Histological and molecular features of the subacromial bursa of rotator cuff tears compared to non-tendon defects: a pilot study.

BACKGROUND: The role of the subacromial bursa in the development or healing of shoulder pathologies is unclear. Due to this limited knowledge, we aimed to understand specific reactions of the subacromial bursa according to rotator cuff (RC) pathologies compared to non-tendon defects of the shoulder. We hypothesized that the tissue composition and inflammatory status of the bursa are likely to vary between shoulder pathologies depending on the presence and the extent of RC lesion. METHOD: Bursa samples from patients with either 1) shoulder instability with intact RC (healthy bursa, control), 2) osteochondral pathology with intact RC, 3) partial supraspinatus (SSP) tendon tear, or 4) full-thickness SSP tear were investigated histologically and on gene expression level. RESULT: Bursae from SSP tears differed from non-tendon pathologies by exhibiting increased chondral metaplasia and TGFß1 expression. MMP1 was not expressed in healthy bursa controls, but strongly increased with full-thickness SSP tears. Additionally, the expression of the inflammatory mediators IL1ß, IL6, and COX2 increased with the extent of SSP tear as shown by correlation analysis. In contrast, increased angiogenesis and nerve fibers as well as significantly upregulated IL6 and COX2 expression were features of bursae from patients with osteochondral pathology. Using immunohistochemistry, CD45+ leukocytes were observed in all examined groups, which were identified in particular as CD68+ monocytes/macrophages. CONCLUSION: In summary, besides the strong increase in MMP1 expression with SSP tear, molecular changes were minor between the investigated groups. However, expression of pro-inflammatory cytokines correlated with the severity of the SSP tear. Most pronounced tissue alterations occurred for the osteochondral pathology and full-thickness SSP tear group, which demonstrates that the bursal reaction is not exclusively dependent on the occurrence of an SSP tear rather than longstanding degenerative changes. The present bursa characterization contributes to the understanding of specific tissue alterations related to RC tears or non-tendon shoulder pathologies. This pilot study provides the basis for future studies elucidating the role of the subacromial bursa in the development or healing of shoulder pathologies.

In Vivo and In Vitro Mechanical Loading of Mouse Achilles Tendons and Tenocytes-A Pilot Study.

Mechanical force is a key factor for the maintenance, adaptation, and function of tendons. Investigating the impact of mechanical loading in tenocytes and tendons might provide important information on in vivo tendon mechanobiology. Therefore, the study aimed at understanding if an in vitro loading set up of tenocytes leads to similar regulations of cell shape and gene expression, as loading of the Achilles tendon in an in vivo mouse model. In vivo: The left tibiae of mice (n = 12) were subject to axial cyclic compressive loading for 3 weeks, and the Achilles tendons were harvested. The right tibiae served as the internal non-loaded control. In vitro: tenocytes were isolated from mice Achilles tendons and were loaded for 4 h or 5 days (n = 6 per group) based on the in vivo protocol. Histology showed significant differences in the cell shape between in vivo and in vitro loading. On the molecular level, quantitative real-time PCR revealed significant differences in the gene expression of collagen type I and III and of the matrix metalloproteinases (MMP). Tendon-associated markers showed a similar expression profile. This study showed that the gene expression of tendon markers was similar, whereas significant changes in the expression of extracellular matrix (ECM) related genes were detected between in vivo and in vitro loading. This first pilot study is important for understanding to which extent in vitro stimulation set-ups of tenocytes can mimic in vivo characteristics.

Is NO the Answer? The Nitric Oxide Pathway Can Support Bone Morphogenetic Protein 2 Mediated Signaling.

