Effects of single and repeated shock wave application on the osteogenic differentiation potential of human primary mesenchymal stromal cells and the osteoblastic cell line MG63 in vitro.

Introduction: Extracorporeal shock wave therapy is a non-invasive and effective option for treating various musculoskeletal disorders. Recent literature indicates that the parameters for extracorporeal shock wave therapy, such as the optimal intensity, treatment frequency, and localization, are yet to be determined. Studies reporting on the effects of shock wave application on primary mesenchymal stromal cells (MSCs) as well as osteoblastic cell lines in vitro are barely available and not standardized. Methods: In this study, we designed a special setup to precisely expose primary MSCs and the osteoblastic cell line MG63 to shock waves and subsequently analyzed the resulting cellular responses using standardized protocols to investigate their viability, proliferation behavior, cytokine secretion, and osteogenic differentiation potential in vitro. The shock wave transducer was coupled to a specifically designed water bath containing a 5 mL tube holder. Primary human MSCs and MG63 cells were trypsinated and centrifuged in a 5 mL tube and exposed to single and repeated shock wave application using different intensities and numbers of pulses. Results: Single treatment of MSCs using intensities 5, 10, 15, and 20 and pulse numbers 100, 250, 500, 750, and 1,000 at a constant pulse repetition frequency of 1 Hz resulted in a decreased viability and proliferation of both cell types with an increase in the intensity and number of pulses compared to controls. No significant difference in the osteogenic differentiation was observed at different time intervals in both cell types when a single shock wave application was performed. However, repeated shock wave sessions over three consecutive days of primary MSCs using low intensity levels 0.1 and 1 showed significant osteogenic differentiation 4-fold higher than that of the extracted Alizarin Red S at day 14, whereas MG63 cells showed no significant osteogenic differentiation compared to their corresponding controls. More specifically, repeated shock wave application triggered a significant downregulation of COL1A1, upregulation of RUNX2, and sustained increase of OCN in primary MSCs but not in the cell line MG63 when induced toward the osteogenic differentiation. Discussion: The effects of shock wave application on MSCs make it an effective therapy in regenerative medicine. We established a protocol to analyze a standardized shock wave application on MSCs and were able to determine conditions that enhance the osteogenic differentiation of MSCs in vitro.

Investigation of Cytotoxicity, Oxidative Stress, and Inflammatory Responses of Tantalum Nanoparticles in THP-1-Derived Macrophages.

Tantalum (Ta) is gaining attention as a biomaterial in bone tissue engineering. Although the clinical advantage of Ta-based implants for primary and revision total joint replacement (TJA) has been well documented, few studies investigated the effect of wear products of Ta implants on peri-implant cells, and their potential contribution to aseptic implant loosening. This study is aimed at examining the cytotoxicity, oxidative stress, and proinflammatory potential of Ta and TiO2 nanoparticles (NPs) on macrophages in vitro. NPs were characterized using scanning electron microscopy, dynamic light scattering, and energy-dispersive X-ray. To test the NP-mediated cellular response in macrophages, THP-1-derived macrophages were challenged with both NPs, and cytotoxicity was analyzed using CCK-8 and LDH assays. Flow cytometry was used to investigate particle uptake and their internalization routes. NP-mediated oxidative stress was investigated by measuring the production of reactive oxygen species, and their proinflammatory potential was determined by quantifying the production of TNFα and IL-1ß in cell culture supernatants using ELISA. We found that both Ta and TiO2 NPs were taken up through actin-dependent phagocytosis, although TiO2 NPs did also show some involvement of macropinocytosis and clathrin-mediated endocytosis. Ta NPs caused no apparent toxicity, while TiO2 NPs demonstrated significant cytotoxicity at a concentration of over 100µg/mL at 24 h. Ta NPs induced negligible ROS generation and proinflammatory cytokines (TNFα, IL-1ß) in macrophages. In contrast, TiO2 NPs markedly induced these effects in a dose-dependent manner. Our findings indicate that Ta NPs are inert, nontoxic, and noninflammatory. Therefore, Ta could be considered an excellent biomaterial in primary and revision joint arthroplasty implants.

Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro.

Osteoporosis is a disease characterized by low bone mass and an increased risk of fractures. Although several cellular players leading to osteoporosis have been identified, the role of mesenchymal stromal cells (MSC) is still not fully elaborated. The aim of this study was, therefore, to isolate and characterize MSCs from vertebral body of healthy non-osteoporotic and osteoporotic patients, with a particular focus on their osteogenic differentiation potential. Isolated MSCs were characterized by their osteogenic, adipogenic, and chondrogenic differentiation, as well as surface marker expression, proliferation behavior, and immunomodulatory capacity. The mineralization process was confirmed using Alizarin Red S and alkaline phosphatase (ALP) stains and further evaluated by determining ALP activity, mineral deposition, and free phosphate ion release. MSCs from both healthy and osteoporotic patients showed common fibroblast-like morphology and similar proliferation behavior. They expressed the typical MSC surface markers and possessed immunomodulatory capacity. Both groups demonstrated solid trilineage differentiation potential; osteogenic differentiation was further confirmed by increased ALP activity, deposition of inorganic crystals, phosphate ion release, and expression of osteoblast marker genes. Overall, MSCs from osteoporotic and non-osteoporotic patients showed neither a difference in general MSC features nor in the detailed analysis regarding osteogenic differentiation. These data suggest that vertebral body MSCs from osteoporotic patients were not impaired; rather, they possessed full osteogenic potential compared to MSCs from non-osteoporotic patients.

