Alexander Kolb

Anti-inflammatory activity of an ethanolic Caesalpinia sappan extract in human chondrocytes and macrophages

ETHNOPHARMACOLOGICAL RELEVANCE Caesalpinia sappan is a common remedy in Traditional Chinese Medicine and possesses diverse biological activities including anti-inflammatory properties. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. In order to provide a scientific basis for the applicability of Caesalpinia sappan in arthritic diseases, the present study aimed to assess the effects of an ethanolic Caesalpinia sappan extract (CSE) on human chondrocytes and macrophages. MATERIALS AND METHODS Primary human chondrocytes were isolated from cartilage specimens of OA patients. Primary cells, SW1353 chondrocytes and THP-1 macrophages were serum-starved and pretreated with different concentrations of CSE prior to stimulation with 10 ng/ml of interleukin-1beta (IL-1β) or lipopolysaccharide (LPS). Following viability tests, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) were evaluated by Griess assay and ELISA, respectively. Using validated real-time PCR assays, mRNA levels of IL-1β, TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were quantified. SW1353 cells were cotransfected with a COX-2 luciferase reporter plasmid and nuclear factor-kappa-B (NF-κB) p50 and p65 expression vectors in the presence or absence of CSE. RESULTS CSE dose-dependently inhibited the expression of pro-inflammatory cytokines IL-1β and TNF-α in IL-1β-stimulated chondrocytes and LPS-stimulated THP-1 macrophages. CSE further suppressed the synthesis of NO in primary OA chondrocytes by blocking iNOS mRNA expression. The inhibition of COX-2 transcription was found to be related with the CSE inhibition of the p65/p50-driven transactivation of the COX-2 promoter. CONCLUSIONS The present report is first to demonstrate the anti-inflammatory activity of CSE in an in vitro cell model of joint inflammation. CSE can effectively abrogate the IL-1β-induced over-expression of inflammatory mediators at the transcriptional level in human chondrocytes and macrophages, most likely by inhibiting NF-κB (p65/p50) signaling. Blockade of IL-1β-induced NF-κB signaling and its downstream pro-inflammatory targets by CSE may be beneficial for reducing cartilage breakdown in arthritis.

IL-1β and TNF-α alter the glycophenotype of primary human chondrocytes in vitro

Despite the significance of glycoproteins for extracellular matrix assembly in cartilage tissue, little is known about the regulation of the chondrocyte glycophenotype under inflammatory conditions. The present study aimed to assess the effect of IL-1beta and TNF-alpha on specific features of the glycophenotype of primary human chondrocytes in vitro. Using LC-MS, we found that both cytokines increased overall sialylation of N- and O-glycans and induced a shift towards alpha-(2-->3)-linked sialic acid residues in chondrocyte glycoproteins. These results were supported by quantitative PCR showing increased expression of alpha-(2-->3) sialyltransferases in treated cells. Moreover, we found that both IL-1beta and TNF-alpha induced a considerable shift from oligomannosidic glycans towards complex-type N-glycans. In contrast, core alpha-(1-->6)-fucosylation of chondrocyte N-glycans was found to be reduced particularly by TNF-alpha. In summary, inflammatory conditions induce specific alterations of the chondrocyte glycophenotype which might affect cell-matrix interactions or the function of endogenous lectins.

Comparison of synovial fluid, urine, and serum ion levels in metal-on-metal total hip arthroplasty at a minimum follow-up of 18 years

Diagnosis of adverse reactions to metal debris in metal‐on‐metal hip arthroplasty is a multifactorial process. Systemic ion levels are just one factor in the evaluation and should not be relied upon solely to determine the need for revision surgery. Furthermore, the correlation between cobalt or chromium serum, urine, or synovial fluid levels and adverse local tissue reactions is still incompletely understood. The hypothesis was that elevated serum and urine metal‐ion concentrations are associated with elevated local metal‐ion concentrations in primary total hip arthroplasties (THA) and with failure of metal‐on‐metal articulations in the long‐term. In our present study, we evaluated these concentrations in 105 cementless THA with metal‐on‐metal articulating surfaces with small head diameter at a minimum of 18 years postoperatively. Spearman correlation showed a high correlation between the joint fluid aspirate concentration of cobalt and chromium with the serum cobalt (r = 0.81) and chromium level (r = 0.77) in patients with the THA as the only source of metal‐ions. In these patients serum metal‐ion analysis is a valuable method for screening. In patients with more than one source of metal or renal insufficiency additional investigations, like joint aspirations are an important tool for evaluation of wear and adverse tissue reactions in metal‐on‐metal THA.

