Tingli Lu

Techniques for fabrication and construction of three-dimensional scaffolds for tissue engineering

Three-dimensional biomimetic scaffolds have widespread applications in biomedical tissue engineering because of their nanoscaled architecture, eg, nanofibers and nanopores, similar to the native extracellular matrix. In the conventional “top-down” approach, cells are seeded onto a biocompatible and biodegradable scaffold, in which cells are expected to populate in the scaffold and create their own extracellular matrix. The top-down approach based on these scaffolds has successfully engineered thin tissues, including skin, bladder, and cartilage in vitro. However, it is still a challenge to fabricate complex and functional tissues (eg, liver and kidney) due to the lack of vascularization systems and limited diffusion properties of these large biomimetic scaffolds. The emerging “bottom-up” method may hold great potential to address these challenges, and focuses on fabricating microscale tissue building blocks with a specific microarchitecture and assembling these units to engineer larger tissue constructs from the bottom up. In this review, state-of-the-art methods for fabrication of three-dimensional biomimetic scaffolds are presented, and their advantages and drawbacks are discussed. The bottom-up methods used to assemble microscale building blocks (eg, microscale hydrogels) for tissue engineering are also reviewed. Finally, perspectives on future development of the bottom-up approach for tissue engineering are addressed.

Pharmacological effects and pharmacokinetic properties of icariin, the major bioactive component in Herba Epimedii☆

Herba Epimedii is an important medicinal plant which has been used in various traditional Chinese formulations for thousands of years as well as in modern proprietary traditional Chinese medicine products. It has extensive clinical indications, especially for the treatment of sexual dysfunction and osteoporosis. There have been more than 260 chemical moieties identified in the genus Epimedium most of which belong to flavonoids. Icariin is the most abundant constituent in Herba Epimedii. Icariin is pharmacologically bioactive and demonstrates extensive therapeutic capacities such as osteoprotective effect, neuroprotective effect, cardiovascular protective effect, anti-cancer effect, anti-inflammation effect, immunoprotective effect and reproductive function. Particularly, the significant osteogenic effect of icariin made it a promising drug candidate in bone tissue engineering. The current review paper aims to summarize the literatures reporting the pharmacological effects of icariin. The pharmacokinetic properties of bioactive ingredients in Herba Epimedii have also been discussed.

Bioactivity assessment of PLLA/PCL/HAP electrospun nanofibrous scaffolds for bone tissue engineering.

AIMS The purpose of this paper was to fabricate PLLA/PCL nanofibrous scaffolds containing HAP to mimic the native bone extracellular matrix for potential applications as bone tissue engineering scaffolds materials and ultimately to help the repairing of bone defects. MATERIALS AND METHODS PLLA (MW 200kDa), PCL (MW 80kDa), HAP, dichloromethane, N,N-dimethylformamide; α-MEM, FBS, trypsin-EDTA, penicillin G, streptomycin, β-sodium glycerophosphate, l-ascorbic acid, dexamethasone; CCK-8, Alkaline Phosphatase Assay Kit, Mouse Osteocalcin ELISA Kit, MC3T3-E1 cells. PLLA, PCL and HAP were dissolved in the solution of DCM and DMF to fabricate nanofibrous scaffolds through electrospinning. The morphology of the scaffolds was investigated with SEM, while the diameter of the fibers, pore size and water uptake of the scaffolds were tested, respectively. TGA was carried out to verify the percentage of HAP in the composite scaffolds fabricated with different HAP concentrations. Cell count kit-8 assay, alkaline phosphatase (ALP) assay, and osteocalcin assay were applied to observe the MC3T3-E1 cells proliferation, differentiation on the composite scaffolds. KEY FINDINGS MC3T3-E1 cells were found to grow actively on the composite scaffolds based on the results of CCK-8 assay. The level of MC3T3-E1 differentiation was evaluated through the ALP activity and osteocalcin concentration, which showed higher value with HAP containing (PLLA/PCL/HAP) than that ones without (PLLA/PCL). SIGNIFICANCE The results demonstrated that the biocomposite PLLA/PCL/HAP nanofibrous scaffold should be a promising candidate for proliferation, differentiation and mineralization of osteoblasts, and potentially can be used for bone tissue regeneration.