The growth factor bone morphogenetic protein 2 (BMP2) plays an important role in bone development and repair. Despite the positive effects of BMP2 in fracture healing, its use is associated with negative side effects and poor cost effectiveness, partly due to the large amounts of BMP2 applied. Therefore, reduction of BMP2 amounts while maintaining efficacy is of clinical importance. As nitric oxide (NO) signaling plays a role in bone fracture healing and an association with the BMP2 pathway has been indicated, this study aimed to investigate the relationship of BMP2 and NO pathways and whether NO can enhance BMP2-induced signaling and osteogenic abilities in vitro. To achieve this, the stable BMP reporter cell line C2C12BRELuc was used to quantify BMP signaling, and alkaline phosphatase (ALP) activity and gene expression were used to quantify osteogenic potency. C2C12BRELuc cells were treated with recombinant BMP2 in combination with NO donors and substrate (Deta NONOate, SNAP & L-Arginine), NOS inhibitor (LNAME), soluble guanylyl cyclase (sGC) inhibitor (LY83583) and activator (YC-1), BMP type-I receptor inhibitor (LDN-193189), or protein kinase A (PKA) inhibitor (H89). It was found that the NOS enzyme, direct NO application, and sGC enhanced BMP2 signaling and improved BMP2 induced osteogenic activity. The application of a PKA inhibitor demonstrated that BMP2 signaling is enhanced by the NO pathway via PKA, underlining the capability of BMP2 in activating the NO pathway. Collectively, this study proves the ability of the NO pathway to enhance BMP2 signaling.

Bone morphogenetic proteins - 7 and - 2 in the treatment of delayed osseous union secondary to bacterial osteitis in a rat model.

BACKGROUND: Bone infections due to trauma and subsequent delayed or impaired fracture healing represent a great challenge in orthopedics and trauma surgery. The prevalence of such bacterial infection-related types of delayed non-union is high in complex fractures, particularly in open fractures with additional extensive soft-tissue damage. The aim of this study was to establish a rat model of delayed osseous union secondary to bacterial osteitis and investigate the impact of rhBMP-7 and rhBMP-2 on fracture healing in the situation of an ongoing infection. METHODS: After randomization to four groups 72 Sprague-Dawley rats underwent a transverse fracture of the midshaft tibia stabilized by intramedullary titanium K-wires. Three groups received an intramedullary inoculation with Staphylococcus aureus (103 colony-forming units) before stabilization and the group without bacteria inoculation served as healing control. After 5 weeks, a second surgery was performed with irrigation of the medullary canal and local rhBMP-7 and rhBMP-2 treatment whereas control group and infected control group received sterile saline. After further 5 weeks rats were sacrificed and underwent biomechanical testing to assess the mechanical stability of the fractured bone. Additional micro-CT analysis, histological, and histomorphometric analysis were done to evaluate bone consolidation or delayed union, respectively, and to quantify callus formation and the mineralized area of the callus. RESULTS: Biomechanical testing showed a significantly higher fracture torque in the non-infected control group and the infected rhBMP-7- and rhBMP-2 group compared with the infected control group (p < 0.001). RhBMP-7 and rhBMP-2 groups did not show statistically significant differences (p = 0.57). Histological findings supported improved bone-healing after rhBMP treatment but quantitative micro-CT and histomorphometric results still showed significantly more hypertrophic callus tissue in all three infected groups compared to the non-infected group. Results from a semiquantitative bone-healing-score revealed best bone-healing in the non-infected control group. The expected chronic infection was confirmed in all infected groups. CONCLUSIONS: In delayed bone healing secondary to infection rhBMP treatment promotes bone healing with no significant differences in the healing efficacy of rhBMP-2 and rhBMP-7 being noted. Further new therapeutic bone substitutes should be analyzed with the present rat model for delayed osseous union secondary to bacterial osteitis.

Different Achilles Tendon Pathologies Show Distinct Histological and Molecular Characteristics.

