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.

An approach to architecture 3D scaffold with interconnective microchannel networks inducing angiogenesis for tissue engineering.

The angiogenesis of 3D scaffold is one of the major current limitations in clinical practice tissue engineering. The new strategy of construction 3D scaffold with microchannel circulation network may improve angiogenesis. In this study, 3D poly(D: ,L: -lactic acid) scaffolds with controllable microchannel structures were fabricated using sacrificial sugar structures. Melt drawing sugar-fiber network produced by a modified filament spiral winding method was used to form the microchannel with adjustable diameters and porosity. This fabrication process was rapid, inexpensive, and highly scalable. The porosity, microchannel diameter, interconnectivity and surface topographies of the scaffold were characterized by scanning electron microscopy. Mechanical properties were evaluated by compression tests. The mean porosity values of the scaffolds were in the 65-78% and the scaffold exhibited microchannel structure with diameter in the 100-200 µm range. The results showed that the scaffolds exhibited an adequate porosity, interconnective microchannel network, and mechanical properties. The cell culture studies with endothelial cells (ECs) demonstrated that the scaffold allowed cells to proliferate and penetrate into the volume of the entire scaffold. Overall, these findings suggest that the fabrication process offers significant advantages and flexibility in generating a variety of non-cytotoxic tissue engineering scaffolds with controllable distributions of porosity and physical properties that could provide the necessary physical cues for ECs and further improve angiogenesis for tissue engineering.

Enhancing the antibacterial activity of biomimetic HA coatings by incorporation of norvancomycin.

BACKGROUND: Bacterial infections associated with the use of biomaterials remain a great challenge for orthopedic surgery. The main purpose of the work discussed in this paper was to improve the antibacterial activity of a biomimetic calcium phosphate (CP) coating widely used in orthopedic biomaterials by incorporation of norvancomycin in the biomimetic process. METHODS: CP coating and CP coating containing norvancomycin were produced on a titanium alloy (Ti6Al4V) surface by a biomimetic process. The morphology, surface crystal structure, and concentrations of elements in the coatings were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDX), respectively. The amount of norvancomycin and its release were investigated by UV-visible spectroscopy. MTT was used to investigate cell behavior. The morphology of adhered bacteria was observed by SEM. Antibacterial activity was expressed as inhibition zone by using Staphylococcus aureus (ATCC 25923) as model bacteria. RESULTS: Results from SEM, EDX, and XRD revealed formation of a hydroxyapatite (HA) coating. The amount of antibiotic in the CP coating increased with increasing concentration of norvancomycin in the coating solution, followed by a plateau when the concentration of norvancomycin in the coating solution reached 600 mg/l. Approximately 2.16 µg norvancomycin per mg coating was co-precipitated with the CP layer onto titanium alloy discs when 600 mg/l norvancomycin coating solution was applied. The norvancomycin had a fast release profile followed by slow release. The MTT test of osteoblast cell cultures suggested that coatings containing norvancomycin did not cause any cytotoxicity compared with the CP coating and control titanium plate. The antibacterial activity test showed that the norvancomycin released from the coatings inhibited the growth of Staphylococcus aureus; more bacteria were found on the CP coating than on the norvancomycin-loaded coating. CONCLUSIONS: A norvancomycin-loaded HA-like coating was successfully obtained on titanium surfaces. The norvancomycin incorporated had no negative effects on osteoblast cell behavior. The released norvancomycin results in excellent antibacterial activity of Ca-P coatings. Therefore, incorporation of norvancomycin can enhance antibacterial activity and the norvancomycin-loaded CP coating can be used to inhibit post-surgical infections in orthopaedics.

[Effects of maleic anhydride-modified poly(D,L-lactic acid) on the adhesion, proliferation and differentiation of osteoblasts].