Local Cellular Responses to Metallic and Ceramic Nanoparticles from Orthopedic Joint Arthroplasty Implants.

Over the last decades, joint arthroplasty has become a successful treatment for joint disease. Nowadays, with a growing demand and increasingly younger and active patients accepting these approaches, orthopedic surgeons are seeking implants with improved mechanical behavior and longer life span. However, aseptic loosening as a result of wear debris from implants is considered to be the main cause of long-term implant failure. Previous studies have neatly illustrated the role of micrometric wear particles in the pathological mechanisms underlying aseptic loosening. Recent osteoimmunologic insights into aseptic loosening highlight the important and heretofore underrepresented contribution of nanometric orthopedic wear particles. The present review updates the characteristics of metallic and ceramic nanoparticles generated after prosthesis implantation and summarizes the current understanding of their hazardous effects on peri-prosthetic cells.

The Effects of Biomaterial Implant Wear Debris on Osteoblasts.

Aseptic loosening subsequent to periprosthetic osteolysis is the leading cause for the revision of arthroplasty failure. The biological response of macrophages to wear debris has been well established, however, the equilibrium of bone remodeling is not only dictated by osteoclastic bone resorption but also by osteoblast-mediated bone formation. Increasing evidence shows that wear debris significantly impair osteoblastic physiology and subsequent bone formation. In the present review, we update the current state of knowledge regarding the effect of biomaterial implant wear debris on osteoblasts. The interaction of osteoblasts with osteoclasts and macrophages under wear debris challenge, and potential treatment options targeting osteoblasts are also presented.

Molecular and Functional Phenotypes of Human Bone Marrow-Derived Mesenchymal Stromal Cells Depend on Harvesting Techniques.

Mesenchymal stromal cells (MSC) harvested in different tissues from the same donor exhibit different phenotypes. Each phenotype is not only characterized by a certain pattern of cell surface markers, but also different cellular functionalities. Only recently were different harvesting and processing techniques found to contribute to this phenomenon as well. This study was therefore set up to investigate proteomic and functional properties of human bone marrow-derived MSCs (hBM-MSC). These were taken from the same tissue and donor site but harvested either as aspirate or bone chip cultures. Both MSC populations were profiled for MSC markers defined by the International Society for Cellular Therapy (ISCT), MSC markers currently under discussion and markers of particular interest. While classic ISCT MSC markers did not show any significant difference between aspirate and outgrowth hBM-MSCs, our additional characterization panel revealed distinct patterns of differentially expressed markers. Furthermore, hBM-MSCs from aspirate cultures demonstrated a significantly higher osteogenic differentiation potential than outgrowth MSCs, which could be confirmed using a transcriptional approach. Our comparison of MSC phenotypes obtained by different harvesting techniques suggests the need of future standardized harvesting, processing and phenotyping procedures in order to gain better comparability in the MSC field.

Characterization and Comparison of Human and Ovine Mesenchymal Stromal Cells from Three Corresponding Sources.

Currently, there is an increasing focus on mesenchymal stromal cells (MSC) as therapeutic option in bone pathologies as well as in general regenerative medicine. Although human MSCs have been extensively characterized and standardized, ovine MSCs are poorly understood. This limitation hampers clinical progress, as sheep are an excellent large animal model for orthopedic studies. Our report describes a direct comparison of human and ovine MSCs from three corresponding sources under the same conditions. All MSCs presented solid growth behavior and potent immunomodulatory capacities. Additionally, we were able to identify common positive (CD29, CD44, CD73, CD90, CD105, CD166) and negative (CD14, CD34, CD45, HLA-DR) surface markers. Although both human and ovine MSCs showed strong osteogenic potential, direct comparison revealed a slower mineralization process in ovine MSCs. Regarding gene expression level, both human and ovine MSCs presented a comparable up-regulation of Runx2 and a trend toward down-regulation of Col1A during osteogenic differentiation. In summary, this side by side comparison defined phenotypic similarities and differences of human and ovine MSCs from three different sources, thereby contributing to a better characterization and standardization of ovine MSCs. The key findings shown in this report demonstrate the utility of ovine MSCs in preclinical studies for MSC-based therapies.

Long-term release of antibiotics by carbon nanotube-coated titanium alloy surfaces diminish biofilm formation by Staphylococcus epidermidis.

