Microbiological and ultrastructural evaluation of bacteriophage 191219 against planktonic, intracellular and biofilm infection with Staphylococcus aureus.

Infections of orthopaedic implants, such as fracture fixation devices and total-joint prostheses, are devastating complications. Staphylococcus aureus (S. aureus) is a predominant pathogen causing orthopaedic-implant biofilm infections that can also internalise and persist in osteoblasts, thus resisting antibiotic therapy. Bacteriophages are a promising alternative treatment approach. However, data on the activity of bacteriophages against S. aureus, especially during intracellular growth, and against in vivo biofilm formation on metals are scarce. Therefore, the present study evaluated the in vitro efficacy of S. aureus bacteriophage 191219, alone as well as in combination with gentamicin and rifampicin, to eradicate S. aureus strains in their planktonic stage, during biofilm formation and after internalisation into osteoblasts. Further, the invertebrate model organism Galleria mellonella was used to assess the activity of the bacteriophage against S. aureus biofilm on metal implants with and without antibiotics. Results demonstrated the in vitro efficacy of bacteriophage 191219 against planktonic S. aureus. The phage was also effective against in vitro S. aureus biofilm formation in a dose-dependent manner and against S. aureus internalised in an osteoblastic cell line. Transmission electron microscopy (TEM) analysis showed bacteriophages on S. aureus inside the osteoblasts, with the destruction of the intracellular bacteria and formation of new bacteriophages. For the Galleria mellonella infection model, single administration of phage 191219 failed to show an improvement in survival rate but appeared to show a not statistically significant enhanced effect with gentamicin or rifampicin. In summary, bacteriophages could be a potential adjuvant treatment strategy for patients with implant-associated biofilm infections.

Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.

Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4',6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.

Cells under pressure - the relationship between hydrostatic pressure and mesenchymal stem cell chondrogenesis.

Early osteoarthritis (OA), characterised by cartilage defects, is a degenerative disease that greatly affects the adult population. Cell-based tissue engineering methods are being explored as a solution for the treatment of these chondral defects. Chondrocytes are already in clinical use but other cell types with chondrogenic properties, such as mesenchymal stem cells (MSCs), are being researched. However, present methods for differentiating these cells into stable articular-cartilage chondrocytes that contribute to joint regeneration are not effective, despite extensive investigation. Environmental stimuli, such as mechanical forces, influence chondrogenic response and are beneficial with respect to matrix formation. In vivo, the cartilage is subjected to multiaxial loading involving compressive, tensile, shear and fluid flow and cellular response. Tissue formation mechanobiology is being intensively studied in the cartilage tissue-engineering research field. The study of the effects of hydrostatic pressure on cartilage formation belongs to the large area of mechanobiology. During cartilage loading, interstitial fluid is pressurised and the surrounding matrix delays pressure loss by reducing fluid flow rate from pressurised regions. This fluid pressurisation is known as hydrostatic pressure, where a uniform stress around the cell occurs without cellular deformation. In vitro studies, examining chondrocytes under hydrostatic pressure, have described its anabolic effect and similar studies have evaluated the effect of hydrostatic pressure on MSC chondrogenesis. The present review summarises the results of these studies and discusses the mechanisms through which hydrostatic pressure exerts its effects.

Achilles tendon elastic properties remain decreased in long term after rupture.

PURPOSE: Rupture of the Achilles tendon results in inferior scar tissue formation. Elastography allows a feasible in vivo investigation of biomechanical properties of the Achilles tendon. The purpose of this study is to investigate the biomechanical properties of healed Achilles tendons in the long term. MATERIALS AND METHODS: Patients who suffered from Achilles tendon rupture were recruited for an elastographic evaluation. Unilateral Achilles tendon ruptures were included and scanned in the mid-substance and calcaneal insertion at least 2 years after rupture using shear wave elastography. Results were compared to patients' contralateral non-injured Achilles tendons and additionally to a healthy population. Descriptive statistics, reliability analysis, and correlation analysis with clinical scores were performed. RESULTS: Forty-one patients were included in the study with a mean follow-up-time of 74 ± 30; [26-138] months after rupture. Significant differences were identified in shear wave elastography in the mid-substance of healed tendons (shear wave velocity 1.2 ±1.5 m/s) compared to both control groups [2.5 ±1.5 m/s (p < 0.01) and 2.8 ±1.6 m/s (p < 0.0001) contralateral and healthy population, respectively]. There was no correlation between the measurements and the clinical outcome. CONCLUSIONS: This study shows that the healed Achilles tendon after rupture has inferior elastic properties even after a long-term healing phase. Differences in elastic properties after rupture mainly originate from the mid-substance of the Achilles tendon, in which most of the ruptures occur. Elastographic results do not correspond with subjective perception. Clinically, sonoelastographical measurements of biomechanical properties can be useful to provide objective insights in tendon recovery.

