Ulrike Ritz

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds.

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

Molecular mechanisms of HLA class I antigen abnormalities following viral infection and transformation

In humans as in other animal species, CD8+ cytotoxic T lymphocytes (CTLs) play an important if not the major role in controlling virus‐infected and malignant cell growth. The interactions between CD8+ T cells and target cells are mediated by human leukocyte antigen (HLA) class I antigens loaded with viral and tumor antigen‐derived peptides along with costimulatory receptor/ligand stimuli. Thus, to escape from CD8+ T‐cell recognition and destruction, viruses and tumor cells have developed strategies to inhibit the expression and/or function of HLA class I antigens. In contrast, cells with downregulated MHC class I surface expression can be recognized by NK cells, although NK cell‐mediated lysis could be abrogated by the expression of inhibiting NK cell receptors. This review discusses the molecular mechanisms utilized by viruses to inhibit the formation, transport and/or expression of HLA class I antigen/peptide complexes on the cell surface. The knowledge about viral interference with MHC class I antigen presentation is not only crucial to understand the pathogenesis of viral diseases, but contributes also to the design of novel strategies to counteract the escape mechanisms utilized by viruses. These investigations may eventually lead to the development of effective immunotherapies to control viral infections and virus‐associated malignant diseases.

Extracorporeal shock wave-mediated changes in proliferation, differentiation, and gene expression of human osteoblasts.

BACKGROUND The goal of this study was to determine whether cell proliferation, differentiation, and gene expression of primary human osteoblasts (hOB) are influenced by shock wave application (SWA). METHODS Osteoblast cultures were isolated from cancellous bone fragments and treated with 500 impulses of energy flux densities of 0.06 mJ/mm, 0.18 mJ/mm, 0.36 mJ/mm, and 0.50 mJ/mm. Twenty-four hours and 96 hours after SWA cell proliferation, alkaline phosphatase activity, and mineralization were analyzed. The global gene expression profiling was determined 96 hours after SWA employing Affymetrix HG-U133A microarrays. RESULTS After 24 hours, hOB showed a dose-dependent increase in cell proliferation from 68.7% (at 0.06 mJ/mm, p = 0.002) up to 81.6% (at 0.5 mJ/mm, p = 0.001), which also persisted after 96 hours. Numbers of alkaline phosphatase-positive hOB increased after SWA treatment with peak levels of response between 0.18 mJ/mm and 0.5 mJ/mm after 24 hours. Mineralization was significantly higher in all groups compared with controls. Microarray analyses revealed SWA-induced differential expression of 94 genes involved in physiologic processes, cell homeostasis, and bone formation. Most intriguing was the up-regulation of multiple genes involved in skeletal development and osteoblast differentiation (e.g., PTHrP, prostaglandin E2-receptor EP3, BMP-2 inducible kinase, chordin, cartilage oligomeric matrix protein, matrillin). CONCLUSION We showed that shock waves have direct dose-dependent stimulatory effects on proliferation and differentiation of osteoblasts from normal human cancellous bone. We demonstrated that several genes critical for osteoblast differentiation and function are regulated after SWA. Overall, data presented herein will aid further understanding of the osteogenic effect of shock waves and, in addition, will enhance current knowledge of the SWA-mediated gene expression.

Cell viability, osteoblast differentiation, and gene expression are altered in human osteoblasts from hypertrophic fracture non-unions

