Electron microscopic stereological study of collagen fibrils in bovine articular cartilage: volume and surface densities are best obtained indirectly (from length densities and diameters) using isotropic uniform random sampling

Monospecific antibodies to bovine cartilage proteoglycan monomer (PG) and link protein (LP) have been used with immunoperoxidase electron microscopy to study the distribution and organization of these molecules in bovine articular cartilage. The following observations were made: (a) The interterritorial matrix of the deep zone contained discrete interfibrillar particulate staining for PG and LP. This particulate staining, which was linked by faint bands of staining (for PG) or filaments (for LP), was spaced at 75- to 80-nm intervals. On collagen fibrils PG was also detected as particulate staining spaced at regular intervals (72 nm), corresponding to the periodicity of collagen cross-banding. The interfibrillar PG staining was often linked to the fibrillar PG staining by the same bands or filaments. The latter were cleaved by a proteinase-free Streptomyces hyaluronidase with the removal of much of the interfibrillar lattice. Since this enzyme has a specificity for hyaluronic acid, the observations indicate that the lattice contains a backbone of hyaluronic acid (which appeared as banded or filamentous staining) to which is attached LP and PG, the latter collapsing when the tissue is fixed, reacted with antibodies, and prepared for electron microscopy. Thishyaluronic acid is anchored to collagen fibrils at regular intervals where PG is detected on collagen. PG and LP detected by antibody in the interterritorial zones are essentially fully extractible with 4 M guanidine hydrochloride. These observations indicated that interfibrillar PG and LP is aggregated with HA in this zone. (b) The remainder of the cartilage matrix had a completely different organization of PG and LP. There was no evidence of a similar latticework based on hyaluronic acid. Instead, smaller more closely packed particulate staining for PG was seen everywhere irregularly distributed over and close to collagen fibrils. LP was almost undetectable in the territorial matrix of the deep zone, as observed previously. In the middle and superficial zones, stronger semiparticulate staining for LP was distributed over collagen fibrils. (c) In the superficial zone, reaction product for PG was distributed evenly on collagen fibrils as diffuse staining and also irregularly as particulate staining. LP was observed as semiparticulate staining over collagen fibrils. The diffuse staining for PG remained after extraction with 4 M guanidine hydrochloride. (d) In pericellular matrix, most clearly identified in middle and deep zones, the nature and organization of reaction product for PG and LP were similar to those observed in the territorial matrix, except that LP and PG were more strongly stained and amorphous staining for both components was also observed. (e) This study demonstrates striking regional variations of ultrastructural organization of PG and LP in articular cartilage...

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Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142669 ◽

1986 ◽

Vol 44 ◽

pp. 208-209

Author(s):

Grace C.H. Yang

Keyword(s):

Chick Embryo ◽

Extracellular Space ◽

Fibroblast Cell ◽

Collagen Fibrils ◽

Electron Microscopic ◽

Voltage Electron ◽

Scanning Electron Microscopic Study ◽

Microscopic Studies ◽

And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

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POS0277 ANABOLIC EFFECT OF LNA043, A NOVEL DISEASE-MODIFYING OSTEOARTHRITIS DRUG CANDIDATE: RESULTS FROM AN IMAGING-BASED PROOF-OF-CONCEPT TRIAL IN PATIENTS WITH FOCAL ARTICULAR CARTILAGE LESIONS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.447 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 363.2-363

Author(s):

S. Trattnig ◽

C. Scotti ◽

D. Laurent ◽

V. Juras ◽

S. Hacker ◽

...