The effect of estrogen on tendon and ligament metabolism and function.

Tendons and ligaments are crucial structures inside the musculoskeletal system. Still many issues in the treatment of tendon diseases and injuries have yet not been resolved sufficiently. In particular, the role of estrogen-like compound (ELC) in tendon biology has received until now little attention in modern research, despite ELC being a well-studied and important factor in the physiology of other parts of the musculoskeletal system. In this review we attempt to summarize the available information on this topic and to determine many open questions in this field.

Periodontal ligament cells as alternative source for cell-based therapy of tendon injuries: in vivo study of full-size Achilles tendon defect in a rat model.

Tendon's natural healing potential is extremely low and inefficient, with significant dysfunction and disability due to hypocellularity and hypovascularity of tendon tissues. The application of stem cells can aid in significantly enhanced repair of tendon rupture; therefore, the main aim of this study is to assess the potential of using periodontal ligament cells (PDL), usually obtained from patients undergoing orthodontic treatment, as a novel cell source for cell-based therapy for tendon injuries in a clinically relevant rat full-size Achilles tendon defect. In addition, the study compares the differences between the healing effects of Achilles tendon-derived cells (AT) versus PDL and, hence, comprises of four experimental groups, native tendon (NT), empty defect (ED), PDL and human AT (hAT). The tendon healing in each group was assessed in the late remodelling phase at 16 weeks after surgery using a combination of methods, including evaluation of gross morphological appearance; various histological and immunohistological stainings; and detailed analyses of cell morphometry. Based on these outcome measures, PDL cell-implanted tendons exhibited not only advanced tissue maturation, less ectopic fibrocartilage formation, more organised collagen fibres, tendon matrix expression corresponding to the final healing stage, and better cell-morphometry parameters when compared with the ED group, but were also very similar to the tendons treated with hAT-derived cells. Taken together, our study clearly demonstrates the feasibility of using PDL cells as a novel cell source for tendon repair and strongly recommends this cell type for the future development of innovative regenerative applications for treatment of different tendon or ligament pathologies.

EGFR-blockade with erlotinib reduces EGF and TGF-ß2 expression and the actin-cytoskeleton which influences different aspects of cellular migration in lens epithelial cells.

INTRODUCTION: After cataract surgery, residual lens epithelial cells migrate and proliferate within the capsular bag resulting in posterior capsule opacification (PCO). The up-regulation of TGF-ß2, EGF and FGF-2 has been identified as a key factor in PCO pathogenesis leading to actin fiber assembly and alterations in the migration pattern. In this in vitro study, the influence of Erlotinib as a selective EGFR inhibitor is investigated on the cellular features indicated, which might promote a future clinical application. METHODS: Expression of EGF, FGF-2 and TGF-ß2 was measured using RT-PCR and ELISA in human lens epithelial cells (HLEC). Computational data of an in vitro time lapse microscopy assay were used for statistical analysis of single cell migration with a particular focus on cell-cell interaction; cell velocity distribution; and displacement before, during and after mitosis. The effect of Erlotinib on the actin-cytoskeleton was evaluated using Alexa Fluor 488 Phalloidin and epifluorescence microscopy. RESULTS: EGF and TGF-ß2 mRNA expression and protein levels are reduced by Erlotinib, while FGF-2 expression remained stable. Overall fluidity of cell-cell interaction is less in the presence of Erlotinib compared to the control and the velocity distribution across all cells becomes less uniform within the cell cluster. After mitosis, HLEC move significantly faster without EGFR inhibition, which can be completely blocked by Erlotinib. Furthermore, Erlotinib diminishes the amount of actin stress fibers and the stress fiber diameter. CONCLUSION: As a novel effect of Erlotinib on HLEC, we describe the down-regulation of EGF and TGF-ß2 expression, both are crucial factors for PCO development. Cellular movement displays complex alterations under EGFR inhibition, which is partly explained by actin fiber depletion. These findings further underline the role of Erlotinib in pharmacologic PCO prophylaxis.