The main objective of this study was to observe the adhesion, proliferation and differentiation of mouse osteblast-like MC3T3-E1 cells cultured on maleic anhydride-modified poly(D,L-lactic acid) (MPLA) and poly(D,L-lactic acid) (PDLLA) polymers, and to evaluate the cytocompatibility of MPLA polymer. The effects of MPLA and PDLLA polymers on the morphology, adhesion, proliferation, the content of total cellular protein, alkaline phosphatase (ALP) activity and the content of Ca of MC3T3-E1 cells were explored. These results indicated that MC3T3-E1 cells on MPLA polymer adhered and spread more fully. On MPLA polymer, the proliferation, total protein content, ALP activity, Ca content of the cells were significantly higher than those of the cells on PDLLA polymer (P < 0.01). It was concluded that MPLA polymer could promote the adhesion, spreading, proliferation and the synthesis of protein of osteoblasts, and also induced the differentiation and mineralization of osteoblasts, suggesting that MPLA polymer might have the better cytocompatibility than PDLLA.

Shape recovery strain and nanostructures on recovered polyurethane films and their regulation to osteoblasts morphology.

Shape memory polyurethanes (SMPUs) have emerged as novel dynamic substrates to regulate cell alignment, in which recovery-induced change in substrates topography has been described as the major contributor. This work, for the first time, confirmed the pivotal roles of recovery strain and phase-separated nanostructures of SMPUs in regulating cell morphology. SMPU films with different stretching ratios (0%, 50%, 100%, and 200%) were found to produce an average recovery strain from 19.41% to 34.04% within 2 h in dulbecco's modified eagle medium (DMEM). Meanwhile, the assembly of hard domains was enhanced during shape recovery, leading to the reorientation of fibrillar apophyses (i.e., nanostructures). Further observation of osteoblast morphology revealed that recovery strain resulted in perpendicular orientation of osteoblasts to strain direction. With the extension of incubation time (24 h), however, the perpendicular orientation was transformed to follow the nanostructures on recovered films, suggesting that the nanostructures might become the determinant of the long-term cell orientation. This study provides a biomechanics-based perspective to understand the dynamic interactions between SMPU and cells, which can help to guide the design of SMPU for specific biomedical applications.

Build up of multilayered thin films with chitosan/DNA pairs on poly(D,L-lactic acid) films: physical chemistry and sustained release behavior.

In an effort to surface engineering of poly(D,L-lactic acid) (PDLLA), layer-by-layer (LbL) self-assembly of chitosan (Chi) and deoxyribonucleic acid (DNA) were employed to build up multilayered films. The formation of multilayers was monitored by using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), water contact-angle measurement, and atomic force microscopy (AFM), respectively. A full coverage of Chi/DNA pair film was formed only after the fifth sequential deposition (PEI/(DNA/Chi)2), which was revealed by contact-angle measurement. Surface chemistry and topography of multilayered films were directly related to the corresponding outmost layer component. Discernable island-like structures on PEI/(DNA/Chi)5/DNA layered PDLLA film was observed. Lysozyme-mediated multilayer degradation and DNA-releasing measurement suggested that DNA was gradually released into the incubation medium over a period of up to 32 h. The approach presented here may be exploited to develop controlled administration of functional DNA constructs from the surfaces of biomedical materials and devices in situ.

PLA-PEG-FA NPs for drug delivery system: Evaluation of carrier micro-structure, degradation and size-cell proliferation relationship.

In this paper, the micro-structure of amphiphilic copolymer Polylactic acid-Polyethylene glycol-Folate (PLA-PEG-FA) was studied firstly by a differential scanning calorimetry (DSC). During the process of nanoparticles (NPs) preparation, we found good inter-structure consistency of polymer was the precondition for forming into stable NPs, and those with micro-phase separation structure were prepared of NPs within limits. Hemolytic test and CCK-8 assay results demonstrated the biotoxicity of both NPs and whose leaching liquor was far below related toxicity standards. Two kinds of cell, human breast cancer cell line (MCF-7) and human umbilical vein endothelial cells (EC), showed different manners in test of NPs size-cell proliferation relationship, respectively. Monitored by a nuclear magnetic resonance (NMR) and a gel permeation chromatography (GPC), the degradation behavior of NPs in aqueous solution indicated amide bond break more difficultly than ester bond, and FA classic proton peak disappeared in the third week, meanwhile lactic acid (LA) unit number became 25% of the initial. Finally the NPs was completely degraded in the eighth week. In the whole process, NPs underwent a change from compact to loose state. We hope these results will benefit to improve design of drug delivery system in nanomedicine, which could offer the selection rule for amphiphilic polymer NPs on material and size.