Caesalpinia sappan extract inhibits IL1β-mediated overexpression of matrix metalloproteinases in human chondrocytes

Exacerbated production of matrix metalloproteinases (MMPs) is a key event in the progression of osteoarthritis (OA) and represents a promising target for the management of OA with nutraceuticals. In this study, we sought to determine the MMP-inhibitory activity of an ethanolic Caesalpinia sappan extract (CSE) in human OA chondrocytes. Thus, human articular chondrocytes isolated from OA cartilage and SW1353 chondrocytes were stimulated with Interleukin-1beta (IL1β), without or with pretreatment with CSE. Following viability assays, the production of MMP-2 and MMP-13 was assessed using ELISA, whereas mRNA levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13 and TIMP-1, TIMP-2, TIMP-3 were quantified using RT-qPCR assays. Chondrocytes were co-transfected with a MMP-13 luciferase reporter construct and NF-kB p50 and p65 expression vectors in the presence or absence of CSE. In addition, the direct effect of CSE on the proteolytic activities of MMP-2 was evaluated using gelatin zymography. We found that CSE significantly suppressed IL1β-mediated upregulation of MMP-13 mRNA and protein levels via abrogation of the NF-kB(p65/p50)-driven MMP-13 promoter activation. We further observed that the levels of IL1β-induced MMP-1, MMP-3, MMP-7, and MMP-9 mRNA, but not TIMP mRNA levels, were down-regulated in chondrocytes in response to CSE. Zymographic results suggested that CSE did not directly interfere with the proteolytic activity of MMP-2. In summary, this study provides evidence for the MMP-inhibitory potential of CSE or CSE-derived compounds in human OA chondrocytes. The data indicate that the mechanism of this inhibition might, at least in part, involve targeting of NF-kB-mediated promoter activation.

Phenotype-related differential α-2,6- or α-2,3-sialylation of glycoprotein N-glycans in human chondrocytes

OBJECTIVE Sialic acids frequently occur at the terminal positions of glycoprotein N-glycans present at chondrocyte surfaces or in the cartilage matrix. Sialic acids are transferred to glycoproteins in either alpha-2,3 or alpha-2,6 linkage by specific sialyltransferases (SiaTs) and can potentially affect cell functions and cell-matrix interactions. The present study aimed to assess the relationship between the expression of the human chondrocyte phenotype and the sialylation of chondrocyte glycoprotein N-glycans. METHODS The transcription of 5 SiaT was quantified using real-time Reverse transcription polymerase chain reaction (RT-PCR) assays. N-glycan analysis was performed using LC-ESI-MS. Primary human chondrocytes were cultured in monolayer or alginate beads and compared to the chondrocyte cell lines C-28/I2 and SW1353. In addition, effects of interleukin-1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) on primary cells were assessed. RESULTS Primary human chondrocytes predominantly express alpha-2,6-specific SiaTs and accordingly, alpha-2,6-linked sialic acid residues in glycoprotein N-glycans. In contrast, the preponderance of alpha-2,3-linked sialyl residues and, correspondingly, reduced levels of alpha-2,6-specific SiaTs are associated with the altered chondrocyte phenotype of C-28/I2 and SW1353 cells. Importantly, a considerable shift towards alpha-2,3-linked sialic acids and alpha-2,3-specific SiaT mRNA levels occurred in primary chondrocytes treated with IL-1beta or tumour necrosis factor-alpha (TNF-alpha). CONCLUSION The expression of the differentiated chondrocyte phenotype is linked to the ratio of alpha-2,6- to alpha-2,3-linked sialic acids in chondrocyte glycoprotein N-glycans. A shift towards altered sialylation might contribute to impaired cell-matrix interactions in disease conditions.

Primary cementless total hip arthroplasty with second-generation metal-on-metal bearings: a concise follow-up, at a minimum of seventeen years, of a previous report.

BACKGROUND Second-generation, metal-on-metal bearings were introduced in 1988, to reduce wear and avoid polyethylene particle-induced osteolysis from total hip arthroplasty. In 2007, we reported the long-term results of ninety-eight patients (105 hips) who underwent primary cementless total hip arthroplasty involving the use of a prosthesis with a high-carbide-concentration, metal-on-metal articulating surface between November 1992 and May 1994. The present study gives an update on this patient cohort. METHODS At a minimum of seventeen years postoperatively, forty-nine patients (fifty-two hips) were available for follow-up examination. We retrospectively evaluated clinical and radiographic results as well as serum metal concentration. The mean patient age at the time of the index arthroplasty was fifty-six years. RESULTS Three cups (6% of the hips) and one stem (2% of the hips) were revised because of aseptic loosening of the implants combined with focal osteolysis. At the time of the latest follow-up evaluation, the mean Harris hip score was 88.8 points, and the mean University of California Los Angeles (UCLA) activity score was 6.7 points. The cumulative rate of implant survival, with aseptic failure as the end point, was 93.0% at 18.8 years. The median serum cobalt concentration in patients whose hip implant was the only source of cobalt was 0.70 μg/L (range, 0.4 to 5.1 μg/L), showing no increase in the value as noted at a minimum of ten years of follow-up. CONCLUSIONS The clinical and radiographic results of our study, which, to our knowledge, represent the longest duration of follow-up for a series of cementless total hip arthroplasties with use of a 28-mm metal-on-metal bearing, continue to be comparable with the results observed for other hard-on-hard bearings.

