Yuanliang Wang

Adsorption force of fibronectin on various surface chemistries and its vital role in osteoblast adhesion.

The amount, type, and conformation of proteins adsorbed on an implanted biomaterial are believed to influence cell adhesion. Nevertheless, only a few research works have been dedicated to the contribution of protein adsorption force. To verify our hypothesis that the adsorption force of protein on biomaterial is another crucial mediator to cell adhesion, fibronectin (FN) adsorbed on self-assembled monolayers (SAMs) with terminal -OH, -CH3, and -NH2 was quantified for FN adsorption force (F(ad)) by utilizing a sphere/plane adsorption model and parallel plate flow chamber. As revealed, F(ad) on SAMs followed a chemistry-dependence of -NH2 > -CH3 ≫ -OH. It is further demonstrated that F(ad) together with FN conformation could regulate the late osteoblast adhesion and the consequent reorganization of the adsorbed FN and fibrillogenesis of the endogenous FN. Our study suggests that protein adsorption force plays a key role in cell adhesion and should be involved for better biomaterial design.

Dodecanol-poly(D,L-lactic acid)-b-poly (ethylene glycol)-folate (Dol-PLA-PEG-FA) nanoparticles: evaluation of cell cytotoxicity and selecting capability in vitro.

Folate-conjugated Dol-poly(D,L-lactic acid)-b-poly (ethylene glycol)-folate (Dol-PLA-PEG-FA), was synthesized from dodecanol-poly(D,L-lactic acid), amino-terminated poly(ethylene glycol) and folate. (1)H NMR proved the successful synthesis of Dol-PLA-PEG-FA. Nanoparticles (NPs) were further fabricated from Dol-PLA-PEG-FA by using solvent evaporation-induced interfacial self-assembly method. The size, critical micelle concentration (CMC), cytotoxicity and selecting capability to cancer cells in vitro were examined for Dol-PLA-PEG-FA NPs. The size of NPs showed polymer concentration-dependent phenomenon in the fabrication process, and its polydispersity index (PDI) was very narrow. The CMC was determined as 1.995×10(-4) g/L in aqueous solution, which is much lower than the reported CMC of block copolymer self-assemble micelles. The cytotoxicity evaluation revealed that the obtained NPs2 are non-toxic to either breast cancer cell or normal endothelial cells, and the cell uptake of NPs indicated that the NPs demonstrated much higher selecting capability to breast cancer cells compared to normal fibroblast cells. The possible receptor-mediated endocytosis pathway mechanism was proposed. Based on the above results, it could be concluded that Dol-PLA-PEG-FA polymer and its nanoparticles can be potentially used as a safe drug carrier with strong tumor cells targeting capability for tumor chemotherapy.

Synthesis, characterization, and biocompatibility of a novel biomimetic material based on MGF-Ct24E modified poly(D, L-lactic acid).

Mechano-growth factor (MGF) is an alternative splicing variant of Insulin-like growth factor I. MGF and its 24 amino acid peptide analog corresponding to the unique C-terminal E-domain (MGF-Ct24E) are the positive regulator for tissue regenesis in bone. A novel biomimetic poly(D, L-lactic acid) (PDLLA) modification was designed and synthesized based on MGF-Ct24E grafted maleic anhydride modified PDLLA (MPLA). MGF-Ct24Es were grafted into the side chain of MPLA via a stable covalent amide bond using 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as the condensing agent to produce biomimetic MPLA materials (MGF-Ct24E-MPLA). Fourier transform infrared spectrometry, amino acid analyzer, and elementary analysis were used to characterize the MGF-Ct24E-MPLA. The hydrophilicity of MGF-Ct24E-MPLA was evaluated by means of the water-uptake ratios and static water contact angle. Data revealed that the grafting efficiency of MGF-Ct24E was about 29.9%. MGF-Ct24E-MPLA had better hydrophilicity than PDLLA and MPLA. The osteoblasts behavior of proliferation, differentiation, and mineralization on PDLLA, MPLA, and MGF-Ct24E-MPLA films was investigated and the results indicated that the introduction of MGF-Ct24E could improve osteoblasts proliferation, mineralization, and delay differentiation. The MGF-Ct24E modified MPLA with higher bioactivity may have potential application for bone tissue engineering.

