Force transmission capacity of the lower limb during walking of amputees with bone-anchored prostheses compared with socket prostheses and persons with hip replacements.

BACKGROUND: Restoring the ability to walk with a prosthesis is considered a fundamental rehabilitation goal after transfemoral amputation. An essential prerequisite for achieving this goal is adequate force transmission between the prosthesis and the body. Does bone anchorage of an artificial limb permit a more normal force transmission? METHODS: Data of 15 healthy subjects, nine amputees fitted with bone-anchored prostheses, nine amputees using socket prostheses, and 18 patients with a total hip replacement were included in this multicenter, observational study. Ground reaction force was measured using Kistler force plates. Kinematics was recorded with 12 Vicon Bonita cameras. Subjects were instructed to walk at three different speeds: first at their self-selected, then at slow, and finally at fast speed. FINDINGS: Self-selected walking speeds of subject groups were significantly different, osseointegrated amputees walked the slowest. The lowest ground reaction force was measured for osseointegrated amputees on the prosthetic side, who also showed the highest force on their contralateral side. Patients with hip replacements showed values similar as healthy subjects. The vertical center of gravity movement was specific for each subject group. INTERPRETATION: The force transmission capacity of the bone-anchored prosthetic leg is limited during walking and is lower than both in socket prostheses users without symptoms and patients with total hip replacement. Therefore, active amputees well fitted with a socket prosthesis who consider a transition to bone-anchorage should be advised that their walking speed may decrease with high probability, and that their self-selected walking speed may even be slower than 3 km/h.

Establishment of a novel, cell-based autotaxin assay.

Autotaxin (ATX) plays an important role in (patho-)physiological lysophosphatidic acid (LPA) signaling. Here we describe the establishment of novel cell-based ATX assay formats. ATX-mediated LPA generation is detected by using a stable LPA receptor reporter cell line. In a first assay variant, ATX-mediated LPA generation is started in the absence of cells and the reaction mix is transferred to the reporter cells after stopping the reaction (two-tube assay). In a second assay variant, ATX is added to the reporter cells expressing the known autotaxin binding partners integrin ß1, integrin ß3 and the LPA receptor 1. LPA generation is started in the presence of cells and is detected in real-time (one-tube assay). Structurally diverse ATX inhibitors with different binding modes were characterized in both cell-based assay variants and were also tested in the well-established biochemical choline release assay. ATX inhibitors displayed similar potencies, regardless if the assay was performed in the absence or presence of cells, and comparable results were obtained in all three assay formats. In summary, our novel cell-based ATX assay formats are well-suited for sensitive detection of enzyme activity as well as for the characterization of ATX inhibitors in the presence and absence of cells.

Novel selective MMP-13 inhibitors reduce collagen degradation in bovine articular and human osteoarthritis cartilage explants.

OBJECTIVE: MMP-13 is highly upregulated in arthritis and therefore strongly implicated in the pathogenesis of osteoarthritis (OA). Selective inhibition of MMP-13 may provide the desired cartilage degradation protection, while overcoming the musculoskeletal toxicity seen with nonselective inhibition of MMPs. METHODS: Activity and selectivity of novel MMP-13 inhibitors were determined in enzymatic and collagenase assays. Inhibition kinetics and competitive binding experiments were performed. The inhibition of collagen degradation was studied in cartilage explants from OA patients and in bovine and human articular cartilage systems. RESULTS: We have identified a new class of very potent and highly selective non-zinc-binding MMP-13 inhibitors. Selective MMP-13 inhibitors completely blocked type II collagen degradation in bovine explants and showed up to 80% inhibition in human OA cartilage. CONCLUSIONS: These results indicate MMP-13 as the primary collagenase in the human OA cartilage and in the IL-1/OSM-induced cartilage degradation process and suggest that selective MMP-13 inhibitors may be a potential treatment of OA.

A new class of potent matrix metalloproteinase 13 inhibitors for potential treatment of osteoarthritis: Evidence of histologic and clinical efficacy without musculoskeletal toxicity in rat models.