Evaluation of the biocompatibility of acellular porcine dermis.

The biocompatibility of an acellular porcine dermis was evaluated by in vitro methods. Endothelia cells (ECV-304) and fibroblasts (NIH-3T3) were seeded on the dermis and cultured for 1 week to assess the cell viability in the skin grafts. Results by morphological assessment and methylthiazolyl tetrazolium assay (MTT assay) indicated good biocompatibility of acellular porcine dermis, which allowed adhesion and proliferation of examined cell types. Flow chamber technique was used to evaluate the adhesive force between cells and biomaterials. There was no significant difference in cells retention ratio between control cells and experimental cells after sheared 24h. The determination of integrin alpha5 expression proved that the acellular porcine dermis did not influence the expression of integrin alpha5 in cells. The results suggested that the dermal equivalent made from pigskin is a promising material for burn treatment.

[Characterization and histocompatibility of acellular bone collagen matrices].

A processing technique has been developed to prepare acellular bone collagen matrix (ABCM) and ABCM-PDLLA composite materials. The properties of these materials were characterized through several different methods. The histocompatibility of the materials were investigated by ELISA (enzyme linked immunosorbent assay) test and healing the defection of New Zealand white rabbit bilateral radius. The spectroscopy indicated that the major inorganic and organic components of the bone blocks were carbonated hydroxyapatite and collagen respectively,and the fatty and cellular components were. completely eliminated. The test results also revealed that the materials had good mechanical property and well-internnected pore structure, and the addition of PDLLA increased the strength of the materials. The ELISA results demonstrated that the materials had low immunogenicity in short order, and the degree of immune response caused by ABCM was greater than that by ABCM-PDLLA. All of the grafts exhibited good osteoconductive ability and a new bone form after the creeping substitution. In conclusion, two kinds of materials with good histocompatibility have been prepared, and owing to its good mechanical performance and low immunogenicity, ABCM-PDLLA is a better candidate for bone substitute and bone tissue engineering scaffold when compared with single ABCM.

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.

Grafting reaction of poly(D,L)lactic acid with maleic anhydride and hexanediamine to introduce more reactive groups in its bulk.

Bioactivity of biomaterials was a new requirement, especially in tissue engineering and drug delivery. As a traditional used biomaterial, polylactide (PLA) had no bioactivity, of course, and it still had few reactive groups to introduce some bioactive molecules in its bulk. Here, we want to introduce carboxyl groups and amino groups in the side chain of PLA to get more reactive groups for incorporating bioactive molecular later and to maintain the structure of main chain to keep its biodegradability, and to settle the acidity of PLA during hydrolysis at the same time. It was performed as follows: first, maleic anhydride was covalently grafted onto the side chain of PLA by a free radical reaction at 100 degrees C for 20 h with BPO as the initiator. Then, by amidation with a maleic anhydride group on PLA at room temperature, hexanediamine was incorporated. The resulting polymers have been characterized via GPC, (13)C NMR, DSC, and TGA. The graft ratio was tested by titration. The pH changes during hydrolysis in 0.1 M PBS with pH 7.4 of PLA, MPLA, and HPLA were investigated. All the results showed that this research has grafted maleic anhydride and then hexanediamine in the bulk of PLA. The molecular weight degradation during reaction was less than 20%. The graft ratios of were 2.68, 2.36, and 1.86%, respectively in 5, 10, and 20% raw MA in MPLA; and the anhydride groups grafted in MPLA can completely react with hexanediamine at room temperature. The pH value of HPLA remained neutral within 12 weeks' hydrolysis compared with the resulted acidity of PLA and MPLA.

[Advances in apoptosis induced by biomaterials].