New proangiogenic activity on vascular endothelial cells for C-terminal mechano growth factor

Angiogenesis is crucial in wound healing. The administration of the C-terminal 24-a.a. peptide of mechano growth factor (MGF24E) has been previously demonstrated to induce more blood vessels in regenerating bone around defective areas compared with the control. Accordingly, this study aims to determine whether MGF24E promotes bone defect healing through MGF24E-increased angiogenesis and whether MGF24E has positive effects on angiogenesis in vitro. The roles of MGF24E on angiogenesis and the underlying mechanisms were investigated. The cell proliferation, migration, and tubulogenesis of the human vascular endothelial EA.hy926 cells co-treated with 2% serum and MGF24E were determined to assess angiogenesis in comparison with 100 ng/ml of vascular endothelial growth factor 165 (VEGF(165))-positive control or vehicle control (phosphate-buffered saline). MGF24E treatment (10 ng/ml) significantly promoted the biological processes of angiogenesis on EA.hy926 cells compared with the vehicle control. The suppression of vascular endothelial growth factor and angiopoietin-I expressions by 2% serum starvation was reversed by the addition of 10 ng/ml of MGF24E in 2% serum medium. This result suggests that MGF24E has a protective effect on angiogenesis. Moreover, the inhibition of ERK due to PD98050 pretreatment completely abolished and mostly blocked MGF24E-induced proliferation and migration, respectively, whereas the MGF24-induced tubulogenesis and the angiogenic factor expression were only partially inhibited. These new findings suggest that MGF24E promotes angiogenesis by enhancing the expression of angiogenic cytokines which involves the MAPK/ERK-signaling pathway.

Molecular biocompatibility evaluation of poly(D,L-lactic acid)-modified biomaterials based on long serial analysis of gene expression.

In this work, long serial analysis of gene expression (LongSAGE) technology was used to investigate the molecular mechanism of the interaction between cells and poly(D,L-lactic acid)-modified biomaterials. After mouse osteoblast-like MC3T3-E1 cells were cultured on poly(D,L-lactic acid) (PDLLA) and a novel maleic anhydride-modified poly(D,L-lactic acid) (MPLA) films, the morphology, proliferation activity and alkaline phosphatase (ALP) activity of MC3T3-E1 cells were assessed by laser confocal microscope, cell counting assay and ALP assay, and the gene expression profiles of the cells were detected and compared at the transcript levels, respectively. 202 tags were found differentially expressed (p<0.05, and fold change >2) between PDLLA and MPLA LongSAGE libraries. Gene ontology functional analysis of the differentially expressed genes indicates that surface modification of MPLA biomaterial has an extensive influence on cells by regulating expression of genes related to cell proliferation, cell cycle, cytoskeleton organization, ossification, bone remodeling, metabolism, and eventually induces osteoblast proliferation and differentiation. The approach presented here provides a new insight in the molecular biocompatibility evaluation of biomaterials, contributing to the development of biomaterials in tissue engineering field.

Endothelin-1 and angiotensin II secretion at different lengths of endothelial cell monolayer in the view of tensile stress accumulation in the upper endothelial cell membrane

Abstract Theoretical analysis and experimental observations have shown that tensile stress inside an endothelial cell membrane is capable of growing in the direction opposite to blood flow and can accumulate to a level that is three or more orders of magnitude higher than flow-induced shear stress on the membrane surface. This phenomenon is called cell membrane tension accumulation (CMTA). We hypothesize that correlation may exist between the endothelial cell monolayer length or CMTA and secretory function of endothelial cells. To verify this hypothesis, a paired experimental study was devised to measure the secretion of endothelin (ET-1) and angiotensin II (Ang II) by two monolayers of cultured human glomerular vascular endothelial cell (HGVEC) monolayers subjected an identical steady shear stress. After replicate cultured HGVEC monolayer with two kinds of length of 6 cm and 10 cm were subjected to the same steady laminar shear stress of 0.45 N/m2 for 24 h, the average secretion rates of ET-1 and Ang II in 6 cm long increased l.7- and 0.5-fold (n=26, P

Surface chemistry modulates osteoblasts sensitivity to low fluid shear stress.

Low fluid shear stress (FSS) is the mechanical environment encountered by osteoblasts in implanted bones or native bones of bed rest patients. High sensitivity of osteoblasts to low FSS is beneficial to osteogenesis. We hypothesize that this sensitivity might be regulated by chemical microenvironment provided by scaffolds. To confirm this hypothesis, self-assembled monolayers (SAMs) were used to provide various surface chemistries including OH, CH3 , and NH2 while parallel-plate fluid flow system produced low FSS (5 dynes/cm(2) ). Alterations in S-phase cell fraction, alkaline phosphatase activity, fibronectin (Fn), and collagen type I (COL I) secretion compared to those without FSS exposure were detected to characterize the sensitivity. Osteoblasts on OH and CH3 SAMs demonstrated obvious sensitivity while on NH2 SAMs negligible sensitivity was observed. Examination of the cell aspect ratio, orientation, and focal adhesions before and after FSS exposure indicates that the full spreading and robust focal adhesions on NH2 SAMs should be responsible for the negligible sensitivity through increasing the cell tolerance to low FSS. Despite the higher sensitivity, the Fn and COL I depositions on both OH and CH3 SAMs after FSS exposure were still less than on NH2 SAMs without FSS exposure. These results suggest that elaborate design of surface chemical compositions is essential for orchestration of surface chemistry with low FSS to realize both high sensitivity and high matrix secretion, facilitating the formation of functional bone tissues in implanted bone.

