Background: Although there are many studies discussing the etiological and pathological factors leading to both, acute and chronic tendon injuries, the pathophysiology of tendon injuries is still not ...clearly understood. Although most lesions are uncomplicated, treatment is long and unsatisfactory due to the poor vascularity of tendon tissue. Platelet mediator concentrate (PMC) contains many growth factors derived from platelets, which can promote wound healing. In this study we investigate the effects of PMC on tenocyte proliferation and differentiation in order to provide an experimental basis for tissue regeneration strategies and to develop new treatment concepts. Methods: Using enzyme linked immunosorbent assay (ELISA) we were able to quantify the several growth factors and cytokines found in PMC. Tenocytes were isolated both from human and from mouse Achilles tendons and stimulated with PMC. CyQuant((R)) and Cell Titer Blue((R)) assays were carried out to analyze tendon growth and viability at different concentrations of PMC. Real time RT-PCR was used to analyze tenocyte gene expression with or without PMC treatment. Immunohistochemistry was carried out to detect the tenocyte-specific antibody tenomodulin (TNMD) and scleraxis (SCX). Results: We were able to detect numerous mediators such as platelet derived growth factor BB (PDGF- BB), interleukin 6 (IL- 6), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF-a), transforming growth factor beta 1 (TGF- beta 1), and bone morphogenetic proteins 2, 4 and 7 (BMP- 4, BMP- 2, BMP- 7) in PMC. It was possible to show a positive effect of PMC on human tendon cell growth and viability in a dose-dependent manner. Furthermore, PMC treatment led to induction of gene expression of scleraxis (SCX), type I collagen A 1 (Col1A1) and TNMD by tenocytes. Conclusions: We suggest that the use of autologous PMC may be a suitable addition to conventional tendon therapy that is capable of increasing and optimizing tendon healing and reducing the risk of recurrence.
Antimicrobial peptides (AP) are important components of the innate immune system, yet little is known about their expression and function in the brain. Our previous work revealed upregulated gene ...expression of cathelicidin-related AP (CRAMP) following bacterial meningitis in primary rat glial cells as well as bactericidal activity against frequent meningitis-causing bacteria. However, the effect of cathelicidin expression on the progression of inflammation and mortality in bacterial meningitis remains unknown. Therefore, we used CRAMP-deficient mice to investigate the effect of CRAMP on bacterial growth, inflammatory responses and mortality in meningitis. Meningitis was induced by intracerebral injection of type 3 Streptococcus pneumoniae. The degree of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time RT-PCR. CRAMP deficiency led to a higher mortality rate that was associated with increased bacterial titers in the cerebellum, blood and spleen as well as decreased meningeal neutrophil infiltration. CRAMP-deficient mice displayed a higher degree of glial cell activation that was accompanied by a more pronounced proinflammatory response. Taken together, this work provides insight into the important role of CRAMP as part of the innate immune defense against pathogens in bacterial CNS infections.
Background The healing potential in the avascular regions of the meniscus is very limited, and improving the vascularity might be a reasonable way to improve healing. Vascular endothelial growth ...factor (VEGF) is one of the most potent proangiogenetic factors. We hypothesized that the local application of VEGF165 would (1) improve the healing of a lesion in the avascular region of the meniscus, (2) induce angiogenesis in both the avascular and vascular regions, and (3) increase the amounts of VEGF mRNA and VEGF. Methods In eighteen sheep, the medial menisci were cut longitudinally in the avascular region and were sutured. Three groups were established depending on the suture material: (1) uncoated Ethibond, (2) Ethibond coated with VEGF165 and its carrier Poly(D,L–Lactide) (PDLLA), and (3) Ethibond coated with PDLLA. The contralateral medial menisci served as a control group. Each of the three suture type groups included six animals. After eight weeks, the sheep were killed, and the menisci were examined macroscopically. Immunohistochemistry of Factor VIII and VEGF and real-time reverse-transcription polymerase chain reaction (RT-PCR) of VEGF mRNA were performed. Additionally, the VEGF release kinetics from the VEGF/PDLLA-coated suture were evaluated in vitro. Results In this model, VEGF did not improve meniscal healing. It did not increase angiogenesis in the avascular or vascular region, the VEGF concentration, or the amount of VEGF mRNA. VEGF release from the coated suture peaked on Day 3 and was nearly zero on Day 9. Conclusions The local application of VEGF165 as eluted from suture did not increase meniscal angiogenesis or improve meniscal healing. In addition, there was no effect on the amount of VEGF mRNA and VEGF. The VEGF carrier (PDLLA) may have been inadequate because of the short duration of VEGF supply. Clinical Relevance This study shows that the application of VEGF at one time point might not be a solution to improve meniscal healing.
Objective
Beta‐defensins are broad‐spectrum antimicrobial peptides (APs) that are components of innate immunity. Recent investigations showed the induction of β‐defensins in synovial membranes of ...osteoarthritic (OA) joints and suggested that they have functions other than the ability to kill microbes. As a result of these findings, we undertook this study to investigate the production of human β‐defensin 3 (HBD‐3) in OA cartilage and to determine its influence on chondrocyte function.