Bacterial biofilms cause a considerable amount of prosthetic joint infections every year, resulting in morbidity and expensive revision surgery. To address this problem, surface modifications of implant materials such as carbon nanotube (CNT) coatings have been investigated in the past years. CNTs are biologically compatible and can be utilized as drug delivery systems. In this study, multi-walled carbon nanotube (MWCNT) coated TiAl6V4 titanium alloy discs were fabricated and impregnated with Rifampicin, and tested for their ability to prevent biofilm formation over a period of ten days. Agar plate-based assays were employed to assess the antimicrobial activity of these surfaces against Staphylococcus epidermidis. It was shown that vertically aligned MWCNTs were more stable against attrition on rough surfaces than on polished TiAl6V4 surfaces. Discs with coated surfaces caused a significant inhibition of biofilm formation for up to five days. Therefore, MWCNT-modified surfaces may be effective against pathogenic biofilm formation on endoprostheses.

The effect of dexamethasone and triiodothyronine on terminal differentiation of primary bovine chondrocytes and chondrogenically differentiated mesenchymal stem cells.

The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal differentiation, they can be used to generate a model of endochondral ossification, but this limitation must be kept in mind when using them in cartilage tissue engineering application.

Tumor-stroma interactions of metastatic prostate cancer cell lines: analyses using microarrays.

Tumor-stroma interactions are of great importance not only for the development and progression of primary prostate carcinoma but probably also for the establishment of metastasis. Fibroblasts are an important stromal cell type encountered by metastatic tumor cells at different sites. In previous investigations, we had found that media conditioned by three metastatic prostate cancer cell lines (LNCaP, PC-3, and DU-145) induced cultured nonprostatic fibroblasts to proliferate or to express matrix-metalloproteinase-1 considered important for tumor invasion. Fibroblast-conditioned media in turn stimulate proliferation of DU-145 cells and migration of PC-3 cells. Both tumor cells and fibroblasts secrete VEGF suggesting that not only metastatic but also stromal cells at metastatic sites contribute to the vascularization of metastasis necessary for continuous growth. In order to better understand the reciprocal tumor-stroma cross-talk in molecular terms we used the mRNA extracted from stimulated and unstimulated neoplastic and fibroblastic stromal cells for cDNA array hybridization using Affymetrix chips. The three prostate cell lines influenced the fibroblasts nearly in the same manner. In particular proteins involved in cell adhesion, cell-cell contact, and cell cycle regulation were downregulated in stimulated fibroblasts. In contrast, fibroblasts affected every prostate cancer cell line in different ways, which may be because of the different origin of the metastatic prostate cancer cell lines.

Tumour-stroma interactions between metastatic prostate cancer cells and fibroblasts.

Previous work has shown the importance of tumour-stroma interactions for prostate cancer development at the primary site. The aim of the present study was to find out whether evidence can be found for a tumour-stroma cross- talk also between metastatic prostate cancer cell lines and non-prostatic stromal fibroblasts which are encountered by metastatic cells at most sites. We addressed this issue in cell culture systems using 3 metastatic human prostate cancer cell lines (LnCaP, PC-3 and DU-145) on the one hand, and a human fibroblast line (HFF, human foreskin fibroblasts) on the other. We incubated fibroblasts with tumour cell- and tumour cells with fibroblast-conditioned media and evaluated several parameters important for the establishment of metastases such as cell proliferation, migration and expression of matrix degrading proteases. We also determined in the conditioned media the concentrations of several growth factors and cytokines which might be responsible for the observed effects. We found that media conditioned by all 3 metastatic prostate cancer cell lines stimulated fibroblast proliferation which corresponds to fibrous stroma induction in vivo. DU-145 cell conditioned media induced in fibroblasts expression of mmp-1 mRNA known to be important for tumour invasion. ELISA assays revealed that tumour cells secrete bFGF, PDGF and TNFalpha known to stimulate fibroblast proliferation and/or MMP-1 expression. Cultivation of DU-145 carcinoma cells in fibroblast conditioned medium resulted in an enhanced proliferation and anchorage-independent growth of this cell line in soft agar. Fibroblast conditioned medium also increased migration of PC-3 cells in the wound assay and slightly augmented mmp-1 expression. KGF (able to stimulate proliferation of normal and neoplastic prostate epithelial cells) was secreted by fibroblasts at higher concentrations than by all 3 tumour cell lines. In addition, fibroblasts secreted TNFalpha, bFGF, PDGF, HGF and also VEGF, the most important factor for tumour vascularization. Our results provide evidence that tumour-stroma interactions do not only exist at the primary site but also between metastatic prostate cancer cell lines and their fibroblastic microenvironment. These interactions, which are mediated through secreted factors, affect several steps of the metastatic cascade including proliferation, anchorage-independent growth, migration and the secretion of matrix-degrading proteases.