Shun Duan

Enhanced osteogenic differentiation of mesenchymal stem cells on poly(l‐lactide) nanofibrous scaffolds containing carbon nanomaterials

Carbon nanomaterials (CNMs), such as carbon nanotube (CNT) and graphene, are highlighted in bone regeneration because of their osteoinductive properties. Their combinations with nanofibrous polymeric scaffolds, which mimic the morphology of natural extracellular matrix of bone, arouse keen interest in bone tissue engineering. To this end, CNM were incorporated into nanofibrous poly(L-lactic acid) scaffolds by thermal-induced phase separation. The CNM-containing composite nanofibrous scaffolds were biologically evaluated by both in vitro co-culture of bone mesenchymal stem cells (BMSCs) and in vivo implantation. The nanofibrous structure itself demonstrated significant enhancement in cell adhesion, proliferation and oseogenic differentiation of BMSCs, and with the incorporation of CNM, the composite nanofibrous scaffolds further promoted osteogenic differentiation of BMSCs significantly. Between the two CNMs, graphene showed stronger effect in promoting osteogenic differentiation of BMSCs than CNT. The results of in vivo experiments revealed that the composite nanofibrous scaffolds had both good biocompatibility and strong ability in inducing osteogenesis. CNMs could remarkably enhance the expression of osteogenesis-related proteins as well as the formation of type I collagen. Similarly, the graphene-containing composite nanofibrous scaffolds demonstrated the strongest effect on inducing osteogenesis in vivo. These findings demonstrated that CNM-containing composite nanofibrous scaffolds were obviously more efficient in promoting osteogenesis than pure polymeric scaffolds.

Effective Bone Regeneration Using Thermosensitive Poly(N-Isopropylacrylamide) Grafted Gelatin as Injectable Carrier for Bone Mesenchymal Stem Cells.

In this study, thermosensitive poly(N-isopropylacrylamide) (PNIPAAm) was grafted onto gelatin via atom transfer radical polymerization (ATRP). The chemical structure of PNIPAAm-grafted gelatin (Gel-PNIPAAm) was confirmed by XPS, ATR-IR, and (1)H NMR characterizations. Gel-PNIPAAm aqueous solution exhibited sol-to-gel transformation at physiological temperature, and was studied as injectable hydrogel for bone defect regeneration in a cranial model. The hydrogel was biocompatible and demonstrated the ability to enhance bone regeneration in comparison with the untreated group (control). With the incorporation of rat bone mesenchymal stem cells (BMSCs) into the hydrogel, the bone regeneration rate was further significantly enhanced. As indicated by micro-CT, histological (H&E and Masson) and immunohistochemical (osteocalcin and osteopontin) staining, newly formed woven bone tissue was clearly detected at 12 weeks postimplantation in the hydrogel/BMSCs treated group, showing indistinguishable boundary with surrounding host bone tissues. The results suggested that the thermosensitive Gel-PNIPAAm hydrogel was an excellent injectable delivery vehicle of BMSCs for in vivo bone defect regeneration.

Electrospun magnetic poly(l-lactide) (PLLA) nanofibers by incorporating PLLA-stabilized Fe3O4 nanoparticles

Magnetic poly(L-lactide) (PLLA)/Fe3O4 composite nanofibers were prepared with the purpose to develop a substrate for bone regeneration. To increase the dispersibility of Fe3O4 nanoparticles (NPs) in the PLLA matrix, a modified chemical co-precipitation method was applied to synthesize Fe3O4 NPs in the presence of PLLA. Trifluoroethanol (TFE) was used as the co-solvent for all the reagents, including Fe(II) and Fe(III) salts, sodium hydroxide, and PLLA. The co-precipitated Fe3O4 NPs were surface-coated with PLLA and demonstrated good dispersibility in a PLLA/TFE solution. The composite nanofiber electrospun from the solution displayed a homogeneous distribution of Fe3O4 NPs along the fibers using various contents of Fe3O4 NPs. X-ray diffractometer (XRD) and vibration sample magnetization (VSM) analysis confirmed that the co-precipitation process had minor adverse effects on the crystal structure and saturation magnetization (Ms) of Fe3O4 NPs. The resulting PLLA/Fe3O4 composite nanofibers showed paramagnetic properties with Ms directly related to the Fe3O4 NP concentration. The cytotoxicity of the magnetic composite nanofibers was determined using in vitro culture of osteoblasts (MC3T3-E1) in extracts and co-culture on nanofibrous matrixes. The PLLA/Fe3O4 composite nanofibers did not show significant cytotoxicity in comparison with pure PLLA nanofibers. On the contrary, they demonstrated enhanced effects on cell attachment and proliferation with Fe3O4 NP incorporation. The results suggested that this modified chemical co-precipitation method might be a universal way to produce magnetic biodegradable polyester substrates containing well-dispersed Fe3O4 NPs. This new strategy opens an opportunity to fabricate various kinds of magnetic polymeric substrates for bone tissue regeneration.