Attenuation of human lens epithelial cell spreading, migration and contraction via downregulation of the PI3K/Akt pathway.

BACKGROUND: Posterior capsule opacification (PCO) represents a major challenge in the postoperative management of cataract patients. Spreading, migration and contraction of residual human lens epithelial cells play a pivotal role in the pathogenesis of PCO. Therefore, we analyzed the effect of the alkylphosphocholine (APC) erufosine on these cellular features as well as on PI3K/Akt, a crucial pathway in PCO pathogenesis. METHODS: Human lens epithelial cells were cultured under standard cell culture conditions. Cell spreading was analyzed on fibronectin-coated wells and chemokinetic migration was assessed by time-lapse microscopy. For evaluation of cell-mediated collagen matrix contraction, the cells were seeded into collagen gels and incubated with an APC in different non-toxic concentrations before the surface area was measured on day 6. The activity of PI3K/Akt was assessed by an ELISA kit after incubation of the cells with different APC concentrations. RESULTS: Human lens epithelial cell spreading and migration were attenuated by APCs as follows: 7 % spreading, 48 % migration (0.1 µM APC), and 32 % spreading, 68 % migration (1.0 µM APC). APC concentrations of 0.1 µM reduced collagen gel diameter by 5 %, and 1.0 µM by less than 1 %, compared to untreated, cell-populated gels that resulted in a cell diameter contraction of 36 %. PI3K was downregulated in a concentration-dependent manner. CONCLUSIONS: The crucial cellular features of PCO pathogenesis are attenuated by the APC erufosine via downregulation of the PI3K pathway. Thus, erufosine might become a valuable tool for pharmacologic PCO prophylaxis in the future.

EGF receptor inhibitor erlotinib as a potential pharmacological prophylaxis for posterior capsule opacification.

BACKGROUND: Posterior capsule opacification (PCO) is the most frequent complication after cataract surgery, leading to a loss of sight if untreated. Erlotinib might be of therapeutic interest as an effective target agent (selective EGF-tyrosin-kinase-1 inhibitor). In this in-vitro study, erlotinib was evaluated for ocular biocompatibility and its effect on cell proliferation, migration, 3D matrix contraction and spreading of human lens epithelial cells. METHODS: To exclude toxic concentrations, erlotinib was assessed for its biocompatibility on five different human ocular cell types in vitro by the tetrazolium dye-reduction assay (MTT) and the Live-Dead assay. To determine its effect on human lens epithelial cell (HLE-B3) proliferation, the MTT test was performed after incubation with different concentrations of erlotinib. Chemotactic migration was analyzed with the Boyden chamber assay and chemokinetic migration was assessed by time lapse microscopy. Contraction was measured by a 3D collagen type 1 matrix contraction assay, and cell spreading was determined by measuring the cell diameter on a fibronectin coated surface. RESULTS: The maximum non-toxic concentration of erlotinib was determined to be 100 µM in cell culture. Erlotinib potently inhibits human lens epithelial cell proliferation, with an IC50 of about 10 µM (8.8 µM ± 0.9 µM SD; r (2) =0.94). Chemotactic migration (p=0.004) and chemokinetic migration (p=0.001) were reduced significantly in a concentration-based manner. Erlotinib prevented human lens epithelial cells from matrix contraction (p=0.001) and cell-spreading (p=0.001). CONCLUSIONS: Erlotinib might become of clinical relevance for PCO prophylaxis in the future since it displayed good biocompatibility on ocular cells and mitigated human lens epithelial cell proliferation, migration, contraction, and spreading in vitro. Further studies are warranted to evaluate its potential for clinical application.

Comparison of tenocytes and mesenchymal stem cells seeded on biodegradable scaffolds in a full-size tendon defect model.

