Novel silk protein barrier membranes for guided bone regeneration.

This study assesses the biocompatibility of novel silk protein membranes with and without modification, and evaluates their effect on facilitating bone formation and defect repair in guided bone regeneration. Two calvarian bone defects 12 mm in diameter were created in each of a total of 38 rabbits. Four different types of membranes, (silk-, hydroxyapatite-modified silk-, ß-TCP-modified silk- and commonly clinically used collagen-membranes) were implanted to cover one of the two defects in each animal. Histologic analysis did not show any adverse tissue reactions in any of the defect sites indicating good biocompatibility of all silk protein membranes. Histomorphometric and histologic evaluation revealed that collagen and ß-TCP modified silk membranes supported bone formation (collagen: bone area fraction p = 0.025; significant; ß-TCP modified silk membranes bone area fraction: p = 0.24, not significant), guided bone regeneration and defect bridging. The bone, which had formed in defects covered by ß-TCP modified silk membranes, displayed a more advanced stage of bone tissue maturation with restoration of the original calvarial bone microarchitecture when compared to the bone which had formed in defects, for which any of the other test membranes were used. Micro-CT analysis did not reveal any differences in the amount of bone formation between defects with and without membranes. In contrast to the collagen membranes, ß-TCP modified silk membranes were visible in all cases and may therefore be advantageous for further supporting bone formation beyond 10 weeks and preventing soft tissue ingrowth from the periphery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2603-2611, 2017.

Strontium doped poly-ε-caprolactone composite scaffolds made by reactive foaming.

In the reconstruction and regeneration of bone tissue, a primary goal is to initiate bone growth and to stabilize the surrounding bone. In this regard, a potentially useful component in biomaterials for bone tissue engineering is strontium, which acts as cationic active agent, triggering certain intracellular pathways and acting as so called dual action bone agent which inhibits bone resorption while stimulating bone regeneration. In this study we established a novel processing for the foaming of a polymer (poly-ε-caprolactone) and simultaneous chemical reaction of a mixture of calcium and strontium hydroxides to the respective carbonates using supercritical carbon dioxide. The resultant porous composite scaffold was optimized in composition and strontium content and was characterized via different spectroscopic (infrared and Raman spectroscopy, energy dispersive X-ray spectroscopy), imaging (SEM, µCT), mechanical testing and in vitro methods (fluorescence vital staining, MTT-assay). As a result, the composite scaffold showed good in vitro biocompatibility with partly open pore structure and the expected chemistry. First mechanical testing results indicate sufficient mechanical stability to support future in vivo applications.

Biocompatible hollow-strut, silica enriched zirconia foams.

BACKGROUND: Porous ceramic biomaterials structures are accepted components in applied research in the field of tissue engineering due to their mechanical properties being closer to structural tissue like bone or other properties related to improved biocompatibility. OBJECTIVE: Hollow-strut, silica enriched zirconia foams were made by replication of polyurethane via impregnation with a suspension of zirconia-particles in polysiloxane. METHODS: Two-step heat treatment allowed conversion of the precursor structures into hollow-strut ceramic foams which were tested for their biocompatibility using an osteoblast cell line. Further, the material was characterized via different spectroscopic (Raman-spectroscopy, EDX) and imaging (SEM, µCT) methods. RESULTS: The material shows open cell porosity with hollow struts and sufficient structural integrity for handling and an expected chemistry as investigated by Raman and EDX spectroscopy. The material further supported cell growth and overall good biocompatibility. CONCLUSIONS: The investigated composite foam shows promising properties and is potentially interesting as candidate material for future bone tissue engineering applications.

Phenotypic redifferentiation and cell cluster formation of cultured human articular chondrocytes in a three-dimensional oriented gelatin scaffold in the presence of PGE2--first results of a pilot study.

