Quantifying osteogenic cell degradation of silk biomaterials

The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins α5 and β1, while the osteoclasts upregulated integrins α2 and β1. There was significant contrast in responses on the silk matrices between osteogenic cells versus undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing, physiological state, and the ability to better understand the role of different cell types is critical to overall regenerative outcomes.     

Osteogenic cell contact with biomaterials influences phenotype expression

Relationship between (1) osteoblast adhesion and spreading, and (2) phenotype expression was investigated. Cellular adhesion and spreading were estimated after short time (24 h), whereas proliferation and other osteoblast functions – after 7 days. Primary human osteogenic cells were seeded on the samples of titanium (T), surgical steel (S) and tissue culture polystyrene (PS), and incubated at 37 °C. After 24 h a number of samples were stained with crystal violet and Hoechst; the average single cell area (spreading) and adhering cell number was measured on each sample. The remaining cultures were supplemented with dexamethasone (10 nM) and -glycerophosphate (5 mM), and incubation was continued for 7 days. The cells on each sample were counted and the following tests were performed: XTT mitochondrial activity assay, total protein content, alkaline phosphatase activity (ALP), Sirius Red test for collagen, osteocalcin and calcium concentration. After 24 h significantly greater cell spreading (p < 0.05) and number (p < 0.05) were on T than on S. After 7 days significantly higher on T than on S were: ALP activity (p < 0.000001), collagen (p < 0.0015) and calcium concentration (p < 0.03). XTT results were bigger on S than on T. In control – XTT results were higher than on the metals; collagen and ALP were lower than on T, and calcium level was significantly lower than on T and S (p < 0.025). After 7 days there were no differences in cell number between T and S. Cell number (24 h) correlated with ALP activity (7 days) on steel (coefficient of correlation, CC = 0.866) and titanium (CC = 0.742). The spreading correlated on steel and on titanium with calcium concentration (CC = 0.645 on S, CC = 0.696 on T) and collagen level (CC = –0.638 on S, CC = –0.69 on T). Conclusions: Better conditions for osteoblast phenotype expression on T after 7 days of culture coincided with greater adhesion and spreading of cells after 24 h on T, as compared with S. The initial contact of cells with underlying surface may influence osteoblast functions and possibly, bone regeneration and implant osteointegration in vivo. Early cell spreading may be an indicator of further expression of osteoblast phenotype and may be important for application of osteogenic cells in reconstructive surgery.     

Histological preparation of implanted biomaterials for light microscopic evaluation of the implant-tissue interaction

A technique is presented for processing implanted biomaterials with surrounding soft tissue for histological assessment of the implant-tissue interaction. Specimens are removed with the implant-tissue interface intact, fixed in formalin, dehydrated in a graded series of ethanol followed by a graded series of acetone in ethanol, and embedded in Spurr's low viscosity epoxy resin. Sections 0.5-1.0 mm thick are cut from the cured blocks using a metallurigical saw with a diamond wafer blade. After being glued to glass microscope slides, they are ground and polished to approximately 75 microns in thickness. The polished sections are treated with 95% ethanol saturated with sodium hydroxide, stained with Gill's hematoxylin and counterstained in eosin Y-phloxine B. The sodium hydroxide solution degrades the resin, allowing the stain to penetrate the tissue. By limiting the time in sodium hydroxide, the depth of staining is controlled and one is able to simulate a thin paraffin section with high resolution of the implant-soft tissue interface.     

Smart biomaterials - regulating cell behavior through signaling molecules

Important advances in the field of tissue engineering are arising from increased interest in novel biomaterial designs with bioactive components that directly influence cell behavior. Following the recent work of Mitchell and co-workers published in BMC Biology, we review how spatial and temporal control of signaling molecules in a matrix material regulates cellular responses for tissue-specific applications.     