OBJECTIVE: Matrix metalloproteinases (MMPs) have long been considered excellent targets for osteoarthritis (OA) treatment. However, clinical utility of broad-spectrum MMP inhibitors developed for this purpose has been restricted by dose-limiting musculoskeletal side effects observed in humans. This study was undertaken to identify a new class of potent and selective MMP-13 inhibitors that would provide histologic and clinical efficacy without musculoskeletal toxicity. METHODS: Selectivity assays were developed using catalytic domains of human MMPs. Freshly isolated bovine articular cartilage or human OA cartilage was used in in vitro cartilage degradation assays. The rat model of monoiodoacetate (MIA)-induced OA was implemented for assessing the effects of MMP-13 inhibitors on cartilage degradation and joint pain. The surgical medial meniscus tear model in rats was used to evaluate the chondroprotective ability of MMP-13 inhibitors in a chronic disease model of OA. The rat model of musculoskeletal side effects (MSS) was used to assess whether selective MMP-13 inhibitors have the joint toxicity associated with broad-spectrum MMP inhibitors. RESULTS: A number of non-hydroxamic acid-containing compounds that showed a high degree of potency for MMP-13 and selectivity against other MMPs were designed and synthesized. Steady-state kinetics experiments and Lineweaver-Burk plot analysis of rate versus substrate concentration with one such compound, ALS 1-0635, indicated linear, noncompetitive inhibition, and Dixon plot analysis from competition studies with a zinc chelator (acetoxyhydroxamic acid) and ALS 1-0635 demonstrated nonexclusive binding. ALS 1-0635 inhibited bovine articular cartilage degradation in a dose-dependent manner (48.7% and 87.1% at 500 nM and 5,000 nM, respectively) and was effective in inhibiting interleukin-1alpha- and oncostatin M-induced C1,C2 release in human OA cartilage cultures. ALS 1-0635 modulated cartilage damage in the rat MIA model (mean +/- SEM damage score 1.3 +/- 0.3, versus 2.2 +/- 0.4 in vehicle-treated animals). Most significantly, when treated twice daily with oral ALS 1-0635, rats with surgically induced medial meniscus tear exhibited histologic evidence of chondroprotection and reduced cartilage degeneration, without observable musculoskeletal toxicity. CONCLUSION: The compounds investigated in this study represent a novel class of MMP-13 inhibitors. They are mechanistically distinct from previously reported broad-spectrum MMP inhibitors and do not exhibit the problems previously associated with these inhibitors, including selectivity, poor pharmacokinetics, and MSS liability. MMP-13 inhibitors exert chondroprotective effects and can potentially modulate joint pain, and are, therefore, uniquely suited as potential disease-modifying osteoarthritis drugs.

A functional role of the membrane-proximal extracellular domains of the signal transducer gp130 in heterodimerization with the leukemia inhibitory factor receptor.

gp130 is the common signal transducing receptor subunit of interleukin (IL)-6-type cytokines. gp130 either homodimerizes in response to IL-6 and IL-11 or forms heterodimers with the leukemia inhibitory factor (LIF) receptor (LIFR) in response to LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) or cardiotrophin-like cytokine resulting in the onset of cytoplasmic tyrosine phosphorylation cascades. The extracellular parts of both gp130 and LIFR consist of several Ig-like and fibronectin type III-like domains. The role of the membrane-distal domains of gp130 (D1, D2, D3) and LIFR in ligand binding is well established. In this study we investigated the functional significance of the membrane-proximal domains of gp130 (D4, D5, D6) in respect to heterodimerization with LIFR. Deletion of each of the membrane-proximal domains of gp130 (Delta 4, Delta 5 and Delta 6) leads to LIF unresponsiveness. Replacement of the gp130 domains by the corresponding domains of the related GCSF receptor either restores weak LIF responsiveness (D4-GCSFR), leads to constitutive activation of gp130 (D5-GCSFR) or results in an inactive receptor (D6-GCSFR). Mutation of a specific cysteine in D5 of gp130 (C458A) leads to constitutive heterodimerization with the LIFR and increased sensitivity towards LIF stimulation. Based on these findings, a functional model of the gp130-LIFR heterodimer is proposed that includes contacts between D5 of gp130 and the corresponding domain D7 of the LIFR and highlights the requirement for both receptor dimerization and adequate receptor orientation as a prerequisite for signal transduction.