Lectin binding patterns reflect the phenotypic status of in vitro chondrocyte models

In vitro studies using chondrocyte cell cultures have increased our understanding of cartilage physiology and the altered chondrocytic cell phenotype in joint diseases. Beside the use of primary cells isolated from cartilage specimens of donors, immortalized chondrocyte cell lines such as C-28/I2 and T/C-28a2 have facilitated reproducible and standardized experiments. Although carbohydrate structures appear of significance for cartilage function, the contribution of the chondrocyte glycocalyx to matrix assembly and alterations of the chondrocyte phenotype is poorly understood. Therefore, the present study aimed to evaluate the glycoprofile of primary human chondrocytes as well as of C-28/I2 and T/C-28a2 cells in culture. First, the chondrocytic phenotype of primary and immortalized cells was assessed using real-time reverse transcriptase polymerase chain reaction, immunofluorescence, and glycosaminoglycans staining. Then, a panel of lectins was selected to probe for a range of oligosaccharide sequences determining specific products of the O-glycosylation and N-glycosylation pathways. We found that differences in the molecular phenotype between primary chondrocytes and the immortalized chondrocyte cell models C-28/I2 and T/C-28a2 are reflected in the glycoprofile of the cells. In this regard, the glycocalyx of immortalized chondrocytes was characterized by reduced levels of high-mannose type and sialic acid-capped N-glycans as well as increased fucosylated O-glycosylation products. In summary, the present report emphasizes the glycophenotype as an integral part of the chondrocyte phenotype and points at a significant role of the glycophenotype in chondrocyte differentiation.

Achskorrektur am wachsenden Skelett – therapeutisches Vorgehen

ru nt er ge la de n vo n: C or ne ll. U rh eb er re ch tli ch g es ch üt zt . ABKÜRZUNGEN BLD Beinlängendifferenz CORA Center of Rotation of Angulation CWOT Closing-Wedge-Osteotomie ED Epiphysiodese HED Hemiepiphysiodese K-Draht Kirschner-Draht MAD Mechanical Axis Deviation (Abweichung der mechanischen Achse) mLDFA mechanischer lateraler distaler Femurwinkel mMPTA mechanischer medialer proximaler Tibiawinkel OS Osteosynthese OT Osteotomie OWOT Opening-Wedge-Osteotomie PETS Percutaneous Epiphysiodesis using transphyseal Screws

Achsdeformitäten am wachsenden Skelett – diagnostisches Vorgehen

ABKÜRZUNGEN AMA anatomisch-mechanischer Winkel BLD Beinlängendifferenz CCD-Winkel Centrum-Collum-Diaphysen-Winkel CORA Center of Rotation of Angulation CPT kongenitale Pseudarthrose der Tibia DMA distale mechanische Achse HKA Hip-Knee Angle JLCA Joint Line Convergence Angle LDFA lateraler distaler Femurwinkel LDTA lateraler distaler Tibiawinkel MAD Mechanical Axis Deviation (Abweichung der mechanischen Achse) MAT Malalignment-Test mLDFA mechanischer lateraler distaler Femurwinkel MPTA medialer proximaler Tibiawinkel NSA Neck Shaft Angle PMA proximale mechanische Achse XLH X-linked hypophosphatemic rickets

Vessel architecture in human knee cartilage in children: an in vivo susceptibility-weighted imaging study at 7 T

ObjectivesTo evaluate the clinical feasibility of ultrahigh field 7-T SWI to visualize vessels and assess their density in the immature epiphyseal cartilage of human knee joints.Methods7-T SWI of 12 knees (six healthy volunteers, six patients with osteochondral abnormalities; mean age 10.7 years; 3 female, 9 male) were analysed by two readers, classifying intracartilaginous vessel densities (IVD) in three grades (no vessels, low IVD and high IVD) in defined femoral, tibial and patellar zones. Differences between patients and volunteers, IVDs in different anatomic locations, differences between cartilage overlying osteochondral abnormalities and corresponding normal zones, and differences in age groups were analysed.ResultsInterrater reliability showed moderate agreement between the two readers (κ = 0.58, p < 0.001). The comparison of IVDs between patients and volunteers revealed no significant difference (p = 0.706). The difference between zones in the cartilage overlying osteochondral abnormalities to corresponding normal zones showed no significant difference (p = 0.564). IVDs were related to anatomic location, with decreased IVDs in loading areas (p = 0.003). IVD was age dependent, with more vessels present in the younger participants (p = 0.001).ConclusionsThe use of SWI in conjunction with ultrahigh field MRI makes the in vivo visualization of vessels in the growing cartilage of humans feasible, providing insights into the role of the vessel network in acquired disturbances.Key Points• SWI facilitates in vivo visualization of vessels in the growing human cartilage.• Interrater reliability of the intracartilaginous vessel grading was moderate.• Intracartilaginous vessel densities are dependent on anatomical location and age.