Microencapsulation of mechano growth factor E peptide for sustained delivery and bioactivity maintenance

Mechano growth factor (MGF) and its C-terminal E-peptide with 24 amino acids, MGF-Ct24E, have superiority in resolving the delayed or failed bone repair derived from shortness of suitable biomechanical stimulation. The chitosan/tripolyphosphate microspheres encapsulated with MGF-Ct24E (CS/TPP/MGF-Ct24E) are prepared using emulsion-ionic cross-linking method in order to achieve the sustained release and preserve the bioactivity of MGF-Ct24E. The microspheres are micron-sized and spherical in shape with smooth surface morphology. The TPP component disintegrates in advance of CS matrix and the MGF-Ct24E maintains sustained delivery during in vitro hydrolytic degradation. With the disappearance of TPP, the total weight loss of CS/TPP/MGF-Ct24E is 32% and the release amount of MGF-Ct24E reaches 84.6% after degrading for 2 weeks. In vitro bioactivity assays reveal that the MGF-Ct24E can accelerate MC3T3-E1 cells proliferation and delay their differentiation as well. The encapsulated MGF-Ct24E shows long-term effects after being loaded in the CS/TPP microspheres and the cells exhibit excellent morphology on the surface of microspheres. The continuous delivery of MGF-Ct24E provides a new perspective on resolving the unsatisfactory bone reconstruction associated with microgravity and stress shielding.

The optimal combination of substrate chemistry with physiological fluid shear stress

Osteoblasts on implanted biomaterials sense both substrate chemistry and mechanical stimulus. The effects of substrate chemistry alone and mechanical stimulus alone on osteoblasts have been widely studied. This study investigates the optimal combination of substrate chemistry and 12dyn/cm(2) physiological flow shear stress (FSS) by examining their influences on primary rat osteoblasts (ROBs), including the releases of ATP, nitric oxide (NO), and prostaglandin E2 (PGE2). Self-assembled monolayers (SAMs) on glass slides with -OH, -CH3, and -NH2 were employed to provide various substrate chemistries, whereas a parallel-plate fluid flow system produced the physiological FSS. Substrate chemistry alone exerted no observable effects on the releases of ATP, NO, and PGE2. Nevertheless, when ROBs were exposed to both substrate chemistry and FSS, the ATP releases of NH2 were upregulated about 12-fold compared to substrate chemistry alone, while the ATP releases of CH3 and OH was similarly increased 7-fold at the peak. Similar trends were observed for the releases of NO and PGE2. The expressions of ATP, NO, and PGE2 followed the pattern of NH2-FSS>Glass-FSS>CH3-FSS≈OH-FSS. ROBs on NH2 produced the optimal combination of substrate chemistry with the physiological FSS. The F-actin organization and focal adhesion (FA) formation of ROBs on various SAMs without FSS were examined. NH2 produced the best results whereas CH3 and OH produced the worst ones. Inhibition of FAs and/or disruption of F-actin significantly decreased the releases of FSS-induced PGE2, NO, and/or ATP. Consequently, a mechanism was proposed that the best F-actin organization and FA formation of ROBs on NH2 lead to the optimal combination of substrate chemistry with the 12dyn/cm(2) physiological FSS. This mechanism gives guidance for the design of implanted biomaterials and bioreactors for bone tissue engineering.

Stretching-induced nanostructures on shape memory polyurethane films and their regulation to osteoblasts morphology.

Programming such as stretching, compression and bending is indispensible to endow polyurethanes with shape memory effects. Despite extensive investigations on the contributions of programming processes to the shape memory effects of polyurethane, less attention has been paid to the nanostructures of shape memory polyurethanes surface during the programming process. Here we found that stretching could induce the reassembly of hard domains and thereby change the nanostructures on the film surfaces with dependence on the stretching ratios (0%, 50%, 100%, and 200%). In as-cast polyurethane films, hard segments sequentially assembled into nano-scale hard domains, round or fibrillar islands, and fibrillar apophyses. Upon stretching, the islands packed along the stretching axis to form reoriented fibrillar apophyses along the stretching direction. Stretching only changed the chemical patterns on polyurethane films without significantly altering surface roughness, with the primary composition of fibrillar apophyses being hydrophilic hard domains. Further analysis of osteoblasts morphology revealed that the focal adhesion formation and osteoblasts orientation were in accordance with the chemical patterns of the underlying stretched films, which corroborates the vital roles of stretching-induced nanostructures in regulating osteoblasts morphology. These novel findings suggest that programming might hold great potential for patterning polyurethane surfaces so as to direct cellular behavior. In addition, this work lays groundwork for guiding the programming of shape memory polyurethanes to produce appropriate nanostructures for predetermined medical applications.