Regulation of cell differentiation by the DNA damage response

When faced with DNA double-strand breaks (DSBs), vertebrate cells activate DNA damage response (DDR) programs that preserve genome integrity and suppress malignant transformation. Three established outcomes of the DDR include transient cell cycle arrest coupled with DNA repair, apoptosis, or senescence. However, recent studies in normal and cancer precursor or stem cells suggest that a fourth potential outcome, cell differentiation, is under the influence of DDR programs. Here we review and discuss the emerging evidence that supports the linkage of signaling from DSBs to the regulation of differentiation, including some of the molecular mechanisms driving this under-appreciated DDR outcome. We also consider the physiologic and pathologic consequences of defects in DDR signaling on cell differentiation and malignant transformation.     

Improved protocols for the isolation and in-situ cryopreservation of cell colonies

Stable transfection and cloning of cells often require physical separation of cell colonies. In order to conveniently isolate cell clones from petri dishes, we developed a protocol starting with a soft agar overlay of cells. This reduces the risk of cell diffusion between different colonies. Cells from individual colonies are mechanically removed, incubated with trypsin, and cell suspensions are seeded onto parallel microtiter plates. The cell clones on one microtiter plate can be cryopreserved in situ using the protocol described here which was tested for a variety of cell lines. Replica plates can be used for screening and further expansion of interesting clones. If screening can also be performed in situ, e.g., by immunocytochemistry, immunofluorescence, or the polymerase chain reaction, it is possible to perform most steps necessary in cell cloning experiments on microtiter plates.     

Biaxial mechanical response of bioprosthetic heart valve biomaterials to high in-plane shear

Utilization of novel biologically-derived biomaterials in bioprosthetic heart valves (BHV) requires robust constitutive models to predict the mechanical behavior under generalized loading states. Thus, it is necessary to perform rigorous experimentation involving all functional deformations to obtain both the form and material constants of a strain-energy density function. In this study, we generated a comprehensive experimental biaxial mechanical dataset that included high in-plane shear stresses using glutaraldehyde treated bovine pericardium (GLBP) as the representative BHV biomaterial. Compared to our previous study (Sacks, JBME, v.121, pp. 551-555, 1999), GLBP demonstrated a substantially different response under high shear strains. This finding was underscored by the inability of the standard Fung model, applied successfully in our previous GLBP study, to fit the high-shear data. To develop an appropriate constitutive model, we utilized an interpolation technique for the pseudo-elastic response to guide modification of the final model form. An eight parameter modified Fung model utilizing additional quartic terms was developed, which fitted the complete dataset well. Model parameters were also constrained to satisfy physical plausibility of the strain energy function. The results of this study underscore the limited predictive ability of current soft tissue models, and the need to collect experimental data for soft tissue simulations over the complete functional range.     

Sensing surfaces: Challenges in studying the cell adhesion process and the cell adhesion forces on biomaterials

A major turning point in the biomaterials field would be to develop tools that can offer greater insight into cell behaviour on material surfaces. Obtaining this information is very important for the development of long-term implantable materials because it can aid in improving cell adhesion and proliferation properties. The amalgamation of multiple disciplines has already produced many interesting techniques and approaches for the characterisation of cell adhesion processes and force adhesion strength determination on biomaterials. In this review, the authors provide an overview of the recent techniques developed for the noninvasive in situ study of the adhesion process as well as systems that allow the measurement of adhesion force strengths over biomaterials. Techniques based on light internal reflection, electrochemical impedance spectroscopy, and the quartz crystal microbalance (QCM) are discussed for their capabilities in investigating the cell adhesion process. Conversely, techniques such as flow cells, centrifugation, and cytodetachers are presented for the adhesion force measurement. An emphasis on atomic force microscopy (AFM) will demonstrate its ability to probe both the cell adhesion process and cell adhesion force, depending on the approach used. A discussion is followed on the strengths and/or weaknesses of these techniques. Finally, new trends and possible long-term directions for determining both adhesion process and force are highlighted.     

