Wolfgang Metzger

The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells.

BACKGROUND AIMS The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques. METHODS We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically. RESULTS The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible. CONCLUSIONS Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.

Reduced myofibroblast differentiation on femtosecond laser treated 316LS stainless steel

In-stent restenosis is a common complication after stent surgery which leads to a dangerous wall narrowing of a blood vessel. Laser assisted patterning is one of the effective methods to modify the stent surface to control cell-surface interactions which play a major role in the restenosis. In this current study, 316 LS stainless steel substrates are structured by focusing a femtosecond laser beam down to a spot size of 50 μm. By altering the laser induced spot density three distinct surfaces (low density (LD), medium density (MD) and high density (HD)) were prepared. While such surfaces are composed of primary microstructures, due to fast melting and re-solidification by ultra-short laser pulses, nanofeatures are also observed as secondary structures. Following a detailed surface characterization (chemical and physical properties of the surface), we used a well-established co-culture assay of human microvascular endothelial cells and human fibroblasts to check the cell compatibility of the prepared surfaces. The surfaces were analyzed in terms of cell adherence, proliferation, cell morphology and the differentiation of the fibroblast into the myofibroblast, which is a process indicating a general fibrotic shift within a certain tissue. It is observed that myofibroblast proliferation decreases significantly on laser treated samples in comparison to non-treated ones. On the other hand endothelial cell proliferation is not affected by the surface topography which is composed of micro- and nanostructures. Such surfaces may be used to modify stent surfaces for prevention or at least reduction of restenosis.

Preparation and characterization of amine functional nano-hydroxyapatite/chitosan bionanocomposite for bone tissue engineering applications

In this study, three different types of scaffolds including a uniquely modified composite scaffold - namely chitosan (CTS), nano-hydroxyapatite/chitosan composite (CTS+nHAP), and amine group (NH2) modified nano-hydroxyapatite/chitosan composite (CTS+nHAP-NH2) scaffolds - were synthesized for bone tissue engineering (BTE) purposes. As results of the study, it was found that all scaffold types were biodegradable with CTS and CTS+nHAP scaffolds losing up to 15% of their initial weight, while the CTS+nHAP-NH2 scaffold showing 10% of weight loss after six weeks of lysozyme treatment. In addition, all three types of scaffolds were shown to be biocompatible, and amongst them CTS+nHAP-NH2 scaffolds supported the most cell proliferation in WST-1 assay and expressed the least and acceptable level of cytotoxicity in lactate dehydrogenase (LDH) test for human bone mesenchymal stem cells (hBM-MSCs). Finally, during osteoinductivity assessment, CTS+nHAP-NH2 nearly tripled initial alkaline phosphatase (ALP) activity when whereas both CTS and CTS+nHAP scaffolds only doubled. These results indicate that all synthesized scaffold types under investigation have certain potential to be used in bone tissue engineering approaches with CTS+nHAP-NH2 scaffold being the most promising and applicable one. In the future, we plan to intensify our studies on osteogenic differentiation on our scaffolds on a detailed molecular level and to include in vivo studies for pre-clinical purposes.

Analysis of cellular composition of co-culture spheroids.

3D spheroids and in particular co-culture spheroids reflect the natural organization of cells in tissues much better than 2D cell cultures as indicated by differences in cellular phyisology. However, most methods to analyze cells were established for 2D cultures and cannot easily be applied to spheroids. This study has aimed to demonstrate the possibility of quantification of the cellular composition of co-culture spheroids without previous dissociation into single cells. Prior to the generation of the spheroids, human endothelial cells, osteoblasts and fibroblasts were stained with fluoresent dyes for living cells. Co-culture spheroids of defined stoichiometric compositions were generated by the liquid overlay technique, cultivated for one, three or six days, respectively, and afterwards snap-frozen in liquid nitrogen. Cryo-sections of co-culture spheroids were analyzed by fluorescence microscopy and a newly established semi-automatic measuring routine. In order to compare the results, spheroids of one group were dissociated and the cellular composition was quantified by FACS-analysis. Staining efficiencies were higher than 95% as quantified in preliminary experiments with 2D cultures. Depending on the staining procedure, variations from uniform to punctate signals were detected. The size of all co-culture spheroids decreased over time and snap-freezing did not lead to shrinkage of the spheroids. We were able to detect organizational patterns of different cell types within the spheroids. It was possible to determine the cellular composition by quantitative microscopic analyses of cryo-sections as it could be confirmed by flow cytometric analyses. Depending on the experimental requirements, a combination of both methods might lead to valuable synergy.