Modern tissue engineering strategies comprise three elemental parameters: cells, scaffolds and growth factors. Articular cartilage represents a highly specialized tissue which allows frictionless gliding of corresponding articulating surfaces. As the regenerative potential of cartilage is low, tissue engineering-based strategies for cartilage regeneration represent a huge challenge. Prostaglandins function as regulators in cartilage development and metabolism, especially in growth plate chondrocytes. In this study, it was analyzed if prostaglandin E2 (PGE2 ) has an effect on the phenotypic differentiation of human chondrocytes cultured in a three-dimensional (3D) gelatin-based scaffold made by directional freezing and subsequent freeze-drying. As a result, it was clearly demonstrated that low doses of PGE2 revealed beneficial effects on the phenotypic differentiation and collagen II expression of human articular chondrocytes in this 3D cell culture system. In conclusion, PGE2 is an interesting candidate for tissue engineering applications since it represents an already well-studied molecule which is available in pharmaceutical quality.

Tetragonal and cubic zirconia multilayered ceramic constructs created by EPD.

The interest in electrophoretic deposition (EPD) for nanomaterials and ceramics production has widely increased due to the versatility of this technique to effectively combine different materials in unique shapes and structures. We successfully established an EPD layering process with submicrometer sized powders of Y-TZP with different mol percentages of yttrium oxide (3 and 8%) and produced multilayers of alternating tetragonal and cubic phases with a clearly defined interface. The rationale behind the design of these multilayer constructs was to optimize the properties of the final ceramic by combining the high mechanical toughness of the tetragonal phase of zirconia together with the high ionic conductivity of its cubic phase. In this work, a preliminary study of the mechanical properties of these constructs proved the good mechanical integrity of the multilayered constructs obtained as well as crack deflection in the interface between tetragonal and cubic zirconia layers.

A method to screen and evaluate tissue adhesives for joint repair applications.

BACKGROUND: Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications. METHOD: Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives. RESULTS: Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa). CONCLUSION: The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings.

Imaging of articular cartilage--data matching using X-ray tomography, SEM, FIB slicing and conventional histology.

The study was aimed at demonstrating a true cellular resolution for articular cartilage using synchrotron radiation-based X-ray microcomputed tomography (SR-µCT) with a sample-specific optimization of the phase contrast. The generated tomographic data were later used to prepare a matching histological sample from the full volume specimen. We used highly coherent and monochromatic X-rays from a synchrotron source to image a tissue sample of bovine articular cartilage after deparaffinization. Phase contrast enhancement was achieved by using five different sample to detector distances for the same X-ray energy. After tomography, the sample was re-embedded into resin while retaining a dedicated sample orientation for subsequent sectioning and polishing, which was conducted until a previously defined spatial position was achieved. The protocol for resin embedding was developed to inhibit morphological changes during embedding. Giemsa staining was applied for better structural and morphological discrimination. Data from tomography and lightmicroscopy were exactly matched and finally compared to results from FIB/SEM imaging. Image detail was achieved at a single cell resolution. Image detail was achieved at a single cell resolution, which has been estimated to be 0.833µm/voxel in the tomographic data. SR-µCT with optimized phase contrast properties represents a method to investigate biological tissues in certain areas of interest, where true cellular resolution or enhanced volumetric imaging is needed. In this study, we demonstrate that this method can compete with conventional histology using light microscopy but even surpasses it due to the possibility of retrieving volumetric data.

Current strategies and future perspectives for intraperitoneal adhesion prevention.

INTRODUCTION: The formation of peritoneal adhesions still is a relevant clinical problem after abdominal surgery. Until today, the most important clinical strategies for adhesion prevention are accurate surgical technique and the physical separation of traumatized serosal areas. Despite a variety of barriers which are available in clinical use, the optimal material has not yet been found. DISCUSSION: Mesothelial cells play a crucial physiological role in friction less gliding of the serosa and the maintenance of anantiadhesive surface. The formation of postoperative adhesions results from a cascade of events and is regulated by various cellular and humoral factors. Therefore, optimization or functionalization of barrier materials by developments interacting with this cascade on a structural or pharmacological level could give an innovative input for future strategies in peritoneal adhesion prevention. For this purpose, the proper understanding of the formal pathogenesis of adhesion formation is essential. Based on the physiology of the serosa and the pathophysiology of adhesion formation, the available barriers in current clinical practice as well as new innovations are discussed in the present review.

