Dental and Orthopaedic Implant Loosening: Overlap in Gene Expression Regulation.

Objectives: Endoprosthetic loosening still plays a major role in orthopaedic and dental surgery and includes various cellular immune processes within peri-implant tissues. Although the dental and orthopaedic processes vary in certain parts, the clinical question arises whether there are common immune regulators of implant loosening. Analyzing the key gene expressions common to both processes reveals the mechanisms of osteoclastogenesis within periprosthetic tissues of orthopaedic and dental origin. Methods: Donor peripheral blood mononuclear cells (PBMCs) and intraoperatively obtained periprosthetic fibroblast-like cells (PPFs) were (co-)cultured with [± macrophage-colony stimulating factor (MCSF) and Receptor Activator of NF-κB ligand (RANKL)] in transwell and monolayer culture systems and examined for osteoclastogenic regulations [MCSF, RANKL, osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)] as well as the ability of bone resorption. Sequencing analysis compared dental and orthopaedic (co-)cultures. Results: Monolayer co-cultures of both origins expressed high levels of OPG, resulting in inhibition of osteolysis shown by resorption assay on dentin. The high OPG-expression, low RANKL/OPG ratios and a resulting inhibition of osteolysis were displayed by dental and orthopaedic PPFs in monolayer even in the presence of MCSF and RANKL, acting as osteoprotective and immunoregulatory cells. The osteoprotective function was only observed in monolayer cultures of dental and orthopaedic periprosthetic cells and downregulated in the transwell system. In transwell co-cultures of PBMCs/PPFs profound changes of gene expression, with a significant decrease of OPG (20-fold dental versus 100 fold orthopaedic), were identified. Within transwell cultures, which offer more in vivo like conditions, RANKL/OPG ratios displayed similar high levels to the original periprosthetic tissue. For dental and orthopaedic implant loosening, overlapping findings in principal component and heatmap analysis were identified. Conclusions: Thus, periprosthetic osteoclastogenesis may be a correlating immune process in orthopaedic and dental implant failure leading to comparable reactions with regard to osteoclast formation. The transwell cultures system may provide an in vivo like model for the exploration of orthopaedic and dental implant loosening.

Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures.

INTRODUCTION: Periprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures. METHODS: PPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 103/cm2) or multilayer cultures (density 8.5 × 105/cm2) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR. RESULTS: Fibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased. CONCLUSION: The multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures.

Chondrogenic Potential of Pellet Culture Compared to High-Density Culture on a Bacterial Cellulose Hydrogel.

Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1-ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13. The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1-ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro.

Impact of Periprosthetic Fibroblast-Like Cells on Osteoclastogenesis in Co-Culture with Peripheral Blood Mononuclear Cells Varies Depending on Culture System.

Co-culture studies investigating the role of periprosthetic fibroblasts (PPFs) in inflammatory osteoclastogenesis reveal contrary results, partly showing an osteoprotective function of fibroblasts and high OPG expression in monolayer. These data disagree with molecular analyses of original periosteolytic tissues. In order to find a more reliable model, PPFs were co-cultivated with peripheral blood mononuclear cells (PBMCs) in a transwell system and compared to conventional monolayer cultures. The gene expression of key regulators of osteoclastogenesis (macrophage colony-stimulating factor (MCSF), receptor activator of NF-κB ligand (RANK-L), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)) as well as the ability of bone resorption were analyzed. In monolayer co-cultures, PPFs executed an osteoprotective function with high OPG-expression, low RANK-L/OPG ratios, and a resulting inhibition of osteolysis even in the presence of MCSF and RANK-L. For transwell co-cultures, profound changes in gene expression, with a more than hundredfold decrease of OPG and a significant upregulation of TNFα were observed. In conclusion, we were able to show that a change of culture conditions towards a transwell system resulted in a considerably more osteoclastogenic gene expression profile, being closer to findings in original periosteolytic tissues. This study therefore presents an interesting approach for a more reliable in vitro model to examine the role of fibroblasts in periprosthetic osteoclastogenesis in the future.

Immobilization of Denosumab on Titanium Affects Osteoclastogenesis of Human Peripheral Blood Monocytes.

Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces.

Effects of single and combined low frequency electromagnetic fields and simulated microgravity on gene expression of human mesenchymal stem cells during chondrogenesis.