Keyword(s):

Articular Cartilage ◽

Weight Bearing ◽

Cartilage Lesion ◽

Cartilage Volume ◽

Lesion Volume ◽

Proof Of Concept ◽

T2 Relaxation Time ◽

T2 Relaxation ◽

Cartilage Lesions ◽

Articular Cartilage Lesions

Background:LNA043 is a modified, recombinant version of the human angiopoietin-like 3 (ANGPTL3) protein acting directly on cartilage-resident cells to transmit its cartilage anabolic effect. A first-in-human study previously demonstrated the favourable safety profile and the modulation of several pathways involved in cartilage homeostasis and osteoarthritis (OA)1. A previous proof-of-mechanism imaging study used high field (7 Tesla) magnetic resonance imaging (MRI) to show formation of hyaline-like tissue after a single injection of 20 mg LNA043 (unpublished data).Objectives:To evaluate non-invasively the chondro-regenerative capacity of multiple intra-articular (i.a.) injections of LNA043 in patients with articular cartilage lesions in the knee (NCT03275064).Methods:This was a randomised, double-blind, placebo (PBO)-controlled, proof-of-concept study in patients with a partial thickness cartilage lesion. In total, 58 patients (43 [20 mg LNA043]; 15 [PBO]), stratified by lesion type (condylar or patellar) were treated with 4 weekly i.a. injections. The primary endpoint was T2 relaxation time measurement as a marker of collagen fiber network, and cartilage lesion-volume was a secondary endpoint, both using 3-Tesla MRI. Assessments were performed at baseline, weeks (wks) 8, 16, 28 and 52 (the latter in 23/58 patients). While lesion-volume for the secondary endpoint was determined from manually segmented images, the cartilage volume of 21 sub-regions spanning the entire knee was also measured from 3D isotropic MR images employing an automated segmentation prototype software (MR Chondral Health 2.1 [MRCH], Siemens Healthcare)2. An exploratory analysis evaluated the treatment effect for the additive volume of the 3 subregions in the weight-bearing area of the medial femur.Results:No change in T2 relaxation time was detected between treatment and PBO groups. Manual segmentation showed continuous filling of the cartilage lesions up to wk 28 in LNA043-treated patients with femoral lesions (p=0.08, vs PBO) while no effect was detected for patients with patellar lesions. Given the limitations of measuring small, irregularly shaped lesions with manual image-analysis, the MRCH approach was used (Figure 1). In the medial femoral weight-bearing region, refilling was detected over time (Δ=123 mm3 at wk 28, N= 37, p= 0.05). No overgrowth was detected in the lateral femoral condyles without cartilage damage. The overall safety profile was favourable; only mild/moderate local reactions were reported, including a higher incidence of joint swelling (9.3% vs 0%) and arthralgia (11.6% vs 6.7%) for LNA043 vs PBO resolving spontaneously or with paracetamol/NSAIDs. No anti-drug antibodies were detected.Conclusion:Treatment with 4 weekly i.a. injections of 20 mg LNA043 resulted in regeneration of damaged cartilage in patients with femoral articular cartilage lesions. Automated measurement of cartilage volume in the femoral index region was able to detect a relevant treatment effect and was found to be more sensitive than the manual segmentation method. No sign of cartilage overgrowth was observed in healthy femoral regions. A Phase 2b study in patients with mild to moderate knee OA is in preparation.References:[1]Scotti et al. ACR Convergence 2020; Abstract #1483[2]Juras et al. Cartilage 2020; Sep 29:1-12Disclosure of Interests:Siegfried Trattnig: None declared, Celeste Scotti Shareholder of: Novartis, Employee of: Novartis, Didier Laurent Shareholder of: Novartis, Employee of: Novartis, Vladimir Juras: None declared, Scott Hacker Grant/research support from: Novartis, Brian Cole: None declared, Libor Pasa: None declared, Roman Lehovec: None declared, Pavol Szomolanyi: None declared, Esther Raithel Employee of: Siemens Healthcare GmbH, Franziska Saxer Shareholder of: Novartis, Employee of: Novartis, Jens Praestgaard Shareholder of: Novartis, Employee of: Novartis, Fabiola La Gamba Shareholder of: Novartis, Employee of: Novartis, José L. Jiménez Employee of: Novartis, David Sanchez Ramos Shareholder of: Novartis, Employee of: Novartis, Ronenn Roubenoff Shareholder of: Novartis, Employee of: Novartis, Matthias Schieker Shareholder of: Novartis, Employee of: Novartis

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