In vivo biocompatibility, vascularization, and incorporation of Integra® dermal regenerative template and flowable wound matrix

Integra® matrix wound dressing (MWD) is used for the reconstruction of full-thickness skin defects. For the treatment of complex wounds, this dermal substitute is available as a flowable wound matrix (FWM) of identical composition. To clarify whether variations in sample preparation and consistency affect the biocompatibility and tissue incorporation, we herein compared MWD and FWM. The matrices were characterized using scanning electron microscopy and histology. Moreover, they were implanted in mouse dorsal skinfold chambers to analyze their in vivo performance over 2 weeks. Scanning electron microscopy showed a planar surface of MWD whereas FWM presented an irregular, fissured morphology. However, histology of the two matrices revealed an identical fiber thickness, fiber length, and interfiber distance. Repetitive stereo-microscopy and immunohistochemical analyses of MWD and FWM showed a comparable epithelialization of the implants in the dorsal skinfold chamber model. At day 14, both matrices exhibited a low collagen content and microvessel density. Moreover, they were infiltrated by a high number of myeloperoxidase (MPO)-positive neutrophilic granulocytes and a lower number of MAC387-positive macrophages and CD3-positive lymphocytes. These findings demonstrate that differences in preparation and consistency do not affect the tissue response to MWD and FWM, indicating a comparable regenerative capacity in wound healing.

Two- and three-dimensional co-culture models of soft tissue healing: pericyte-endothelial cell interaction

The demographic change in western countries towards an older population is being shadowed by an increased appearance of chronic diseases influencing soft tissue healing in a negative manner. Although various promising therapeutic approaches are available for treating chronic wounds, no in vitro model exists that successfully allows the analysis of interacting cells and of the effect of therapeutic drugs within a wound. Granulation tissue assures wound stability, neo-angiogenesis and revascularization finally leading to functional soft tissue repair. As one of the first steps in developing a model for human granulation tissue, we examined microvascular endothelial cells and pericytes in conventional 2D and in 3D spheroid co-cultures. We determined which parameters could be used in a standardized manner and whether the cultures were responsive to hypoxia and to erythropoietin supplementation. The read-out parameters of cell migration, cell density, rate of apoptotic cells, spatial cell distribution in the spheroid and spheroid volume were shown to be excellent analytic measures. In addition, quantification of hypoxia-related genes identified a total of 13 genes that were up-regulated in spheroids after hypoxia. As these parameters delivered reliable results in the present approach and as the general morphological distribution of pericytes and endothelial cells within the spheroid occurred in a typical manner, we believe that this basic in vitro model will serve for the future study of diverse aspects of soft tissue healing.

Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements.

Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbeccos modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.

Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture.

Abstract Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as “mini tissues” to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D3 induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D3 has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D3 and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.

Evaluation of cell-surface interaction using a 3D spheroid cell culture model on artificial extracellular matrices

Since decades, cell-surface interactions are studied in 2D cell culture approaches, but cells organized in 3D (spheroids) reflect the normal situation of cells in tissues much better due to intense cell-cell-contacts. Accordingly, this study aimed to prove, if spheroids could be used to study cell-surface interaction. Spheroids consisting of fibroblasts and/or osteoblasts were seeded on artificial extracellular matrices. Here, non-sulfated hyaluronan as a biological relevant compound of the extracellular matrix was chemically sulfated to different extents and co-fibrillised with collagen. The changes of the spheroid diameters and the migration distance of outgrown cells after seeding on the matrices were used as parameters to evaluate cell-surface interaction quantitatively. Fibroblast-based spheroids reacted in the initial phase of adhesion with different spheroid sizes on the contact with the matrices. In contrast, the reaction of osteoblasts was more pronounced at later time points exhibiting a decrease of the size of the spheroids with increasing sulfation degree of the matrix. The migration of the cells was impaired by increasing sulfation degree, which might be caused by an increased expression of focal adhesion relevant proteins. In summary, spheroids can be used in cell-surface interaction studies and additional analytical tools could be implemented.

Surface modification by plasma etching impairs early vascularization and tissue incorporation of porous polyethylene (Medpor(®) ) implants.

Porous polyethylene (Medpor®) is commonly used in craniofacial reconstructive surgery. Rapid vascularization and tissue incorporation are crucial for the prevention of migration, extrusion, and infection of the biomaterial. Therefore, we analyzed whether surface modification by plasma etching may improve the early tissue response to Medpor®. Medpor® samples were treated in a plasma chamber at low (20 W; LE-PE) and high energy levels (40 W; HE-PE). The samples and non-treated controls were implanted into mouse dorsal skinfold chambers to analyze angiogenesis, inflammation, and granulation tissue formation over 14 days using intravital fluorescence microscopy, histology, and immunohistochemistry. Scanning electron microscopy (SEM) analyses revealed that elevating energy levels of plasma etching progressively increase the oxygen surface content and surface roughness of Medpor®. This did not affect the leukocytic response to the implants. However, LE-PE and HE-PE samples exhibited an impaired vascularization. This was associated with a reduced formation of a collagen-rich granulation tissue at the implantation site. Additional in vitro experiments showed a reduced cell attachment on plasma-etched Medpor®. Thus, plasma etching may not be recommended to improve the clinical outcome of reconstructive interventions using Medpor®. However, it may be beneficial for temporarily implanted polyethylene-based biomedical devices for which tissue incorporation is undesirable.