Recent studies have provided evidence that the number and proliferation capacity of bone marrow-derived mesenchymal stem cells, as well as the number of osteoprogenitor cells are reduced in patients with fracture non-unions. For fracture non-unions that do not heal after appropriate surgical intervention, the question arises as to what extent systemic cellular dysfunctions should be considered as being pathogenetic factors. For this purpose, we have examined the hypothesis that the cell function of osteoblasts isolated from patients with fracture non-unions may differ from those of normal control individuals in an identical and controlled in vitro situation. We analyzed the osteoblast cell viability, formation of alkaline phosphatase-positive (CFU-ALP) and mineralization-positive (CFU-M) colony forming units, as well as global differences of gene expression in osteoblasts from patients with fracture non-unions and from control individuals. We found that cell viability and CFU-M-formation were significantly reduced in non-union osteoblasts. This was accompanied by significant differences in osteoblast gene expression as revealed by Affymetrix-microarray analysis and RT-PCR. We identified a set of significantly down-regulated factors in non-union osteoblasts that are involved in regulation of osteoblast proliferation and differentiation processes (canonical Wnt-, IGF-, TGF-beta-, and FGF-signaling pathways). The results of the present study strongly support the hypothesis that cell viability, differentiation, and gene expression of osteoblasts may be altered in patients who develop recurrent and recalcitrant fracture non-unions. Proteins involved in Wnt-, IGF, TGF-beta-, and FGF-signaling pathways may be of particular interest and may unveil new potential therapies.

The Transporter Associated With Antigen Processing (TAP): Structural Integrity, Expression, Function, and Its Clinical Relevance

BackgroundThe transporter associated with antigen processing (TAP), a member of the family of ABC transporters, plays a crucial role in the processing and presentation of the major histocompatibility complex (MHC) class I restricted antigens. TAP transports peptides from the cytosol into the endoplasmic reticulum, thereby selecting peptides matching in length and sequence to respective MHC class I molecules. Upon loading on MHC class I molecules, the trimeric MHC class I/β2-microglobulin/peptide complex is then transported to the cell surface and presented to CD8+ cytotoxic T cells. Abnormalities in MHC class I surface expression have been found in a number of different malignancies, including tumors of distinct histology, viral infections, and autoimmune diseases, and therefore represent an important mechanism of malignant or virus-infected cells to escape proper immune response. In many cases, this downregulation has been attributed to impaired TAP expression, which could be due to structural alterations or dysregulation. This review summarizes the physiology and pathophysiology of TAP, thereby focusing on its function in immune responses and its role in human diseases.

Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly.

Within the adaptive immune system the transporter associated with antigen processing (TAP) plays a pivotal role in loading of peptides onto major histocompatibility (MHC) class I molecules. As a central tool to investigate the structure and function of the TAP complex, we created cysteine‐less human TAP subunits by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2. After expression in TAP‐deficient human fibroblasts, cysteine‐less TAP1 and TAP2 are functional with respect to adenosine triphosphate (ATP)‐dependent peptide transport and inhibition by ICP47 from herpes simplex virus. Cysteine‐less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface.

Impaired Transporter Associated with Antigen Processing (TAP) Function Attributable to a Single Amino Acid Alteration in the Peptide TAP Subunit TAP1

The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1−/− cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu263 was comparable to that of TAP1−/− cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe265 of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu263 of the TAP1 protein, but not the Phe265, is critical for proper TAP function.

Photocrosslinkable dextran hydrogel films as substrates for osteoblast and endothelial cell growth

Functional hydrogel films on solid supports are versatile materials with large potential for cell growth and tissue engineering. Here, we report on a modular approach to generate functional hydrogel composite films for endothelial and osteoblast cell co-culture. The polymer network of the parent hydrogel was formed by a dextran derivative (BP-CMD), which contained carboxymethyl (CM) groups for further chemical functionalization and benzophenone (BP) moieties as a photocrosslinkable unit. BP-CMD could be synthesized by three different routes, first with the benzophenone unit attached via an amide bond, or second by an ether bond, or third as an ion pair between the benzophenone ammonium salt and the carboxylate groups on the dextran backbone. For composite formation, BP-functionalized silica nanoparticles, as well as gelatin particles were mixed with BP-CMD and permanently fixed in the hydrogel matrix by photocrosslinking for mechanical reinforcement and to facilitate cell growth. In the last preparation step the BMP-2 growth factor was covalently coupled to the polymer backbone, which enhanced osteoblast and endothelial cell growth. The swelling behavior and successful BMP-2 immobilization of the hydrogel composite films were investigated with surface plasmon resonance and optical waveguide mode spectroscopy coupled with fluorescence detection. This modular approach allows independent selection and optimization of each component in the hydrogel composite for a wide range of potential applications for targeted cell growth, as successfully shown here with osteoblast–endothelial cell co-culture for bone tissue regeneration.

