4 weeks, after implantation of Sr-HA/CPC in 10 rats compared to implantations of CPC or empty defects in the same rats (n = 30, ANOVA P \u3c 0.05). Methods and results pertaining to SIM-loaded PLGA microspheres + nanostrontium-CPC composite After SEM assessment, the produced composite of microspheres and enhanced CPC were implanted for 8 weeks in 10 rabbits, along with positive and negative controls, enhanced CPC, and enhanced CPC plus SIM (n = 50). In the control group, only a small amount of bone had been regenerated (localized at the boundary of the defect); whereas, other groups showed new bone formation within and around the materials. A significant difference was found in the osteogenesis induced by the groups sham control (16.96 ± 1.01), bone materials (32.28 ± 4.03), nanostrontium-CPC (24.84 ± 2.6), nanostrontium-CPC-simvastatin (40.12 ± 3.29), and SIM-loaded PLGA + nanostrontium-CPC (44.8 ± 6.45) (ANOVA P \u3c 0.001). All the pairwise comparisons were significant (Tukey P \u3c 0.01), except that of nanostrontium-CPC-simvastatin and SIM-loaded PLGA + nanostrontium-CPC. This confirmed the efficacy of the SIM-loaded PLGA + nanostrontium-CPC composite, and its superiority over all materials except SIM-containing nanostrontium-CPC

Critical-Sized Bone Defects Regeneration Using a Bone-Inspired 3D Bilayer Collagen Membrane in Combination with Leukocyte and Platelet-Rich Fibrin Membrane (L-PRF): An \u3cem\u3eIn Vivo\u3c/em\u3e Study

Objectives We aim to develop a 3D-bilayer collagen (COL) membrane reinforced with nano beta-tricalcium-phosphate (nβ-TCP) particles and to evaluate its bone regeneration in combination with leukocyte-platelet-rich fibrin (L-PRF) in vivo. Background data L-PRF has exhibited promising results as a cell carrier in bone regeneration in a number of clinical studies, however there are some studies that did not confirm the positive results of L-PRF application. Methods Mechanical & physiochemical characteristics of the COL/nβ-TCP membrane (1/2 & 1/4) were tested. Proliferation and osteogenic differentiation of seeded cells on bilayer collagen/nβ-TCP thick membrane was examined. Then, critical-sized calvarial defects in 8 white New Zealand rabbits were filled with either Col, Col/nβ-TCP, Col/nβ-TCP combined with L-PRF membrane, or left empty. New bone formation (NBF) was measured histomorphometrically 4 & 8 weeks postoperatively. Results Compressive modulus increases while porosity decreases with higher β-TCP concentrations. Mechanical properties improve, with 89 % porosity (pore size ∼100 μm) in the bilayer-collagen/nβ-TCP membrane. The bilayer design also enhances the proliferation and ALP activity. In vivo study shows no significant difference among test groups at 4 weeks, but Col/nβ-TCP + L-PRF demonstrates more NBF compared to others (P \u3c 0.05) after 8 weeks. Conclusion The bilayer-collagen/nβ-TCP thick membrane shows promising physiochemical in vitro results and significant NBF, as ¾ of the defect is filled with lamellar bone when combined with L-PRF membrane

Outcome of Different Processing Methods on Mechanical and Physicochemical Properties of Human Dentin as a Potential Natural Scaffold

Dentin has been considered a promising scaffold for bone regeneration. This study investigated the effects of two different demineralization and deproteinization methods on the mechanical and physicochemical properties of dentin as a potential scaffold for tissue engineering approaches. Eighty dentin discs were divided into five groups according to the treatment process: control (C), no treatment; DEM1, complete demineralization with HCl; DEM2, partial demineralization with different concentrations of EDTA; DEP1, complete deproteinization using NaOCl solution; and DEP2, partial deproteinization by boiling water. The treated dentin discs were characterized using ATR-FTIR and SEM. The compressive strength, elastic modulus, and microhardness values of all C and treated samples were measured. They were analyzed using one-way ANOVA, Kruskal-Wallis, and Weibull analysis. FTIR showed significantly reduced mineral/matrix ratio in demineralized groups (DEM1 and 2) (p \u3c 0.001), while increase of mineral/matrix ratio in DEP1 was not significant (p = 0.31). SEM observations revealed open dentinal tubules in DEM1 group and lower amounts in the other groups. The C showed the highest compressive strength (78.18 ± 16.19 MPa), and the DEM1 showed the lowest (0.84 ± 0.32 MPa). The treated groups showed lower Weibull moduli (m), when compared with the C group. The DEP2 (642 ± 318.9) and DEM1 (2.9 ± 1.37) groups showed the highest and lowest modulus of elasticity, respectively. DEM1 showed the least hardness values (10.27 ± 3.09) compared to the other groups (p \u3c 0.001). Based on the results, the DEM1 showed confirmed demineralization and lowest mechanical properties. The highest mechanical properties belonged to DEM2 and DEP2 groups which both were partially treated. Depending on the purpose of the regeneration, the dentin processing method can be selected. Whenever the high mechanical properties are more important in scaffold selection, DEM2 and DEP2 are the best choices