Reasons for the development of chronic tendon pathologies are still under debate and more basic knowledge is needed about the different diseases. The aim of the present study was therefore to characterize different acute and chronic Achilles tendon disorders. Achilles tendon samples from patients with chronic tendinopathy (n = 7), chronic ruptures (n = 6), acute ruptures (n = 13), and intact tendons (n = 4) were analyzed. The histological score investigating pathological changes was significantly increased in tendinopathy and chronic ruptures compared to acute ruptures. Inflammatory infiltration was detected by immunohistochemistry in all tendon pathology groups, but was significantly lower in tendinopathy compared to chronic ruptures. Quantitative real-time PCR (qRT-PCR) analysis revealed significantly altered expression of genes related to collagens and matrix modeling/remodeling (matrix metalloproteinases, tissue inhibitors of metalloproteinases) in tendinopathy and chronic ruptures compared to intact tendons and/or acute ruptures. In all three tendon pathology groups markers of inflammation (interleukin (IL) 1ß, tumor necrosis factor α, IL6, IL10, IL33, soluble ST2, transforming growth factor ß1, cyclooxygenase 2), inflammatory cells (cluster of differentaition (CD) 3, CD68, CD80, CD206), fat metabolism (fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, adiponectin), and innervation (protein gene product 9.5, growth associated protein 43, macrophage migration inhibitory factor) were detectable, but only in acute ruptures significantly regulated compared to intact tendons. The study gives an insight into structural and molecular changes of pathological processes in tendons and might be used to identify targets for future therapy of tendon pathologies.

Time-Dependent Alterations of MMPs, TIMPs and Tendon Structure in Human Achilles Tendons after Acute Rupture.

A balance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) is required to maintain tendon homeostasis. Variation in this balance over time might impact on the success of tendon healing. This study aimed to analyze structural changes and the expression profile of MMPs and TIMPs in human Achilles tendons at different time-points after rupture. Biopsies from 37 patients with acute Achilles tendon rupture were taken at surgery and grouped according to time after rupture: early (2-4 days), middle (5-6 days), and late (≥7 days), and intact Achilles tendons served as control. The histological score increased from the early to the late time-point after rupture, indicating the progression towards a more degenerative status. In comparison to intact tendons, qRT-PCR analysis revealed a significantly increased expression of MMP-1, -2, -13, TIMP-1, COL1A1, and COL3A1 in ruptured tendons, whereas TIMP-3 decreased. Comparing the changes over time post rupture, the expression of MMP-9, -13, and COL1A1 significantly increased, whereas MMP-3 and -10 expression decreased. TIMP expression was not significantly altered over time. MMP staining by immunohistochemistry was positive in the ruptured tendons exemplarily analyzed from early and late time-points. The study demonstrates a pivotal contribution of all investigated MMPs and TIMP-1, but a minor role of TIMP-2, -3, and -4, in the early human tendon healing process.

New insights into tenocyte-immune cell interplay in an in vitro model of inflammation.

Inflammation plays an important role in the development and resolution of tendon diseases, but underlying mechanisms are poorly understood. We therefore aimed to analyze the response of human tenocytes to inflammatory stimuli and to uncover their interplay with macrophages in vitro. Tenocytes from human ruptured supraspinatus tendons (n = 10) were treated for three days with a stimulation mixture derived from activated mononuclear cells isolated from healthy human peripheral blood. Significantly increased expression levels of selected adhesion- and human leukocyte antigen (HLA)-molecules, and enhanced interleukin (IL)-6 release were detected by flow cytometry. Tenocyte stimulation with the pro-inflammatory cytokines interferon gamma, tumor necrosis factor alpha and IL-1ß triggered similar changes in surface markers and enhanced the release of IL-6, IL-8 and monocyte chemoattractant protein 1 (MCP-1). In co-cultures of macrophages with pre-stimulated tenocytes, macrophages significantly increased CD80 expression, but simultaneously decreased HLA-DR-expression, which are both typical pro-inflammatory polarization markers. Co-cultures also released more IL-6, IL-8, MCP-1 than tenocyte-cultures alone. We demonstrate that tenocytes respond to inflammatory environments in vitro with altered surface marker and cytokine profiles and influence macrophage polarization. Importantly, all changes detected in direct co-cultures were also present in a transwell setting, implicating that communication between the cells involves soluble factors.

An investigation of BMP-7 mediated alterations to BMP signalling components in human tenocyte-like cells.