Biomedical materials are the biomaterials that, used in physiological system for diagnosis, treatment, plerosis or replacement of tissues and organs. Apoptosis, also known as PCD or ACD, is a normal physiological mechanism of cell in organism and a process of automatic cell death in which multicell organism modulates the development of organism and maintains the stability of internal environment. The human beings are able to understand the interaction between the material and organism at the molecular level due to the widely-used biomedical material and the development of material science, life science and biological technology. The research of that interaction is mainly focused on biocompatibility, while much attention has been drawn to the apoptosis induced by biomaterial concerning that apoptosis could be caused by inducing factor, and many therapies of diseases are closely related to inducing apoptosis. Based on the recent research advances of apoptosis in life science and the development of biomaterials, the pathways of apoptosis induced by biomaterials were reviewed; from the different views, the pathways of signal transduction of apoptosis include traditional pathway of signal transduction, the pathway of death receptor, and the pathway through mitochondrion. By the other way, the pathways of apoptosis caused by reactive oxygen species induced by biomaterials and apoptosis by affecting cell adhesion to biomaterials and so forth were discussed also. It indicates that the pathways to apoptosis due to biomaterials possess the characteristics of variety, intercrossing and multiplicity. It is essential for a research to inquire into the mechanism of apoptosis that is induced by biomaterials, and further into the manufacturing of biomaterials. This review is devoted to shedding light on the wide application of biomaterials in the therapy of human diseases, especially in the therapy of cancer that is closely related to apoptosis.

[Compatibility of a novel ethylenediamine modified polylactic acid with osteoblasts].

Biocompatibility of a newly developed ethylenediamine modified poly (DL-latic acid) (EMPLA) with osteoblasts was investigated by means of cell morphology and cell proliferation. Films of PLA and EMPLA were made by solvent casting. Osteoblasts obtained from crania of neonatal Wistar rats were cultured on surfaces of PLA and EMPLA, with glass as control. The cell morphology was observed by phase contrast microscope and the cell proliferation was determined by MTT assay. The morphology observations revealed that the osteoblasts cultured on EMPLA spread wider than those on PLA, and much more cells were confluent on EMPLA, compared to those on PLA and glass. The growth curves showed the osteoblasts on EMPLA grew faster than did those on PLA and glass. The results exhibited that the biocompatibility of EMPLA with osteoblasts is better than that of PLA and glass, which suggested wide applications of EMPLA in biomedical area, especially in tissue engineering.

[Advance in research of osteoblast adhesion to bioactive materials].

In the research field of bone tissue engineering, the interaction of osteoblast and substrate is pivotal and the adhesion of osteoblast to biomaterials is the basic condition. Firstly, osteoblast must adhere to biomaterials, then it can migrate, proliferate and differentiate. This paper introduces the proteins relating to the adhesion of osteoblast and the influences of relating surface character and modification of biomaterials on the adhesion ability of osteoblast. These could serve as basic data and useful reference for the development of bone tissue engineering and tissue engineering scaffold materials.

[Preparation and characterization of bovine bone collagen matrix].

A process of preparing bovine cortical bone in order to form materials suitable for biomedical xenograft implants was described. Fresh bone samples cut from the middiaphyseal region of bovine femora were obtained from a local slaughterhouse. The bovine bone collagen matrix (BBCM) of various shapes fabricated from bovine bone by defatting and deproteination procedure may be implanted surgically for various purposes. The bone cubes were first defatted in a mixture of defatting agent; subsequently, the samples were extracted to release noncollagenous proteins, followed by digestion using a proteolytic enzyme to remove the telopeptide portions of collagen and residual noncollagenous proteins. Finally,the samples were dried in vacuum, packed and sterilized by gamma irradiation. The bone specimens were characterized by a suite of analytical techniques involving FTIR spectroscopy, X-ray diffraction spectroscopy, differential scanning calorimetry (DSC), uniaxial tension mechanical tests and scanning electron microscopy (SEM). The result showed that BBCM occurred as a white structure with suitable porosity. It contains reasonable proprotion of mineral and organic components in the original osseous architecture of the bovine bone, which is beneficial to keeping the mechanic property and weaker immunogenicity; therefore, it can serve as a potential bone implantable material and extracellular matrix material in bone tissue engineering.

Preparation and degradation characteristic study of bone repair composite of DL-polylactic acid/hydroxyapatite/decalcifying bone matrix.