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning.

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray µCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses.

Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: investigation for cartilage tissue engineering.

Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage.

Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells.

Current light microscopic methods such as serial sectioning, confocal microscopy or multiphoton microscopy are severely limited in their ability to analyse rather opaque biological structures in three dimensions, while electron optical methods offer either a good three-dimensional topographic visualization (scanning electron microscopy) or high-resolution imaging of very thin samples (transmission electron microscopy). However, sample preparation commonly results in a significant alteration and the destruction of the three-dimensional integrity of the specimen. Depending on the selected photon energy, the interaction between X-rays and biological matter provides semi-transparency of the specimen, allowing penetration of even large specimens. Based on the projection-slice theorem, angular projections can be used for tomographic imaging. This method is well developed in medical and materials science for structure sizes down to several micrometres and is considered as being non-destructive. Achieving a spatial and structural resolution that is sufficient for the imaging of cells inside biological tissues is difficult due to several experimental conditions. A major problem that cannot be resolved with conventional X-ray sources are the low differences in density and absorption contrast of cells and the surrounding tissue. Therefore, X-ray monochromatization coupled with a sufficiently high photon flux and coherent beam properties are key requirements and currently only possible with synchrotron-produced X-rays. In this study, we report on the three-dimensional morphological characterization of articular cartilage using synchrotron-generated X-rays demonstrating the spatial distribution of single cells inside the tissue and their quantification, while comparing our findings to conventional histological techniques.

Stability of prostaglandin E(2) (PGE (2)) embedded in poly-D,L: -lactide-co-glycolide microspheres: a pre-conditioning approach for tissue engineering applications.

Prostaglandin E(2) (PGE(2)) is involved in angiogenesis, bone repair and cartilage metabolism. Thus, PGE(2) might represent a suitable signaling molecule in different tissue engineering applications. PGE(2) also has a short half-life time. Its incorporation into poly-D: ,L: -lactide-co-glycolide (PLGA) microspheres was demonstrated in a previous study. However, the stability of bioactive PGE(2) in these microspheres is unknown. With an adjusted mass spectrometry assay we investigated the amount of incorporated PGE(2) and the stability of PGE(2) in conventional cell culture medium and in PLGA microspheres. The stability of PGE(2) was closely pH dependent. Strong acidic or basic environments reduced the half-life from 300 h (pH 2.6-4.0) to below 50 h at pH 2.0 or pH 8.8. The half-life of PGE(2) incorporated into poly-D: ,L: -lactide-co-glycolide increased drastically to 70 days at 37 degrees C and to 300 days at 8 degrees C. Analysis with scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated a distinct nanostructure of the polymeric phase and both nano- and microporosity.

Immobilization and controlled release of prostaglandin E2 from poly-L-lactide-co-glycolide microspheres.

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite involved in physiological homeostasis and numerous pathophysiological conditions. Furthermore, it has been demonstrated that prostaglandins have a stimulating effect not only on angiogenesis in situ and in vitro but also on chondrocyte proliferation in vitro. Thus, PGE(2) represents an interesting signaling molecule for various tissue engineering strategies. However, under physiological conditions, PGE(2) has a half-life time of only 10 min, which limits its use in biomedical applications. In the present study, we investigated if the incorporation of PGE(2) into biodegradable poly-L-lactide-co-glycolide microspheres results in a prolonged release of this molecule in its active form. PGE(2)-modified microspheres were produced by a cosolvent emulsification method using CHCl(3) and HFIP as organic solvents and PVA as emulsifier. Thirteen identical batches were produced; and to each batch 1.0 mL of serum-free medium was added. The medium was removed at defined time points and then analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) to measure the residual PGE(2) content. In this study we demonstrated the prolonged release of PGE(2), showing a linear increase over the first 12 h, followed by a plateau and a slow decrease. The microspheres were further characterized by scanning electron microscopy.