Macroporous and nanofibrous poly(lactide-co-glycolide)(50/50) scaffolds via phase separation combined with particle-leaching.

Poly(lactide-co-glycolide) (PLGA) copolymers are the most prevalent materials for tissue engineering applications. To mimic the real microenvironment of extracellular matrix (ECM) for cell growth, nanofibrous PLGA scaffolds are preferred. PLGA5050 (in which the molar ratio of lactidyl to glycolidyl units is 50:50), which is an utterly amorphous polymer, was first reported to be made into nanofibrous networks (fiber diameter around 500 nm) using phase separation from PLGA5050/THF solutions in this study. The concentration of polymeric solution had significant effects on fiber diameter and unit length. Nonsolvent (e.g. H2O) was unnecessary to form the PLGA5050 gel, which was critical to nanofibrosis, as if the environmental temperature for gelation occurrence was low enough (-70 °C). The physical crosslinks to stabilize the PLGA5050/THF gel were believed to be GA segments along the backbone owing to their inferior solubility in THF. The addition of H2O would cause adverse effects of liquid-liquid phase separation and nanofibrosis failure owing to the hydrophilicity of glycolidyl units. Associating with the phase separation method, particle-leaching technique was applied to fabricate three-dimensional scaffolds with macroporous and nanofibrous structures. To ensure the occurrence of nanofibrosis on macropore walls, the temperature of salt particles should be best lowed to -70 °C beforehand. Accordingly, scaffolds prepared under varied parameters exhibited different nanofiber and pore morphologies, which affected the pore size, porosity, specific surface area, water contact angle and protein adsorption ability etc. The preliminary cell (MC3T3-E1) culture confirmed the cell ingrowth into the macroporous and nanofibrous PLGA5050 scaffolds in comparison with the solely nanofibrous matrixes. This kind of bi-scaled three dimensional matrixes can be superior candidate scaffolds for tissue engineering applications.

Osteocompatibility evaluation of poly(glycine ethyl ester-co-alanine ethyl ester)phosphazene with honeycomb-patterned surface topography.

Biodegradable amino acid ester-substituted polyphosphazenes are unique biomaterials for tissue engineering. Considering the surface properties as topography and chemical composition having vital roles in regulating cellular response, in this study, a kind of micropatterned polyphosphazene films were prepared and subjected to osteoblasts culture. Briefly, poly(glycine ethyl ester-co-alanine ethyl ester)phosphazene (PGAP) was synthesized, and its solution in chloroform was cast under high (80%) or low (20%) environmental humidity. Honeycomb-patterned or flat PGAP films were resulted. By analyzing with scanning electron microscope, atomic force microscope, X-ray photoelectron spectroscope, and water contact angle measurement, the honeycomb-patterned PGAP films demonstrated higher surface roughness, phosphorous and nitrogen content, and hydrophilicity than the flat one. Although the initial cell attachment and proliferation on PGAP films were inferior to those on conventional poly(lactic-co-glycolic acid) films, P-containing PGAP was a sort of bone-binding bioactive polymer. With these alternations, honeycomb-patterned PGAP films had accordingly enhanced protein adsorption and apatite deposition in simulated body fluid and showed great advantages in promoting osteogenous differentiation. The results suggested a potential way to make polyphosphazenes as good choices for bone tissue regeneration by increasing their surface roughness and phosphorous content.

Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

Electrospun biodegradable polyorganophosphazene fibrous matrix with poly(dopamine) coating for bone regeneration

Biodegradable polyphosphazenes were categorized as osteoinductive materials because of their phosphorus-containing feature; however, they were less supportive in cell attachment and proliferation at earlier points in comparison with biodegradable aliphatic polyesters. Therefore, mussel-inspired surface modification of poly(alanine ethyl ester-co-glycine ethyl ester)phosphazene (PAGP) was studied, intending to circumvent the above-mentioned disadvantage of polyphosphazene. To this end, PAGP and poly(L-lactide) (PLLA) were electrospun into nanofibrous substrates and surface treated with dopamine aqueous solution. With the analysis of scanning electron microscope, transmission electron microscope, X-ray photoelectron spectroscope, and Fourier transform infrared spectroscope, the successful poly(dopamine) coating was identified on both PAGP and PLLA nanofibers. MC3T3-E1 osteoblasts were found attaching and proliferating much well on poly(dopamine)-modified nanofibrous substrates in comparison with the pristine ones. In addition, the poly(dopamine) coating demonstrated high activity in promoting osteogenous differentiation. Because the phosphorus content on nanofiber surface was decreased with the poly(dopamine) coating, the poly(dopamine)-coated PAGP nanofibrous substrate was slightly inferior to pure PAGP nanofibrous substrate in osteogenous differentiation. In a summary, the results confirmed that poly(dopamine)-modified polyphosphazenes were promising scaffold materials with both high cell affinity and high osteocompatibility for bone regeneration.

Osteogenic differentiation of MC3T3-E1 cells on poly(l-lactide)/Fe3O4 nanofibers with static magnetic field exposure

Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1 mT-1 T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(L-lactide) (PLLA) and ferromagnetic Fe3O4 nanoparticles (NPs). The PLLA/Fe3O4 composite nanofibers demonstrated homogeneous dispersion of Fe3O4 NPs, and their magnetism depended on the contents of Fe3O4 NPs. SMF of 100 mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe3O4 composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe3O4 NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering.

In vitro and in vivo drug release behavior and osteogenic potential of a composite scaffold based on poly(ε-caprolactone)-block-poly(lactic-co-glycolic acid) and β-tricalcium phosphate

To cure serious bone tuberculosis, a novel long-term drug delivery system was designed and prepared to satisfy the needs of both bone regeneration and antituberculous drug therapy. An antituberculous drug (rifampicin, RFP) was loaded into a porous scaffold, which composed of a newly designed polylactone, poly(ε-caprolactone)-block-poly(lactic-co-glycolic acid) (b-PLGC) copolymer, and β-tricalcium phosphate (β-TCP). The releasing results demonstrated that RFP could be steadily released for as long as 12 weeks both in vitro and in vivo. During the in vivo experimental period, the drug concentration in tissues surrounding implants was much higher than that in blood which was still superior to the effective value to kill mycobacterium tuberculosis. MC3T3-E1 osteoblasts proliferated well in extracts and co-cultures on composite scaffolds, indicating good cytocompatibility and cell affinity of the scaffolds. The results of a rabbit radius repair experiment displayed that scaffolds have good bone regeneration capacity. The RFP-loaded b-PLGC/TCP composite scaffold thus could be envisioned to be a potential and promising substrate in clinical treatment of bone tuberculosis.

Visualization of in vivo degradation of aliphatic polyesters by a fluorescent dendritic star macromolecule

In tissue engineering, most polymeric scaffolds should degrade along with the formation of the new tissues. Therefore, it is necessary to look into the in vivo degradation of scaffolds. In this study, a fluorescent perylenediimide-cored (PDI-cored) dendritic star macromolecule bearing multiple amines (d-p48) was incorporated into biodegradable polyester nanofibrous scaffolds by eletrospinning as an indicator. The polyester/d-p48 blend nanofibers could emit strong red fluorescence when they were irradiated under exciting light. Initially, using slowly degradable polyester, poly(L-lactide) (PLLA)/d-p48 nanofibers were soaked in phosphate buffered saline for various lengths of time to determine the possible diffusing release of d-p48 macromolecule from nanofibers. The PLLA/d-p48 nanofibers were then implanted subcutaneously into mice and left for up to 2 weeks. In both cases, no undesirable release of the incorporated d-p48 macromolecule was detected, and the nanofibers were clearly visualized in vivo by fluorescence microscopy. Using a fast degradable polyester, poly(lactide-co-glycolide) (PLGA)/d-p48 nanofibers were electrospun and implanted subcutaneously to determine the possibility of monitoring in vivo degradation by fluorescence during 12 weeks. The results showed that the location and the contour of PLGA/d-p48 nanofibrous scaffolds could be clearly visualized using an animal fluorescent imaging system. The fluorescent intensities decreased gradually with the degradation of the scaffolds. No side effects on liver and kidney were found during the detection. This study indicates that the fluorescent PDI-cored dendritic star macromolecule can be used as a stable bioimaging indicator for biodegradable aliphatic polyesters in vivo.