Novel polymer biomaterials and interfaces inspired from cell membrane functions

Background

Materials with excellent biocompatibility on interfaces between artificial system and biological system are needed to develop any equipments and devices in bioscience, bioengineering and medicinal science. Suppression of unfavorable biological response on the interface is most important for understanding real functions of biomolecules on the surface. So, we should design and prepare such biomaterials.

Scoop of review

One of the best ways to design the biomaterials is generated from mimicking a cell membrane structure. It is composed of a phospholipid bilayered membrane and embedded proteins and polysaccharides. The surface of the cell membrane-like structure is constructed artificially by molecular integration of phospholipid polymer as platform and conjugated biomolecules. Here, it is introduced as the effectiveness of biointerface with highly biological functions observed on artificial cell membrane structure.

Major conclusions

Reduction of nonspecific protein adsorption is essential for suppression of unfavorable bioresponse and achievement of versatile biomedical applications. Simultaneously, bioconjugation of biomolecules on the phospholipid polymer platform is crucial for a high-performance interface.

General significance

The biointerfaces with both biocompatibility and biofunctionality based on biomolecules must be installed on advanced devices, which are applied in the fields of nanobioscience and nanomedicine.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

In vitro effects of calcium phosphate biomaterials on fibroblastic cell behavior

The effect of synthetic granular hydroxyapatite (HAP) on cultured fibroblastic cells (L929, human bone and gingiva cells) was studied. Phagocytosis of HAP particles and resulting morphological cell changes were demonstrated by microscopic examinations. Cell counts and [3H]thymidine uptake indicated significant increases in cell proliferation and DNA synthesis. These results could account for some of the alterations of the fibroblast behavior induced by changes in intracellular levels of calcium ions released from the material.     

Characterization of bioreactors by in-situ fluorometry

A new microfluorometer with a special fiberoptic for the simultaneous detection of two different wavelengths was developed. Several tracers were tested for reactor characterization at different wavelengths, and the influence of the pH value and culture medium components on fluorescence was studied. In CST bioreactors, the effect of aeration rate and stirrer speed on the fluorescence can be used for reactor characterization. Mixing time experiments were performed in two different bubble columns.     

Electromechanical imaging of biomaterials by scanning probe microscopy

The majority of calcified and connective tissues possess complex hierarchical structure spanning the length scales from nanometers to millimeters. Understanding the biological functionality of these materials requires reliable methods for structural imaging on the nanoscale. Here, we demonstrate an approach for electromechanical imaging of the structure of biological samples on the length scales from tens of microns to nanometers using piezoresponse force microscopy (PFM), which utilizes the intrinsic piezoelectricity of biopolymers such as proteins and polysaccharides as the basis for high-resolution imaging. Nanostructural imaging of a variety of protein-based materials, including tooth, antler, and cartilage, is demonstrated. Visualization of protein fibrils with sub-10nm spatial resolution in a human tooth is achieved. Given the near-ubiquitous presence of piezoelectricity in biological systems, PFM is suggested as a versatile tool for micro- and nanostructural imaging in both connective and calcified tissues.     

Study of the corrosion of metallic biomaterials by surface physical methods

The authors investigated the corrosion mechanism of metallic implants inserted into the human body in contact with bone. It was shown that a significant degree of corrosion occurred after several months. The dissolution seems to be non-selective but can be different for each individual. The corrosion mechanism is complex, starting with an inter-granular process, but pitting also occurred . The oxidation of Fe2+ to Fe3+ favours the elimination of iron oxides and is followed by the destruction of the local alloy structure. An estimation of the quantity of each type of element released in the organism was also realized.     