In order to investigate cell-based tendon regeneration, a tendon rupture was simulated by utilizing a critical full-size model in female rat achilles tendons. For bridging the defect, polyglycol acid (PGA) and collagen type I scaffolds were used and fixed with a frame suture to ensure postoperatively a functional continuity. Scaffolds were seeded with mesenchymal stem cells (MSC) or tenocytes derived from male animals, while control groups were left without cells. After a healing period of 16 weeks, biomechanical, PCR, histologic, and electron microscopic analyses of the regenerates were performed. Genomic PCR for male-specific gene was used to detect transplanted cells in the regenerates. After 16 weeks, central ossification and tendon-like tissue in the superficial tendon layers were observed in all study groups. Biomechanical test showed that samples loaded with tenocytes had significantly better failure strength/cross-section ratio (P < 0.01) compared to MSC and the control groups whereas maximum failure strength was similar in all groups. Thus, we concluded that the application of tenocytes improves the outcome in this model concerning the grade of ossification and the mechanical properties in comparison to the use of MSC or just scaffold materials.

Multiscale computational and experimental approaches to elucidate bone and ligament mechanobiology using the ulna-radius-interosseous membrane construct as a model system.

An in vivo axial loading model of the rat ulna was developed almost two decades ago. As a minimally invasive model, it lends itself particularly well for the study of functional adaptation in bone and the interosseous membrane, a ligament spanning between the radius and ulna. The objective of this paper is to review computational and experimental approaches to elucidate its applicability for the study of multiscale bone and ligament mechanobiology. Specifically, this review describes approaches, including i) measurement of strains on bone tissue surfaces, ii) development of a three-dimensional finite element (FE) mesh of a skeletally mature rat ulna, iii) parametric study of the relative influence of mechanical constants and materials properties on computational model predictions, iv) comparison of experimental and computational strain distribution data, and analysis of the radius and interosseous membrane (IOM) ligament's effect on axial load distribution through the ulna of the rat, and v) the effect of mechanical loading on transport through the IOM using different molecular weight fluorescent tagged dextrans. In the first stage of the study a computational stress analysis was performed after applying a 20 N single static load at the ulnar extremities, corresponding to values of experimental strain gauge measurements. To account for the anisotropy of the bone matrix, transverse isotraopic, elastic material properties were applied. In a parametric study, we analyzed the qualitative effect of different material properties on the global load and displacement behavior of the computational model. In a second stage, the same ulnar model used in the parametric study was extended to account for the interaction between the ulna, radius and IOM. The three-dimensional FE model of the rat forelimb confirms the influence of ulnar curvature on its deformation and underscores the influence of the radius and IOM on strain distribution through the ulna. The mode of strain, {i.e.} compression or tension, and strain distribution along the bone diaphysis correspond to those measured experimentally in vivo. When the radius and, indirectly, the IOM were loaded, the bone deformation shifted distally with respect to the diaphysis. In a final stage, the aforementioned ulnar model was used to study the permeability of fluorescent tagged dextrans with different molecular weights in the presence and absence of ulnar compression. Small molecular weight dextrans (3,000 Da) were distributed throughout the IOM in the absence of as well as after mechanical loading. Interestingly, no gradient in distribution was observed in either case. In contrast, very high molecular weight dextrans (1,000,000 Da) were observed only within vascular and lymphatic spaces in the bone (as well as periosteum) and IOM, both in the absence of and after the application of mechanical loading via end load compression. Between the two extremes, both 10 and 70 kDa tracers were distributed throughout the IOM after application of compressive loading. Loading appears to dissipate the steep gradient of fluorescent 70 kDa tracer observed along the lateral surface of the unloaded IOM and its insertion into the radius and ulna. Hence, this combined computational and experimental analysis of the ulna compression model provides new insight into multiscale mechanobiology of the ulna-radius-interosseous membrane construct and may provide new avenues for elucidation of ligament's remarkable structure-function relationships.

[Alterations of cell phenotype in Dupuytren's disease--an in vitro analysis]. / Veränderung des Zell-Phänotyps durch Morbus Dupuytren--eine in-vitro-Analyse.