INTRODUCTION: Low frequency electromagnetic fields (LF-EMF) and simulated microgravity (SMG) have been observed to affect chondrogenesis. A controlled bioreactor system was developed to apply LF-EMF and SMG singly or combined during chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in 3D culture. MATERIAL AND METHODS: An external motor gear SMG bioreactor was combined with magnetic Helmholtz coils for EMF (5 mT; 15 Hz). Pellets of hMSCs (±TGF-ß3) were cultured (P5) under SMG, LF-EMF, LF-EMF/SMG and control (1 g) conditions for 3 weeks. Sections were stained with safranin-O and collagen type II. Gene expression was evaluated by microarray and real-time polymerase chain reaction analysis. RESULTS: Simulated microgravity application significantly changed gene expression; specifically, COLXA1 but also COL2A1, which represents the chondrogenic potential, were reduced (p < 0.05). Low frequency electromagnetic fields application showed no gene expression changes on a microarray basis. LF-EMF/SMG application obtained significant different expression values from cultures obtained under SMG conditions with a re-increase of COL2A1, therefore rescuing the chondrogenic potential, which had been lowered by SMG. CONCLUSIONS: Simulated microgravity lowered hypertrophy but also the chondrogenic potential of hMSCs. Combined LF-EMF/SMG provided a rescue effect of the chondrogenic potential of hMSCs although no LF-EMF effect was observed under optimal conditions. The study provides new insights into how LF-EMF and SMG affect chondrogenesis of hMSCs and how they generate interdependent effects.

Effect of electromagnetic fields on human osteoarthritic and non-osteoarthritic chondrocytes.

BACKGROUND: Studies of the effects of electromagnetic fields (EMFs) on cartilaginous cells show a broad range of outcomes. However EMFs are not yet clinically applied as standard treatment of osteoarthritis, as EMF effects are showing varying outcomes in the literature. The aim of this study was to examine effects of EMFs (5 mT or 8 mT) on osteoarthritic (OA) and non-OA chondrocytes in order to investigate whether EMF effects are related to chondrocyte and EMF quality. METHODS: Pellets of human OA and non-OA chondrocytes were exposed to a sinusoidal 15 Hz EMF produced by a solenoid. Control groups were cultivated without EMF under standard conditions for 7 days. Cultures were examined by staining, immunohistochemistry and quantitative real-time PCR for RNA corresponding to cartilage specific proteins (COL2A1, ACAN, SOX9). RESULTS: OA chondrocytes increased the expression of COL2A1 and ACAN under 5 mT EMF compared to control. In contrast no changes in gene expression were observed in non-OA chondrocytes. OA and non-OA chondrocytes showed no significant changes in gene expression under 8 mT EMF. CONCLUSION: A 5 mT EMF increased the expression of cartilage specific genes in OA chondrocytes whereas in non-OA chondrocytes no changes in gene expression were observed. An 8 mT EMF however showed no effect altogether. This suggests that EMF effects are related to EMF but also to chondrocyte quality. Further studies about the clinical relevance of this effect are necessary.

Factors regulating bone remodeling processes in aseptic implant loosening.

This study was undertaken to screen periprosthetic tissues (PPTs) under specified conditions for a series of molecular components and describe them in bone remodeling processes within aseptic loosening. PPT samples were obtained from patients undergoing revision surgery of endoprostheses (n = 24) and synovial tissues from patients with OA (control) (n = 18), patients with any form of inflammatory arthritides were excluded. Tissue samples were examined via microbiology, histology (H&E, TRAP), immunohistochemistry (CD68/anti-S100a4), quantitative real-time PCR (ALP, COL1A1, cathepsin K, M-CSF, MMP13, OPG, RANK, RANKL, TNF-α, and TRAP) and an endotoxin-assay. PPT samples contained a variety of cellular components and stained positive for TRAP (56%), CD68 (100%), and S100a4 (100%). Wear debris were found in cells staining positive for CD68 and S100a4. In PPTs significantly higher ALP, COL1A1, MMP-13, RANK, RANKL, and TRAP expression were found along with a significantly higher RANKL/OPG ratio and a significantly lower OPG expression. No significant difference was observed for M-CSF, TNF-α, cathepsin K, and endotoxin levels. In conclusion we found osteogenic proteins (ALP, COL1A1), a proteolytic enzyme (MMP-13), markers for osteoclast differentiation (RANK, RANKL), and osteoclast activity (TRAP) to be increased in PPT, whereas OPG expression decreased significantly in comparison to control. We present data about a large series of molecular components in PPT and describe novel and key findings about their expression levels in regards to aseptic implant loosening. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:248-257, 2017.

Comparing effects of perfusion and hydrostatic pressure on gene profiles of human chondrocyte.

Hydrostatic pressure and perfusion have been shown to regulate the chondrogenic potential of articular chondrocytes. In order to compare the effects of hydrostatic pressure plus perfusion (HPP) and perfusion (P) we investigated the complete gene expression profiles of human chondrocytes under HPP and P. A simplified bioreactor was constructed to apply loading (0.1 MPa for 2 h) and perfusion (2 ml) through the same piping by pressurizing the medium directly. High-density monolayer cultures of human chondrocytes were exposed to HPP or P for 4 days. Controls (C) were maintained in static cultures. Gene expression was evaluated by sequencing (RNAseq) and quantitative real-time PCR analysis. Both treatments changed gene expression levels of human chondrocytes significantly. Specifically, HPP and P increased COL2A1 expression and decreased COL1A1 and MMP-13 expression. Despite of these similarities, RNAseq revealed a list of cartilage genes including ACAN, ITGA10 and TNC, which were differentially expressed by HPP and P. Of these candidates, adhesion related molecules were found to be upregulated in HPP. Both HPP and P treatment had beneficial effects on chondrocyte differentiation and decreased catabolic enzyme expression. The study provides new insight into how hydrostatic pressure and perfusion enhance cartilage differentiation and inhibit catabolic effects.

Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells.

PURPOSE: During in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) hypertrophy is an inadvertent event associated with cell differentiation toward the osteogenic lineage. Up to now, there is no stringent experimental control mechanism to prevent hypertrophy of MSCs. Microgravity is known to have an impact on osteogenesis. In this study, the influence of simulated microgravity (SMG) on both chondrogenesis and hypertrophy of hMSCs was evaluated. METHODS: A bioreactor using a rotating wall vessel was constructed to simulate microgravity. Pellet cultures formed from hMSCs (P5) were supplemented with human transforming growth factor-ß3 (TGF-ß3). The hMSC pellet cultures treated with TGF-ß3 were either kept in SMG or in a control system. After three weeks of culture, the chondrogenic differentiation status and level of hypertrophy were examined by safranin-O staining, immunohistochemistry and quantitative real-time PCR. RESULTS: SMG reduced the staining for safranin-O and collagen type II. The expression of collagen type X α1 chain (COL10A1) and collagen type II α1 chain (COL2A1) were both significantly reduced. There was a higher decrease in COL2A1 than in COL10A1 expression, resulting in a low COL2A1/COL10A1 ratio. CONCLUSIONS: SMG reduced hypertrophy of hMSCs during chondrogenic differentiation. However, the expression of COL2A1 was likewise reduced. Even more, the COL2A1/COL10A1 ratio decreased under SMG conditions. We therefore assume that SMG has a significant impact on the chondrogenic differentiation of hMSCs. However, due to the high COL2A1 suppression under SMG, this culture system does not yet seem to be suitable for a potential application in cartilage repair.

Comparison of ropivacaine and fentanyl toxicity in human fibroblasts.

INTRODUCTION: Although ropivacaine and fentanyl are commonly administered intra-articularly after knee or shoulder arthroscopy for postoperative pain control, there are no studies investigating the toxicity of ropivacaine and fentanyl on human fibroblasts (hF). MATERIAL AND METHODS: Human fibroblasts were seeded in monolayer triple flasks at a density of 10(4) cells/cm(2) and plated into 96 plates at a density of 5000 cells per well. After fully aspirating the culture medium 200 µl of ropivacaine or fentanyl in its corresponding concentration or culture medium only was added to each well. After 30 min ropivacaine or fentanyl was removed and fresh culture medium was added. Fibroblast mitochondrial activity and apoptosis marker level were evaluated after 1 h, 24 h and 7 days. RESULTS: We found a significant decrease in mitochondrial activity after 7 days when exposed to 0.5% ropivacaine. Mitochondrial activity after 1 h, 24 h and 7 days was significantly decreased when fibroblasts were exposed to 0.05% fentanyl. Also, a significant decrease in mitochondrial activity was observed 1 h after exposure to 0.025% fentanyl. Cell viability remained unchanged at any other point in time. A significant increase of caspase-3, as a marker of apoptosis, was only present after exposure to 0.5% ropivacaine after 7 days. CONCLUSIONS: These data suggest that both drugs have a concentration-dependent effect on mitochondrial activity in hF in vitro. This effect is more pronounced with fentanyl. Because the cytotoxicity of fentanyl, without the anticipated increase of caspase-3 as an apoptosis marker, remains unclear, we cannot support fentanyl as an alternative to ropivacaine.

An evaluation of medications commonly used for epidural neurolysis procedures in a human fibroblast cell culture model.

BACKGROUND AND OBJECTIVES: Epidural injections are popular therapies for sciatica and low back pain. Local anesthetics and corticosteroids are commonly used for most injections techniques, but some treatments use a specific combination of several agents. The epidural lysis of adhesions procedure (Racz) uses a combination of bupivacaine, hyaluronidase, a corticosteroid, and hypertonic saline. Because severe complications, some with permanent neurologic deficits, have been observed, we considered the possibility that individual agents or a combination thereof might be capable of damaging or destroying cells in primarily the epidural tissues. METHODS: We used monolayer cell cultures of human fibroblasts in Dulbecco modified Eagle medium to study these pharmacological agents alone or in combination. Cell viability and proliferation were assessed by Trypan blue staining, cell counts, and the WST-1 assay. Time and concentration series were performed. RESULTS: With the corticosteroid, we observed the previously described proliferation-retarding effects. Hyaluronidase was not found to have a relevant effect on fibroblast proliferation. Bupivacaine and hypertonic saline were found to have a time- and concentration-dependent effect on cell viability and proliferation. Both were found to be toxic at concentrations well below the ones used clinically. CONCLUSIONS: We identified a potential for harm caused by commonly used pharmacological agents when applied epidurally. Animal studies will have to show whether the same can be observed in living tissues.