Enhanced bactericidal potency of nanoliposomes by modification of the fusion activity between liposomes and bacterium

Background Pseudomonas aeruginosa represents a good model of antibiotic resistance. These organisms have an outer membrane with a low level of permeability to drugs that is often combined with multidrug efflux pumps, enzymatic inactivation of the drug, or alteration of its molecular target. The acute and growing problem of antibiotic resistance of Pseudomonas to conventional antibiotics made it imperative to develop new liposome formulations to overcome these mechanisms, and investigate the fusion between liposome and bacterium. Methods The rigidity, stability and charge properties of phospholipid vesicles were modified by varying the cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and negatively charged lipids 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG), 1,2-dimyristoyl-sn-glycero-3-phopho-L-serine sodium salt (DMPS), 1,2-dimyristoyl-sn-glycero-3-phosphate monosodium salt (DMPA), nature phosphatidylserine sodium salt from brain and nature phosphatidylinositol sodium salt from soybean concentrations in liposomes. Liposomal fusion with intact bacteria was monitored using a lipid-mixing assay. Results It was discovered that the fluid liposomes-bacterium fusion is not dependent on liposomal size and lamellarity. A similar degree of fusion was observed for liposomes with a particle size from 100 to 800 nm. The fluidity of liposomes is an essential pre-request for liposomes fusion with bacteria. Fusion was almost completely inhibited by incorporation of cholesterol into fluid liposomes. The increase in the amount of negative charges in fluid liposomes reduces fluid liposomes-bacteria fusion when tested without calcium cations due to electric repulsion, but addition of calcium cations brings the fusion level of fluid liposomes to similar or higher levels. Among the negative phospholipids examined, DMPA gave the highest degree of fusion, DMPS and DMPG had intermediate fusion levels, and PI resulted in the lowest degree of fusion. Furthermore, the fluid liposomal encapsulated tobramycin was prepared, and the bactericidal effect occurred more quickly when bacteria were cultured with liposomal encapsulated tobramycin. Conclusion The bactericidal potency of fluid liposomes is dramatically enhanced with respect to fusion ability when the fusogenic lipid, DOPE, is included. Regardless of changes in liposome composition, fluid liposomes-bacterium fusion is universally enhanced by calcium ions. The information obtained in this study will increase our understanding of fluid liposomal action mechanisms, and help in optimizing the new generation of fluid liposomal formulations for the treatment of pulmonary bacterial infections.

Influence of polymer size, liposomal composition, surface charge, and temperature on the permeability of pH-sensitive liposomes containing lipid-anchored poly(2-ethylacrylic acid).

Background Liposomes containing pH-sensitive polymers are promising candidates for the treatment of tumors and localized infection. This study aimed to identify parameters influencing the extent of contents release from poly(ethylacrylic acid) (PEAA) vesicles, focusing on the effects of polymer size, lipid composition, vesicle surface charge, and temperature. Methods Anchored lipid pH-sensitive PEAA was synthesized using PEAA with a molecular weight of 8.4 kDa. PEAA vesicles were prepared by insertion of the lipid-anchored PEAA into preformed large unilamellar vesicles. The preformed liposomes were manipulated by varying the phosphocholine and cholesterol content, and by adding negative or positive charges to the liposomes. A calcein release assay was used to evaluate the effects of polymer size, liposome composition, surface charge, and temperature on liposomal permeability. Results The release efficiency of the calcein-entrapped vesicles was found to be dependent on the PEAA polymer size. PEAA vesicles containing a phosphatidylcholine to cholesterol ratio of 60:40 (mol/mol) released more than 80% of their calcein content when the molecular weight of PEAA was larger than 8.4 kDa. Therefore, the same-sized polymer of 8.4 kDa was used for the rest of study. The calcein release potential was found to decrease as the percentage of cholesterol increased and with an increase in the phosphocholine acyl chain length (DMPC DPPC DSPC). Negatively charged and neutral vesicles released similar amounts of calcein, whereas positively charged liposomes released a significant amount of their contents. pH-sensitive release was dependent on temperature. Dramatic content release was observed at higher temperatures. Conclusion The observed synergistic effect of pH and temperature on release of the contents of PEAA vesicles suggests that this pH-sensitive liposome might be a good candidate for intracellular drug delivery in the treatment of tumors or localized infection.

The importance of the prenyl group in the activities of osthole in enhancing bone formation and inhibiting bone resorption in vitro.

Osteoporosis treatment always aimed at keeping the balance of bone formation and bone resorption. Recently, prenyl group in natural products has been proposed as an active group to enhance the osteogenesis process. Osthole has both the prenyl group and bone-protective activities, but the relationship is still unknown. In this study we found that osthole exerted a potent ability to promote proliferation and osteogenic function of rat bone marrow stromal cells and osteoblasts, including improved cell viability, alkaline phosphatase activity, enhanced secretion of collagen-I, bone morphogenetic protein-2, osteocalcin and osteopontin, stimulated mRNA expression of insulin-like growth factor-1, runt-related transcription factor-2, osterix, OPG (osteoprotegerin), RANKL (receptor activator for nuclear factor-κB ligand), and the ratio of OPG/RANKL, as well as increasing the formation of mineralized nodules. However, 7-methoxycoumarin had no obvious effects. Osthole also inhibited osteoclastic bone resorption to a greater extent than 7-methoxycoumarin, as shown by a lower tartrate-resistant acid phosphatase activity and lower number and smaller area of resorption pits. Our findings demonstrate that osthole could be a potential agent to stimulate bone formation and inhibit bone resorption, and the prenyl group plays an important role in these bone-protective effects.