Mobilization of CD34+-Progenitor Cells in Patients with Severe Trauma

Circulating CD34+ progenitor cells () gained importance in the field of regenerative medicine due to their potential to home in on injury sites and differentiate into cells of both endothelial and osteogenic lineages. In this study, we analyzed the mobilization kinetics and the numbers of CD34+, CD31+, CD45+, and CD133+ cells in twenty polytrauma patients (n = 13 male, n = 7 female, mean age 46.5±17.2 years, mean injury severity score (ISS) 35.8±12.5 points). In addition, the endothelial differentiation capacity of enriched CD34+cells was assessed by analyzing DiI-ac-LDL/lectin uptake, the expression of endothelial markers, and the morphological characteristics of these cells in Matrigel and spheroid cultures. We found that on days 1, 3, and 7 after a major trauma, the number of CD34+cells increased from 6- up to 12-fold (p<0.0001) over the number of CD34+cells from a control population of healthy, age-matched volunteers. The numbers of CD31+ cells were consistently higher on days 1 (1.4-fold, p<0.01) and 7 (1.3-fold, p<0.01), whereas the numbers of CD133+ cell did not change during the time course of investigation. Expression of endothelial marker molecules in CD34+cells was significantly induced in the polytrauma patients. In addition, we show that the CD34+ cell levels in severely injured patients were not correlated with clinical parameters, such as the ISS score, the acute physiology and chronic health evaluation II score (APACHE II), as well as the sequential organ failure assessment score (SOFA-2). Our results clearly indicate that pro-angiogenic cells are systemically mobilized after polytrauma and that their numbers are sufficient for the development of novel therapeutic models in regenerative medicine.

Surface Functionalization of Orthopedic Titanium Implants with Bone Sialoprotein

Orthopedic implant failure due to aseptic loosening and mechanical instability remains a major problem in total joint replacement. Improving osseointegration at the bone-implant interface may reduce micromotion and loosening. Bone sialoprotein (BSP) has been shown to enhance bone formation when coated onto titanium femoral implants and in rat calvarial defect models. However, the most appropriate method of BSP coating, the necessary level of BSP coating, and the effect of BSP coating on cell behavior remain largely unknown. In this study, BSP was covalently coupled to titanium surfaces via an aminosilane linker (APTES), and its properties were compared to BSP applied to titanium via physisorption and untreated titanium. Cell functions were examined using primary human osteoblasts (hOBs) and L929 mouse fibroblasts. Gene expression of specific bone turnover markers at the RNA level was detected at different intervals. Cell adhesion to titanium surfaces treated with BSP via physisorption was not significantly different from that of untreated titanium at any time point, whereas BSP application via covalent coupling caused reduced cell adhesion during the first few hours in culture. Cell migration was increased on titanium disks that were treated with higher concentrations of BSP solution, independent of the coating method. During the early phases of hOB proliferation, a suppressive effect of BSP was observed independent of its concentration, particularly when BSP was applied to the titanium surface via physisorption. Although alkaline phosphatase activity was reduced in the BSP-coated titanium groups after 4 days in culture, increased calcium deposition was observed after 21 days. In particular, the gene expression level of RUNX2 was upregulated by BSP. The increase in calcium deposition and the stimulation of cell differentiation induced by BSP highlight its potential as a surface modifier that could enhance the osseointegration of orthopedic implants. Both physisorption and covalent coupling of BSP are similarly effective, feasible methods, although a higher BSP concentration is recommended.