Methods
Healthy and OA cartilage were assessed for HBD‐3 expression by reverse transcriptase–polymerase chain reaction (RT‐PCR) and immunohistochemistry. HBD‐3 expression in C28/I2 chondrocytes after administration of tumor necrosis factor α (TNFα) and interleukin‐1 (IL‐1) was determined by real‐time RT‐PCR and immunodot blot. Enzyme‐linked immunosorbent assay experiments were used to study the effects of HBD‐3 in cultured articular chondrocytes and in healthy and OA cartilage discs. Immunohistochemical analyses were performed to study the expression of mouse β‐defensins (MBDs) in OA cartilage of STR/Ort mice.
Results
HBD‐3 was induced in OA cartilage without bacterial challenge. Cytokines involved in the pathogenesis of OA, namely, TNFα and IL‐1, were strong inducers of HBD‐3 in cultured chondrocytes. Application of the recombinant HBD‐3 protein to cultured chondrocytes and cartilage discs resulted in increased production of cartilage‐degrading matrix metalloproteinases and in down‐regulation of their endogenous regulators, tissue inhibitors of metalloproteinases 1 and 2. Furthermore, STR/Ort mice, which are genetically predisposed to develop OA‐like lesions in the knee joint, demonstrated an increased expression of MBDs 3 and 4 in cartilage compared with that in healthy animals.
Conclusion
These findings widen our knowledge of the functional spectrum of APs and demonstrate that HBD‐3 is a multifunctional AP with the ability to link host defense mechanisms and inflammation with tissue‐remodeling processes in articular cartilage. Moreover, our data suggest that HBD‐3 is an additional factor in the pathogenesis of OA.
Objective
Pleiotrophin (PTN), a 15.3‐kd heparin‐binding peptide, is expressed in mesodermal and neuroectodermal cells during development, but rarely in adult tissues. Since developmentally regulated ...factors often reappear during disease, we sought to determine whether there was PTN expression in the synovial membranes of patients with rheumatoid arthritis (RA).
Methods
PTN messenger RNA expression was assayed by quantitative reverse transcriptase–polymerase chain reaction. The protein was localized by immunohistochemistry and quantified by enzyme‐linked immunosorbent assay (ELISA). Effects of PTN on cell proliferation in vitro were determined by DNA measurements.
Results
PTN expression in normal adult synovial membranes and cartilage was barely detectable. However, PTN was strongly up‐regulated in synovial tissues from patients with RA. In contrast, samples from patients with pyogenic arthritis had moderate PTN levels, and those from patients with osteoarthritis had only a slight increase in PTN, as measured by ELISA. In RA patients, PTN was localized primarily in synoviocytes but was also found in endothelial cells of blood vessels. In cultured mouse fibroblasts used as a model, PTN expression was up‐regulated by tumor necrosis factor α and was more weakly up‐regulated by epidermal growth factor. Recombinant PTN stimulated the proliferation of cultured human synoviocytes and the monocyte cell line THP‐1, but not human dermal fibroblasts, in which PTN increased the synthesis of vascular endothelial growth factor.
Conclusion
In addition to certain types of cancer, the embryonic growth and differentiation factor PTN is expressed in adults with inflammatory diseases, in particular, RA. Proinflammatory cytokines enhance the expression of PTN. Thus, we propose that PTN is a further paracrine angiogenesis and growth factor for synovial cells in RA.
Objective
Defensins are broad‐spectrum antimicrobial peptides that are components of innate immunity. To date, only epithelial surfaces and blood cells have been shown to produce these cationic ...peptides in bactericidal concentrations when challenged with microorganisms or inflammatory cytokines. Infections caused by gram‐negative pathogens occur only infrequently in association with joint surgery. The present study was undertaken to investigate whether this may be explained by intraarticular production of gram‐negative–specialized antimicrobial peptides.
Methods
Healthy articular cartilage and cultured T/C‐28a2 chondrocytes were assessed, by reverse transcriptase–polymerase chain reaction (RT‐PCR) and immunohistochemistry, for expression of various antimicrobial peptides. The expression of human β‐defensin 2 (HBD‐2) was studied in cultured chondrocytes after exposure to bacterial supernatants and proinflammatory cytokines and was assayed by real‐time RT‐PCR and immunoblot analysis. A septic arthritis mouse model was used to investigate the regulation of the murine homolog of HBD‐2 in articular cartilage after bacterial inoculation.
Results
Healthy articular cartilage and T/C‐28a2 chondrocytes were able to produce different antimicrobial peptides. After exposure to gram‐negative bacteria and proinflammatory cytokines, expression of cartilage‐derived HBD‐2 strongly increased. Immunoblot analysis revealed up‐regulation of the gram‐negative–specialized HBD‐2 in microbicidal doses. Immunohistochemistry analysis revealed induction of the murine homolog of HBD‐2 in vivo after intraarticular injection of bacteria.
Conclusion
This study demonstrated a previously unrecognized function of human chondrocytes. In addition to its biomechanical properties, articular cartilage has the ability to produce antimicrobial substances when challenged with microorganisms. The expression of HBD‐2 in microbicidal doses suggests that antimicrobial peptides may contribute to host defense mechanisms in articular joints.
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