INTRODUCTION: Despite its potential complications, partial aponeurectomy still is the mainstay of treatment whenever it comes to significant contracture in Dupuytren's disease. With the goal in mind to identify new therapeutic strategies we isolated and characterised cells from healthy palmar aponeurosis (Kon) and compared them to cells isolated from palmar aponeurosis of patients with a primary manifestation of Dupuytren's disease (PrimDup) as well as from patients with recurrent Dupuytren's disease (RezDup). As cells from palmar aponeurosis from patients with Dupuytren's disease share characteristics with stem cells, such as the ability to differentiate into other cell types, we analysed the stemness, morphology and integrin receptor profiles of the cells. MATERIALS AND METHODS: A total of 15 Dupuytren samples were collected from regular partial aponeurectomy procedures. From these, 3 donors without extrinsic risk factors were selected per group (RezDup, PrimDup). Cells were isolated and expanded under standard cell culture conditions. Cells from healthy patients served as control (Kon). Growth curves were produced. Cells were subjected to osteogenic and adipogenic differentiation using standard protocols. Semiquantitative PCR analysis of the integrins α2, ß3, ß5 and fibronectin was performed. RESULTS: PrimDup cells proliferated significantly faster than control cells, which in turn proliferated faster than RezDup cells. Both PrimDup and control cells went into senescence after approximately 40 days whereas RezDup cells proliferated over the entire period of 100 days. Osteogenic and adipogenic differentiation was best in cells derived from Dupuytren patients while Kon cells differentiated poorly. PCR analysis revealed that fibronectin-binding integrins ß3 and ß5 are upregulated in Dupuytren's disease. CONCLUSIONS: PrimDup cells grow faster than the other cell types suggesting that their growth regulation may be altered. The fact that RezDup cells do not reach senescence over 100 days in culture indicates that senescence regulating factors may be altered. As cells from Dupuytren patients differentiate better along the osteogenic and adipogenic lineages, they probably possess a higher level of stemness. Their modified integrin profile may be a key to future therapies.

Integrins α2ß1 and α11ß1 regulate the survival of mesenchymal stem cells on collagen I.

Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors - α1ß1, α2ß1 and α11ß1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2ß1- or α11ß1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I.

Establishment of immortalized periodontal ligament progenitor cell line and its behavioural analysis on smooth and rough titanium surface.

Periodontal ligament (PDL) can be obtained from patients undergoing orthodontic treatment. PDL contains progenitor cells that can be expanded and differentiated towards several mesenchymal lineages in vitro. Furthermore, PDL-derived cells have been shown to generate bone- and PDL-like structures in vivo. Thus, PDL cells, combined with suitable biomaterials, represent a promising tool for periodontitis-related research and PDL engineering. Here, a new PDL cell line using lentiviral gene transfer of human telomerase reverse transcriptase (hTERT) was created. HTERT-expressing PDL cells showed similar morphology and population doubling time but an extended lifespan compared to the primary cells. In addition, PDL-hTERT cells expressed several characteristic genes and upon osteogenic stimulation produced a calcified matrix in vitro. When cultivated on two topographically different titanium scaffolds (MA and SLA), PDL-hTERT cells exhibited augmented spreading, survival and differentiation on smooth (MA) compared to rough (SLA) surfaces. These findings differ from previously reported osteoblast behaviour, but they are in agreement with the behaviour of chondrocytes and gingival fibroblasts, suggesting a very cell type-specific response to different surface textures. In summary, we report the testing of titanium biomaterials using a new PDL-hTERT cell line and propose this cell line as a useful model system for periodontitis research and development of novel strategies for PDL engineering.

In vitro study on cytotoxic effect of some chemicals on serum McCoy and serum-free McCoy-Plovdiv cell culture systems.

The cytotoxicity of test agents on serum-free McCoy cultures has not been studied at all. The cytotoxic effect of EDTA, methanol, DMSO, and cycloheximide on serum-free McCoy-Plovdiv cell culture (SF) was detected visually on inverted microscope and quantitatively by tests for viability (NR) and total protein (KBP). The IC50 values for the tested chemicals were calculated. SF showed the lowest IC50 values for cycloheximide, DMSO and EDTA and the highest for methanol according to both tests. EDTA, methanol, DMSO and cycloheximide had dose-effect relationship in the cell test systems after treatment. The data indicate that McCoy-Plovdiv cell line is a suitable serum-free cell system for in vitro cytotoxic studies.