Polyethylenimine–DNA solid particles for gene delivery

Polyethylenimine (PEI), a cationic polymer, was used to develop a non-viral vector for gene delivery. A simple, reproducible process is described with which to condense plasmid DNA with PEI. When prepared at the optimum charge ratio of 6.3 ( ± ; PEI:DNA, 5:1 w/w), PEI–DNA complexes were 30–60 nm in diameter and excluded intercalating dyes from the plasmid DNA. The particles were stable for more than one month at 4°C with respect to size and transfection activity. PEI–condensed DNA transfected a broad range of murine and human tumor cell lines (B16, Lewis Lung, SK-OV-3 and LS180) in vitro in the presence of fetal calf serum. Intraperitoneal administration of PEI–condensed DNA resulted in significant gene expression in a human ovarian cancer peritoneal xenograft model.

Preparation of amifostine polylactide-co-glycolide microspheres and its irradiation protective to mouse through oral administration

The objectives of this study were to prepare the amifostine polylactide-co-glycolide (PLGA) microsphere and investigate its irradiation protective to mouse through oral administration. Amifostine-loaded PLGA microsphere was formulated using a modified double emulsion-solvent evaporation technique. The microsphere particle was spherical with a mean diameter of 2.8 ± 0.1 µm. Release data of amifostine PLGA microsphere was tested in phosphate-buffered saline at 37°C using a dialysis method and its release profiles was biphasic, showing a relatively large burst effect (50%) over the first 6 h, followed by a slower release phase, which sustained with 80% amifostine released in 48 h and almost 100% release till 6 days (144 h). A diffusion-controlled release model (Higuchi equation, R2 = 0.9725) was obtained for amifostine releasing from PLGA microsphere. The radiation experiment was performed by applied cobalt-60 γ-radiation source. One hour before γ-radiation exposure, the mouse was orally given free amifostine and PLGA microsphere, respectively. The irradiation effects, such as blood cell concentration, superoxidase dismutase (SOD) activity and malondialdehyde (MDA) level were monitored. The results indicated that amifostine PLGA microsphere was more irradiation protective to mouse than that of free amifostine under the same oral administration route.

Radix Dipsaci total saponins stimulate MC3T3-E1 cell differentiation via the bone morphogenetic protein-2/MAPK/Smad-dependent Runx2 pathway

Radix Dipsaci total saponins (RTS) are primary active components of Radix Dipsaci, which is administered orally for the treatment of osteoporosis according to Chinese Medicine. RTS have also been shown to reduce the risk of bone fractures in rats. However, the detailed molecular mechanisms underlying their action remain elusive. In the present study, the ability of RTS to increase alkaline phosphatase activity, osteocalcin levels and the degree of mineralization was investigated in MC3T3‑E1 mouse osteoblast precursor cells. In addition, the associated molecular mechanism was detected. The results revealed that RTS exerted an effect on osteoblastic maturation and differentiation. Induction of differentiation by RTS was associated with an increase in the expression levels of bone morphogenetic protein‑2 (BMP‑2), phosphorylated (P)‑Smad1/5/8, P‑ERK1/2, P‑p38 and Runt‑related transcription factor 2 (Runx2). Blocking BMP‑2 expression with noggin significantly reduced the levels of osteoblastic differentiation and subsequently attenuated the expression levels of P‑Smad1/5/8, P‑ERK1/2, P‑p38 and Runx2. This indicated that RTS induced osteoblastic differentiation through BMP‑2/mitogen‑activated protein kinase/Smad1/5/8‑dependent Runx2 signaling pathways and that it may be a promising agent for enhancing bone formation.

Designer Functionalized Self-Assembling Peptide Scaffolds for Adhesion, Proliferation, and Differentiation of MC3T3-E1

Induction of some specific cell signaling molecules in peptide scaffold play a critical role in developing of functional substitutes for damaged tissue. In this study, a new peptide ELK8 (Ac-LELELKLK-CONH2) was designed by replacing Ala in EAK16 (Ac-AEAEAKAKAEAEAKAK-CONH2) with more hydrophobic residue Leu in order to enhance the strength of peptide. The functionalized peptides were obtained through directly coupling three functional motifs to C-terminal of ELK8, such as osteogenic growth peptide, osteopontin cell adhesion motif, and 2-unit RGD binding sequence. Then, the new self-assembling peptide scaffolds were fabricated by mixing three functionalized peptides severally with pure ELK8 at the ratio of 1:1 (v:v) and the nanostructures of self-assembling peptides were investigated with AFM and DLS. The osteogenic efficacy of designed peptides on mouse pre-osteoblast MC3T3-E1 were examined after cells were incubated in scaffolds for 14 d. The results showed that these functionalized peptides, ALK mix particularly, promoted the attachment, proliferation, and osteogenic differentiation of MC3T3-E1 cells by increasing in cell numbers, ALP activities and osteocalcin concentrations.