Cyclooxygenases (COX-1 and COX-2) for tissue engineering of articular cartilage--from a developmental model to first results of tissue and scaffold expression.

Tissue engineering of articular cartilage remains an ongoing challenge. Since tissue regeneration recapitulates ontogenetic processes the growth plate can be regarded as an innovative model to target suitable signalling molecules and growth factors for the tissue engineering of cartilage. In the present study we analysed the expression of cyclooxygenases (COX) in a short-term chondrocyte culture in gelatin-based scaffolds and in articular cartilage of rats and compared it with that in the growth plate. Our results demonstrate the strong cellular expression of COX-1 but only a focal weak expression of COX-2 in the seeded scaffolds. Articular cartilage of rats expresses homogeneously COX-1 and COX-2 with the exception of the apical cell layer. Our findings indicate a functional role of COX in the metabolism of articular chondrocytes. The expression of COX in articular cartilage and in the seeded scaffolds opens interesting perspectives to improve the proliferation and differentiation of chondrocytes in scaffold materials by addition of specific receptor ligands of the COX system.

Emulsion-based synthesis of PLGA-microspheres for the in vitro expansion of porcine chondrocytes.

The in vitro cell expansion of autologous chondrocytes is of high interest in regenerative medicine since these cells can be used to treat joint cartilage defects. In order to preserve chondrocyte phenotype, while optimizing adhesion on microspheres, several processing parameters for the microsphere synthesis were varied. In this study three different polylactide-co-glycolides were used with differing lactide-glycolide ratios (85:15 and 50:50) and differing inherent viscosities. An emulsion route was established, where the polymer was dissolved in chloroform and then injected into a stirred polyvinyl alcohol-water solution at different polymer concentrations and different stirring velocities to produce microspheres with varying diameters. The sphere size distribution and morphology was analyzed using image processing software on SEM pictures. Based on previous experiments with commercial microspheres, three optimum samples were selected for further investigations. The degradation of the microspheres was determined in a long-term experiment in culture medium for 3 months. Adherent cells were characterized after 3 and 5 days by FDA+EB vital staining and in SEM.

Inverse inkjet printed gold micro electrodes for the structured deposition of epithelial cells and fibrin.

The micro structured deposition of vital cells is an important challenge in tissue engineering, biosensor technology, and in all research dealing with cell-cell and cell-substrate contacts. Hence, an inkjet printing technology has been developed to manufacture Au-based micro electrodes by sputter coating inversely printed polyester-foils. These electrodes feature minimal structure sizes of 35 microm and consist of an anode and a cathode part. They were used with fibrinogenic epithelial cell suspensions to deposit human keratinocytes (HaCaT), mouse fibroblasts (L-929) and the protein fibrin by applying DC voltage. Subsequently cells were electrophoretically attracted to the anode, following exactly its shape, while the insoluble fibrin was simultaneously precipitated due to the electrically mediated polymerization of the soluble fibrinogen molecule. Furthermore, it was demonstrated that this technique is suitable to co-deposit both cell types in a layered fashion. The lower voltage boundary for successful deposition was set at approximately 0.8 V needed for the conversion of fibrinogen into fibrin, while the upper voltage boundary was set at approximately 1.85 V, when commencing electrolysis inhibited the deposition of vital cells. Subsequent to the anodic cell-fibrin deposition, cells were cultivated for up to 4 days and then characterized by FDA+EB staining, methyl violet staining, MNF staining and SEM. The conversion from fibrinogen into fibrin was studied using ATR/FTIR.