The incidence of tendon re-tears post-surgery is an ever present complication. It is suggested that the application of biological factors, such as bone morphogenetic protein 7 (BMP-7), can reduce complication rates by promoting tenogenic characteristics in in vitro studies. However, there remains a dearth of information in regards to the mechanisms of BMP-7 signalling in tenocytes. Using primary human tenocyte-like cells (hTLCs) from the supraspinatus tendon the BMP-7 signalling pathway was investigated: induction of the BMP associated Smad pathway and non-Smad pathways (AKT, p38, ERK1/2 and JNK); alterations in gene expression of BMP-7 associated receptors, Smad pathway components, Smad target gene (ID1) and tenogenic marker scleraxis. BMP-7 increases the expression of specific BMP associated receptors, BMPR-Ib and BMPR-II, and Smad8. Additionally, BMP-7 activates significantly Smad1/5/8 and slightly p38 pathways as indicated by an increase in phosphorylation and proven by inhibition experiments, where p-ERK1/2 and p-JNK pathways remain mainly unresponsive. Furthermore, BMP-7 increases the expression of the Smad target gene ID1, and the tendon specific transcription factor scleraxis. The study shows that tenocyte-like cells undergo primarily Smad8 and p38 signalling after BMP-7 stimulation. The up-regulation of tendon related marker genes and matrix proteins such as Smad8/9, scleraxis and collagen I might lead to positive effects of BMP-7 treatment for rotator cuff repair, without significant induction of osteogenic and chondrogenic markers.

Longitudinal analysis of osteogenic and angiogenic signaling factors in healing models mimicking atrophic and hypertrophic non-unions in rats.

Impaired bone healing can have devastating consequences for the patient. Clinically relevant animal models are necessary to understand the pathology of impaired bone healing. In this study, two impaired healing models, a hypertrophic and an atrophic non-union, were compared to physiological bone healing in rats. The aim was to provide detailed information about differences in gene expression, vascularization and histology during the healing process. The change from a closed fracture (healing control group) to an open osteotomy (hypertrophy group) led to prolonged healing with reduced mineralized bridging after 42 days. RT-PCR data revealed higher gene expression of most tested osteogenic and angiogenic factors in the hypertrophy group at day 14. After 42 days a significant reduction of gene expression was seen for Bmp4 and Bambi in this group. The inhibition of angiogenesis by Fumagillin (atrophy group) decreased the formation of new blood vessels and led to a non-healing situation with diminished chondrogenesis. RT-PCR results showed an attempt towards overcoming the early perturbance by significant up regulation of the angiogenic regulators Vegfa, Angiopoietin 2 and Fgf1 at day 7 and a further continuous increase of Fgf1, -2 and Angiopoietin 2 over time. However µCT angiograms showed incomplete recovery after 42 days. Furthermore, lower expression values were detected for the Bmps at day 14 and 21. The Bmp antagonists Dan and Twsg1 tended to be higher expressed in the atrophy group at day 42. In conclusion, the investigated animal models are suitable models to mimic human fracture healing complications and can be used for longitudinal studies. Analyzing osteogenic and angiogenic signaling patterns, clear changes in expression were identified between these three healing models, revealing the importance of a coordinated interplay of different factors to allow successful bone healing.

Stimulation of bone healing by sustained bone morphogenetic protein 2 (BMP-2) delivery.

The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (µCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p=0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p=0.006) with reduced cartilage at Day 84 (p=0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p=0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms.

Efficacy of two different demineralised bone matrix grafts to promote bone healing in a critical-size-defect: a radiological, histological and histomorphometric study in rat femurs.