OBJECTIVE: To explore the preparative method and study the degradation characteristics of bone repair composite of DL-polylactic acid (PDLLA)/hydroxyapatite(HA)/decalcifying bone matrix (DBM) in vitro. METHODS: An emulsion blend method was developed to prepare the composite of PDLLA/HA/DBM in weight ratio of PDLLA:HA:DBM = 1.5-2:1-1.5:1. The dynamic changes of weight, biomechanical property and pH value of PDLLA/HA/DBM and PDLLA in phosphate buffered saline (PBS, pH 7.4) were studied respectively through degradation tests in vitro. RESULTS: Without being heated, PDLLA, HA and DBM could be synthesized with the emulsion blend method as bone composite of PDLLA/HA/DBM, which had both osteoconductive and osteoinductive effects. The diameter of the aperture was 100-400 microm and the gap rate was 71.3%. During degradation, the pH value of PDLLA solution decreased lightly within 2 weeks, but decreased obviously at the end of 4 weeks and the value was 4.0. While the pH value of PDLLA/HA/DBM kept quite steady and was 6.4 at the end of 12 weeks. The weight of PDLLA changed little within 4 weeks, then changed obviously and was 50% of its initial weight at the end of 12 weeks. While the weight of PDLLA/HA/DBM changed little within 5 weeks, then changed obviously and was 60% of the initial weight at the end of 12 weeks. The initial biomechanical strength of PDLLA was 1.33 MPa, decreased little within 3 weeks, then changed obviously and kept at 0.11 MPa at the end of 12 weeks. The initial biomechanical strength of PDLLA/HA/DBM was 1.7 MPa, decreased little within 4 weeks, then changed obviously and kept at 0.21 MPa at the end of 12 weeks. CONCLUSIONS: The emulsion blend method is a new method to prepare bone repair materials. As a new bone repair material, PDLLA/HA/DBM is more suitable for regeneration and cell implantation, and the environment during its degradation is advantageous to the growth of bone cells.

[The application and advancement of rapid prototyping technology in bone tissue engineering].

In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is essential to the attachment, proliferation and differentiation of bone cells (osteoblast, osteoclast and osteocytes) and the formation of bone tissue. However, conventional scaffold materials for bone tissue engineering proved less valuable for actual applications because they lack mechanical strength, interconnected channel network, and controllable porosity or channel size. Therefore,to explore the ideal scaffold materials is one of the popular studies on current bone tissue engineering. In this paper, we review, the application and advancement of a newly-developed technology generally known as rapid prototyping (RP) techniques in bone tissue engineering.

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.

In vitro cytocompatibility evaluation of MGF-Ct24E chemically grafted and physically blended with maleic anhydride modified poly(D, L-lactic acid).

As a growth repair factor, mechano-growth factor (MGF) and its 24 amino acid peptide analogs corresponding to the unique C-terminal E-domain (MGF-Ct24E) positively regulate bone regeneration. MGF-Ct24E was introduced into the poly(D, L-lactic acid) (PDLLA) to improve bone regeneration in our previous study. MGF-Ct24E-grafted PDLLA was chemically characterized and showed potential as a biofunctional polymer. In this study, we evaluated the cytocompatibility of MGF-Ct24E chemically grafted and physically blended with maleic anhydride modified PDLLA, relative to maleic anhydride modified PDLLA (MPLA) as the control. The surface properties of these three polymer films were characterized with scanning electron microscopy and X-ray photoelectron spectroscopy. Rat calvarial osteoblasts were seeded on the three polymer films, and cell adhesion, spreading, and proliferation were assessed with an inverted microscope, laser scanning confocal microscope, and a cell counting kit-8, respectively. The alkaline phosphatase activity and extracellular calcium production were exploited to characterize the differentiation and mineralization of rat calvarial osteoblasts on various polymer films. The results revealed that compared with MPLA, MGF-Ct24E-MPLA, and MGF-Ct24E/MPLA blends promoted adhesion, spreading, proliferation, and the later differentiation and mineralization process of rat calvarial osteoblasts. In addition, the positive effect of MGF-Ct24E-MPLA on rat calvarial osteoblasts was maintained longer than the MGF-Ct24E/MPLA blends. In conclusion, MGF-Ct24E-MPLA synthesized chemically might be a promising biomaterial for bone tissue engineering.