The role of macrophages in the tissues regeneration stimulated by the biomaterials

Allogenic grafted tissues are subjected to biodegradation and replaced by the regenerate. To minimize the immune response and improve the rebuilding of tissues there was developed a technology to treat tissues with a cells elimination and dosed out extraction of proteoglycanes (Alloplant^®). With aim to clarify the role of macrophages in the tissues regeneration resulting implantation the biomaterials 112 rats were injected the allogenic and xenogenic (rabbits) pulverized biomaterials in the form of suspension. Injections were performed subcutaneously into the animals back by the base of the tail. The control group (14 rats) were injected a physiologic saline. Animals were killed by ether inhalation on day 2, 4, 7, 14, 30, 90 and 180 and tissue sections were studied by light and electron microscopy. The study showed the key role of the macrophages in resorption of the allogenic biomaterial and formation of the newly-formed tissue. Implantation of the biomaterial induced activity a great number of the mature macrophages, which completely lysed and resorbed the biomaterial particles. Expression TNF was significantly higher whereas expression TGF-1 was significantly lower. With xenogenic biomaterial implantation there were less macrophages, their activity was restricted. Macrophages containing large vacuoles with an active endo- and exocytosis were revealed in the allogenic biomaterial implantation and were named matrix-forming macrophages. We may suppose that these macrophages synthesize (or re-synthesize) proteoglycan component of the newly-formed collagen fibers. There was put forward a hypothesis about the two component mechanism of the collagen fibers formation.     

Application of biomaterials to advance induced pluripotent stem cell research and therapy

Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation.     

In vitro model systems for evaluation of smooth muscle cell response to cryoplasty

Restenosis is a major health care problem, with approximately 40% of angioplasties resulting in restenosis. Mechanisms related to elastic recoil, cell proliferation, and extracellular matrix (ECM) synthesis are implicated. In vivo studies have demonstrated the potential for cryotherapy to combat the process of restenosis, but the mechanisms whereby freezing and/or cooling can reduce or eliminate smooth muscle cell (SMC) proliferation and ECM synthesis are not well known. While in vivo testing is ultimately necessary, in vitro models can provide important information on thermal parameters and mechanisms of injury. However, it is important to carefully choose the model system for in vitro work on cryoinjury characterization to adequately reflect the clinical situation. In this study, we examined the differences in response to cryoinjury by SMCs from different species (rat, pig, and human) and in different cellular environments (suspension vs. tissue equivalent). Tissue equivalents, composed of cells embedded in collagen or fibrin gel, provide a 3-D tissue-like environment, while allowing for controlled composition. As reported here, all SMCs showed similar trends, but rat cells appeared less sensitive to cooling at faster cooling rates in suspension, while human SMCs were less sensitive to temperatures just above freezing when embedded in collagen. In addition, the SMCs were less sensitive in suspension than they were in collagen. Cells in suspension exhibited 70% viability at -11 degrees C, whereas cells in the tissue equivalent model showed only 30% survival. Future studies will aim to more adequately represent the conditions in restenosis by providing inflammatory and proliferative cues to the cells.     

Antigen-specific suppression of the plaque-forming cell response induced polyclonally by lipopolysaccharides

Horse erythrocytes (HRBC) were added with LPS in mouse spleen cell cultures, and the effects of HRBC on the LPS-induced polyclonal PFC response were investigated by enumerating total IgM-secreting PFC, anti-HRBC PFC, and PFC against sheep erythrocytes (SRBC). The addition of HRBC influenced the frequencies of anti-HRBC PFC in the total IgM-secreting PFC, but did not influence those of anti-SRBC PFC. The augmentation of the frequencies of anti-HRBC PFC occurred only when an appropriate dose of HRBC was added in the cultures containing T cells. Higher doses of HRBC decreased the frequencies of anti-HRBC PFC whether T cells were present or absent. The degree of reduction of the frequencies of anti-HRBC PFC was dependent on the dose of HRBC, but independent of the dose of LPS. The addition of HRBC at 1 day after LPS stimulation also decreased the frequency of anti-HRBC PFC, though the addition of 2 or 3 days hardly suppressed it. These results suggest that the antigen-specific augmentation occurs via helper T cells, and the suppression is ascribed to the direct action of antigen on the antigen-specific B cells.