PURPOSE: The aim of the study was to compare two different demineralised bone matrices used clinically regarding their ability to induce bone healing in a critical-size-defect rat model. METHODS: We stabilised 4 mm femur defects with a custom-made plate and filled them either with demineralised bone matrix (DBM) or DBX (DBX Putty®). Bone morphogenetic protein 2 (BMP-2)-loaded collagen and an empty defect served as controls. The outcome was followed after 21 and 42 days by radiology (Faxitron; microCT) and histology. RESULTS: Defect healing did not occur in any animal from the empty control, DBM or DBX group. Residuals of the implanted material were still found after six weeks, but only limited callus formation was visible. In contrast, the BMP-2 control demonstrated enhanced formation of callus tissue and undisturbed healing. After 21 days, 11 out of 16 and after 42 days, 7 out of 8 BMP-2-treated animals showed complete defect bridging by cancellous bone tissue. CONCLUSIONS: Demineralised bone grafts were not capable of defect reconstruction; only BMP-2 was able to provide sufficient stimulus to induce uneventful bridging under the specific experimental conditions.

Human epidermal stem cell function is regulated by circadian oscillations.

Human skin copes with harmful environmental factors that are circadian in nature, yet how circadian rhythms modulate the function of human epidermal stem cells is mostly unknown. Here we show that in human epidermal stem cells and their differentiated counterparts, core clock genes peak in a successive and phased manner, establishing distinct temporal intervals during the 24 hr day period. Each of these successive clock waves is associated with a peak in the expression of subsets of transcripts that temporally segregate the predisposition of epidermal stem cells to respond to cues that regulate their proliferation or differentiation, such as TGFß and calcium. Accordingly, circadian arrhythmia profoundly affects stem cell function in culture and in vivo. We hypothesize that this intricate mechanism ensures homeostasis by providing epidermal stem cells with environmentally relevant temporal functional cues during the course of the day and that its perturbation may contribute to aging and carcinogenesis.

Rapid binding of electrostatically stabilized iron oxide nanoparticles to THP-1 monocytic cells via interaction with glycosaminoglycans.

Magnetic resonance imaging (MRI) with contrast agents that target specific inflammatory components of atherosclerotic lesions has the potential to emerge as promising diagnostic modality for detecting unstable plaques. Since a high content of macrophages and alterations of the extracellular matrix are hallmarks of plaque instability, these structures represent attractive targets for new imaging modalities. In this study, we compared in vitro uptake and binding of electrostatically stabilized citrate-coated very small superparamagnetic iron oxide particles (VSOP) to THP-1 cells with sterically stabilized carboxydextran-coated Resovist(®). Uptake of VSOP in both THP-1 monocytic cells and THP-derived macrophages (THP-MΦ) was more efficient compared to Resovist(®) without inducing cytotoxicity or modifying normal cellular functions (no changes in levels of reactive oxygen species, caspase-3 activity, proliferation, cytokine production). Importantly, VSOP bound with high affinity to the cell surface and to apoptotic membrane vesicles. Inhibition of glycosaminoglycan (GAG) synthesis by glucose deprivation in THP-MΦ was associated with a significant reduction of VSOP attachment suggesting that the strong interaction of VSOP with the membranes of cells and apoptotic vesicles occurs via binding to negatively charged GAGs. These in vitro experiments show that VSOP-enhanced MRI may represent a new imaging approach for visualizing high-risk plaques on the basis of targeting pathologically increased GAGs or apoptotic membrane vesicles in atherosclerotic lesions. VSOP should be investigated further in appropriate in vivo experiments to characterize accumulation in unstable plaque.

The Aspergillus niger RmsA protein: A node in a genetic network?

Many cells and organisms go through polarized growth phases during their life. Cell polarization is achieved by local accumulation of signaling molecules which guide the cytoskeleton and vesicular trafficking to specific parts of the cell and thus ensure polarity establishment and maintenance. Polarization of signaling molecules is also fundamental for the lifestyle of filamentous fungi such as Aspergillus niger and essential for their morphogenesis, development and survival under environmental stress conditions. Considerable advances in our understanding on the protagonists and processes mediating polarized growth in filamentous fungi have been made over the past years. However, how the interplay of different signaling pathways is coordinated has yet to be determined. We found that the A. niger RmsA protein is central for the polarization of actin at the hyphal tip but also of vital importance for the metabolism, viability and stress resistance of A. niger. This suggests that RmsA could occupy an important position in the global network of pathways that balance growth, morphogenesis and survival of A. niger.