DEVELOPMENT AND RAMAN SPECTROSCOPIC CHARACTERIZATION OF AN IN VITRO MODEL OF CALCIFIED CARTILAGE
Notice bibliographique
Résumé
Osteoarthritis is a disease that affects millions of Canadians and for which there is no cure or disease-modifying drug to halt or reverse progression. While one of the primary characteristics of osteoarthritis is the degeneration of articular cartilage, histological evidence has pointed to thickening of the zone of calcified cartilage as a hallmark of its progression, the result of mechanisms which are not yet fully elucidated. It is the purpose of this work to develop an in vitro model of articular cartilage with a calcified zone, to be used in characterizing the effects of various stimuli on the mineralization process in cartilage. In this work, we aim to identify culture conditions that lead to the formation of a stable calcified layer. Deep zone cartilage was extracted from bovine metacarpophalangeal joints within 24h of death. Tissue was digested enzymatically to yield a suspension of deep zone chondrocytes, which was seeded at high density onto tissue culture membranes. Cultures were kept for up to eight weeks in media containing 10mM β-glycerophosphate (βGP), with select samples’ supplementation reduced to 0 or 3mM βGP after four weeks. In vitro-grown tissues were harvested for analysis at four-week intervals. Tissues harvested for quantification of mineral were digested in hydrochloric acid and assayed for calcium and phosphate content. Additional harvested tissues were fixed in formalin for 72 hours and histologically stained for mineral and matrix (von Kossa with hematoxylin counterstain) or mounted on plastic for Raman microspectroscopic analysis. Raman maps were collected using a 532 nm laser from a 200x200µm region in the deepest aspect of tissues, and peak masks for bands corresponding to relevant organic (Amide I at 1650cm-1) and inorganic (ν1(PO43-) at 960cm-1, ν1(CO32-) at 1060cm-1) vibrational modes were used to discriminate the zonal distribution of mineral. Calcium content of tissues revealed that mineral deposits steadily up to 8 weeks when maintained at 10mM βGP. Deposition of mineral slows significantly when supplementation is reduced to 3mM and when it is removed entirely. Von Kossa staining indicated that mineral deposits form a relatively uniform calcified layer in close proximity with the substrate and restricted to the deeper aspect of the tissues. While Raman spectroscopic analysis of tissues is ongoing, preliminary peak quantification shows that the mineral-to-matrix ratio (ν1(PO43-)/Amide I) is comparable to that in native tissue. Raman mappings indicate that removal or reduction of βGP supplementation results in reorganization of mineral visible at four weeks into a clearly-delimited uniform layer at eight weeks. The results summarized here demonstrate that a stable mineral zone may be achievable by tuning the supplementation of βGP at 4 weeks, after a contiguous layer has formed. Raman spectral mappings proved a useful tool in determining the structure and physicochemical properties of the zonal distribution in the model. Further studies with the model will include quantifying responses in the mineral layer to compressive forces, as well as inflammatory cytokine signaling.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».