Yanfeng Luo

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.

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.

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.

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.

Fallopian tube occlusion with a shape memory polymer device: evaluation in a rabbit model

BACKGROUND The present study evaluates the feasibility of a shape memory polymer (SMP) device for fallopian tube occlusion in rabbits. STUDY DESIGN The SMP contraceptive device is made of poly(dl-lactic acid)-based poly(urethane urea) SMP in the shape of a spiral cylinder that was 10 mm long and had a diameter of 2.6 mm. Using this device, bilateral transuterine fallopian tube occlusions were performed in 78 New Zealand white female rabbits. Forty-eight female rabbits (group 1) were chosen as the experimental group and were implanted with the SMP devices. The remaining 30 female rabbits (group 2) served as the control group, which only received an incision in the abdomen but no SMP device. Follow-up consisted of hysterosalpingography, histologic evaluation and contraceptive effect. In addition, the shape memory behavior and in vivo degradation characterization of the SMP device were observed in this study. RESULTS Under heat (37 °C) stimulation, the temporary shape SMP device returned to its permanent shape within 60 s. The average weight loss percentage of SMP devices was 7.0% at 2 weeks and 72.5% at 12 weeks. The inflammatory reactions caused by SMP devices were aseptic and nonspecific at 2 and 12 weeks, respectively. The SMP device boundaries and the surrounding tissues were obscured by fiber hyperplasia in 11/12 tubes at 24 weeks. Hysterosalpingography showed an occluded fallopian tube of Group 1 in 6/6 rabbits at 12 weeks and 6/6 rabbits at 24 weeks. No pregnancy was found in all 18 rabbits of group 1 (contraceptive rate of 100%); all 20 rabbits in the control group were pregnant. CONCLUSION Biodegradable and biocompatible SMP devices could provide reliable, instant and permanent tubal occlusion.

The responses of osteoblasts to fluid shear stress depend on substrate chemistries

Natural bone tissue receives chemical and mechanical stimuli in physiological environment. The effects of material chemistry alone and mechanical stimuli alone on osteoblasts have been widely investigated. This study reports the synergistic influences of material chemistry and flow shear stress (FSS) on biological functions of osteoblasts. Self-assembled monolayers (SAMs) on glass slides with functional groups of OH, CH3, and NH2 were employed to provide various material chemistries, while FSS (12 dynes/cm(2)) was produced by a parallel-plate fluid flow system. Material chemistry alone had no obvious effects on the expressions of ATP, nitric oxide (NO), and prostaglandin E2 (PGE2), whereas FSS stimuli alone increased the production of those items. When both material chemistry and FSS were loaded, cell proliferation and the expressions of ATP, NO and PGE2 were highly dependent on the material chemistry. Examination of the focal adhesion (FA) formation and F-actin organization of osteoblasts before FSS exposure indicates that the FA formation and F-actin organization followed similar chemistry-dependence. The inhibition of FAs and/or disruption of F-actins eliminated the material dependence of FSS-induced ATP, PGE2 and NO release. A possible mechanism is proposed: material chemistry controls the F-actin organization and FA formation of osteoblasts, which further modulates FSS-induced cellular responses.

Incorporating isosorbide as the chain extender improves mechanical properties of linear biodegradable polyurethanes as potential bone regeneration materials

One key limitation to the application of linear biodegradable polyurethanes (LBPUs) in scaffold materials for bone regeneration is their insufficient mechanical properties. In this study, isosorbide (ISO), a rigid two-ring small diol is selected as a chain extender to produce a series of new poly (D,L-lactide)-based polyurethanes (ISO-PUs). It is confirmed that incorporating ISO as the chain extender can significantly reduce the crosslinking degree of ISO-PUs and thus increase their molecular weight and mechanical properties. ISO-PUs with Mn values of 72.09 kDa and 84.79 kDa demonstrate higher tensile strength & modulus (39.87 MPa & 2.19 GPa and 42.68 MPa & 2.57 GPa) than PDLLA with a Mn of 100 kDa (36.15 MPa & 2.07 GPa). All these results, together with the sound cytocompatibility of ISO-PUs with MC3T3-E1 cells based on the morphology observation and cell proliferation, suggest that ISO-PU should be a promising scaffold material for bone regeneration.

Surface chemistry regulates the sensitivity and tolerability of osteoblasts to various magnitudes of fluid shear stress.

Scaffolds provide a physical support for osteoblasts and act as the medium to transfer mechanical stimuli to cells. To verify our hypothesis that the surface chemistry of scaffolds regulates the perception of cells to mechanical stimuli, the sensitivity and tolerability of osteoblasts to fluid shear stress (FSS) of various magnitudes (5, 12, 20 dynes/cm2 ) were investigated on various surface chemistries (-OH, -CH3 , -NH2 ), and their follow-up effects on cell proliferation and differentiation were examined as well. The sensitivity was characterized by the release of adenosine triphosphate (ATP), nitric oxide (NO) and prostaglandin E2 (PGE2 ) while the tolerability was by cellular membrane integrity. The cell proliferation was characterized by S-phase cell fraction and the differentiation by ALP activity and ECM expression (fibronectin and type I collagen). As revealed, osteoblasts demonstrated higher sensitivity and lower tolerability on OH and CH3 surfaces, yet lower sensitivity and higher tolerability on NH2 surfaces. Observations on the focal adhesion formation, F-actin organization and cellular orientation before and after FSS exposure suggest that the potential mechanism lies in the differential control of F-actin organization and focal adhesion formation by surface chemistry, which further divergently mediates the sensitivity and tolerability of ROBs to FSS and the follow-up cell proliferation and differentiation. These findings are essentially valuable for design/selection of desirable surface chemistry to orchestrate with FSS stimuli, inducing appropriate cell responses and promoting bone formation.